carbohydrates in grain legume seeds improving nutritional quality and

CARBOHYDRATES IN GRAIN LEGUME SEEDS

Improving Nutritional Quality and Agronomic Characteristics

A3867: AMA: Hedley: First Proof:30-Oct-00

1Z:\Customer\CABI\A3867 - Hedley - Carbohydrates\A3933 - Hedley - Carbodhydrates #L.vp30 October 2000 14:56:07

Color profile: DisabledComposite Default screen

Carbohydrates in Grain LegumeSeedsImproving Nutritional Quality and AgronomicCharacteristics

Edited by

C.L. HedleyDepartment of Applied GeneticsJohn Innes CentreNorwichUK

Associate EditorsJane Cunningham and Alan Jones

CABI Publishing




CABI Publishing is a division of CAB International

CABI PublishingCAB InternationalWallingfordOxon OX10 8DEUK

Tel: +44 (0)1491 832111Fax: +44 (0)1491 833508Email: [email protected] site: http://www.cabi.org

CABI Publishing10 E 40th Street

Suite 3203New York, NY 10016

USA

Tel: +1 212 481 7018Fax: +1 212 686 7993

Email: [email protected]

© CAB International 2001. All rights reserved. No part of this publicationmay be reproduced in any form or by any means, electronically, mechanically,by photocopying, recording or otherwise, without the prior permission of thecopyright owners.

A catalogue record for this book is available from the British Library, London, UK.

Library of Congress Cataloging-in-Publication DataCarbohydrates in grain legume seeds : improving nutritional quality and

agronomic characteristics / edited by C.L. Hedley.p. cm.

Includes bibliographical references.ISBN 0-85199-467-9 (alk. paper)

1. Legumes--Seeds--Composition. 2. Carbohydrates. 3. Seed technology.I. Hedley, C.L. (Cliff)

SB177.L45 C27 2000633.3′D421--dc21 00-041354

ISBN 0 85199 467 9

Typeset by AMA DataSet Ltd, UK.Printed and bound in the UK by Biddles Ltd, Guildford and King’s Lynn.




ContentsContentsContents

Contents

Contributors xi

Preface xv

1 Introduction 1Editor: Cliff Hedley1.1 The Grain Legumes 11.2 Grain Legume Production 11.3 Grain Legume Consumption 71.4 Grain Legume Carbohydrates 11

2 Carbohydrate Chemistry 15Editor: Pavel Kadlec2.1 The Carbohydrates 15

2.1.1 Soluble carbohydrates 162.1.2 Polysaccharides 222.1.3 Other carbohydrate components 28

2.2 Chemical Analysis of the Carbohydrates 312.2.1 Soluble carbohydrates (monosaccharides, sucrose,

α-galactosides, cyclitols) 312.2.2 Polysaccharides 452.2.3 Other carbohydrate components 56

v

A3867: AMA: Hedley: First Proof:30-Oct-00 Contents



3 Nutrition 61Editor: Halina Kozlowska3.1 Introduction 613.2 The Content of Carbohydrates in Grain Legumes Utilized in

Europe 623.2.1 The content of carbohydrates in grain legumes used for

human nutrition 623.2.2 The content of carbohydrates in grain legumes used for

animal nutrition 673.3 Physiological Effect of Grain Legume Carbohydrates in Animal

Nutrition 693.3.1 Consumption of grain legume carbohydrates in feed 693.3.2 Effect of mono- and disaccharides in animal nutrition 713.3.3 Effect of oligosaccharides in animal nutrition 713.3.4 Effect of starch in animal nutrition 743.3.5 Effect of non-starch polysaccharides (NSP) in animal

nutrition 763.3.6 Effect of grain legume carbohydrates in ruminant nutrition 78

3.4 Physiological Effect of Grain Legume Carbohydrates in HumanNutrition 79

3.4.1 Nutritional classification of grain legume carbohydrates 793.4.2 Consumption of grain legume carbohydrates in food 823.4.3 Physiological effect of available carbohydrates from grain

legumes 843.4.4 Physiological effect of unavailable carbohydrates from grain

legumes 85

4 Processing 89Editor: Bálint Czukor4.1 Native Starch 89

4.1.1 Isolation 894.1.2 Granular structure 934.1.3 Functional properties 98

4.2 Modified Starch 1014.2.1 Physical methods 1024.2.2 Chemical methods 1044.2.3 Biotechnological methods 108

4.3 Food Application of Native and Modified Legume Starches 1094.4 Effect of Processing on Starch and Other Carbohydrates in

Foods 1104.4.1 Resistant starch formation 1104.4.2 Content, composition and digestibility 112

4.5 Legume Seeds as a Source of Raw Materials 116

vi Contents




5 Seed Physiology and Biochemistry 117Editor: Ryszard J. Górecki5.1 The Legume Seed 117

5.1.1 Seed components 1175.1.2 Seed development 119

5.2 The Accumulation and Biosynthesis of Carbohydrates 1225.2.1 Accumulation of soluble carbohydrates 1225.2.2 Biosynthesis of soluble carbohydrates 1255.2.3 Accumulation of starch 1285.2.4 Biochemistry of starch 130

5.3 Physiological Role of Carbohydrates in Legume Seeds 1315.3.1 During seed development 1315.3.2 During temperature stress 1365.3.3 During seed storage 1375.3.4 During germination 138

6 Biotechnology 145Editor: Nickolay Kuchuk6.1 Introduction 1456.2 In vitro Cultures and Plant Regeneration of Grain Legumes 146

6.2.1 Introduction to in vitro culture 1466.2.2 Plant regeneration systems 1486.2.3 Pioneering studies on pea regeneration 1496.2.4 Regeneration via somatic embryogenesis 1506.2.5 Regeneration via organogenesis and multiple shoot

formation 1516.2.6 Recent studies to produce more efficient, fast and reliable

systems for regeneration 1536.2.7 Factors effecting regeneration 1546.2.8 Advantages of the different developmental pathways for

in vitro manipulation 1556.3 Isolated Protoplasts from Grain Legumes 156

6.3.1 Introduction to protoplast cultures 1566.3.2 Protoplast cultures from leguminous species 1576.3.3 Application of grain legumes protoplasts to the study of

carbohydrates 1586.4 Somaclonal Variation in Grain Legumes 162

6.4.1 Introduction 1626.4.2 Factors causing variation 1636.4.3 Mechanisms of somaclonal variation 1636.4.4 Potential and disadvantages of somaclonal variation 1646.4.5 Variation in grain legumes at the cell and tissue culture

level in vitro 1656.4.6 Variation in grain legumes at the whole plant level 172

Contents vii

A3867: AMA: Hedley: First Proof:14-Nov-00 Contents

7Z:\Customer\CABI\A3867 - Hedley - Carbohydrates\A3933 - Hedley - Carbodhydrates #L.vp14 November 2000 14:12:37


6.5 Transformation Methods in Grain Legumes 1816.5.1 Introduction 1816.5.2 Gene delivery systems used in agronomically important

legumes 1826.5.3 Methods giving positive results – transgenic plants 1836.5.4 Transgenic plants and useful genes/traits transformed

into grain legumes 1846.5.5 Field trials with transgenic grain legume plants and

commercialized transgenic legume crops 1926.5.6 Future prospects 193

6.6 The Availability and Possible Manipulation of Genes Involvedin Starch Biosynthesis 195

6.6.1 Biochemical pathways of starch biosynthesis 1956.6.2 The availability of genes involved into starch biosynthesis 1966.6.3 The availability of other genes influencing starch

biosynthesis and starch quality 1986.7 The Availability and Possible Manipulation of Genes Involved

in α-Galactoside Accumulation and Degradation 1996.7.1 Biochemical pathways of α-galactoside biosynthesis 1996.7.2 The availability of genes involved in α-galactoside

accumulation and degradation and their possiblemanipulation 199

6.8 Cell Suspension Culture as a Model for Studying CarbohydrateMetabolism 201

6.8.1 Introduction 2016.8.2 Composition of plant cell walls 2026.8.3 Biosynthesis of the cell wall components 2026.8.4 Oligosaccharides as signals and substrates in the plant

cell wall 2036.8.5 Plant cell suspension cultures – a powerful tool in

investigating cell wall metabolism 204

7 Breeding and Agronomy 209Editor: Goran Engqvist7.1 Current Breeding Goals 2097.2 Breeding Techniques 211

7.2.1 Pedigree breeding 2117.2.2 Bulk selection 2127.2.3 Deviations from the pedigree and bulk methods 212

7.3 Access to Genetic Variation 2137.3.1 Germplasm banks 2137.3.2 Existing variation for the carbohydrates 2147.3.3 Newly identified genetic variation 214

7.4 Selection Methods 220

viii Contents




7.5 Physical Screening Methods 2227.5.1 Near-infrared (NI) spectroscopy 2247.5.2 Mid-infrared spectroscopy 225

7.6 Some Agronomic Considerations of Carbohydrates 2257.6.1 During plant growth and development 2257.6.2 During seed development 226

7.7 European Registration Requirements for New Varieties 2277.7.1 Background 2277.7.2 Agronomic characters 2287.7.3 Technological characters 2317.7.4 Chemical characters 232

8 Strategies for Manipulating Grain Legume Carbohydrates 233Editor: Cliff Hedley8.1 The Problems 2338.2 Strategies for Overcoming the Problems 235

8.2.1 The soluble carbohydrates 2358.2.2 Starch 2378.2.3 Fibre 237

8.3 Conclusions 238

References 241

Index 315

Contents ix




ContributorsContributorsContributors

Contributors

Mr Mike Ambrose, John Innes Centre, Norwich Research Park, Colney,Norwich NR4 7UH, UK. Tel: +44 1603 450630; fax: +44 1603 450045;Email: [email protected]

Dr Pilar Aranda, Institute of Nutrition, Pharmacy Faculty, CampusUniversitario de Cartuja, 18071 Granada, Spain. Tel: +34 58 243885;fax: +34 58 243879; Email: [email protected]

Dr Charlotte Bjergegaard, Royal Veterinary and Agricultural University,Department of Chemistry, Thorvaldsensvej 40, 1871 Frederiksberg,Denmark. Tel: +45 35 282432; fax: +45 35 282398

Dr Tatiana Bogracheva, John Innes Centre, Norwich Research Park,Colney, Norwich NR4 7UH, UK. Tel: +44 1603 450233; fax: +44 1603450045; Email: [email protected]

Prof. Nikolai Chekalin, Breeding Firm ‘NIVA-1’, Lomany str 14-53, 314 022Poltava, Ukraine. Tel: +380 5322 70889; fax: +380 5322 22957; Email:[email protected]

Dr Peter Chekrygin, Plant Production Institute, Moskovskiy Prospect 142,310060 Kharkov, Ukraine. Tel: +38 0572 921285/924343; fax: +38 0572920354

Dr Zsuzsanna Cserhalmi, Central Food Research Institute, Herman Otto ut15, PO Box 393, H-1022 Budapest, Hungary. Tel: +36 1 355 8244; fax:+36 1 355 8928

Dr Balint Czukor, Central Food Research Institute, Herman Otto ut 15, POBox 393, H-1022 Budapest, Hungary. Tel: +36 1 355 8244; fax: +36 1355 8928; Email: [email protected]

xi

A3867: AMA: Hedley: First Proof:30-Oct-00 Contributors



Dr Jana Dostalova, Dept of Food Chemistry and Analysis, PragueInstitute of Chemical Technology, Technicka 5, 166 28 Prague 6, CzechRepublic. Tel: +4202 2435 3264; fax: +4202 311 9990

Mr Goran Engqvist, Svalof Weibull AB, Forage Crop Dept, S-268 81 Svalov,Sweden. Tel: +46 418 667159; fax: +46 418 667102; Email: [email protected]

Prof. Gabriel Fordonski, Prorector, Department of Plant Diagnostics andPathophysiology, University of Warmia and Mazury, 10-718 Olsztyn,Plac Lodzki 3, Poland. Tel: +48 89 523 49 52; fax: +48 89 523 48 81

Prof. Jozef Fornal, Institute of Animal Reproduction and Food Research,Division of Food Science, PO Box 55, ul Tuwima 10, 10-718 Olsztyn,Poland. Tel: +48 89 523 63 13; fax: +48 89 523 78 24; Email:[email protected]

Dr Juana Frias, Instituto de Fermentaciones Industriales CSIC, calle Juande la Cierva 3, 28006 Madrid, Spain. Tel: +34 1 5622900; fax: +34 15644853; Email: [email protected]

Prof. Ryszard Gorecki, Rector, University of Warmia and Mazury,10-718 Olsztyn, Plac Lodzki 3, Poland. Tel: +48 89 523 49 52; fax: +4889 523 48 81; Email: [email protected]

Dr Miroslav Griga, AGRITEC Research, Breeding & Services Ltd, 787 01Sumperk, Zemedelska 16, Czech Republic. Tel: +420 649 382126; fax:+420 649 382999; Email: [email protected]

Dr Krzysztof Gulewicz, Institute of Bioorganic Chemistry, ul Noskowskiego12/14, 61-704 Poznan, Poland. Tel: +48 618 528503; fax: +48 618520532; Email: [email protected]

Dr Horia Halmajan, Bucharest University of Agronomical Science andVeterinary Medicine, Department of Phytotechnics, Bd Marasti nr 59,71331 Bucharest, Romania. Tel: +40 1 2223700/248; fax: +40 12300195; Email: [email protected]

Prof. Cliff Hedley, John Innes Centre, Norwich Research Park,Norwich NR4 7UH, UK. Tel: +1603 450234; fax: +1603 450045; Email:cliff.hedley@bbsrc. ac.uk

Dr Marcin Horbowicz, Research Institute of Vegetable Crops, Konstytucji 3Maja 1/3, 96-100 Skierniewice, Poland. Tel: +48 46 332604; fax: +48 46333186; Email: [email protected]

Mr Alan Jones, John Innes Centre, Norwich Research Park, NorwichNR4 7UH, UK. Tel: +1603 450253; fax: +1603 450027; Email:alan.jones@bbsrc. ac.uk

Mr Rupert Jones, John Innes Centre, Norwich Research Park, NorwichNR4 7UH, UK. Tel: +1603 450234; fax: +1603 450045; Email:rupert.jones@ bbsrc.ac.uk

Prof. Pavel Kadlec, Institute of Chemical Technology, Prague, Technicka5, 166 28 Prague 6, Czech Republic. Tel: +420 2 311 7070; fax: +420 2311 9990; Email: [email protected]

xii Contributors




Dr Saima Kalev, Jogeva Plant Breeding Institute, EE 2350 Jogeva, Estonia.Tel: +372 77 22565; fax: +372 77 60126

Prof. Pavel Kintia, Institute of Genetics, Moldavian Academy of Sciences,20 Padurilor Str., 2002 Chisinau, Moldova. Fax: +373 2 542823; Email:[email protected]

Dr Georgina Kosturkova, Department of Cell Genetics, Institute ofGenetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.Tel: +359 2 759042, x246 or 239; fax: +359 2 757087; Email:[email protected]

Dr Elisabeth Kovacs, Jozsef Attila University, Szeged College of FoodIndustry, Szeged Higher Education Federation, Mars ter 7, 6724Szeged, Hungary. Tel: +36 62 456022; fax: +36 62 456005; Email:[email protected]

Prof. Halina Kozlowska, Institute of Animal Reproduction and FoodResearch, Division of Food Science, PO Box 55, ul Tuwima 10, 10-718Olsztyn, Poland. Tel: +48 89 524 03 13; fax: + 48 89 524 01 24; Email:[email protected]

Prof. Christo Kratchanov, Laboratory of Biological Active Substances, 95 V.Aprilov Str., 4002 Plovdiv, PO Box 27, Bulgaria. Tel: +359 32 452140;fax: +359 32 440102

Dr Maria Kratchanova, Laboratory of Biological Active Substances, 95 V.Aprilov Str., 4002 Plovdiv, PO Box 27, Bulgaria. Tel: +359 32 452140;fax: +359 32 440102

Dr Nickolay Kuchuk, International Institute of Cell Biology, NASU,Zabolotnogo str. 148, 252022 Kiev, Ukraine. Tel/fax: +380 44 252 1786;Email: [email protected]

Dr Leslaw Lahuta, Department of Plant Physiology and Biotechnology,University of Warmia and Mazury, 10-718 Olsztyn, Plac Lodzki 3,Poland. Tel: +48 89 523 48 24; fax: +48 89 523 48 81; Email:[email protected]

Dr Grazyna Lewandowicz, Starch and Potato Products, ResearchLaboratory, ul. Zwierzyniecka 18, 60-814 Poznan, Poland. Tel: +48 618668045; fax: +48 618 417610; Email: [email protected]

Dr Maria Lopez-Jurado, Institute of Nutrition, Pharmacy Faculty, CampusUniversitario de Cartuja, 18071 Granada, Spain. Tel: +34 58 243885;fax: +34 58 243879; Email: [email protected]

Ing Martin Mrskos, UKZUZ, Central Institute for Supervising and Testingin Agriculture, Variety Testing Dept., Hroznova 2, 656 06 Brno, CzechRepublic. Tel: +420 5 4332 1304 x224; fax: +420 5 4321 2440; Email:[email protected]

Prof. Jan Pokorny, Department of Food Chemistry and Analysis, PragueInstitute of Chemical Technology, Technicka 5, 166 28 Prague 6,Czech Republic. Tel: +4202 2435 3264; fax: +4202 311 9990; Email:[email protected]

Contributors xiii




Dr Paolo Ranalli, Istituto Sperimentale per le Colture Industriali, Via diCorticella 133, 40129 Bologna, Italy. Tel: +39 51 6316847; fax: +39 51374857; Email: [email protected]

Dr Ion Scurtu, Research Institute for Vegetable and Flower Growing, 8268Vidra, S.A.I., Romania. Tel/fax: +40 13139282/6395; Email: [email protected]

Dr Ildiko Schuster-Gajzago, Central Food Research Institute, HermanOtto ut 15, PO Box 393, H-1022 Budapest, Hungary. Tel: +36 1 3558244; fax: +36 1 355 8928; Email: [email protected]

Dr Maria Soral-Smietana, Institute of Animal Reproduction and FoodResearch, PO Box 55, ul. Tuwima 10, 10-718 Olsztyn, Poland. Tel: +4889 523 46 51; fax: +48 89 524 01 24

Prof. Hilmer Sorensen, Royal Veterinary and Agricultural University,Department of Chemistry, Thorvaldsensvej 40, 1871 Frederiksberg,Denmark. Tel: +45 35 282432; fax: +45 35 282398; Email: [email protected]

Prof. Mladenka Ilieva-Stoilova, Institute of Microbiology, Laboratory ofBiotechnology and Microbiology, 26 Maritza Blvd., 4002 Plovdiv,Bulgaria. Tel: +359 2 438130; fax: +359 2 700109; Email: [email protected]

Mr Jan Urban, AGRITEC Research, Breeding & Services Ltd, 787 01Sumperk, Zemedelska 16, Czech Republic. Tel: +420 649 382126;fax: +420 649 382999

Prof. Gloria Urbano, Institute of Nutrition, Pharmacy Faculty, CampusUniversitario de Cartuja, 18071 Granada, Spain. Tel: +34 58 243885;fax: +34 58 243879; Email: [email protected]

Prof. Concepcion Vidal, Instituto de Fermentaciones Industriales CSIC,calle Juan de la Cierva 3, 28006 Madrid, Spain. Tel: +34 1 5622900;fax: +34 1 5644853; Email: [email protected]

Prof. Zenon Zdunczyk, Institute of Animal Reproduction and FoodResearch, Division of Food Science, PO Box 55, ul Tuwima 10, 10-718Olsztyn, Poland. Tel: +48 89 523 63 13; fax: +48 89 523 78 24

xiv Contributors




PrefacePrefacePreface

Preface

Grain legumes have been an important part of the social evolution ofmankind over the past 10,000 years, carbonized remains having beendiscovered in Neolithic settlements. In more recent times, however, thesecrops have become less fashionable, particularly in Europe and NorthAmerica, as the consumption of meat has increased and economicpressures have promoted the growth of cereal monocultures in agriculturalsystems. The increasing awareness of environmental problems caused bypollution from the over use of fertilizers and a growing interest in morehealthy diets is again focusing interest on to this important group of plants.The neglect of grain legumes has resulted in crops that are relativelyunderdeveloped compared with cereals such as maize, wheat and rice.These crops have been subjected to intensive scientific and technologicalinvestigations following substantial public and private financial investment.It is hoped that this book will begin the process of rectifying this imbalance.

The book is the result of a combined effort from scientists coveringmany disciplines interacting within a European Union-funded Copernicusproject entitled ‘Carbohydrate Biotechnology Network for Grain Legumes’(CABINET; contract number IC15-CT96-1007). The CABINET projectlinked 30 participants from 14 countries across Europe, including statesassociated within the former Soviet Union.

I would like to thank all members of CABINET for their friendship andfor making the past 3 years so enjoyable and rewarding. With regard to thebook I would like to give my special thanks to those members of CABINETwho took on the task of sub-editing the various chapters and for integratingthe work within each of the disciplines. Finally I would like to thank my two

xv

A3867: AMA: Hedley: First Proof:30-Oct-00 Preface



colleagues at the John Innes Centre, Jane Cunningham and Alan Jones.Jane has been at the hub of the CABINET project, responsible for allcommunication relating to meetings and to the book. Jane’s cheerful andefficient way of administering the project is a major reason why it has beenso successful. Alan has been a constant source of support in the running ofthe project and has taken on major responsibilities for editing the wholebook, including constructing and redrawing all of the tables and figures.

Cliff Hedley

xvi Preface

A3867: AMA: Hedley: First Proof:30-Oct-00 Preface



IntroductionC. Hedley1

1IntroductionEditor: Cliff Hedley

There are two things which I am confident I can do very well: one is anintroduction to any literary work, stating what it is to contain, and how itshould be executed in the most perfect manner . . .

Boswell Life, vol. 1, p. 2 (1755)Samuel Johnson (1709–1784), English poet, critic and lexicographer

1.1 The Grain Legumes

There are about 60 domesticated grain legume species throughout theworld, the major ones being summarized in Table 1.1. The nutritionalpotential of the seeds from this group of plants is universally recognizedsince they contain high levels of protein and, depending on the species, ahigh proportion of either starch or oil. Legumes play a very importantrole in sustainable agriculture, particularly in developing countries, mainlybecause of the ability of legume plants to fix atmospheric nitrogen by asymbiotic relationship with nitrogen-fixing bacteria (Rhizobium spp.).

1.2 Grain Legume Production

On a world scale, the area of grain legumes excluding soybean (often cate-gorized as an oil seed) has been static for many years at about 68 millionhectares (Table 1.2). Likewise, the total production in the world (excludingsoybean) has stabilized at between 55 and 57 million t (Table 1.2). On this

©CAB International 2001. Carbohydrates in Grain and Legume Seeds(ed. C.L. Hedley) 1

A3867: AMA: Hedley: First Proof:30-Oct-00 1



2 C. Hedley

A3867: AMA: Hedley: First Proof:14-Nov-00 1

Com

mon

nam

eLa

tinna

me

Des

crip

tion

Soyb

ean

Gly

cine

max

(L.)

Mer

rill

Econ

omic

ally

and

agri

cultu

rally

the

mos

tim

port

antl

egum

ein

the

wor

ld,p

rovi

ding

prot

ein

and

oilt

oth

efo

odan

dan

imal

feed

indu

stry

and

the

base

ingr

edie

nts

for

hund

reds

ofch

emic

alpr

oduc

ts.N

otag

ricu

ltura

llyim

port

anti

nEu

rope

.Als

oco

nsum

edas

tofu

and

soy

sauc

ein

Far

East

ern

cook

ery.

Whi

te(fl

ower

ed)

lupi

nLu

pinu

sal

bus

L.O

rigi

natin

gin

the

Med

iterr

anea

nar

eaan

dfo

und

wid

ely

inN

orth

Am

eric

aov

er20

0sp

ecie

sar

ekn

own.

The

nam

elu

pin

(som

etim

essp

elle

das

lupi

ne)c

omes

from

the

Latin

for

‘wol

f’an

dde

rive

sfr

omth

em

ista

ken

belie

ftha

tthe

plan

t‘w

olfe

d’m

iner

als

from

the

soil.

The

cont

rary

istr

ue,l

upin

sai

dso

ilfe

rtili

tyan

dar

edr

ough

ttol

eran

t.A

gric

ultu

rally

they

are

grow

nfo

ran

imal

feed

.

Yel

low

lupi

nLu

pinu

slu

teus

L.

Lupi

nus

mut

abili

sL.

Swee

tlup

inLu

pinu

san

gust

ifoliu

sL.

Chi

ckpe

aC

icer

arie

tinum

L.A

lso

calle

dB

enga

lgra

m,b

oot,

chan

ach

ola,

chol

e,gr

am,h

omm

es,p

ois

chic

hean

dga

rban

zobe

an.G

roup

edin

totw

oty

pes

base

don

seed

colo

uran

dge

ogra

phic

dist

ribu

tion;

desi

type

are

ofIn

dian

orig

inan

dka

buli

type

are

ofM

edite

rran

ean,

Nor

thA

fric

anan

dW

est

Asi

anor

igin

.Des

isee

dsar

eab

out1

20g,

wri

nkle

dat

the

beak

with

brow

n,fa

wn,

yello

w,

oran

ge,b

lack

orgr

een

colo

ur,n

orm

ally

dehu

lled

and

split

toob

tain

dhal

.Kab

ulis

eeds

are

abou

t400

g,ar

ew

hite

/cre

amco

lour

edan

dus

edex

clus

ivel

yby

cook

ing

who

lese

eds.

Thir

dm

osti

mpo

rtan

tgra

inle

gum

ecr

opaf

ter

bean

san

dpe

as.

Mun

gbe

anV

igna

radi

ata

L.W

ilcze

kC

omm

only

grow

nin

Sout

hA

sia,

Chi

naan

dIn

dia,

used

asgr

een

vege

tabl

eor

assp

rout

ing

shoo

ts.

Pige

onpe

aC

ajan

usca

jan

(L.)

Mill

sp.

Use

dw

orld

-wid

efo

rhu

man

cons

umpt

ion,

inIn

dia

seed

and

plan

tuse

das

anim

alfe

ed.

Tabl

e1.

1.A

listo

fthe

com

mon

grai

nle

gum

esgr

own

inth

ew

orld

and

thei

rus

es.



Introduction 3


Jack

bean

Can

aval

iaen

sifo

rmis

(L.)

D.C

.A

lso

calle

dsw

ord

bean

.Gro

wn

intr

opic

s,N

orth

and

East

Afr

ica,

Far

East

and

Indi

a.U

sed

asa

dry

bean

,als

oea

ten

asa

vege

tabl

ean

dus

edas

agr

een

man

ure

and

cove

rcr

op.Y

ield

sco

mpa

refa

vour

ably

with

the

com

mon

bean

and

nutr

ition

ally

they

are

sim

ilar.

Com

mon

bean

Phas

eolu

svu

lgar

isL.

Als

oca

lled

Fren

ch,g

arde

n,ha

rico

t,ki

dney

,pin

to,n

avy

(bak

edbe

an),

blac

k,pi

nk,b

lack

eye,

cran

berr

y,gr

eatn

orth

ern

ordr

ybe

an.B

eans

harv

este

dgr

een

inpo

dsor

dry

for

hum

anco

nsum

ptio

nor

used

for

cann

ing

orfr

eezi

ng.O

rigi

nate

din

Cen

tral

and

Sout

hA

mer

ica.

Faba

bean

(fiel

dbe

an)

Vic

iafa

baL.

Als

oca

lled

broa

d,ho

rse

ortic

kbe

an.H

arve

sted

fres

hfo

rhu

man

cons

umpt

ion

orus

edfo

rca

nnin

gor

free

zing

.The

seed

isha

rves

ted

dry

for

anim

alfe

ed.

Lent

ilLe

nscu

linar

isM

edic

.L.

One

ofth

em

osta

ncie

ntof

culti

vate

dfo

ods.

Ofu

nkno

wn

orig

in,t

hele

ntil

isw

idel

ygr

own

thro

ugho

utEu

rope

,Asi

aan

dN

orth

Afr

ica

buti

slit

tlegr

own

inth

eW

este

rnH

emis

pher

e.So

calle

dbe

caus

eof

the

lens

shap

eof

the

seed

s.

Cow

pea

Vig

naun

guic

ulat

a(L

.)W

alp

Als

oca

lled

Poon

ape

a,ya

rd-l

ong

bean

,cat

jang

.An

impo

rtan

tfoo

dso

urce

inA

fric

a(N

iger

ia),

part

sof

Asi

aan

dM

edite

rran

ean

area

.Typ

ical

lya

dhal

-typ

epa

ste

ism

ade

from

the

soak

edde

hulle

dse

eds.

Pea

Pisu

msa

tivum

L.O

neof

the

mos

tanc

ient

ofcu

ltiva

ted

food

s.Th

ough

tto

have

orig

inat

edin

seve

ralc

entr

es;

Ethi

opia

,Asi

aM

inor

,Cau

casu

sar

eaan

dA

fgha

nist

an.G

arde

npe

asw

ere

used

byM

ende

l(1

865)

inhi

sst

udie

sof

inhe

rita

nce.

The

whi

teflo

wer

edor

field

pea

isgr

own

asa

vini

ngcr

opfo

rhu

man

cons

umpt

ion

oras

aco

mbi

ning

crop

for

anim

alfe

ed.C

olou

red

flow

ered

vari

etie

sar

egr

own

for

anim

alfe

ed.



4 C. Hedley


1997

1998

1999

Are

aPr

oduc

tion

Are

aPr

oduc

tion

Are

aPr

oduc

tion

Wor

ldto

tal

68,3

24,1

3755

,512

,759

66,9

13,5

5656

,143

,600

68,3

20,5

6857

,514

,450

Euro

peto

tal

4,78

0,84

910

,260

,753

4,55

0,67

09,

258,

881

4,18

1,10

78,

225,

785

Alb

ania

68,3

28,4

5968

,322

,546

68,3

27,7

1068

,324

,683

68,3

32,3

0068

,330

,000

Aus

tria

68,3

27,7

8368

,386

,282

68,3

27,0

4368

,385

,254

68,3

27,3

3368

,385

,600

Bel

arus

68,2

62,2

0068

,492

,000

68,2

69,0

0068

,334

,000

68,2

12,0

0068

,253

,000

Bel

gium

–Lux

embo

urg

68,1

34,1

9968

,318

,958

68,3

24,0

4068

,316

,300

68,3

24,0

5068

,315

,900

Bos

nia

and

Her

zego

vina

68,3

12,6

2068

,314

,740

68,3

12,6

2068

,314

,740

68,3

12,6

2068

,314

,740

Bul

gari

a68

,354

,537

68,3

40,0

1468

,351

,309

68,3

37,2

6368

,355

,330

68,3

39,1

64C

roat

ia68

,311

,471

68,3

24,0

3968

,327

,417

68,3

24,6

8468

,328

,150

68,3

26,0

50C

zech

Rep

ublic

68,3

51,4

2868

,104

,697

68,3

59,3

9568

,133

,391

68,3

52,6

9768

,106

,602

Den

mar

k68

,397

,800

68,3

87,0

0068

,106

,800

68,3

87,8

5768

,106

,800

68,3

57,8

57Es

toni

a68

,328

,700

68,3

17,0

0068

,326

,200

68,3

12,3

9068

,235

,700

68,3

10,6

08Fi

nlan

d68

,326

,000

68,3

13,1

0068

,324

,900

68,3

11,0

0068

,324

,900

68,3

11,0

00Fr

ance

68,6

29,2

693,

121,

076

68,6

30,3

073,

305,

621

68,4

97,7

002,

657,

000

Ger

man

y68

,185

,000

68,5

87,9

0068

,235

,315

68,7

79,8

5368

,227

,061

68,7

92,6

74G

reec

e68

,326

,374

68,3

42,2

5268

,326

,016

68,3

41,4

1968

,325

,870

68,3

41,2

50H

unga

ry68

,361

,936

68,1

18,4

3668

,363

,294

68,1

40,9

0768

,362

,365

68,1

40,9

07

Tabl

e1.

2.To

talc

ultiv

atio

nar

ea(h

a)an

dpr

oduc

tion

(t)of

grai

nle

gum

esin

the

Wor

ldan

dEu

rope

(FA

O,2

000)

.



Introduction 5


Irel

and

68,3

24,2

0068

,319

,000

68,3

24,2

0068

,319

,000

68,2

34,2

0068

,319

,000

Italy

68,3

74,6

7468

,115

,902

68,3

78,3

4768

,127

,408

68,3

80,1

2468

,137

,938

Latv

ia68

,323

,200

68,3

21,6

5068

,324

,946

68,3

21,2

7068

,324

,946

68,3

21,8

50Li

thua

nia

68,3

52,3

0068

,106

,000

68,3

66,1

0068

,104

,100

68,3

52,3

0068

,106

,400

Mac

edon

ia68

,312

,123

68,3

30,9

1068

,314

,698

68,3

30,4

8768

,314

,698

68,3

30,4

87M

alta

68,3

21,4

5068

,321

,200

68,3

22,4

5068

,321

,200

68,2

2,34

5068

,321

,200

Mol

davi

a68

,346

,563

68,3

63,8

8068

,353

,483

68,3

71,0

2768

,353

,100

68,3

72,5

11N

ethe

rlan

ds68

,324

,200

68,3

16,8

0068

,324

,200

68,3

16,8

0068

,324

,200

68,3

16,8

00Po

land

68,1

44,9

2968

,260

,052

68,1

48,9

2868

,289

,017

68,1

48,9

2868

,289

,017

Port

ugal

68,3

54,8

7668

,330

,699

68,3

52,6

1068

,331

,810

68,3

52,6

1068

,331

,810

Rom

ania

68,3

48,4

4768

,375

,334

68,3

59,0

6468

,372

,282

68,3

38,0

7468

,346

,572

Rus

sian

Fede

ratio

n1,

239,

840

1,80

2,22

01,

068,

850

68,8

88,5

501,

037,

700

68,8

77,0

00Sl

ovak

ia68

,339

,038

68,3

97,8

0768

,341

,443

68,1

03,8

5568

,343

,676

68,3

97,7

35Sl

oven

ia68

,324

,267

68,2

36,6

1468

,324

,280

68,3

27,5

1368

,234

,280

68,3

27,5

13Sp

ain

68,6

13,1

0068

,390

,000

68,5

32,4

0068

,382

,800

68,5

04,1

0068

,291

,400

Swed

en68

,353

,400

68,1

68,8

0068

,345

,900

68,1

42,3

0068

,343

,900

68,1

39,6

00Sw

itzer

land

68,2

34,6

7968

,314

,500

68,2

34,2

6668

,312

,100

68,3

24,4

0068

,311

,100

Uni

ted

Kin

gdom

68,1

96,8

0068

,748

,000

68,2

01,2

0068

,701

,000

68,1

88,5

0068

,373

,200

Ukr

aine

68,6

35,3

001,

072,

800

68,5

55,9

0068

,770

,000

68,5

03,0

0068

,585

,500

Yug

osla

via

68,3

81,0

4568

,142

,000

68,3

78,0

4568

,137

,000

68,3

63,0

4568

,118

,000



basis, the most important grain legume is dry bean, covering many varietiesof Phaseolus vulgaris and amounting to about 28 million hectares. The mostimportant countries for dry bean production are India and Brazil, withEurope accounting for only about 2% of the world production.

In Europe, the area of grain legumes (excluding soybean) hasdecreased from about 5 million hectares in 1997 to about 4 million in 1999(Table 1.2). In 1999 the total production of grain legumes in Europewas about 8 million t, the highest proportion being produced in France(c. 32%), followed by Russia (c. 11%), Germany (c. 10%), the UK (c. 9%)and the Ukraine (c. 7%; Table 1.2). The dominant legume within Europeis pea, with a total growing area in 1997 of about 1 million hectares and aproduction of about 4 million t, more than 70% of which was produced inFrance.

Soybean is by far the most economically important legume in the worldand is cultivated on around 60 million hectares. The main producers ofsoybean are the USA, Brazil, China and Argentina, which together accountfor 82% of the total world area. The cultivation of this species in Europe israther small and is only about 1% of the world area (FAO, 1998). In the lastfew years the area of soybean in Europe has decreased from about 1 millionhectares in 1989 to about 0.7 million hectares in 1995, with Italy, Romaniaand France being the largest producers.

There are large differences across the world in the proportion ofcultivated land occupied by legumes (Table 1.3). In the USA, legumesaccount for about 16% of the total arable land, the great majority of whichis due to soybean production. In Europe, this proportion is much lower,amounting to about 7% in Portugal, 5% in Austria and Denmark, 4% inFrance, Italy and the UK, about 3% in Hungary and 2% in the CzechRepublic. Legumes play an even less important role in the agriculture ofIreland, Finland, Germany and Belgium.

In the world, the average seed yield of grain legumes has been at asimilar level for many years, amounting to about only 0.8 t ha−1 (Table 1.4).On average, seed yields in Europe are about three times higher than thisworld average and generally higher than in the USA. France has the highestseed yields at about 4.7 t ha−1, with Ireland, Belgium, The Netherlands,Denmark, the UK and Austria below this, but still at satisfactory levels(3.3–4.7 t ha−1). In Europe, the lowest yields of legumes are found inPortugal, Spain, Bulgaria and Romania. The high yields found in mostEuropean countries can be attributed to more productive varieties beinggrown within a more intensive agricultural system.

Also, there is variation between legume species for yield across theworld. The average yield of peas on a world scale is 1.7 t ha−1, which is halfthe average yield achieved in Europe. The highest pea yields in Europe arefound in The Netherlands, Belgium, France and Ireland, with yields from4.2 to 4.6 t ha−1 followed by Denmark, UK, Austria and Italy, with yieldsfrom 3.2 to 3.8 t ha−1. The Czech Republic, Hungary and Poland have lower

6 C. Hedley




yields in the range 2.1–2.6 t ha−1, which is similar to the yields obtained inthe USA.

The average yield of dry beans is low throughout the world, thehighest yields being found in France, Greece and Italy (1.7–1.9 t ha−1),while Portugal, which is the main producer of dry beans in Europe,achieves very low yields.

Average world yields of soybeans are relatively high, at about 2 t ha−1, inspite of the large area of this species under cultivation. The average yieldwithin Europe is about 2.4 t ha−1, with the main producer, Italy, and Greeceattaining more than 3 t ha−1. In Romania, where the acreage of soybean isrelatively high, yields of soybeans are about half those of Italy. By far themajor producer of soybeans in the world is the USA, with a total productionof about 60 million t, which is about half of the total world production.

1.3 Grain Legume Consumption

Of those grain legume species that have been domesticated, only a few findwider application in human food production and animal feed. In Europe,

Introduction 7


CountryGrain legumes

(excluding soybean)Soybean

productionTotal legume production

(including soybean)

EuropeAustria 2.1 3.0 5.1Belgium 0.8 – 0.8Bulgaria 1.4 0.4 1.8Czech Republic 2.2 0.1 2.3Denmark 4.6 – 4.6Finland 0.4 – 0.4France 3.6 0.4 4.0Germany 0.7 – 0.7Greece 0.8 0.1 0.9Hungary 2.1 0.5 2.6Ireland < 0.1< – < 0.1<Italy 1.1 2.5 3.6Netherlands 1.2 – 1.2Poland 1.7 – 1.7Portugal 6.6 – 6.6Romania 0.8 1.3 2.1Spain 1.4 0.1 1.5Sweden 1.1 – 1.1UK 3.5 – 3.5

USA 0.4 15.5 15.9

World total 0.5 0.4 0.9

Table 1.3. The proportion of cultivated land used for legume production (%).



most of the home-produced and imported seed (about 95%) of chickpea,lentil, vetch and bean, and a considerably smaller proportion of faba bean(17%) and pea seeds (4%), are used for human food (Table 1.5).

8 C. Hedley


Country 1990 1991 1992 1993 1994 1995

Austria 3.6 3.5 3.5 2.4 3.4 3.7Belgium 4.5 3.8 4.1 4.4 4.4 4.6Bulgaria 0.9 1.0 1.2 0.8 0.9 1.0Czech Republic – – – 2.4 2.3 2.4Denmark 4.8 4.2 2.6 3.8 3.7 3.3Finland 2.9 2.5 1.8 2.4 2.2 2.2France 5.1 4.7 4.7 5.0 5.0 4.7Germany 2.6 3.1 2.5 2.7 2.7 –Greece 1.4 1.5 1.6 1.5 1.6 1.6Hungary 2.2 2.2 2.1 1.5 2.3 2.2Ireland 4.4 4.7 4.7 4.7 4.7 –Italy 1.3 1.7 1.7 1.6 1.6 1.6Netherlands 4.4 3.6 4.2 4.2 4.0 4.0Poland 1.9 2.1 1.1 1.9 1.4 1.8Portugal 0.3 0.3 0.3 0.3 0.3 0.3Romania 0.9 1.0 1.1 1.2 1.1 1.1Spain 0.8 0.7 0.6 0.7 0.7 0.6Sweden 2.5 2.5 2.5 2.5 2.4 –UK 3.6 3.3 3.2 3.9 3.2 3.2

Europe total 2.6 2.5 2.5 2.8 2.7 2.5

USA 1.7 2.0 1.7 1.6 1.8 1.9

World total 0.9 0.8 0.8 0.9 0.8 0.8

Table 1.4. Grain legume yields in the World and Europe (t ha−1) (Carrouee, 1995).

SpeciesProduction(× 1000 t)

Import(× 1000 t)

Food use(%)

Feed use(%)

Pea (P. sativum) 4800 800 4 91Faba bean (V. faba) 1020 350 17 80Lupin (Lupinus spp.)a 19 300 2 97Chickpea (C. arietinum) 40 105 95 –Lentil (L. culinaris) 35 210 95 –Beansb and other grain legumes 155 390 95 –

aLupins – sweet cultivars of L. albus, L. luteus and L. angustifolius.bBeans – including; common bean (P. vulgaris), Lima bean (P. lunatus), mungobean (V. mungo) and others.

Table 1.5. Food and feed uses of grain legumes for 12 EU member states(Carouee, 1995).



Overall, however, five times more grain legume seed is used for animalfeed than for human food. Within the European Union (EU), the produc-tion of grain legumes accounts for about 25% of the total protein requiredfor animal feed. The contribution of grain legumes to the total amount ofanimal feed, however, is only about 8% (Table 1.6).

The pea is by far the most important grain legume in Europe, com-prising about 77% of the total, followed by faba bean (c. 19%) and lupin(c. 4%). Within the EU in 1996–1997, about 3.5 million t of dry peas(c. 88%) and about 0.5 million t of faba beans were utilized in animalfeed stuff (Bourdillon, 1998), amounting to about 5% of the total rawingredients used by the EU compound feed industry (Table 1.7). In Francethe proportion of grain legumes in the concentrate mixture is about10% and in Belgium about 12%, while in Germany the proportion is onlyabout 3%. The major part is made up of cereals and other high-proteincomponents, in particular soybean (Pahl, 1998).

With the exception of Spain, which imports most of its grain legumesfrom non-European countries (above 40%), most (greater than 80%) ofthe grain legume seed consumed within Europe is European in origin –

Introduction 9


Grain legumes Other protein sources

Share in protein crops 25.0 75Share of protein requirement 8.0 92Animal consumption of grain legumes

Pea 76.8 –Faba bean 18.8 –Lupin 4.4 –

Table 1.6. Grain legume share (as a percentage) in protein crops and proteinrequirement for animal production within the EU (Carouee, 1995).

CountryTotal

consumption (t)Compounded feed

production (t)Share in feed

production (%)

Belgium 5,661 5,325 12.4Denmark 5,222 5,666 3.9France 2,114 21,998 9.6Germany 5,859 19,326 4.4Italy 5,598 11,700 5.1Netherlands 5,586 16,495 3.6Spain 5,969 15,215 6.4UK 5,450 12,657 3.6

EU 5,409 1,248 5.4

Table 1.7. Grain legumes used for animal feed in Europe (Gatel and Champ,1998).



France, Russia, Germany, the UK and the Ukraine being the largestproducers (Gatel and Champ, 1998). In most EU countries grain legumesare produced by farmers in quantities that are too small and with qualitythat is too variable. For this reason, the animal feed industry usually prefersto use protein crops from abroad (e.g. soybean meal), which are morehomogenous.

In 1996, the average consumption of grain legumes in the world was6.36 kg per person, made up of 2.51 kg of dry bean, 0.61 kg of pea and3.26 kg of other grain legumes (Table 1.8). Human consumption of grainlegumes in European countries is relatively low. According to FAO data(1996) in central, northern and western continental Europe the consump-tion of grain legumes in 1996 averaged 2.37 kg per person, with a range of0.2–9.3 kg depending on the country (Table 1.8). In many regions of theworld, including Mediterranean countries, the Middle East, North Americaand East Africa, the consumption of grain legumes is three to four timeshigher (ranging from 7.0 to 10.9 kg per person).

There has been a decline in the consumption of grain legumes withinEurope for several decades to its present very low level. The recent develop-ment of vegetarian habits mainly in northern Europe, however, has stoppedthis trend. The consumption of legumes is not necessarily affected by eco-nomic factors. For example, according to household budget surveys in theCzech Republic (Stikova et al., 1997), poor families (with small children)had a similar consumption of legumes to average families, and amongpoor, retired persons it was only slightly higher. It is interesting (Stikovaet al., 1996), that the highest consumption of grain legumes was found in

10 C. Hedley


Region Peas Beans OtherTotalrange

Totalmean

Northern and Western Europe 1.37 0.53 0.41 1.0–4.9 2.43Central and Eastern Europe 1.65 0.54 0.06 0.2–9.3 2.32Mediterranean countries 0.58 1.90 6.33 4.2–13.6 8.85Middle East 0.58 2.75 7.03 3.0–12.8 10.42Far East 0.48 1.33 1.15 1.0–11.4 2.88North America 0.52 5.29 0.04 3.7–13.4 6.50Central America 0.14 10.06 0.74 5.4–15.7 10.88The Caribbean 0.44 3.13 7.31 2.1–13.6 10.71South America 0.58 9.41 0.76 1.0–16.8 7.03West Equatorial Africa 0.24 0.76 6.03 0.2–26.4 10.37Eastern Africa 0.14 3.25 6.97 2.0–20.3 7.03South Africa 0.58 2.62 0.89 2.9–11.0 4.08Oceania 0.87 0.64 0.95 0.4–8.0 2.40

World 0.61 2.51 3.26 0.2–26.4 6.36

Table 1.8. Consumption of grain legumes for human nutrition in regions,expressed as kg per person (FAO, 1996).



the group of families with the highest income, which could be due to ahigher level of education.

1.4 Grain Legume Carbohydrates

In general, grain legume seeds are characterized by having a relatively highprotein content (c. 20–30%) and a very high proportion of carbohydrate(c. 50–65%) in their seeds (Table 1.9). The exceptions are soybean and thevarious lupin species, all of which have higher protein contents (c. 35–45%)and a lower proportion of carbohydrate (c. 30–40%) in their seeds (Table1.9). The difference between the seeds of soybean and lupin and those ofmost other grain legumes lies in the fact that these two legumes do notstore starch as their main energy source and both have an increased oilcontent in their seeds. The oil content in seeds of the starch storinglegumes is not usually greater than about 2%. The oil content in soybeanseeds, however, can reach more than 20% and in lupin seeds can rangefrom about 4 to 15% oil, depending on the species (Table 1.9).

For each legume species, the carbohydrate fraction of the seed canbe broadly divided into three groups of compounds: starch, mono- anddisaccharides plus low molecular weight oligosaccharides and a groupcontaining structural cell wall polysaccharides. This last group includescellulose, lignin and pectin, together with cell wall components, such asgalactose, arabinose, fucose and xylose. Much of this latter group isincluded in the non-digestible material often referred to as the ‘fibre’fraction of the seed (Table 1.10).

As mentioned earlier, for most grain legumes the largest part of thiscarbohydrate fraction is starch, accounting for about 35–45% of the seedweight depending on the legume species. In seeds of soybean and those ofthe various types of lupin, however, starch only makes up about 1.5% andless than 0.5%, respectively, of the seed weight.

The most important low molecular weight soluble carbohydrates aresucrose and the individual members of the raffinose family of oligosaccha-rides (RFO): raffinose, stachyose and verbascose. All grain legume seedscontain these compounds to a greater or a lesser extent and considerablegenetic variation exists, both between and within species, for their contentand composition, in particular for the RFO.

There is an even greater variation within the group of carbohydratescontaining the ‘fibre’ material, much of which still remains to be character-ized and quantified for the different species. It is also apparent that the twonon-starch storing legumes represented here have a greater proportion oftheir carbohydrate fraction in this ‘fibre’ group of compounds.

The fact that the carbohydrates make up the largest proportion of theseed in itself makes this group of compounds of paramount importancewhen considering the quality and potential uses of grain legumes.

Introduction 11




Nutritionally, they contain starch, which is the major energy source forhumans and animals, plus many compounds, including the RFO and thosein the ‘fibre’ group, that either enhance or reduce nutritional value, orhave a positive or negative effect on health. Carbohydrates are also veryimportant, however, to the growth and development of the seed, formingthe main structural elements and the main translocation and storagecompounds. The consequences to the seed and to nutritional uses must

12 C. Hedley


Protein Oil Carbohydrates

Legume species Min. Max. Min. Max. Min. Max.

Soybean 38.4 19.7 32.5G. max 35.1 – 42.0 17.7 – 21.0 30.2 – 35.5

LupinL. albus 38.1 11.1 36.5

34.3 – 44.9 8.0 – 14.5 31.0 – 42.0L. luteus 41.7 5.3 32.0

39.0 – 47.0 4.0 – 7.1 26.0 – 37.0L. angustifolius 34.1 5.7 41.5

28.0 – 37.9 4.6 – 7.0 36.0 – 47.0

Chickpea 21.8 5.2 65.3C. arietinum 15.5 – 28.2 3.1 – 7.0 59.9 – 70.8

Mung bean 23.3 1.2 60.0V. radiata 22.9 – 23.6 1.2 – 1.2 58.2 – 61.8

Pigeon pea 21.2 2.6 64.9C. cajan 19.5 – 22.9 1.3 – 3.8 63.0 – 66.8

Jack bean 29.6 2.4 47.8C. ensiformis 26.9 – 32.2 1.8 – 2.9 46.1 – 49.5

Common bean 23.4 1.5 61.3P. vulgaris 20.9 – 27.8 0.9 – 2.4 58.2 – 63.4

Faba bean 29.0 2.0 59.8V. faba 22.4 – 36.0 1.2 – 4.0 57.8 – 61.0

Lentil 26.8 1.4 64.4L. culinaris 23.0 – 32.0 0.8 – 2.0 60.5 – 68.2

Cowpea 23.5 1.3 60.0V. unguiculata

Pea 25.3 2.7 65.5P. sativum 18.3 – 31.0 0.6 – 5.5 60.7 – 70.7

aSources from where this data was derived are given with Table 1.10.

Table 1.9. Protein, oil and carbohydrate composition (% of seed) of grain legumeseeds.a



be taken into consideration, therefore, in any programme designed tomanipulate the content and/or composition of the carbohydrates. It isevident that such a programme would require a multidisciplinary approachencompassing nutritionists, plant biologists, chemists, technologists,geneticists and plant breeders.

This book sets out in a limited way what is known about the roleof these compounds in the seed and their potential use in nutrition.Information is presented on the chemical composition and analysis of thevarious carbohydrates and how they can be manipulated genetically, usingconventional breeding and modern molecular techniques, or by processingtechnology. Since each of these areas could form the basis of a book inits own right, we have outlined within each chapter the main areas to betaken into consideration and appended a comprehensive literature list forfurther reading if necessary. The book is deliberately aimed at thestarch-storing grain legumes, because starch is an important nutritionalcomponent and also because the main oil storing grain legume, soybean,is already covered comprehensively in the literature. Information ispresented on soybean and lupin, however, to make specific points andwhen this is the only available source in the literature for comparativepurposes.

Introduction 13


Legume species Total Starch Sucrose Raffinose Stachyose Verbascose ‘Fibre’a

Soybean 32.5 1.5 6.2 0.9 4.3 0.1 20Lupin spp. 36.7 0.4 2.5 0.7 6.8 0.6 26Chickpea 65.3 44.4 2.0 1.5 5.5 3.0 9Mung bean 60.0 45.0 1.1 1.7 2.0 3.0 7Pigeon pea 64.9 44.3 2.5 1.0 3.0 4.0 10Jack bean 47.8 35.0 1.5 0.7 1.5 0.1 9Common bean 61.3 41.5 5.0 0.3 4.1 0.1 10Faba bean 59.8 41.0 3.3 0.2 0.7 2.5 12Lentil 64.4 46.0 2.9 0.5 2.4 0.9 12Pea 65.5 45.0 2.1 0.9 2.4 3.2 12

aFibre – this category includes other soluble and insoluble carbohydrates.Sources of information for Tables 1.9 and 1.10: Proceedings of 1st EuropeanConference on Grain Legumes (1992); Proceedings of the International ConferenceEuro Food Tox IV on Bioactive substances in food of plant origin (1994); Nwokoloand Smartt (1996); Proceedings of 3rd European Conference on Grain Legumes(1998).

Table 1.10. Carbohydrate composition (% of seed) of grain legumes.



Carbohydrate ChemistryP. Kadlec et al.2

2Carbohydrate ChemistryEditor: Pavel Kadlec

Contributors: Charlotte Bjergegaard, KrzysztofGulewicz, Marcin Horbowicz, Alan Jones, PavelKadlec, Pavel Kintia, Christo Kratchanov, MariaKratchanova, Grazyna Lewandowicz, MariaSoral-Smietana, Hilmer Sorensen and Jan Urban

He was a practical electrician but fond of whisky, a heavy, red-hairedbrute with irregular teeth. He doubted the existence of the Deity butaccepted Carnot’s cycle, and he had read Shakespeare and found himweak in chemistry.

Complete Short Stories (1927) ‘Lord of the Dynamos’H.G. Wells (1866–1946), English novelist

2.1 The Carbohydrates

Carbohydrates – hydrates of carbon – were historically so called becausethey contain the elements of water, Cx(H2O)y; however, there are nowmany that have recently been discovered to be exceptions to this formula.Carbohydrates or saccharides can simply be defined as polyhydroxy alde-hydes or ketones and their derivatives. These compounds are an importantsource of energy for living organisms as well as a means by which chemicalenergy can be stored. In addition, some carbohydrates function asstructural components within the cell. They can be divided into two groups:(i) soluble carbohydrates and (ii) polysaccharides, and further subdividedas shown in Fig. 2.1.





2.1.1 Soluble carbohydrates

Monosaccharides and disaccharidesMonosaccharides, the simplest of all sugars, are the building blocks ofcarbohydrate chemistry. Their general formula is (CH2O)n, where n = 3or some larger number. Monosaccharides cannot be hydrolysed to formsimpler or smaller entities. The three most commonly found mono-saccharides that are measurable in any quantity in grain legume seeds areglucose, galactose and fructose.

Disaccharides consist of two sugars joined by a glycosidic bond – anoxygen bridge. The three abundant naturally occurring disaccharides aresucrose, maltose and lactose, and these are widely distributed in livingorganisms. Sucrose (common table sugar) is the only one present inany appreciable quantity in legume seeds. Sucrose was first obtainedcommercially from sugar-cane (Saccharum officinarum L.) and hence sugarswere given the scientific name of saccharides. Confusingly, when peopletalk of sugar they invariably mean sucrose, whereas to scientists sugar is aname given to a group of compounds such as those we are describing here.As a consequence of a glycosidic bond joining the anomeric carbon atomsof the glucose and a fructose moieties, sucrose lacks a free reducing group(i.e. there is no aldehyde or ketone end group), in contrast with most othersugars.

The hydrolysis of sucrose to glucose and fructose is catalysed bythe enzyme invertase (EC 3.2.1.26, so named because hydrolysis changesthe optical activity from dextro- to laevorotatory), also known as saccharase.A mixture of glucose and fructose so obtained is called ‘invert sugar’.

In maltose, two glucose units are joined by an α(1→4) glycosidiclinkage. Maltose derives from the hydrolysis of starch and is in turnhydrolysed to glucose by the enzyme maltase (EC 3.2.1.20).

16 P. Kadlec et al.


Fig. 2.1. Classification of carbohydrates.



Monosaccharides are present in dried, mature legume seeds inrelatively small amounts. Traces of fructose and glucose are found in pea,lentil and lupin seeds at a level of 0–1% of dry weight. Glucose is present indeveloping pea embryos in slightly higher quantities (1–3%), together withoccasional traces of galactose. Although monosaccharides are not a majorcomponent of legume seeds, sucrose can accumulate in appreciable quanti-ties in some legume species. For example, mature dry seeds of the starchlesspea mutant, rug3, accumulates about 7–8% of the seed dry weight assucrose compared with the wild-type value of 2–3%. Sucrose is present insimilar quantities in mature faba bean seeds to the wild-type pea and inlesser amounts (1–2%) in lupin seeds (Frias et al., 1996a).

a-GalactosidesOligosaccharides (from Greek oligos, a few) are compounds that giveonly monosaccharide units after complete hydrolysis. Depending on thenumber of monosaccharide residues per mole, oligosaccharides areclassified as trisaccharides, tetrasaccharides and so forth.

The α-galactosyl derivatives of sucrose are the most common group ofα-galactosides found in the plant kingdom. They are the most abundantsoluble sugars in plants and rank only second to sucrose in importance.The most ubiquitous group of galactosyl sucrose oligosaccharides arethe raffinose family of oligosaccharides (RFO), so named after the firstmember of this homologous series of α-galactosides.

The RFO are α(1→6) galactosides linked to C-6 of the glucose moietyof sucrose. They are low molecular weight non-reducing sugars that aresoluble in water and water–alcohol solutions (Arentoft and Sorensen, 1992;Arentoft et al., 1993). In addition to raffinose, this group of α-galactosidesincludes stachyose, verbascose, ajugose and unnamed longer-chain oligo-saccharides up to nonasaccharide (Cerning-Beroard and Filiatre-Verel,1976). Chemically, the RFO may be considered as derivatives of sucrose.Their IUPAC (International Union of Pure and Applied Chemistry) namesare listed below.

• raffinose α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside

• stachyose α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside

• verbascose α-D-galactopyranosyl-(1→6)-[α-D-galactopyranosyl-(1→6)-]2-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside

• ajugose a-D-galactopyranosyl-(1→6)-[α-D-galactopyranosyl-(1→6)-]3-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside

Structures of these oligosaccharides are shown in Fig. 2.2.The α-galactosides are often considered to be antinutritional factors,

because they are not hydrolysed by mucosal enzymes in the small intestineof monogastric animals and pass into the lower gut where they are

Carbohydrate Chemistry 17




fermented with the production of gas (Cristofaro et al., 1974; Saini andGladstones, 1986; Price et al., 1988). Conversely, their ingestion in the formof pure compounds in the diet increases the bifidobacteria population inthe colon, which in turn contributes positively to human health in manyways (Minami et al., 1983; Tomomatsu, 1994; see Chapter 2).

CyclitolsThere are nine isomers of inositol, the prevalent natural form is cis-1,2,3,5-trans-4,6-cyclohexanehexol (trivial name myo-inositol; Greek mys, muscle).Myo-inositol is widely distributed in plants and animals and is a growthfactor for animals and microorganisms.

There are three other forms of underivatized inositol present in seedsof some legume species: D-chiro-inositol, muco-inositol, and scyllo-inositol.These naturally occurring isomers are synthesized from myo-inositol byepimerization (Loewus and Dickinson, 1982; Loewus, 1990) and have thesame molecular formula (C6H12O6), and formula weight (180.16; forfurther information see Hudlicky and Cebulak, 1993).

Myo-inositol is the primary source for the biosynthesis of many naturallyoccurring derivatives including methyl-cyclitols (Hoffmann-Ostenhof andPittner, 1982). In plants of the Leguminosae family, myo-inositol is convertedby a specific O-methyl transferase into D-ononitol (4-O-methyl-myo-inositol),

18 P. Kadlec et al.


Fig. 2.2. Raffinose family of oligosaccharides (RFO).



which is a precursor for the formation of D-pinitol, a methyl derivative ofD-chiro-inositol (3-O-methyl-D-chiro-inositol). Other methylation products ofmyo-inositol are sequoyitol (5-O-methyl-myo-inositol) and D-bornesitol (1-O-methyl-myo-inositol). Only one methyl derivative of muco-inositol occurs inLeguminosae seeds, methyl-muco-inositol, where the methyl group is attachedto oxygen in position 1 (Dittrich and Brandl, 1987; Keller and Ludlow,1993). The solubility of methyl-cyclitols is similar to that of the correspond-ing cyclitols. The occurrence of cyclitols and methyl-cyclitols in legumeseeds is presented in Table 2.1.

Among the cyclitol galactosides only galactinol (galacto-myo-inositol) iscommon in seeds and particularly widespread in legume seeds (Fig. 2.3). Itserves as the galactose donor to form galactosyl sucrose oligosaccharides,the RFO (Lehle and Tanner, 1972). Galactinol is formed from UDP-galactose and myo-inositol, and can then add a galactose residue to sucroseforming raffinose, then to raffinose to form stachyose, etc. (Dey, 1990). Inaddition, galactinol may contribute galactose to another molecule ofgalactinol to form the digalactosyl derivative of myo-inositol (Petek et al.,1966). When the accumulation of sucrose galactosides is limited,galactinol and di-galactosides of myo-inositol can accumulate to higherlevels (Horbowicz et al., 1995). Galactinol is also the galactose donor toD-ononitol to form galacto-ononitol (Fig. 2.4), found in seeds of adzukibean (Yasui, 1980; Obendorf, 1997).

Other cyclitol galactosides common in legume seeds are the galacto-pinitols. In these galactosides, the galactose molecule can be attached toD-pinitol in position 1, 2 or 5. So-called galactopinitols [A (O-α-D-galacto-pyranosyl-(1→2)-4-O-methyl-D-chiro-inositol) (Fig. 2.5) and B (O-α-D-galactopyranosyl-(1→2)-3-O-methyl-D-chiro-inositol) (Fig. 2.6)] are presentin many legume seeds (Schweizer et al., 1978). Another galactopinitolisomer called leucaenitol (O-α-D-galactopyranosyl-(1→1)-3-O-methyl-D-chiro-inositol) has been recently discovered in seeds of a tropical legume,leucaena (Leucaena leucocephala Lam.) (Chien et al., 1996).

Less common in legume seeds is the galactoside of chiro-inositol,fagopyritol B1, so called because of its abundance in seeds of buck-wheat, Fagopyrum esculentum (Obendorf, 1997). Fagopyritol B1 (O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol; Fig. 2.7) was first identified insoybean seeds (Schweizer and Horman, 1981), but has now been reportedin seeds of lupin, pigeon pea, cowpea and lentil (Horbowicz and Obendorf,1994; Górecki et al., 1996).

D-Ononitol is an intermediate in D-pinitol biosynthesis, the free formand the galactoside of which is present in small or trace amounts. Largerquantities of galacto-ononitol (O-α-D-galactopyranosyl-(1→5)-4-O-methyl-myo-inositol), however, have been found in the seeds of adzuki bean (Yasui,1980).

Among the di- and tri-galactosides of cyclitols only ciceritol (O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→2)-4-O-methyl-D-chiro-





20 P. Kadlec et al.

A3867: AMA: Hedley: First Proof:14-Nov-00 2

Spec

ies

Latin

nam

e

Lupi

nLu

pinu

slu

teus

L.+

++

++

++

++

++

++

++

++

Lupi

nLu

pinu

sal

bus

L.+

++

++

++

++

++

+So

ybea

nG

lyci

nem

ax[L

.]M

erri

ll+

++

++

++

++

++

Pige

onpe

aC

ajan

usca

jan

[L.]

Mill

sp.

++

++

++

++

++

++

++

+C

owpe

aV

igna

ungu

icul

ata

[L.]

Wal

p+

++

++

Mun

gbe

anV

igna

radi

ata

[L.]

Wilc

zek

++

++

++

++

++

++

++

++

++

Faba

bean

Vic

iafa

baL.

++

++

Dry

bean

Phas

eolu

svu

lgar

isL.

++

++

Gar

den

bean

Phas

eolu

svu

lgar

isL.

++

+Pe

a(r

ound

)Pi

sum

sativ

umL.

++

++

+Le

ntil

Lens

culin

aris

Med

ic.L

.+

++

++

++

++

++

++

++

Chi

ckpe

aC

icer

arie

tinum

L.+

++

++

++

++

++

++

++

++

+Lu

cern

eM

edic

ago

sativ

aL.

++

++

++

++

++

++

++

++

++

Adz

ukib

ean

Vig

naan

gula

ris

L.+

++

++

++

+

+,V

alue

betw

een

0.05

and

0.10

%of

dry

wei

ght;

++

,val

uebe

twee

n0.

10an

d0.

50%

ofdr

yw

eigh

t;+

++

,val

ueab

ove

0.5%

ofdr

yw

eigh

t.A

nem

pty

cell

mea

nsth

ele

veli

sbe

low

the

limit

ofde

tect

ion,

trac

eam

ount

sor

noda

taav

aila

ble.

The

tabl

eis

com

pila

tion

ofda

tafr

om:A

man

(197

9),F

ord

(198

2),F

rias

etal

.(19

96c)

,Gór

ecki

etal

.(19

96),

Hor

bow

icz

and

Obe

ndor

f(1

994)

,Hor

bow

icz

etal

.(19

95),

Uen

oet

al.(

1973

),Y

asui

(198

0),Y

asui

etal

.(19

85).

Tabl

e2.

1.O

ccur

renc

eof

cycl

itols

,met

hyl-

cycl

itols

and

gala

cto-

cycl

itols

inle

gum

ese

eds.

myo-inositol

methyl-chiro-inositol(D-pinitol)

D-chiro-inositol

methyl-scyllo-inositol

methyl-myo-inositol(D-ononitol)

galacto-myo-inositol(galactinol)

galacto-pinitol A

galacto-pinitol B

galacto-chiro-inositolB1 (fagopyritol B1)

galacto-ononitol

di-galacto-inositol

di-galacto-pinitol A(ciceritol)

di-galacto-chiro-inositol (fagopyritol B2)

tri-galacto-pinitol(galacto-ciceritol)



inositol; Fig. 2.5) is relatively common and has been fully identified(Quemener and Brillouet, 1983; Bernabé et al., 1993). Ciceritol is presentin the seeds of chickpea, lupin, lentil, soybean, kidney bean and lucerne.The chickpea seed (Cicer arietinum, from which ciceritol is named), alsocontains galacto-ciceritol (O-α-D-galactopyranosyl-(1→6)-O-α-D-galacto-pyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→2)-4-O-methyl-D-chiro-inositol)(Nicolas et al., 1984). Ciceritol is a digalactosidic derivative of galactopinitolA. Mimositol (O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→2s)-3-O-methyl-D-chiro-inositol) an isomer ofgalacto-ciceritol, is a digalactoside derivative of galactopinitol B, and hasbeen reported and isolated from seeds of the Brazilian legume tree, Mimosascabrella (Ganter et al., 1991).



Fig. 2.3. Galactinol series.

Fig. 2.4. Galactosyl ononitol series.



2.1.2 Polysaccharides

StarchStarch is the major storage carbohydrate (polysaccharide) in higher plants.It exists in the form of granules, which are deposited as a reserve or storagecarbohydrate in plant organs such as seeds, tubers and roots. Starch isunique among carbohydrates because it occurs naturally as discrete

22 P. Kadlec et al.


Fig. 2.5. Galactopinitol A series.

Fig. 2.6. Galactopinitol B series.



granules (see Chapter 4). Starch granules are relatively dense, insolubleand hydrate only slightly in cold water. They are also unique because, ingeneral, they are composed of a mixture of two polymers, an essentiallylinear polysaccharide, amylose, and a highly branched polysaccharide,amylopectin (BeMiller and Whistler, 1996).

AMYLOSE Amylose is essentially a linear chain of (1→4)-linked α-D-glucopyranosyl units (Fig. 2.8). Most amylose molecules have a limitednumber of α-D-(1→6) branches, perhaps 1 in 180–320 units, or 0.3–0.5% ofthe linkages (Takeda et al., 1990). The branches in branched amylosemolecules are either very long or very short, and the branch points areseparated by large distances so that the physical properties of amylosemolecules are essentially those of linear molecules. The axial-equatorialposition coupling of the (1→4)-linked α-D-glucopyranosyl units in amylosechains gives the molecules a right-handed spiral or helical shape. Theinterior of the helix contains only hydrogen atoms and is lipophilic, whilethe hydroxyl groups are positioned on the exterior of the coil. Moststarches contain about 25% amylose (BeMiller and Whistler, 1996). The



Fig. 2.7. Fagopyritol B series.

Fig. 2.8. Linear chain structure of amylose.



amylose found in pea starch shows a wide distribution of molecular weight,with an average value of approximately 500,000 daltons. A typical amylosemolecule from pea probably consists of two or three long chains of glucoseunits (Colonna and Mercier, 1984).

AMYLOPECTIN Amylopectin is a very large, highly branched molecule, withbranch-point linkages constituting 4–5% of the total linkages (Fig. 2.9).Amylopectin consists of a C-chain containing the only reducing end-group with numerous branches, or B-chains, each of which have severalsmaller branches, or A-chains, attached. Overall, therefore, A-chains areunbranched and B-chains are branched with A-chains or other B-chains(Fig. 2.10). The branches of amylopectin molecules are believed to beclustered and to occur as double helices. Molecular weights of from 107

to 5 × 108 daltons make amylopectin molecules among the largest, if notthe largest, molecules found in nature. Amylopectin usually constitutesabout 75% of most common starches (BeMiller and Whistler, 1996).

Heating starch suspensions in excess water results in disturbance of theordered structures in the starch granules, a process known as gelatinization(see Chapter 4). The gelatinization process is dependent on the organiza-tion of starch granules, which contain both crystalline and amorphousdomains (Bogracheva et al., 1997; see Chapter 4).

24 P. Kadlec et al.


Fig. 2.9. Linear structure of amylopectin showing a side chain branching point.

Fig. 2.10. Amylopectin – cluster model showing the organization of the differentchain types within the molecule (Manners, 1989).



Fibre fractionThe first definition of dietary fibre (DF) was ‘the skeletal remains of plantcells that are resistant to hydrolysis by the enzymes of man’. This definitionincludes a wide spectrum of compounds within the DF fraction (Trowell,1972). DF was redefined later to include ‘plant polysaccharides and ligninwhich are resistant to hydrolysis by the digestive enzymes of man’ (Trowell,1976). This chemically more precise definition restricted DF to polysaccha-rides and lignin, but on the other hand expanded the definition to includecompounds outside the plant cell wall.

For the purpose of this book, the chemical structure of DF is describedfor the cellulosic and non-cellulosic polysaccharides, including hemicellu-loses, pectins and some associated components. In addition, the complexstructure of lignin is discussed, whereas the proteins and various amphi-philic compounds, which also are considered as important components ofthe cell wall and, therefore, DF (Andersen et al., 1997; Bjergegaard et al.,1997a,b), are not described. An excellent review of the definition of termsused to describe DF is given by Hall (1989).

CELLULOSE AND HEMICELLULOSE Cellulose is composed of linear β(1→4)-D-glucans with a very high degree of polymerization resulting in molecularweights ranging from about 0.5 to 1 million daltons. The fibrillar appear-ance of cellulose in the plant cell wall arises from the side-by-side alignmentof cellulose chains, which are stabilized in a crystalline structure by inter-and intramolecular hydrogen bonds (Southgate, 1995a).

Non-crystalline regions occur at regular intervals in the fibrils. Tracesof sugars other than glucose, found in preparation of cellulose, probablyoriginate from non-cellulose polysaccharides, e.g. mannans or xylanspresent in these regions (Heredia et al., 1995). The cellulose content istypically about 35% in cotyledon cell walls of legume seeds (Selvendranand Robertson, 1990).

Hemicellulose is not chemically or structurally similar to cellulose, as itsname may imply. The traditional classification of hemicelluloses comprisescell wall polysaccharides, preferentially solubilized by aqueous alkali afterremoval of water-soluble polysaccharides. Hemicelluloses cover a widespectrum of complex hetero-polysaccharides, containing a minimum oftwo types of sugar residues. The dominating constituent monosaccharidesare of neutral character, although some uronic acid may be present inminor amounts (McDougall et al., 1993).

Xyloglucans (also called amyloids) are the predominant hemicellulosicpolysaccharides in the primary cell wall of dicotyledons. Xyloglucans arecommonly composed of D-glucose, D-xylose and D-galactose residues in amolar ratio of 4 : 3 : 1 (Hayashi, 1989). The polysaccharides have a repeatedstructure of characteristic oligosaccharides with β(1→4)-D-glucose back-bones, regularly branched with D-xylose at C-6 (α) for the majority of theglucose residues. Part of the xylose residues may be further substituted by a





disaccharide (α-L-fucose-1,2-β-D-galactose) and sometimes by β(1→2)-linked L-arabinose (Selvendran, 1983; Gibeaut and Carpita, 1994).

Xylans have a β(1→4)-backbone of D-xylose with some residuesacetylated or substituted with different sugars at C-2 or C-3, e.g. dominatedby α-linked L-arabinose (arabinoxylans), or 4-O-methyl-D-glucuronic/D-glucuronic acid (glucuronoxylans). Xylans also exist with ferulic acidsubstituents, which may participate in cross-linking of the plant cell wall(Brett and Waldron, 1990; Heredia et al., 1995). Glucomannans consist of abackbone of β(1→4)-linked D-glucose and D-mannose residues (about 1 : 3,depending on the plant species) without any regularity in its sequence.α(1→6)-D-Galactose residues are often found as side chains, either inglucomannans (galactoglucomannans), or attached to a pure β(1→4)-linked mannose backbone (galactomannans). The group of pure mannanscomprises unsubstituted homopolymeric chains of β(1→4)-linkedmannose. Glucuronomannans contain a backbone of α(1→4)-linkedD-mannose and β(1→4)-linked D-glucuronic acid residues with side chainsincluding D-xylose or D-galactose linked to the mannose by β(1→6) links orof L-arabinose linked to mannose by β(1→3) links (Brett and Waldron,1990; Heredia et al., 1995).

In cotyledon cell walls of legume seeds, the hemicellulose content istypically about 15% (Selvendran and Robertson, 1990). A detailed study ofthe chemical composition of certain dehulled legume seeds and their hullshas been performed, with special reference to carbohydrates, by Davebyand Aman (1993). The legume seeds studied comprised pea (Pisum sativumL.), soybean (Glycine max L.), broad bean (Vicia faba L.), sweet white lupin(Lupinus albus L.) and brown bean (Phaseolus vulgaris L.). The study did notdistinguish between cellulose, hemicelluloses and pectins. It gave, however,a detailed picture of the dominating monomeric residues in the non-starchpolysaccharide fraction by determining the content of rhamnose, fructose,arabinose, xylose, mannose, galactose, glucose and uronic acids, respec-tively. Fractionation of non-starch polysaccharides from the cotyledons andhulls of lupin (L. albus L.) into pectic and hemicellulosic polysaccharideshave been performed by Mohamed and Rayas-Duarte (1995). In thisspecies arabinose and xylose were found to be the major sugars in thehulls, whereas galactose was predominant in the cotyledons.

Covalent cross-linking between cell wall polymers is a physiologicallysignificant strategy contributing to the termination of the extensibility andstrengthening of the cell wall. Xyloglucans in the hemicellulosic fractionare closely linked to the cellulose microfibrils by means of hydrogenbonds, and phenolic carboxylic acids and proteins are also well known tocontribute to the cross-linking of cell wall components.

PECTIN The term ‘pectic substances’ is generally used to describe thegroup of complex plant heteropolysaccharides in which D-galacturonicacid is esterified to various extents with methanol. The great diversity in

26 P. Kadlec et al.




composition and forms of occurrence of pectic substances in plants hasled to the development of several more restrictive definitions. The nomen-clature of pectins is essentially based on the degree of methoxylation ordegree of esterification of the carboxyl groups of the polygalacturonanchain. The degree of methoxylation is defined as the proportion ofgalacturonic-acid units esterified with methanol and is expressed as apercentage.

Pectic substances can be classified as follows as defined by Jeltema andZabik (1980):

• pectic acid – pectic substances mostly free of methyl ester groups(degree of methoxylation less than 5%), the salts of pectic acid arecalled pectates;

• pectinic acid – pectic substances mostly composed of polygalacturonicacids carrying more than a negligible proportion of methyl estergroups, the salts of pectinic acid are called pectinates;

• pectin – name is derived from the Greek pectos, which means coagulumand is mainly used to designate those water-soluble pectic substanceswhich are capable of forming gels under suitable conditions;

• protopectin – pectic substances in plants, insoluble in water and con-sidered to be the parent pectic substances that can, upon restrictedhydrolysis, yield pectin.

It is evident that pectin is not a homologous polysaccharide and that it has achain structure of α(1→4)-linked D-galacturonic acid units interrupted bythe insertion of α(1→2)-linked L-rhamnopyranosyl residues in adjacent oralternate positions (Sathe and Salunkhe, 1981; Ravindran and Palmer,1984; Ross et al., 1985). Homologous galacturonans consisting solely orpredominantly of α(1→4)-linked D-galacturonosyl residues have beenisolated from various plant tissues such as sunflower heads and seeds(Shehata et al., 1985), sisal (Reid et al., 1986), rice endosperm cell walls(Champ et al., 1986), from apple pectin (Goldberg et al., 1986) and otherplant cell walls. Such galacturonans, however, were obtained by extractiontreatments likely to cleave covalent bonds, so that they may have beenreleased from a heterogeneous pectic polysaccharide. The homologousgalacturonan type of pectin contains no side chains and therefore, is alsoreferred to as ‘smooth regions’ of pectin. In contrast, the second major typeof pectic polysaccharide, rhamnogalacturonan, contains many side chainsand is often referred to as ‘hairy regions’ (Ross et al., 1985; Bhatty, 1990;Vidal-Valverde et al., 1992a; Ralet et al., 1993).

Various sugars are attached in side chains, the most common beingD-galactose, L-arabinose and D-xylose, while D-glucose, D-mannose, L-fructose and D-glucuronic acid are found less frequently. D-galactose andL-arabinose are present in more complex chains with structures similar tothose of arabinans and arabinogalactans and with chain lengths that canbe considerable. Side chains are glycosidically linked to C-4 and/or C-3 of





α-L-rhamnopyranose or C-2 and C-3 of some of the galacturonosyl residues(Goldberg et al., 1994). There is little or no evidence to suggest whatstructural forms are present in grain legume seeds. The occurrence ofpectic substances in legume seeds is presented in Table 2.2.

2.1.3 Other carbohydrate components

LigninThe term lignin is used now to refer not to a single chemical compound,but rather to a group of structurally related amorphous, high molecularweight, aromatic polymer compounds. They typically consist of monomericunits of oxygen derivatives of phenylpropane with different degrees ofmethoxylation of the aromatic nucleus. Lignin substances have a complexthree-dimensional structure and are insoluble both in water and in organicsolvents. Lignin is one of the chief constituents of plant cell walls andDF, performing the role of a cementing substance with regard to theother biopolymers of the cell walls. Lignin accounts for about 25% of thecomposition of wood and occupies the second place in occurrence oforganic substances in nature after cellulose. It has been established thatlignins are heterogeneous in terms of chemical structure and molecularmass, the molecular heterogeneity depending both on the age and the kindof plant. They are normally linked by covalent and hydrogen bonds tocarbohydrates.

Lignification serves two main functions. It cements and anchors thecellulose microfibrils and other matrix polysaccharides (pectins, hemicellu-loses) and because the lignin–polysaccharide complexes are hard, theystiffen the walls, thus preventing biochemical degradation and physicaldamage to the walls. These properties of lignified walls are important in theDF context, because they minimize the bacterial degradation of the wallsin the human colon. The occurrence of lignin in legume seeds is shown inTable 2.2.

SaponinsSaponins are naturally occurring glycosides widely distributed in plants,including soybean and pea seeds. Each saponin consists of a sapogenin,which constitutes the aglycon moiety of the molecule and a sugar. Thesapogenin may be a steroid or a triterpene (the later type being most com-mon form of saponin found in cultivated crop plants) and the sugar moietymay be glucose, galactose, a pentose or a methyl pentose. The name comesfrom Saponaria, soapwort, the root of which has been used as a soap (sapo,Latin for soap). All saponins foam strongly when shaken in water. They

28 P. Kadlec et al.






Legumes Pectic substances % Lignin % References

Faba beans seedsWater-soluble 0.8 Shehata et al. (1985)EDTA-soluble 0.4 Shehata et al. (1985)NaOH-soluble 0.4 Shehata et al. (1985)

Green beans 15.4 Vazquez-Blanco et al. (1995)Blanched 0.5 Margareta et al. (1994)Canned 2.5 2.4 Ross et al. (1985)Canned 0.4 Margareta et al. (1994)Canned 8.1 2.6 Weightman et al. (1994)Fresh from store 2.7 2.1 Ross et al. (1985)Freshly cooked 3.8 1.4 Ross et al. (1985)Frozen 5.0 4.4 Ross et al. (1985)Microwave heating 0.4 Margareta et al. (1994)

Kidney beans, canned 5.3 3.4 Weightman et al. (1994)

Lima beans, canned 3.8 0.8 Weightman et al. (1994)

Mung beans 10.0–18.0 Goldberg et al. (1986)

Navy bean, dried cooked 7.8 1.2 Weightman et al. (1994)

Pinto beansCanned 4.5 3.3 Weightman et al. (1994)Dried, then cooked 7.5 2.7 Weightman et al. (1994)Dried raw 8.2 1.6 Weightman et al. (1994)

Red kidney beans 12.0 Moscoco et al. (1984)

Runner beans 14.0 Selvendran and King (1989)

White beansCanned 6.3 1.4 Weightman et al. (1994)Dried, then cooked 5.3 1.0 Weightman et al. (1994)Dried raw 4.5 1.0 Weightman et al. (1994)

Lentils 17.7–18.1 1.2–1.7 Bhatty (1990)

Lentils 1.2–4.8 1.2–3.0 Vidal-Valverde et al. (1992a)Dried, then cooked 1.7 3.1 Weightman et al. (1994)Dried raw 1.3 2.1 Weightman et al. (1994)

Peas

Black-eyed peas, canned 1.2 2.2 Weightman et al. (1994)

Green peas 3.7 0.6 Theander (1995)Blanched 0.1 Margareta et al. (1994)Canned 3.0 0.9 Weightman et al. (1994)Canned 0.1 Margareta et al. (1994)Microwave heating 0.9 Margareta et al. (1994)

Pea hulls 15.0 Weightman et al. (1994)

Soybean okara 22.0 Yamaguchi et al. (1996a)

Soybean okara 4.6 Yamaguchi et al. (1996b)

Table 2.2. Occurrence of pectic substances and lignin in various legumes.



form oil–water emulsions and act as protective colloids. Triterpenoidaglycones contain glucuronic acid in place of the sugar moiety and have abitter taste. Saponins cause growth depression in poultry and pigs, bloatin ruminants. Some aglycone moieties increase the permeability of cellmembranes and cause haemolysis by destroying the membranes of redblood cells, releasing haemoglobin into the bloodstream. The haemolyticactivity of saponins varies between plant species.

Another property of saponins is their toxicity to fish and lower forms oflife, because of their capacity to bind with cholesterol and their antibioticactivity. Along with the above properties, which are common to all ofthe triterpene glycosides (saponins), each of them possesses specificpharmaceutical properties (Turova and Gladkych, 1964; Hiller et al., 1966;Woitke et al., 1970a,b; Vecherko et al., 1973).

Triterpene aglycones can be subdivided into two groups, accordingto their structure, either with peptocyclic or tetracyclic carbohydrateskeletons. The first group includes aglycones based on carbohydratessuch as oleonon, ursan, lupan and honone, and the second group is basedon dommoran, epostan and holostan (Fenwick et al., 1991; Tsukamotoet al., 1993). The saponin contents of 13 types of legume seeds are shown inTable 2.3. Saponins are expressed as a weight percentage of the defattedflour, assuming that the aglycone : carbohydrate ratio is equal to 1.0. Thiscan only be an approximation, however. For example, in soybean the fivereported saponins (I, II, III, A1 and A2) have aglycone : carbohydrate ratiosof 0.9 : 1, 1 : 1, 1.3 : 1, 0.6 : 1 and 0.7 : 1, respectively, while their relativecomposition in the saponine mixture has been reported to be 60, 6, 1, 30and 3%, respectively (Kitagawa et al., 1984a,b). Other workers havereported more complex saponins in P. vulgaris (Chirva et al., 1970), whichwill further reduce this ratio.

30 P. Kadlec et al.


Species Latin name Saponin content (g kg-1)

Butter bean Phaseolus lunatus 1.0Chick pea Cicer arietinum 2.3Field bean Vicia faba 0.1Green pea Pisum sativum 1.8Haricot bean Phaseolus vulgaris 2.3Kidney bean Phaseolus vulgaris 3.5Lentil Lens culinaris 1.1Mung bean Phaseolus aureus 0.5Peanut Arachis hypogaea < 0.1<Runner bean Phaseolus coccineus 3.4Soybean Glycine max 6.5Yellow split pea Pisum sativum 1.1

Table 2.3. Saponin content of legume seeds.



2.2 Chemical Analysis of the Carbohydrates

2.2.1 Soluble carbohydrates (monosaccharides, sucrose, a-galactosides,cyclitols)

StandardsAccurate and precise chemical analysis demands a comparison with theknown purified compound. Fortunately some of these compounds areavailable commercially at a high purification, around 99% in some cases.Sugar standards readily available from the major suppliers of laboratorychemicals include: D(−)fructose (cat. no. F2543), D(+)galactose (G6404),D(+)glucose (G7528), maltose (M5885), sucrose (S7903), raffinose(R0250), and stachyose (S4001) (Sigma-Aldrich, St Louis, Missouri, USA).Verbascose (O-VER) is available from Megazyme International IrelandLtd. (www.megazyme.com). Phenyl α-D-glucoside (P6626), D(+) melezitose(M5375) or D(+) lactose (L1768) are suitable for use as internal standards,since they are unlikely ever to be found in legume seeds in any significantquantity.

Some of the more ‘exotic’ carbohydrates cannot be purchased and willhave to be prepared by each laboratory from biological sources. Among thecommonly found cyclitols and methylcyclitols in legume seeds, only two arecommercially available: myo-inositol, and D-pinitol. The others described inSection 2.1.3 are unavailable. Details of procedures for the isolation andpurification of cyclitols are published in Schweizer et al. (1978) and Binderand Haddon (1984). Table 2.4 summarizes suitable plant sources forextracting and isolating cyclitols and galactocyclitols.

Extraction of soluble carbohydrates from seeds

SAMPLE PREPARATION For low molecular weight sugars, water is the optimalextraction solvent. Unfortunately, it is also an excellent solvent forinterfering hydrophilic components such as polysaccharides, proteins, etc.In addition, α-amylases and α-galactosides, present in the plant material,may degrade starch and the raffinose oligosaccharides if not inactivatedduring, or prior, to extraction. These problems are minimized byextraction in aqueous alcohols. Alcohol type and concentration, extractiontemperature and procedure vary considerably among the methodsdescribed: 80% ethanol or methanol (v/v) is most commonly used, butthere are indications that in some cases these solvents lead to incompleteextraction. Increasing the alcohol concentration above 80% (v/v) hasbeen shown greatly to reduce the amount of RFO extracted from plantmaterial (Cegla and Bell, 1977; Shukla, 1996; Bach Knudsen and Li, 1991).Furthermore, marginally higher extraction yields have been noted withmethanol compared with ethanol (Shukla, 1996). This is in contrast toother studies showing no difference between 80% methanol and water in





32 P. Kadlec et al.


Com

mon

nam

eSo

urce

Ref

eren

ce

D-P

inito

lSo

ybea

nse

ed(G

lyci

nem

ax)

Schw

eize

ret

al.(

1978

)D

-Ono

nito

lLu

cern

ele

aves

(Med

icag

osa

tiva)

Bin

der

and

Had

don

(198

4)O

-Met

hyl-

scyl

lo-i

nosi

tol

Mun

gbe

an(V

igna

radi

ata)

Bin

der

and

Had

don

(198

4)O

-Met

hyl-

muc

o-in

osito

lR

edw

ood

(Seq

uoia

sem

perv

iren

s)B

inde

ran

dH

addo

n(1

984)

Sequ

oyito

lG

inkg

obi

loba

Bin

der

and

Had

don

(198

4)sc

yllo

-Ino

sito

lC

hem

ical

isom

eriz

atio

nof

myo

-ino

sito

lSa

saki

etal

.(19

88)

chir

o-In

osito

l1.

Che

mic

alis

omer

izat

ion

ofm

yo-i

nosi

tol

1.Sa

saki

etal

.(19

88)

2.B

uckw

heat

(Fag

opyr

umes

cule

ntum

)2.

Hor

bow

icz

etal

.(19

98)

3.D

emet

hyla

tion

ofD

-pin

itol

3.B

inde

ran

dH

addo

n(1

984)

Gal

actin

ol1.

Cuc

umbe

rle

aves

(Cuc

umis

sativ

us)

1.Ph

arr

etal

.(19

87)

2.C

omm

onbu

gle

(Aju

gare

ptan

s)2.

Bac

hman

n(1

993)

3.C

asto

rbe

anse

eds

(Ric

cinu

sco

mm

unis

)3.

Kuo

(199

2)4.

Jojo

babe

ans

(Sim

mon

dsia

chin

ensi

s)4.

Oga

wa

etal

.(19

97)

Gal

acto

-ono

nito

lA

dzuk

ibea

n(V

igna

angu

lari

s)Y

asui

(198

0)G

alac

topi

nito

lASo

ybea

nse

eds

(Gly

cine

max

)Sc

hwei

zer

and

Hor

man

(198

1)G

alac

topi

nito

lBSo

ybea

nse

eds

(Gly

cine

max

)Sc

hwei

zer

and

Hor

man

(198

1)Fa

gopy

rito

lB1

1.So

ybea

nse

eds

(Gly

cine

max

)1.

Schw

eize

ret

al.(

1978

);Sc

hwei

zer

and

Hor

man

(198

1)2.

Buc

kwhe

at(F

agop

yrum

escu

lent

um)

2.H

orbo

wic

zet

al.(

1998

)3.

Jojo

babe

ans

(Sim

mon

dsia

chin

ensi

s)3.

Oga

wa

etal

.(19

97)

Cic

erito

l1.

Chi

ckpe

a(C

icer

arie

tinum

),le

ntil

(Len

scu

linar

is)

1.Q

uem

ener

and

Bri

lloue

t(19

83)

2.Le

ntil

(Len

scu

linar

is)

2.B

erna

béet

al.(

1993

)G

alac

to-c

icer

itol

Chi

ckpe

a(C

icer

arie

tinum

)N

icol

aset

al.(

1984

)M

imos

itol

Seed

sof

Mim

osa

scab

rella

Gan

ter

etal

.(19

91)

Tabl

e2.

4.So

urce

sof

stan

dard

ofcy

clito

ls,m

ethy

lcyc

litol

san

dth

eir

gala

ctos

ides

.



the extraction of low molecular weight sugars (Li and Schuhmann, 1980;Li et al., 1985). It has been found that water extraction at 60°C and boilingin aqueous ethanol (80%, v/v) gave comparable results. Muzquiz et al.(1992) used two methods for the extraction of carbohydrates. Firstly,extraction in 60% methanol at boiling temperature under reflux for 2 h,and secondly, homogenization with 70% methanol for 1 min at room tem-perature. Both methods gave satisfactory recoveries, but higher amountswere recovered using the second method (Table 2.5).

Kvasnidka et al. (1996) compared the extraction efficiency of the RFOfrom different varieties of pea, using sonication (80% ethanol (v/v), for 30min) and boiling (80% ethanol v/v, for 30, 60, 120 min under reflux) andfound that the latter technique was twice as efficient compared with



RFO Amount added (mg) Total amount found (mg) Recovery (%)

Method I (in 2 g of flour)Sucrose 0.00 29.29 –

20.21 46.15 83.4240.08 69.29 99.8080.23 101.43 89.84

Raffinose 0.00 21.64 –20.14 37.15 77.0140.25 56.43 86.4380.20 98.22 95.49

Stachyose 0.00 20.36 –20.07 37.86 87.1940.06 57.15 91.5480.21 87.72 83.98

Method II (in 500 mg of flour)Sucrose 0.00 8.10 –

5.21 13.70 107.4910.30 18.26 98.6420.18 27.32 95.24

Raffinose 0.00 2.485.22 7.30 92.34

10.19 11.36 87.1420.36 20.38 87.92

Stachyose 0.00 27.38 –5.04 34.06 132.54

10.19 37.46 98.9220.09 45.52 90.29

Method I – extraction in 60% methanol under reflux for 2 h.Method II – homogenization with 70% methanol for 1 min at ambient temperature.

Table 2.5. Recovery of raffinose family of oligosaccharides (RFOs) using differentextraction methods (Muzquiz et al., 1992).



sonication and that the optimum extraction time for the boiling methodwas 60 min.

Johansen et al. (1996) studied the effect of extraction solvents andtemperature on the extraction yields of monosaccharides, sucrose andRFO from toasted soybean meal, cottonseed meal, field peas and a feedmixture. They found that extraction in 80% (v/v) alcohol was stronglyinfluenced by the extraction temperature and that the maximumextraction was only achieved at the boiling point. Extraction in waterand 50% (v/v) methanol or ethanol was less heat sensitive and gavecomparable results. It has also been shown that aqueous ethanol 50%(v/v) was as effective as 50% (v/v) methanol, whereas lower yieldswere observed at higher alcohol percentages. There was no consistentdifference in the extraction yield when comparing reflux with constantstirring and water bath with occasional mixing, for any of the extractionsolvents used.

Comparison of five methods for extraction of oligosaccharides fromsoybean and cottonseeds has been described by Bach Knudsen and Li(1991).

In light of the above results, it can be concluded that the extractionprocedure for the analysis of soluble carbohydrates is always going tobe a compromise between the optimal extraction of a group of differentcompounds, the level of recovery and the possible interaction of othernon-carbohydrate components present in the seed.

RECOMMENDED EXTRACTION PROCEDURE The following method can beapplied to whole seed, or seed with the seed coat (testa) removed. Thiscan be arrived at on individual seeds by picking off the seed coat with asharp dissecting needle (Jones et al., 1995), or for larger samples by usinga small-scale industrial de-huller. For a single seed, or part of a single seed,the seed has to be first finely ground to produce flour with a mean particlesize that will pass through a 75-µm test sieve (Jones et al., 1995).

Extraction of soluble carbohydrates from legume seeds (0.1–0.3 g offlour in 5 ml of 50% v/v ethanol or methanol, containing either phenylα-D-glucoside or D(+) melezitose at 0.1 mg ml−1 as an internal standard),can be performed either at 50°C with constant stirring for 1 h under reflux,or in an ultrasonic bath at ambient temperature for 60 min. After thistime the mixture is centrifuged at 6000 rpm for 20 min and the residuere-extracted as before and washed with deionized water until the Molischreaction test is negative (Pearson, 1976). In practice only three or fourcycles are needed to extract all the available carbohydrate from thesample (Jones, 1999). Combined supernatants are then heated at 80°C for20 min to inactivate endogenous enzymes and centrifuged at 6000 rpmfor 20 min. The supernatant is evaporated to dryness in a rotary vacuumevaporator at 40°C. The residue is dissolved in 4.0 ml of pure water andstored at 4°C.

34 P. Kadlec et al.




High performance gas chromatography (GC)Since its inception some 40 years ago GC has become a highly sophisticatedand sensitive analytical tool, with new developments and technologiesbeing continuously introduced (Bartle, 1993). Carbohydrate analysis lendsitself well to this technique. The advent of capillary columns, reliabletemperature and gas flow control arguably makes gas chromatographythe analytical method of choice for sugar analysis (Tipler, 1993).

GC determination of carbohydrates, cyclitols and polyols is possibleafter their conversion into volatile derivatives. In general the most usefulmethod of polyol derivatization is trimethylsilylation (TMS; Bierman,1988). Reducing sugars (fructose, glucose, galactose, mannose and others)produce multiple peaks on gas chromatograms because they occur in theiranomeric forms. Simultaneous determination of such sugars and cyclitols,therefore, can be difficult because retention times of TMS derivatives ofboth classes of carbohydrate are similar. Phenyl α-D-glucoside is commonlyused as an internal standard.

RECOMMENDED GC METHOD FOR QUANTIFYING SOLUBLE CARBOHYDRATES Thefollowing procedure according to Horbowicz and Obendorf (1994) issuitable for the determination of many carbohydrate classes (glucose,fructose, cyclitols, sucrose, mono-, di-, tri-galactosides of cyclitols, raffinose,stachyose and verbascose).

Seed tissues are twice homogenized in a mortar with a solution ofethanol : water (1 : 1 v/v) containing phenyl α-D-glucoside (normal work-ing concentration is between 50 and 100 mg l−1) as internal standard. Thehomogenate is heated at 80°C for 45 min in a microfuge tube and then cen-trifuged. The residue is re-extracted and the combined supernatants arepassed through a 10,000 MWCO (molecular weight cut-off) filter. Aliquotsof the filtrate are transferred to reaction vials and evaporated to dryness in astream of nitrogen. The residues are left overnight (16 h) in a desiccatorover phosphorus pentoxide to remove traces of water. The dry residues arederivatized with trimethylsilylimidazole : pyridine (Sigma cat. nos. T7510,P4036, 1 : 1, v/v) and analysed by high resolution GC. Alternatively, thedrying stage can be omitted when using Tri-Sil®Z (Pierce Chemical Co.),since this derivatizing reagent will work in the presence of water.

There are many different manufacturers and models of GC to choosefrom. Even the most basic can be configured to analyse TMS sugar deriva-tives. Horbowicz and Obendorf (1994) used a Hewlett Packard 5890Series II gas chromatograph equipped with a flame ionization detectorand a Hewlett Packard 3396A integrator. A DB-1 capillary column (15 mlength, 0.25 mm ID and 0.25 µm film thickness; J & W Scientific, Folsom,California, USA) operated with a programmed initial temperature of150°C, adjusted to 200°C at 3°C min−1, adjusted to 325°C at 7°C min−1, andthen held at 325°C for 20 min. The injector port was operated at 335°Cand the detector at 350°C. The carrier gas was helium at 3.0 ml min−1,





split 1 : 50. The detector gas was hydrogen at 30 ml min−1 and air at300 ml min−1. It should be noted that these operating conditions can betransferred to other systems and used as a starting point for optimizing theGC conditions. J & W Scientific are now able to supply high temperatureversions (DB-1ht) of the column used above. The advantages of this newtechnology is that high boiling point TMS derivatives (e.g. TMS-verbascose)will elute faster and produce sharper peaks, when hydrogen is used as thecarrier gas and it is possible to complete a GC analysis in 20 min! A typicalchromatogram using the above system is shown in Fig. 2.11 (from D.A.Jones, personal communication).

Many analyses of cyclitols, galactocyclitols and other carbohydratecontents in seeds of several Leguminosae species (and other species) havebeen performed using this procedure (Horbowicz and Obendorf, 1994;Horbowicz et al., 1995; Górecki et al., 1996; Horbowicz et al., 1998).

36 P. Kadlec et al.


Fig. 2.11. A chromatogram of trimethylsilylation (TMS) sugar derivatives extractedfrom a single round (RR RbRb) pea.



CALCULATION AND STATISTICAL ANALYSIS Quantities of soluble carbohydratescan be determined by extrapolation from standard curves; the ratios ofthe area of peaks for each known component to the area of the internalstandard peak, phenyl α-D-glucoside, are plotted against known amountsof each component. Amounts of unknown carbohydrates are estimated bycalculation using the nearest known standard. Amounts below the level ofdetection are presented as zero. Alternatively the peak area normalizationmethod can be used. This method assumes that the relative response factorfor each component is the same or very nearly the same as that of theinternal standard. From the peak area data obtained it is possible tocalculate the amount (as a percentage of dry matter) of each componentdetected and the total amount of the soluble carbohydrates in the sampleanalysed.

For individual components:

Concentration of component A AA B C D

=+ + +

×AreaTotal area ( )

%100

(Note that the area of the internal standard (IS) is not included in the totalarea sum)

Internal standard concentration =+ + +

×Area ISTotal area (A B C D)

%100

(Note that the area of the IS is not included in the total area sum)

Total amount of components in sample (as % of dry matter)

=÷Amount of IS in extraction medium (mg) Concentration of IS

Sample weight (mg)×100%

For further explanation see Burchfield and Storrs (1962) and Chapman(1985).

Samples would normally be analysed in triplicate.

ADVANTAGES AND DISADVANTAGES OF GC METHODS

Advantages:

1. Analytical stability: GC capillary columns can remain stable over manyyears even with intensive use, thousands of samples can be run withoutchanges in the column resolution.2. High resolution: it is possible to detect all the soluble carbohydratecomponents found in legume seeds from one injection.3. High sensitivity: analyses are possible with 5–10 mg of plant material,sensitivity can be increased by using a mass selective detector.4. The time consuming clean-up step can be omitted, at the expense ofcolumn life.5. It is possible to separate D and L optical isomers of compounds usingspecial columns.





6. A wide range of stationary phases are available for columns to custom-ize a method for a particular class of soluble carbohydrate.7. GC equipment is more common in laboratories and tends to becheaper to purchase.

Disadvantages:

1. Preparing the TMS derivatization of -OH groups can be difficult, but iscrucial for successful results.2. The chemicals involved are toxic, hazardous and expensive and mustbe disposed of safely.3. The multiple peaks produced by TMS derivatives of reducing sugarscan interfere with peaks of free and methylated cyclitols.4. A supply of compressed gases is needed. The gas has to be clean, dryand pure to produce consistent results.5. An analytical run can sometimes take 1 h to complete, although shorterrun times are possible.

High performance liquid chromatography (HPLC)

SAMPLE CLEAN-UP The water extract of soluble carbohydrates from biologi-cal material, (containing an internal standard) should be cleaned beforeHPLC analysis to improve reliability and resolution, using one of the follow-ing procedures.

1. The extract is filtered through a Sep-Pak C18 cartridge, pre-wettedwith methanol and pure water. The effect of sample pre-treatment car-tridges on the carbohydrate analytes themselves should be predeterminedusing standard solutions. (It may be found that some carbohydrates have astrong affinity for particular cartridge packing materials. This is obviouslyimportant if dealing with low levels of carbohydrate in the sample.)The eluate is collected and further filtered through a 0.45-µm PTFE filterto remove particulate matter. Soluble protein and polysaccharides are pre-cipitated by adding an equal volume of absolute ethanol and centrifuged.The clear supernatant is dried at 50°C under nitrogen and finallyredissolved in 0.015 M, Na2SO4. The standard sugar solutions are formu-lated to simulate concentrations in the material under study and aresubjected to the same clean-up procedure as used for the sample (Johansenet al., 1996; Frias et al., 1996b).2. The carbohydrate extract is filtered through a column containingDowex 50 WX8 (H+ form, 200–400 mesh) and Dowex 1X8 (Cl− form,100–200 mesh). The sugar fraction is eluted with deionized water. Theeluate is filtered through a JSO-DISC N-252 nylon membrane, 0.2 µmpore size (Muzquiz et al., 1992; Górecki et al., 1997).3. The dry extract is dissolved in double-deionized water, vortexed andpassed through a DEAE cellulose minicolumn, equilibrated and later

38 P. Kadlec et al.




eluted with deionized water to remove anionic substances from the sample.The eluant is filtered through a Uniflo membrane prior to HPLC analysis(Kuo et al., 1988).

HPLC (NORMAL PHASE) Supelcosil LC-NH2 column 250 × 4.6 mm (SupelcoInc., Sigma, St Louis, MO, USA), acetonitrile–water eluent (75 : 25 v/v),RID-6A refractive index detector (Shimadzu, Kyoto, Japan; www.shimadzu.com) (Górecki et al., 1997). Spherisorb-5-NH2 column (250 × 4.6 mm)(Teknokroma, Bellefonte, Pennsylvania, USA), acetonitrile–water eluent(72 : 28 and 65 : 35 v/v), flow rate 1 ml min−1, an ERMA 7520 RID(Barcelona, Spain) (Muzquiz et al., 1992).

RP-HPLC (REVERSE PHASE) µ-Bondapack/Carbohydrate column (300 × 3.9mm) (Waters Associates) with a precolumn (4.0 cm × 3.2 mm) packed withC18 Porasil B, acetonitrile–water eluent (75 : 25 and 85 : 15 v/v), flowrate 2 and 3 ml min−1, respectively, temperature 35°C, model 132 opticalreflection type differential RID (Gilson Associates) (Vidal-Valverde et al.,1992a, 1993a; Vidal-Valverde and Frias, 1992; Frias et al., 1994a).

Or, an analytical column 250 × 4 mm Separon SGX RPS 7 µm orSeparon SGX C18 5 µm with guard columns (Separon RPS or SeparonC18) at ambient temperature, deionized water eluent, flow rate 1 or0.7 ml min−1, respectively, RID (Kvasnidka et al., 1996).

IMP-HPLC (ION MODERATE PARTITION) Shodex Ionpak KS-801 resin-basedcolumn in sodium form (Waters, Milford, Massachusetts, USA), deionizedwater eluent, flow rate 0.6 ml min−1, temperature 85°C, RID (Johansenet al., 1996).

Aminex HPX-87N (300 × 7.8 mm) resin-based column in the sodiumform (Bio-Rad, Richmond, California, USA), eluent 0. 015 M, Na2SO4, flowrate 0.5 ml min−1, temperature 85°C, a model 156 RID (Bach Knudsen andLi, 1991).

Column 250 × 8 mm filled with strong cation exchanger OSTION LGKS 0803, 17–20 µm in Ca2+ form fitted with desalting guard columns,temperature 80°C, eluent demineralized water, flow rate 0.4 ml min−1, RID(Kvasnidka et al., 1996).

HPAC-PAD (HIGH PERFORMANCE ANION CHROMATOGRAPHY WITH PULSED

AMPEROMETRIC DETECTION, DIONEX SYSTEM) CarboPak PA-100 pellicularanion exchange resin column 250 × 4.0 mm with a CarboPak PA-100 guardcolumn 25 × 3 mm (Dionex Corporation, Sunnyvale, California, USA),flow rate 1 ml min−1, at ambient temperature. The mobile phase: 145 mMsodium hydroxide solution, prepared with deionized water and fresh 50%NaOH solution (cat. no. 19154, BDH, Merck Ltd, Poole, UK), a ModelPAD-II detector equipped with a solvent-compatible electrode (Frias et al.,1996; Kvasnidka et al., 1996).





AC-HPLC (AFFINITY CHROMATOGRAPHY) Sugar-Pack 1 column (Waters Asso-ciates Milford, Massachusetts, USA) at 90°C connected with a guardcolumn (cation cartridge, Pierce Chemical Company, Rockford, Illinois,USA). The elution is monitored by a Waters Model 401 refractometer,mobile phase – 0.1 mM CaNa2/EDTA/H2O, flow rate 0.5 ml min−1 (Kuoet al., 1988).

CALCULATION AND STATISTICAL ANALYSIS Concentration of sugars is calcu-lated from the peak height of detector response as:

Content of sugars (% dry matter) = ×H R WH R W

s s IS

IS IS s

100

where Hs and HIS are peak heights or areas, and Ws and WIS are dry weightsof sample (s) and internal standard (IS), respectively. Rs and RIS areresponse factors (amount/height) for sugar and internal standard insolution containing a known amount of each component (Bach Knutsenand Li, 1991; Johansen et al., 1996). For each sample, data are analysedusing a two-way analysis of variance model (Snedecor and Cochran, 1973):

Xijk = µ + αi + βj + (αβ)ij + εijk

where Xijk is a dependent variable (i.e. content of sugar); µ an overallmean; αi the effect of extraction medium; βj the effect of temperature orextraction procedure and εijk a random variable.

According to Johansen et al. (1996) the detector response of raffinoseand stachyose are linear in the range 0.05–9.0 mg ml−1 extract, correspond-ing to an injected amount of 1–180 µg. The correlation coefficients (r) ofdetector response versus concentration are 0.9991 and 0.9940 for raffinoseand stachyose respectively, both when calibrated on basis of height andarea. In the range tested (0.06–6.0 mg ml−1), verbascose gave a linearresponse (r = 0.9997).

RECOMMENDED HPLC METHODS OF QUANTIFYING SOLUBLE CARBOHYDRATES Forroutine analysis of individual RFOs:

• HPLC (Muzquiz et al., 1992; Górecki et al., 1997);• Dionex HPAC-PAD (Anonymous, 1994; Frias et al., 1994; 1996a,b);• IMP-HPLC (Bach Knudsen and Li, 1991; Johansen et al., 1996;

Kvasnidka et al., 1996);• RP-HPLC (Vidal-Valverde and Frias, 1992; Vidal-Valverde et al., 1993a;

Frias et al., 1994a);• AC-HPLC (Kuo et al., 1988).

Rapid (10 min) method of determination of total RFOs:

• RP-HPLC using the method of Kvasnidka et al. (1996), see section onIMP-HPLC (pp. 39).

40 P. Kadlec et al.




ADVANTAGES AND DISADVANTAGES OF HPLC METHODS

Advantages:

1. The water-based extract can be analysed directly, without any chemicalderivatization.2. The analysis can be conducted relatively quickly.3. Pure compounds can be recovered from the sample, using a fractioncollector, if required.4. Usually a single peak is detected from each component.5. The chemicals used (in extraction and sample clean-up) are relativelycheap and non-toxic.

Disadvantages:

1. Clean-up of extracts can be sometimes time consuming, involvingseveral chemicals and extra disposable equipment, such as filter discs.2. There is no one method that will analyse all of the soluble carbohy-drates normally found in legume seeds.3. HPLC columns are expensive and have a relatively short life time.4. HPLC column properties slowly change with time.5. HPLC equipment is expensive, especially for the gradient elutionsystem.6. An analytical run can sometimes take 1 h to complete, although shorterrun times are possible.

COMPARISON BETWEEN GC AND HPLC Bach Knudsen and Li (1991) deter-mined mean values (percentage of dry weight) for the RFO in protein-richfeedstuffs using both HPLC and GC methods. The regression equationsand the standard error of slope (±) for the sugar determinations by HPLC(X) and GC (Y) for these studies were:-

sucrose Y = 0.094 + 0.985 X ± 0.017 R2 = 0.995raffinose Y = −0.097 + 1.015 X ± 0.025 R2 = 0.989stachyose Y = −0.355 + 1.034 X ± 0.025 R2 = 0.990total Y = −0.262 + 1.006 X ± 0.026 R2 = 0.988

It was apparent that for sucrose and raffinose there was an excellentagreement between the two methods, while the value for stachyoseobtained with the GC method was on average 0.4% (absolute units) lowerthan with HPLC. This difference is almost within the analytical error forthis type of analysis. The coefficient of variation, for the GC method rangedfrom 1.5 to 5.0% and for the HPLC method from 0.6 to 1.3% for sucroseand stachyose, respectively. Compared with literature values the analyticalprecision was quite acceptable. For a GC method this was reported as1.3–3.9% (Sosulki et al., 1982; Molnar-Perl et al., 1984) and for HPLCmethods as 3.5–10.5% and 1.5–24.0% (Kuo et al., 1988). The coefficient ofvariation found for soybean using the HPLC method (Bach Knudsen and





Li, 1991) is comparable to the values found by other groups using GC(Sosulki et al., 1982; Molnar-Perl et al., 1984) and HPLC (Kuo et al., 1988).

Bach Knudsen and Li (1991) have also shown that the injection porttemperature (300–325°C) and overlapping between peaks, in the case ofHPLC, had no effect on the lower GC value obtained for stachyose. In theopinion of these researchers, the lower value for stachyose and the fact thatanalytical precision of the GC method is lower indicates that HPLC shouldbe the technique of choice for this type of feed material.

Thin layer chromatography (TLC)Thin layer chromatography is based on separation of carbohydrates onpaper chromatography or silica gel. The separated zones can be elutedwith water and the soluble carbohydrate components quantified bya colorimetric or densitometric method. Standard curves should beconstructed from eluates obtained after similar analysis using knownconcentrations of the marker carbohydrates (Pearson, 1976).

RECOMMENDED TLC METHODS FOR QUANTIFYING SOLUBLE CARBOHYDRATES Forseparation of the RFO the following mobile phases are used: isopropanol–ethyl acetate–water (5 : 2 : 3); n-butanol–acetic acid–water (5 : 2 : 1);chloroform–methanol–water (6.5 : 3.5 : 1); isopropanol–25% ammonia–water (7 : 1 : 2). The RFO are visualized by spraying with 80 mgnaphthoresorcinol in 40 ml ethanol containing 0.8 ml concentratedsulphuric acid (Jones et al., 1999b). For preparative separation of the RFO,the PSC Fertigplatten Kieselgel 60 F254, (20 × 20 × 0.2 cm plates, cat. no.5717, Merck Ltd, Poole, UK) is used (Stahl, 1969; Pearson, 1976; Joneset al., 1999b).

ADVANTAGES OF TLC METHOD TLC can be a useful method for the initialand rapid screening of material for soluble carbohydrate content before amore detailed study is undertaken, as demonstrated by Jones et al. (1999b).

Capillary zone electrophoresis (CZE)The determination of soluble carbohydrates by capillary zone electro-phoresis is based on the separation of borate complexes of the saccharidesin an electric field. Arentoft et al. (1993) have shown that increasedborate concentration (20–100 mM Na2B4O7) and pH favour the complexformation, which improves the UV absorption at 195 nm. Increasedelectrophoretic mobility of the compounds result in improved separationand longer migration times. The running conditions that were foundto provide the best compromise between acceptable separation, detectionefficiency and duration of analysis were 100 mM Na2B4O7, pH 9.9, 50°C,10 kV and omission of 2-propanol modifier.

42 P. Kadlec et al.




RECOMMENDED CZE METHODS FOR QUANTIFYING SOLUBLE CARBOHYDRATES Thefollowing description and specification of two CZE systems is fairlyrepresentative of the technique.

1. P/ACE 2210 Series capillary electrophoresis system (Beckman Instru-ments (UK) Ltd, High Wycombe, UK), 500 mm × 50 µm I.D. fused-silicacapillary, the injection time is 4 s, the detector window (432 mm) from theinjection end (anode). On-column UV detection is performed at 200 nmand a rise time of 2.0 s. The electrophoresis is conducted at 50°C and ata field strength of 10 kV. The buffer details are disodium tetraborate at aconcentration of 100 mM in pure water, adjusted to pH 9.9 (Frias et al.,1996a,b).2. Capillary electrophoresis instrument ABI Model 270 A-HT (AppliedBiosystems, Warrington, UK), the fused silica capillary is 720 mm × 50 µmI.D. × 360 µm OD, including coating material. The injection time is 2.0 s,the detector rise time 0.5 s. The detector window is 500 mm from injectionend (anode). On-column detection is performed at 195 nm, operatingconditions; temperature (30–60°C), field strength voltage 10–20 kV, with aconcentration of 2-propanol modifier 0–15%, v/v (Arentoft et al., 1993).

CALCULATION AND STATISTICAL ANALYSIS The quantification of each sugar isaccomplished by plotting the normalized peak areas obtained from thesample, against those obtained from the standard solutions. Lactose is usedas a reference peak with computer software normalizing the times duringsubsequent runs to allow for migration time variation. Relative responsefactors are calculated by dividing the slope of the calibration graph forlactose by the corresponding slope for individual analytes.

ADVANTAGES AND DISADVANTAGES OF CZE COMPARED WITH GC AND HPLC

Advantages:

1. The method is easier to use and set up.2. The equipment is less expensive as HPLC or GC equipment.3. Uses non-toxic chemicals.4. Can produce faster analysis times.

Disadvantages:

1. CZE is less sensitive than HPLC or GC, requiring concentrations ofmicrograms per millilitre of sugars for detection. The HPAC-PAD is moresensitive, detecting concentrations at the nanogram per millilitre level,high performance GC with FID being the most sensitive of all, working atthe picograms per millilitre level.2. Sample preparation requires a purification step using cation/anionexchange in the form of Sep-Pak C18 cartridges, which adds time and costto the analysis.





Comparison of results of the CZE method with those obtained by anionexchange HPLC coupled to a triple-pulsed amperometric detection(HPAC-PAD) showed a high degree of precision and reproducibility forthe RFO compositions of a number of pea strains. No statistically significantdifferences (P ≥ 0.05) were found between the two analytical techniquesusing paired Student t-tests (Frias et al., 1996a,b).

Other analytical methods

OPTICAL ROTATION When sugars or their derivatives are reasonably pureand, in particular, free of optically active impurities, the measurement ofthe optical rotation can provide a simple method for their identificationand analysis (Pearson, 1976). One of the most important sugar mixturesthat can be analysed by this method is sucrose and its products of hydrolysis,fructose and glucose. The method is based on the rotation of plane-polar-ized light using a polarimeter or saccharimeter to measure the change inthe angle of polarized light. This method, dating from the 1840s, is notwidely used today, not least because large volumes of the samples to betested are needed. Also, this technique is not very sensitive at low concen-trations of sugars.

REDUCING SUGAR METHOD There are several well known tests which makeuse of the reducing action of sugars in alkaline solutions in the presence ofcertain metallic salts, e.g. copper, silver, mercury and bismuth. Those ofcopper have been employed by far the most extensively in sugar analysis(Pearson, 1976). The basic form of the reaction is:

RCHO + Ag2O → 2 Ag + RCOOHRCHO + 2 CuO → Cu2O + RCOOH

Unfortunately, for quantitative work these reactions do not proceedstoichiometrically. This is because of the ability for many sugars tomutarotate. This causes the carbon chain eventually to break and the freealdehyde and ketone groups are lost. Close control of the reaction condi-tions is needed along with calibration using standards. Fehling originallydevised the most widely known test based on this method, in 1848.

MEDICAL/FOOD DIAGNOSTIC TEST KITS There are various diagnostic kitsavailable for detecting fructose, glucose or sucrose in food substances,and so would be suitable for legume seed flour samples (Sigma-AldrichCompany and Boehringer Mannheim GmbH, Mannheim, Germany).These tests make use of the reducing capacity of the carbonyl grouppresent (or not present) in these sugars. Diagnostic test kits work quitewell but are generally expensive on a per sample basis. The BoehringerMannheim glucose test kit, cat. no. 124036, using the GOD-Perid method,forms the basis of a reliable starch assay (described in a later section). See

44 P. Kadlec et al.




the Official Methods of Analysis of the Association of Official AnalyticalChemists (AOAC, 1984) and American Association of Cereal Chemists(AACC, 1995) for more detailed information.

ENZYMATIC METHOD The enzymatic method of RFO determination involvesincubation with α-galactosidase followed by measuring the liberatedD-galactose, using either a chemical method or D-galactose dehydrogenase.For determining reducing carbohydrates, a useful colorimetric methodcan be used (Honda et al., 1982). Alternatively, the total oligosaccharidecontent can be expressed as sucrose units, which would be the endproduct of α-galactosidase (EC 3.2.1.22) action. Sucrose can be readilyestimated in the same digest by first reacting with invertase followed bythe colorimetric assay of glucose, using glucose oxidase reagent. However,the α-galactosidase method is not adequate for determining an individualoligosaccharide in a mixture containing other homologous sugars.

SPECTROPHOTOMETRIC METHOD The spectrophotometric method is oneof the simplest ways of determining the total soluble sugars in biologicalmaterial. In the case of oligosaccharides, the results are overestimatedbecause the analyses include other sugars like mono- and disaccharides.According to the data of Muzquiz et al. (1999), the content of sucrose asa proportion of the total sugar in legume seeds is in the range from7.1% (Lupinus luteus cv. Piast) to 53.0% (V. faba cv. Nadwiœlañski). For thisreason, the spectrophotometric method is burdened with a large error andis not very useful, therefore, for the determination of α-galactosides.

In connection with the TLC method, however, this method can bevery useful for the RFO. The content of individual RFO components (afterseparation by means of TLC, see p. 42) can be determined by thespectrophotometric method described by Pearson (1976) and Fry (1994).

2.2.2 Polysaccharides

StarchMany different ways for the determination of starch have been described inthe literature. In principle, two groups of methods can be distinguished.Firstly, there are the polarimetric methods in which the starch is quantifiedas a dissolved and partly degraded polymer (determination according toEwers and the calcium chloride method of the AOAC, see below). In thesecond group of methods, the starch is fully hydrolysed into glucose andthen quantified by measuring the glucose content.

POLARIMETRIC METHODS This is termed the Ewers method (AOAC method14.031) and is based on acid hydrolysis of starch, followed by the measure-ment of the optical rotation of the resulting solution.





Weigh 1 g of flour into a 50 ml flask, add 25 ml of 1.125% HCl. Cap andboil for 15 min in a water bath with stirring, add pure water to give a totalvolume of 40 ml and cool the flask to 20°C. Add 1 ml Carréz solution I(30% ZnSO4) to the solution. After stirring, add 1 ml Carréz solution II(15% K4[Fe(CN)6]) and adjust the volume to 50 ml. Filter the solution, andmeasure the optical rotation by polarimetry, using a 100 mm tube.

Calculate the total starch content:

Starch (%)[ ]

=× ×

× ×50 34 66. p

L mαwhere p is the measured value (°S), [α] the specific rotation power of peastarch (187.7°S), L the length of the tube (dm) and m the weight of sample(g).

CALCIUM CHLORIDE METHOD The flour under test (2–2.5 g) is passedthrough a 150-µm mesh sieve and defatted with diethyl ether, 10 ml of65% ethanol (v/v) is then added, the mixture is centrifuged and thesupernatant discarded. The residue is taken up in 10 ml of pure water,transferred to a 500 ml flask and mixed with 60 ml of a 33% (w/w) solutionof CaCl2 containing 2 ml of 0.8% acetic acid. The mixture is then cooled,transferred to a 100-ml flask and made up to 100 ml with CaCl2 solution.After filtration, the optical rotation of the resulting solution is determinedby polarimetry, using a 100-mm tube. Duplicate determinations (each con-sisting of the mean of ten separate optical rotation measurements) mustnot differ by greater than 0.006 units.

METHODS IN WHICH THE STARCH IS FULLY HYDROLYSED INTO GLUCOSE Differentprotocols for sample preparation and starch solubilization have beendescribed in the literature, e.g. autoclaving, treatment with hydrochloricacid in a boiling waterbath, treatment with dimethyl sulphoxide (DMSO)solution and treatment with DMSO/hydrochloric acid mixtures (Bruntet al., 1997; Jones et al., 1999a).

For seeds, sample solubilization is best carried out using 90% DMSO.The conversion of starch into glucose can be performed by acid orenzymatic hydrolysis. For acid hydrolysis different concentrations ofhydrochloric acid are used in a waterbath, for enzymatic hydrolysis, anenzymic mixture containing amyloglucosidase (EC 3.2.1.3) and α-amylase(EC 3.2.1.1) is used. The resulting glucose can be measured by either,titration, enzymatic determination (hexokinase, glucose oxidase) or HPLCdetermination. See AOAC method 920.40 and AACC methods 76–11.

DETERMINATION OF RESISTANT STARCH Because resistant starch (RS) has areduced content of energy and is characterized by physiological effects thatmake it comparable to DF, it is logical that the question be asked whetheror not it should be included in DF analytical figures (Lee and Prosky,

46 P. Kadlec et al.




1994). Since RS can be degraded to free glucose by acid hydrolysis, it can beeasily mistaken for the glucan polymers associated with the plant cell wall inthe chemical determination of DF (Wen et al., 1996). The ideal situationwould be to determine DF and RS contents of foods independently(Sambucetti and Zuleta, 1996).

Two main approaches for determination of resistant starch can be usedin vivo and in vitro. In this chapter only in vitro methods are described. Thein vitro analysis of RS implies the performance of an enzymatic hydrolysis(α-amylase in most cases), which is usually supposed to mimic the hydrolysisof starch by endogenous enzymes in the upper part of the digestive tract(mouth, stomach and small intestine).

The first attempt to analyse RS was performed by Englyst et al. (1982).Their method was only able to analyse retrograded ‘enzyme resistantstarch’ (R III). Indeed the grinding of the sample and the subsequentthermal treatment at 100°C made the quantification of the other two majortypes of RS, physically entrapped starch (RS I) and RS granules (RS II)impossible. Englyst later identified the fraction quantified by this methodas retrograded amylose. The main modifications introduced by Berry(1986) and then by the collaborators of the EURESTA inter-laboratorystudy (Champ, 1992, 1995) concerned the elimination of the gelatinizationstep and of the pullulanase hydrolysis. Consequently, both RS III and RS IIcould be quantified using this new method. Independently, Englyst et al.(1992) developed a more sophisticated methodology set up to analyserapidly digestible starch (RDS), slowly digestible starch (SDS) and RS.Minor modifications to the method of Berry (1986) were then proposed asdescribed by Champ (1992) and Faisant et al. (1995). These modificationswere undertaken to improve the slight underestimation of RS. One of themodifications is the use of sodium azide to prevent bacterial proliferationduring amylase hydrolysis, and secondly to introduce a de-proteinizationstep with pepsin. Both proposed the elimination of the drying step beforethe solubilization with potassium hydroxide.

There were also attempts to develop an in vitro RS assay by applyingchewing as the initial disintegration step (Muir and O’Dea, 1992). Thismethod was validated against in vivo studies in human ileostomates (Muirand O’Dea, 1993; Muir et al., 1995). The validation of the in vitro methodsagainst the in vivo methods showed quite good reproducibility (Table 2.6).

Two methods have been recommended for analysing RS in foods invitro, one developed by Englyst et al. (1992) and one described by Champ(1992) and Faisant et al. (1995), which has been developed from the modi-fied Berry method. After tests for validation in vitro methods against in vivostudies, the following conclusions were drawn (Champ, 1995; Asp, 1996).

Firstly, the two methods give very similar values with a high level ofRS. They both give an estimation of RS that does not take into account,however, the potentially digestible starch or starch fragments found in vivoat the end of intestine. Secondly, the modified Berry method is quicker and





easier to reproduce than the Englyst method. Thirdly, the Englyst methodmay reflect better the in vivo physiology than the other methods.

With all of the methods for RS analysis there is a fundamental problemrelated to the definition of RS. None of these methods, including the onedeveloped by Englyst et al., takes into account the whole amount of RSdefined as ‘starch and products of starch degradation not absorbed inthe small intestine of healthy individuals’, since low molecular weightfragments, soluble in aqueous ethanol, are not determined.

MEGAZYME KIT This method determines the total starch in a sample using a‘kit’ sold by Megazyme International Ireland Ltd, Co. Wicklow, Ireland(www.megazyme.com). It is based on the principles described in AACCmethod 76–12 and is listed as AACC method 76–13. For samples that donot contain high levels of resistant starch (e.g. wheat flour), completesolubilization of starch is achieved by cooking the sample in the presenceof thermostable α-amylase. Samples that contain high levels of RS (e.g.high-amylose maize) are completely solubilized by pre-treatment withDMSO at 100°C. Glucose produced by the enzymatic hydrolysis of thesolubilized starch is measured using glucose oxidase/peroxidase reagent.Samples containing high levels of glucose or maltodextrins have to bewashed with aqueous ethanol before analysis.

Fibre fractionSome of the earliest analytical methods of DF include crude fibre, aciddetergent fibre (ADF) and neutral detergent fibre (NDF) as the mostcommonly used methods. The principle of the crude fibre method impliesboiling and extraction of the sample by dilute acid and dilute alkali, withsubsequent isolation of the insoluble residue by filtration (AOAC methods920.86, 962.09). The crude fibre method essentially determines thecellulose and lignin content, however, the recovery may vary markedly.

48 P. Kadlec et al.


Quantification of resistant starch (% RS/TS)

in vitro in vivo

Origin of the RSChampmethoda

Englystmethodb Ileostomyc Incubationd

Beans 17 – – 17Raw green banana 61 71 68 84Retrograded high amylose corn starch 30 34 – 49

aChamp (1992); bEnglyst et al. (1992); cGöteborg (1995); dFaisant et al. (1993,1995).

Table 2.6. In vitro and in vivo quantification of resistant starch (RS) as a propor-tion of the total starch (TS).



In the ADF method, boiling of a sample is performed for 1 h inan acidic solution containing the detergent cetyltrimethylammoniumbromide (CTAB), and the residue is obtained by filtration (Van Soest,1963). The ADF method aims at determining cellulose and ligninwith higher precision than the crude fibre method, but remnants ofhemicellulose and pectin have been reported (Asp and Johansson, 1984).

The NDF method implies extraction of a sample for 1 h in a hot neutralsolution, containing the detergent sodium dodecyl sulphate (SDS) andethylenediamine tetraacetic acid (EDTA) (Van Soest and Wine, 1967). Thismilder treatment leaves most of the hemicellulose, in addition to celluloseand lignin in the residue, whereas pectins are efficiently removed. Instarchy material, e.g. peas, filtration problems of the residue may occur dueto residual starch. This problem may be solved by including a thermostableamylase in the NDF-reagent or by pre-incubation with amylase (Robertsonand Horvath, 1993). Common for the detergent methods is the removalof detergent soluble components, some being part of the enlarged DFconcept, including indigestible components other than the carbohydrateand lignin DF constituents (NSP and lignins).

Current methodologies in DF determination comprise the enzymatic–gravimetric and the enzymatic–chemical procedures. The enzymatic–gravimetric methods are based on enzymatic degradation of polymericmaterial such as starch, proteins and other components, with subsequentisolation and weighing of the undegraded residue equal to DF. A numberof procedures differing in types of enzyme, duration of incubation, pH ofbuffers, temperature and methods of DF isolation have been proposed.AOAC Official Enzymatic Gravimetric Methods comprise Methods 985.29,991.42, 993.16, 993.19, 993.21 and 991.43. Common for the enzymatic–gravimetric methods is the general inclusion of minor levels of a wide rangeof indigestible components, in addition to NSP and lignins (see Section2.2.3). DF values are thus usually corrected for the content of ash andprotein in the residue, although the correction of, in particular, protein isquestionable, as close association exists between proteins and polysaccha-rides/lignins in the plant cell wall. Insoluble (IDF) and soluble (SDF) DFmay be determined separately or pooled and determined as total DF(TDF). The traditional DF components present in the insoluble and solu-ble DF fraction differs depending on the DF source and specific isolationconditions, but basically lignin and cellulose is present in IDF, whereas SDFcomprise pectins. Hemicelluloses are found in both fractions dependingon their actual molecular structure and thereby solubility properties.

The idea behind the enzymatic–chemical methods is analysis of theindividual monomeric constituents of polysaccharides in the DF fraction(NSP, non-starch polysaccharides). The methods comprise, as a first step,partly removing non-DF components by means of enzymes, followed byacid hydrolysis of DF polysaccharides. The analysis of neutral mono-saccharides is generally performed by GC, but other chromatographic





systems may be used, e.g. HPLC (Quigley and Englyst, 1992) and HPCE(Rassi and Mechref, 1996). Alternatively, colorimetry may be used fordetermination of groups of pentoses, hexoses and uronic acids; howeverthis technique is seldom used as routine. Uronic acids also may be analysedby decarboxylation (Theander et al., 1994). Lignin is not included in themethod, but can be determined separately. The NSP may be separated intoa soluble and insoluble part in some of the methods. Monosaccharidelosses during the polysaccharide acid catalysed hydrolysis step are aproblem of major concern in enzymatic–chromatographic methods. Theconditions in acid hydrolysis are actually a compromise between completeliberation and destruction of monosaccharides. Various methods exist,differing in type and concentration of acid as well as temperature and timeused for hydrolysis. An AOAC Official Method has now been accepted(Method 994.13).

The analytical methods for determination of DF described abovegive only limited information on the level and nature of individual DFcomponents. More specific studies were originally performed usingthe classical fractionation schemes evolved in the 1930s (Southgate,1995a).

The experimental conditions used in these fractionations wereextremely vigorous and generally caused some severe modifications of thestructures of the components. Modern techniques for fractionation aregenerally more gentle, although the possibility still exists, that a certainnumber of bonds must be broken in order to extract components from thecell wall, leading to incorrect conclusions about the chemistry, especially ofcell wall polysaccharides. Moreover, the specific procedure for extractionof a particular type of component may result in only partial recovery of theexpected polysaccharide.

The fractionation is performed on purified plant material, and notdirectly on the fresh tissue. Methods for preparation of plant material priorto fractionation are described in the following sections, examples of theanalytical approaches for determination of the subfractions: cellulose,hemicellulose, pectins and lignins are given, extraction of minor DFcompounds such as, e.g., phenolics are described briefly, and more detailsare given elsewhere (Andersen et al., 1997; Bjergegaard et al., 1997a,b).

PREPARATION OF PLANT CELL WALL MATERIAL Although the definition of DFincludes components from outside the plant cell wall, the quantitativelydominating part in land plants is found inside the plant cell wall, and plantcell wall polysaccharides and lignins will, therefore, be considered. Analysisof individual non-starch polysaccharides may be disturbed by the presenceof other plant tissue components. On the other hand, some of these com-ponents may be found in close association with the cell wall polysaccha-rides, affecting the properties of the DF fraction. This apparent conflict ofinterest must be taken into consideration when planning the study of the

50 P. Kadlec et al.




individual DF components (Andersen et al., 1997; Bjergegaard et al.,1997a,b).

As a first step, it is important to consider the starting material to beused for the fractionation. The TDF fraction produced by the enzymatic–gravimetric procedure could be a possible starting point for the fraction-ation, whenever additional information on the level of some non-traditionalDF components is considered relevant. Any reserve polysaccharides fromoutside the plant cell wall will also be included. Another more commonpossibility is to use isolated plant cell walls, which may be more or less pure,depending on the exact procedure for preparation. Examples of suchpreparative methods are presented below.

A very simple cell wall preparation method consists of washing thehomogenized tissue in 70% ethanol. This procedure leaves polymersinsoluble (AIR, alcohol insoluble residue), whereas low molecular weightcompounds such as sugars, amino acids, organic acids and many inorganicsalts are solubilized (Fry, 1988). In addition to the cell wall polymers, otherhigh molecular weight components such as intracellular proteins, RNA,starch, reserve polysaccharides, etc., will be present in AIR and one mayconsider whether this will constitute a problem in the following DF studies.A problem with AIR is the dehydration effect of the alcohol possibly leadingto formation of various artefacts.

A more comprehensive method is as follows: 50 g fresh weight of sam-ple is homogenized in 100 ml 1% (w/v) aqueous sodium deoxycholate(SDC) or 1.5% (w/v) aqueous SDS containing 5 mM Na2S2O5. The slurry isfiltered and washed (twice). The resulting residue, R1, is ground in a wetball-mill in 100 ml 0.5% SDC (w/v) or SDS (w/v) containing 3 mMNa2S2O5 at 2°C for 15 h. The residue/slurry is centrifuged and washed.

Alternately the residue R2 is extracted twice with 50 ml phenol : aceticacid : water (PAW) (2 : 1 : 1, w/v/v) at 20°C and washed twice.

Sodium metabisulphite is included in order to diminish oxidation ofpolyphenolics. SDC/SDS solubilizes intracellular compounds as well assome cold-water-soluble pectins. PAW efficiently removes residual intra-cellular proteins together with some starch, adsorbed detergent, lipids andpigments. The amount of cell wall components solubilized in this step islow. The DMSO treatment extracts starch, whereas the amount ofco-extracted cell wall material depends on the kind of starting material.The method provides a useful procedure to obtain relatively pure cell walls,with about 90% of the total cell wall constituents remaining in the finalpreparation (Selvendran et al., 1985; Southgate, 1995b).

The diversity of method for the preparation of cell wall material isillustrated above and one has to consider what is needed in each specificcase. A simple isolation technique may thus be adequate, wheneverimpurities are not expected to interfere with the planned analysis, whereasin other situations very pure cell wall preparations may be needed. Forextraction of lipids and amphiphilic compounds it is more efficient, simple,





fast and cheap to use supercritical fluid techniques (super critical extrac-tion (SCE)/super critical chromatography (SFC); Andersen et al., 1997;Bjergegaard et al., 1997a,b).

CELLULOSE AND HEMICELLULOSE Determination of cellulose is generally partof a more comprehensive fractionation procedure, in which α-celluloseis obtained as the residue after sequential extraction of pectic material(Fig. 2.12), possibly lignins (delignification in lignified tissues) and hemi-celluloses. α-Cellulose is insoluble in 17.5% NaOH (w/v), whereas a minorpart of native cellulose may be solubilized here. Cellulose is virtuallyinsoluble in water. Selvendran (1983) considered α-cellulose as the fibrillarpart of the plant cell wall and that the solubilized native cellulose probablyoriginated from more amorphous regions.

Fry (1988) describes a method for direct extraction of cellulose by thehighly basic reagent cadoxen. Cadoxen is prepared by stirring a solutionconsisting of 1,2-aminoethane (310 ml), H2O (710 ml) and cadmium oxide(100 g) at 20°C for 3 h followed by 4°C for 18 h. The supernatant obtained

52 P. Kadlec et al.


Fig. 2.12. Sequential extraction of pectin material from purified cell walls.



after centrifugation is used for the extraction. The procedure convertscellulose into a soluble fraction by heating in dry DMSO and paraformalde-hyde. Free cellulose can then be regenerated as a precipitate by addition ofH2O or methanol.

The classical procedure for solubilization and subsequent hydrolysis ofcellulose is a two-step procedure involving the use of 72% H2SO4 (w/w)(e.g. 1 h at 35°C) as the first step followed by hydrolysis in dilute acid (e.g.2 M H2SO4 for 1 h at 100°C). The solubility of cellulose in strong sulphuricacid is due to disruption of hydrogen bonds caused by sulphonation ofhydroxyl groups at C-6 of glucose (Selvendran and Robertson, 1990). Thisprocedure is also used for hydrolysis of total NSP in the enzymatic–chemical methods and, according to Englyst et al. (1982) and Englyst andCummings (1988), omission of the first step gives a hydrolysate of non-cellulosic polysaccharides (NCP), whereas only cellulose is included by thetwo-step procedure. This division of cellulose and NCP has, however, beenquestioned (Bingham and Selvendran, 1983). Further studies of thecellulose fraction are generally restricted to determination of the degree ofpolymerization and studies of associated polysaccharides.

Quantification of the hemicellulose fraction as a difference betweenthe NDF and ADF residue has been a formerly accepted method (Asp andJohansson, 1984). This procedure gives an indirect measure of hemicellu-lose. The value of the method is questionable, however, due to the losses ofhemicellulosic polysaccharides by the NDF procedure and the incompleteremoval of hemicellulosic polysaccharides by the ADF procedure. Thisresults in an underestimation of the fraction and, in addition, there is nopossibility for a closer examination of the individual components.

A more preferable method is the extraction of hemicelluloses fromdepectinated sample material. This extraction is usually performed sequen-tially with extractions at increasing alkali strength and/or temperature.The extraction of hemicelluloses is carried out in the absence of oxygenusing potassium or sodium hydroxide following the saturation of theextraction medium with nitrogen or argon gas (Selvendran et al., 1985;Southgate, 1991). Moreover, a strong reducing agent such as NaBH4 maybe added. These precautions are taken to prevent the formation ofpolyphenolic complexes forming with the polysaccharides, making themdifficult to extract. Another function of NaBH4 is protection of thereducing sites in the carbohydrate molecule, as C-3 bound aldoses andC-4 bound ketoses may otherwise undergo β-elimination under alkalineconditions.

Various schemes exist for the extraction of hemicelluloses and specificprocedures have been developed for different types of sample. Pecticsubstances, which remain in the residue after a previous treatment withchelating agents, may be extracted with the hemicellulose fraction in step 1.The proportion of solvent to depectinated material may vary, as may thechoice of using argon or nitrogen gases. The scheme for the sequential





extraction of hemicelluloses by KOH is described by Selvendran andO’Neill (1987) and Southgate (1995b).

If heavily or moderately lignified tissue is used, it will be necessary toinclude a delignification step. It is debatable whether legume seeds couldbe classed as containing moderately or lightly lignified material. In order tocompare data from legumes with that obtained from other sources it wouldbe wise to consider including the following delignification step anyway.Delignification in moderately lignified tissue is carried out after the firstextraction with KOH, by treatment with sodium chlorite and acetic acidat 70°C for 2–4 h (Selvendran and O’Neill, 1987; Southgate, 1995b). Theaddition of the delignification step after the first alkali treatment isperformed in order to preserve native hemicellulosic polysaccharide–protein and polysaccharide–protein–polyphenol complexes, which maybe partially modified by the delignification treatment. The duration of thedelignification procedure may be varied depending on the type of materialbeing analysed. Heavily lignified tissue requires longer treatment anddelignification is generally performed prior to extraction of the hemi-celluloses. As delignification may lead to some modification of plant cellwall proteins and polysaccharides it should be avoided in only lightlylignified tissue (Selvendran and O’Neill, 1987).

The solubilization of polysaccharides by use of alkali is brought aboutby cleavage of ester linkages between polysaccharides (uronic acids) aswell as polysaccharides and non-carbohydrate constituents (e.g. phenolicacids). Hydrogen bonds will also be disrupted (Selvendran et al., 1985).Highly polymerized polysaccharides, glucomannans and slightly branchedxyloglucans, being strongly hydrogen bound to cellulose, require the use ofstrong alkali for solubilization (Selvendran and Robertson, 1990), and theextraction of glucomannans is enhanced by the inclusion of boric acid.Other hemicellulose extractants are the aqueous chaotropic agents: per-chlorate, urea and guanidinum thiocyanate. Common to these extractantsis that they are effective protein solvents, unlikely to cause any degradation.Only a minor part of the hemicellulosic compounds, however, will beextracted (Fry, 1988). The chaotropic agents are stated to be useful forsolubilization of mannose-rich polymers (Selvendran et al., 1985), probablybecause of the concurrent extraction of proteins.

Further studies of the extracted components comprise a wide rangeof techniques including gel filtration, anion exchange chromatography,affinity chromatography, precipitation by complex formation with inor-ganic salts (e.g. iodine and copper complexes), stepwise precipitationwith ethanol, precipitation by Ba(OH)2 and quaternary ammonium salts.More details of these techniques can be found in Wilkie (1985), Selvendranand O’Neill (1987) and Fry (1988). Fry also gives a thorough descriptionof the methods for structural elucidation, such as different hydrolysisprocedures, methylation analysis, periodate-oxidation studies, specificenzymatic degradations, etc.

54 P. Kadlec et al.




Some examples from characterization of polysaccharides in variousgrain legumes can be found for pea (Talbott and Ray, 1992; Ralet et al.,1993; Weightman et al., 1994) and lupin (Mohamed and Rayas-Duarte,1995). The commonly used preparation of TDF is described elsewhere(Andersen et al., 1997). The fractionation procedure for pea TDF intopectic material, hemicelluloses, cellulose and lignins is described below.

1. Pectins – add 25 ml 1% ammonium oxalate to 1 g TDF and adjust pHto 5.0 using 1 M HCl. Extract for 2 h at 80°C in a shaking waterbath. Cooland centrifuge (34,000 × g ; 20 min), freeze-dry the sediment, weigh anduse this sediment for extraction of hemicellulose (procedure 2). Keep 5 mlof the supernatant (−20°C) for further analyses and dialyse the rest (knownvolume) against water overnight (5°C). Save 5 ml dialysed supernatant(−20°C). Freeze-dry the remaining dialysed supernatant (known volume),weigh and save in desiccator (pectins). The weight of pectic materialshould be corrected for reduction in starting material.2. Hemicelluloses – add 25 ml 2 M NaOH to the sediment from proce-dure 1 and extract (under N2) for 2 h in a 30°C waterbath with occasionalshaking. Centrifuge (34,000 × g ; 20 min), freeze-dry the sediment, weighand use this sediment for extraction of cellulose (procedure 3). Make thesupernatant weakly acidic (with 3–4 ml acetic acid) and save 5 ml of the pHadjusted supernatant (−20°C) for further analyses. Dialyse the supernatant(known volume), including any sedimented material occurring after thepH adjustment, overnight (5°C). Collect the sedimented material bycentrifugation (3000 × g ; 3 min), freeze-dry, weigh and keep it in desicca-tor (hemicellulose A). Save 5 ml dialysed supernatant (−20°C). Freeze-drythe remaining dialysed supernatant (known volume), weigh and store in adesiccator (hemicellulose). The weight of the hemicellulose fractionsshould be corrected for reduction in starting material.3. Cellulose and lignin – add 1 ml 12 M H2SO4 to the sediment from2 and leave it for 1 h at 35°C with occasional mixing. Dilute with 11 mlH2O to give a 1 M H2SO4 solution, and continue extraction in a boilingwaterbath under reflux for 18 h. Cool and centrifuge (3000 × g ; 3 min),freeze-dry the sediment, weigh and store in a desiccator (lignin). Neutralizethe supernatant (known volume) with saturated Ba(OH)2 and centrifugeto remove BaSO4. Keep 5 ml of the neutralized supernatant (−20°C)for further analyses. Freeze-dry the remaining supernatant (knownvolume), weigh and store in a desiccator (cellulose). The weight of thecellulose fractions should be corrected for reduction in starting materialand for the weight reduction brought about by hydrolysis of thepolysaccharide.

The extraction conditions may be varied depending on the plant materialconsidered and the method presented here is only one of several possibili-ties as also indicated in the preceding sections. It should be noted that theresults obtained are exclusively dependent on the actual fractionation





procedure and this must be taken into consideration when the method isevaluated.

2.2.3 Other carbohydrate components

PectinCommon methods exist for the isolation of pectic substances from plantmaterials, and the main steps are outlined below.

1. Grind the plant material in warm ethanol or acetone.2. Wash with ethanol, to inactivate endogenous enzymes.3. Wash with sodium deoxycholate (SDC) (or enzyme treatment), toremove proteins.4. Wash with phenol–acetic acid–water mixture, to remove lipids andpigments.5. Treat with aqueous 90% DMSO (or enzyme treatment) to removestarch.6. Wash with ethanol, to remove the other organic solvents.

DETERMINATION OF GALACTURONIC ACID (GA) The titration method is widelyused for total pectin determination. In this method the galacturonic acidcontent and the degree of esterification (by methylation and acetylation)of a pectin preparation are calculated from the neutralization andsaponification equivalents of the pectic and acetic carboxyl groups. Prior toanalysis, the pectin must be converted to the free acid form by mixing witha strong cation-exchanger, or by washing with an alcohol/HCl mixture,followed by washing with alcohol until the washings are neutral. If acetylgroups are present in the pectins, a saponification equivalent that is toohigh is obtained.

By using the copper-binding method (Keijbets and Pilnik, 1974),interference by acetyl groups is overcome. In this method the quantity ofcopper ion (Cu2+) that binds to the pectin before and after saponification isdetermined stoichiometrically or by atomic absorption spectrometry andthe dry matter is calculated from the ratio of these values.

Colorimetric procedures, such as those based on carbazole (Bitter andMuir, 1962), have been widely used. Blumenkrantz and Asboe-Hansen(1973) significantly reduced the interference of neutral sugars by addingm-hydroxydiphenyl as a chromogen to heated solutions of uronidesin a sulphuric acid/boric acid mixture. Ahmed and Labavitch (1977)modified and tested this procedure for native and extracted pectins. Them-hydroxydiphenyl assay was automated by Thibault (1979). Garleb et al.(1991) described an anion exchange HPLC method with pulsed ampero-metric detection for the determination of galacturonic acid, after acidhydrolysis.

56 P. Kadlec et al.




DETERMINATION OF NEUTRAL SUGARS The most widely used method for sugardetermination involves acid hydrolysis of the sample with 2 M trifluoro-acetic or 1 M sulphuric acid, or by Saeman hydrolysis, which uses 72%sulphuric acid for solubilization and subsequent hydrolysis with 0.4 Msulphuric acid (Selvendran et al., 1979). After hydrolysis, the sugarsreleased are reduced to corresponding alditols and then converted toalditol acetates. These volatile derivatives can be reliably analysed by GC assingle peaks (Blakeney et al., 1983). Uronides are not determined by thismethod, but may be assayed together with neutral sugars by making TMSderivatives of the anomeric methyl glycosides obtained by methanolysis.In order to resolve the many peaks obtained from all sets of isomers, it isnecessary to use capillary columns in the GC.

The monosaccharides are subsequently analysed by high performanceanion exchange chromatography without the need for derivatization. It isalso possible to determine galacturonic acid and neutral sugars by a combi-nation of enzymic hydrolysis and methanolysis, followed by HPLC separa-tion on a C18 column eluted by water (Quemener and Thibault, 1990).

The types of glycosidic linkages between sugar residues are in generaldetermined by methylation analysis. Glycosyl-linkage analysis of uronosylresidues in polysaccharides is possible only after reduction to thecorresponding neutral sugar units.

LigninThe methods of lignin determination are essentially the same or similar tothose used to analyse the fibre, hemicellulose and cellulose fractions inlegume seeds. Suitable methods are summarized below.

VAN SOEST METHOD The residue of ADF is added to 72% H2SO4 at 0–4°Cfor 3 h with stirring. After hydrolysis, the residue is filtered, and washedwith hot water, then acetone and dried in a 100°C oven for 12 h, cooled in adesiccator and weighed.

MORRISON METHOD Digestion with acetyl bromide (Morrison, 1972).

KLASON METHOD In this procedure lignin is the residue after all theenzyme and chemical treatments of the Englyst DMSO method have beencompleted.

DELIGNIFICATION METHOD The residue is delignified by treatment withsodium chloride–acetic acid at 70°C for 4 h.

GOERING AND VAN SOEST METHOD The residue from the ADF is treated withpotassium permanganate solution, containing trivalent iron and mono-valent silver as catalysts. Deposited manganese and iron oxides are dissolvedwith an alcoholic solution of oxalic and hydrochloric acids, leaving





cellulose and insoluble minerals. Lignin is measured as the weight loss bythese treatments, while cellulose is determined as the weight loss uponashing of the residue.

NIR SPECTROSCOPIC ANALYSIS See Reeves (1988).

FT-IR ANALYSIS See Buta and Galletti (1989).

REVISED METHOD FOR QUANTIFYING DF COMPONENTS This method involveslipid removal with diethyl ether; removal of water-soluble componentsand quantification of water-soluble fibre components, removal of water-insoluble hemicellulose and cellulose (Jeltema and Zabik, 1980).

GRAVIMETRIC METHOD After starch extraction, the residue is washed, dried,then hydrolysed by heating with 10 ml 1 M H2SO4 for 2 h at 100°C to hydro-lyse the remaining cellulose fraction. The samples are then diluted with11 ml of water and heated for 2 h at 100°C. The residue is washed withwater and ethanol. Samples are extracted twice with warm diethyl ether andthen with acetone and air dried at 45°C. The residue is then weighed andashed at 500°C for 8 h to constant weight. The loss in weight is assumed tobe lignin (Anderson and Bridges, 1988).

Saponins

EXTRACTION A general method for the extraction of saponins from legumeseeds is as follows.

Seeds are ground to a flour (30 g) and extracted with chloroform(800 ml) for 16 h in a Soxhlet extractor. The chloroform extract is evapo-rated in vacuo and the air-dried defatted flour is then extracted withmethanol (800 ml) for 30 h. The methanol extract is evaporated to drynessin vacuo, dissolved in distilled water and run through a column of reversedphase octasilane (C-8) bonded to silica gel. The column is successivelyeluted with distilled water (150 ml) and methanol (150 ml) and thefraction evaporated to dryness.

The residue is hydrolysed with dry hydrochloric acid in methanol(5 ml, 5% solution) and refluxed for 3 h, the solution is neutralized andevaporated to dryness. After redissolving in water (5 ml), the sapogenolsare extracted with ethyl acetate (3 × 5 ml). The combined ethyl acetateextracts are then dried over anhydrous sodium sulphate, filtered andevaporated to dryness. For more information see Fenwick et al. (1991) andTsukamoto et al. (1993).

TLC ANALYSIS The dried sapogenol hydrolysis products are redisolved inmethanol (1 g ml−1) and loaded on to TLC plates together with standardsof soyasapogenol A and B. The plates are run with a chloroform : methanol

58 P. Kadlec et al.




(76 : 4 v/v) for a distance of 5.5 cm. Plates are air-dried and visualized witha p-anisaldehyde reagent.

GC ANALYSIS The methanol solution of hydrolysed saponins (1 ml), equiva-lent to 1 g of defatted flour, is evaporated to dryness in a vial by a streamof nitrogen. After further drying over phosphorus pentoxide in a vacuumdesiccator (12 h), a TMS derivatizing reagent is added (pyridine : bistrimethylsilyl trifluoroacetamide (BSTFA) and the samples heated for20 min at 50°C; 1 µl of the sample is injected (with 1 µl of cholesteryl-n-decylate, 1.87 mg ml−1 as an internal standard) on to a Perkin ElmerSigma 3B GC fitted with a glass column (1 m × 2 mm I.D.), packed with 3%OVl on Diatomite CQ AW DMCS, 60–80 mesh fitted with FID. The columntemperature was 280°C, injector temperature 285°C and detector tempera-ture 300°C. The carrier gas (He) flow rate is 35 ml min−1.

The identities of soyasapogenols A, B and C can be confirmed by directcomparison with authentic standards as well as by combined GC–MS usingthe TMS-ether derivatives.

HPLC ANALYSIS Aliquots (10 µl) of each extract can be injected directlyon to a 250 × 4.6 mm Spherisorb S5 ODS 2 column and eluted using anacetonitrile : water : trifluoracetic acid mixture with the compositionchanging from 80 : 20 : 0.1 v/v to 20 : 30 : 0.1 v/v over a time span of25 minutes using an eluant flow rate of 1 ml min−1. Detection is by UVabsorption at 210 nm.

For further details of experimental systems, see Fenwick et al. (1991)and Tsukamoto et al. (1993).





NutritionH. Kozlowska et al.3

3NutritionEditor: Halina Kozlowska

Contributors: Pilar Aranda, Jana Dostalova,Juana Frias, Maria Lopez-Jurado, HalinaKozlowska, Jan Pokorny, Gloria Urbano,Concepcion Vidal-Valverde and ZenonZdyunczyk

Food is an important part of a balanced diet.Metropolitan Life, p. 110 (1978)

Fran Lebowitz (1946–), American writer

3.1 Introduction

The carbohydrate fraction of grain legumes is a major source of foodand feed energy. The optimal composition of grain legume carbohydrates,however, depends on a number of factors, for example, consumer demandand the requirements for animal feedstuff.

The diets for intensively farmed animals are required to have a highenergy value. The desired direction for grain legume breeders and proces-sors, therefore, is to decrease the content of those carbohydrates that havelow energy values, or, which act as antinutrients. In this context manyauthors classify α-galactosides in grain legumes as antinutrients (Eskin et al.,1980; Saini, 1989). It has been known for many centuries that peas andbeans, although nutritional wholesome foods, produce wind (Gerarde,1633). To quote from the British Herbal (Hill, 1756) ‘The fruits of these sev-eral kinds are all of the same quality, wholesome as food, but apt to breedwind’. High quantities of α-galactosides and non-starch polysaccharides arebelieved to cause flatulence and reduce the net energy of seeds (Fernandezand Batterham, 1995). From the point of view of animal nutrition, lowbioavailability of starch is also a disadvantage of grain legumes.





Consumer perception of grain legumes depends on national andcultural differences, often related to a country’s wealth. In developingcountries, for example, grain legumes are looked on as ‘poor man’s meat’and are of great importance, therefore, as a protein source. In developedcountries there is often an excessive consumption of animal products (richin saturated fats) and a reduced intake of vegetables and grain legumes,which are, therefore, of far less importance as a protein source. Also,grain legume seeds are becoming more associated in developed countrieswith preventative or therapeutic effects on some diseases such as diabetes,hypercholesterolaemia, cancer, etc., rather than as a source of nutrition(Frühbeck et al., 1997).

Despite their relatively infrequent consumption of legumes (only 10%of the population include them in their diet on any one day), legumesprovide 14% of the dietary fibre (DF) intake in the USA, 25% in GreatBritain and Belgium, and 5–10% in France (Benamouzig et al., 1994).Although legumes are a good source of DF, as mentioned above, they alsohave significant amounts of oligosaccharides associated with flatulence,which is claimed as a major reason for their limited consumption (Sainiand Gladstones, 1986; Price et al., 1988). There are, however, positiveeffects of these compounds and possible health benefits in humannutrition have commanded considerable attention in recent years (Oku,1994; Cummings and Englyst, 1995). The possible elimination of oligo-saccharides from grain legumes, therefore, requires a renewed discussion.Numerous works published recently make it possible to characterize betterthe nutritional and physiological value of grain legumes, allowing therequirements for improving carbohydrates in the seed to be set out moreclearly.

3.2 The Content of Carbohydrates in Grain Legumes Utilizedin Europe

3.2.1 The content of carbohydrates in grain legumes used for humannutrition

The most common legumes for human consumption are bean (Phaseolusvulgaris), lentil (Lens culinaris), pea (Pisum sativus), chickpea (Cicer arie-tinum) and faba bean (Vicia faba). The carbohydrate fraction in the seeds ofthese legumes is composed of soluble carbohydrates (mainly fructose,sucrose and low molecular weight oligosaccharides such as ciceritol,raffinose, stachyose and verbascose), starch and longer chain oligosaccha-rides and polysaccharides constituting dietary fibre. Table 3.1 collates theinformation found in the literature for mono- and disaccharides, lowmolecular weight oligosaccharides (or α-galactosides), total soluble sugarsand starch from bean, pea, lentil, chickpea and faba bean.

62 H. Kozlowska et al.




According to different authors the total soluble sugar content of ninevarieties of bean (Phaseolus vulgaris) is very similar to that found in lentil,although some differences were observed in the individual sugar content(Table 3.1; Salunkhe et al., 1989; Vidal-Valverde et al., 1993a; Troszynska etal., 1995). Fructose was not present, however, and the content of sucrose(1.6–3.9%) was higher than in lentil but lower than in chickpea. Theaverage α-galactoside content was similar for lentil and chickpea, but thehighest range (0.4–8.0%) was found among bean varieties. Ciceritol wasnot present and higher amounts of raffinose, stachyose and verbascose,(average value of 0.7, 2.7 and 0.6% respectively) were detected in beancompared with lentil and chickpea. The information found in the literaturefor the starch content of bean was for three varieties only and this value,51–59%, is the highest of all the legumes used for human consumption.

The information obtained from the literature for 36 pea varieties(Cerning-Beroard and Filiatre, 1976; Cerning-Beroard and Filiatre-Verel,1979; Adsule et al., 1989; Van Lonkhuijsen et al., 1992; Troszyska et al., 1995;Frias et al., 1996d; Vicente, 1998) indicates that the content of total solublesugars is similar to that found in lentils, although once again the individualcomposition is very different (Table 3.1). Fructose was not present and thecontent of sucrose (0.9–5.4%) is much higher than in lentils. The range oftotal α-galactosides content found among the pea varieties was 2.3–9.6%.Raffinose (0.4–2.3%) and stachyose (0.3–4.2%) were present in all of thepea varieties and verbascose only in 24 varieties, with a mean value of 1.8%.

Nutrition 63


Beans Peas Lentils Chickpeas Faba beans

Fructose (mean) – – 0.1 – 0.4(range) .30–0.2

Sucrose 2.5 2.1 1.7 4.7 2.21.6–3.9 0.9–5.4 1.1–3.0 2.8–6.9 0.1–3.8

Raffinose 0.7 0.9 0.3 0.3 0.50.2–2.5 0.4–2.3 0.1–0.8 .30–0.3 0.1–1.5

Ciceritol – – 0.7 2.20.2–2.1 1.2–3.1

Stachyose 2.7 2.0 1.9 1.3 0.90.2–3.9 0.3–4.2 1.1–4.0 0.4–2.0 0.2–1.6

Verbascose 0.6 1.8 0.3 trace 1.80.1–1.8 .30–4.3 .30–6.4 trace–0.4 1.1–2.4

Total α-galactosides 3.8 4.6 3.2 3.8 3.00.4–8.0 2.3–9.6 1.8–7.5 2.0–7.6 1.0–4.5

Total soluble sugars 5.2 6.7 5.0 8.4 5.62.0–9.6 3.5–13.8 3.3–9.5 4.6–14.2 2.2–8.5

Starch 54.0 39.0 47.4 50.4 43.051.0–59.0 24.7–57.4 40.1–57.4 43.0–59.0 39.2–47.2

Table 3.1. Soluble carbohydrate and starch content (average and range as % drymatter) of some common grain legumes, used mainly for human consumption.



The starch content in the 12 pea varieties described in the literaturediffered widely, wrinkled peas showing quite low starch contents (mean of24.7%), while in smooth peas the starch content was very high (meanof 49.6%).

For lentil seeds (Table 3.1), information found in the literaturecovered about 20 varieties (Vidal-Valverde et al., 1992a,b, 1993a,b; Friaset al., 1994, 1995, 1996a,b,c; Troszynska et al., 1995; Urbano et al., 1995;Sotomayor, 1997). This showed a large difference in the total soluble sugarcontent, ranging from 3.9 to 9.5%, which was mainly due to differences inthe range of α-galactoside content (1.8–7.5%). Fructose was found in verysmall amounts (0.01–0.2%), while sucrose was present in all varieties inquantities ranging from 1.1 to 3%. With regard to the individualα-galactoside content, ciceritol was found in all varieties of lentil, theamount ranging from 0.2 to 2%. With the exception of chickpea, thisoligosaccharide does not occur in the other grain legumes. Raffinose wasalso present in all lentil varieties with quantities ranging from 0.1 to 0.8%.Stachyose was the most abundant α-galactoside, ranging between 1.1 and4.0%. Verbascose is not present in some lentil varieties, while it can reachup to 1.8% in others. Information on starch content in lentil has beenfound for only 19 varieties. The average starch content in lentil was 47%with a range of 40–57%.

For chickpea (Table 3.1), the information collected for 24 varietiesfrom the literature (Rossi et al., 1984; Saini and Knights, 1984; Chavan et al.,1989; Vidal-Valverde et al., 1993a; Sotomayor, 1997; Frias et al., 1999) showsthat soluble sugar content has the highest value within the grain legumes(4.6–14.2%), mainly due to the high content of sucrose (2.8–6.9%). Of theindividual α-galactosides, ciceritol was present in the largest amount(1.2–3.1%) followed by stachyose (0.4–2.0%) with only two of the varietieshaving very high amounts of this oligosaccharide (4–6.5%). Raffinosewas present in chickpea in very small amounts (traces – 0.3%), but thosevarieties with very high amounts of stachyose also had quite high amountsof raffinose (1.0–2.0%). The pentasaccharide, verbascose, was present intrace amounts, but in those varieties with the highest content of raffinoseand stachyose, the verbascose content increased to 0.2–0.4%. The contentof starch has been reported for only six chickpea varieties and ranged from43 to 59%.

According to the information from different authors, for 10 varieties offaba bean (Cerning-Beroard and Filiatre, 1976; Kozlowska et al., 1992; VanLonkhuijsen et al., 1992; Troszynska et al., 1995; Frias et al., 1996d; Frejnagelet al., 1997; Vidal-Valverde et al., 1998) the total soluble sugar content(2.2–8.5%) is similar to that found in bean (Table 3.1). Fructose contentwas not referred to in most of the faba bean varieties, but for those in whichit has been analysed the amount was relatively high (0.4%). The sucrosecontent was very wide with values ranging from 0.1 to 3.8%. The content ofα-galactosides (1.0–4.5%) was similar to that present in common bean.





Ciceritol was not present in either faba bean or pea. The content ofraffinose in faba bean was lower than in bean and pea and the stachyosecontent was the lowest amongst the grain legumes reported (Table 3.1).The starch content of the six varieties of faba bean reported in theliterature ranged from 39.2 to 47.2%.

DF is defined physiologically as the total amount of polysaccharidesand lignin not digested by endogenous enzymes of the human gastro-intestinal tract. Since the composition of DF is complex, different methodshave been used to quantify its content and constituents. The compositionof DF reported can vary widely, therefore, depending on the methodologyused. Taking this consideration into account, a large amount of data hasbeen recorded on the content and composition of DF in legumes used forhuman consumption.

The DF content in legume seeds depends on many factors, includingthe species and the variety (Table 3.2). The DF content of common beansindicated by different authors (Naivikul and D’Appolonia, 1978; Fleming,1980; Chen and Anderson, 1982; Fidanza et al., 1982; Reddy et al., 1984;Garcia-Olmedo et al., 1985; Paul and Southgate, 1985; Souci et al., 1986;Lintas et al., 1992; Mongeau and Brassard, 1994, 1995; Vidal-Valverde et al.,1992c) ranged from 11.2 to 27.5%, the contribution of the soluble compo-nent being the highest for the grain legumes (8.1–10%). The DF content inbean, noted by different authors, showed considerable differences for thesame types of seeds (Table 3.3). The results presented refer to the raw seedsof bean. During cooking, changes in the DF content and composition canbe observed. Acevedo et al. (1994) noted that the DF content in black beansdiffered according to the type of processing; it reached 26.5% in cookedseeds, 28.1% in blended seeds and 29% in fried seeds. The proportion ofsoluble DF in cooked, blended and fried beans reached 31.7, 26.7 and

Nutrition 65


Beans Peas Lentils Chickpeas Faba beans

NDF 8.9–12.8 13.2–25.6 9.7–24.1 7.5–19.2 13.0–19.5ADF 3.5–7.2 no information 2.0–6.8 3.8–14.7 10.3–11.4Cellulose 3.2–13.1 0.9–13.3 3.5–14.8 1.1–13.7 8.3–14.3Hemicellulose 0.5–5.6 0.9–12.4 1.2–15.7 0.6–16.0 1.6–8.9Lignin 0.1–3.1 0.3–2.1 trace–2.6 trace–7.1 0.7–2.0TDF 11.2–27.5 16.1–21.6 11.0–21.4 8.2–24.0 17.1–23.8SDF 8.1–10.0 4.6–6.0 1.2–4.4 3.7 6.0–8.7IDF 9.1–11.6 11.6–16.1 8.8–13.7 7.9 8.3–15.5NSP 6.4–20.4 no information 6.9–14.7 5.5–35.4 17.5

NDF, neutral detergent fibre; ADF, acid detergent fibre; TDF, total dietary fibre;SDF, soluble dietary fibre; IDF, insoluble dietary fibre; NSP, non-starch polysaccha-rides.

Table 3.2. Content of dietary fibre and its components (as % dry matter) of somecommon grain legumes, used mainly for human consumption.



22.8%, respectively, of total DF. In raw seeds, the soluble DF content isusually higher, up to 35% of total DF (Acevedo and Bressani, 1989; Lintaset al., 1992).

The DF content of pea, according to information from differentauthors (Van Soest, 1978; Chen and Anderson, 1982; Reddy et al., 1984;Paul and Southgate, 1985; Souci et al., 1986; Maltese et al., 1995; Zdunczyket al., 1997; Kmita-Glazewska and Kostyra, 1998) ranged between 16.1 and21.6%, the insoluble DF content being higher than the soluble DF (Table3.2). In the seeds of 15 Polish cultivars of white-flowered spring pea (Pisumsativum) the average DF content was 18.8% of the dry matter (DM) witha range from 16.2 to 21% (Zdunczyk et al., 1997). A highly significantnegative correlation r = −0.815 (P = 0.01) was found between the weight ofseeds and DF content. Igbasan et al. (1997) reported that in seeds of 12Canadian pea cultivars, the average DF content was 20.3%, including 14.1%of NSP, 2.5% cell wall protein, 0.4% of cell wall ash and 3.3% of lignin andpolyphenols.

The DF content of lentil found in the literature (Naivikul andD’Appolonia, 1978; Chen and Anderson, 1982; Fleming, 1981; Fidanza,1982; Reddy et al., 1984; Garcia Olmedo et al., 1985; Paul and Southgate,1985; Shekib et al., 1985; Souci et al., 1986; Lintas et al., 1992; Vidal-Valverdeand Frias, 1992; Vidal-Valverde et al., 1992c, 1993a; Pizzoferrato et al., 1995)ranged from 11 to 21.4%, the highest amount being insoluble DF(8.8–13.7%) and the smallest amount (1.2–4.4%) for soluble DF. On thebasis of the analyses from many authors, Savage (1988) states that theaverage DF content in lentil seed is 21.4%, including 4.5% of soluble DF.Other authors mention much lower DF contents ranging from 11 to 15%of DM (Garcia-Olmedo et al., 1985; Paul and Southgate, 1985; Lintas et al.,1992; Pizzoferrato et al., 1995).



Type DF content (%DM) References

Great northern 20.7 Mongeau and Brassard (1994)Light red kidney 14.3–18.1 Mongeau and Brassard (1995)Red kidney (Goya) 20.7–21.3 Mongeau and Brassard (1994)Red kidney (Townhouse) 21.3–21.9 Mongeau and Brassard (1994)White navy 15.8–19.2 Mongeau and Brassard (1995)No name 12.7–12.8 Mongeau and Brassard (1995)Mottled 19.1 Lintas et al. (1992)White 19.6 Lintas et al. (1992)No name 11.6 Lintas et al. (1992)Pinto 27.0 Chen and Anderson (1982)No name 16.8 Paul and Southgate (1985)No name 28.1 Paul and Southgate (1985)

Table 3.3. Dietary fibre (DF) (as a percentage of dry matter, DM) content in beans(Phaseolus vulgaris).



The information obtained from the literature on the DF content ofchickpea (Fleming, 1980; Vitaladasa and Belavady, 1980; Reddy et al., 1984;Rossi et al., 1984; Saini and Knights, 1984; Garcia-Olmedo et al., 1985; Pauland Southgate, 1985; Souci et al., 1986; Lintas et al., 1992; Vidal-Valverdeet al., 1992c; Mongeau and Brassard, 1994, 1995; Modgil and Mehta, 1996;Nestares et al., 1997) ranged from 8.0 to 24.0%. As in the case of lentil, theinsoluble components were much higher than the soluble components.

The DF content for faba beans, according to the literature (Spiller,1986; Wang et al., 1991; Gdala et al., 1995; Pizzoferrato et al., 1995; Vidal-Valverde et al., 1998) ranged from 17.1 to 23.8, where the content ofinsoluble DF was very high (8.3–15.5%) and the soluble DF was 6.0–8.7%.

It is apparent, therefore, that the amount of soluble carbohydratesfound in most common legumes used for human consumption rangesfrom about 3.3 to 13.8%, depending on the variety and the type of legume.Fructose is present in very small amounts (traces – 0.4%) and is only foundin lentil and faba bean. All of the legumes contain sucrose in a rangebetween 0.1 and 6.9%, the lowest content being found in some varieties offaba bean and the highest in some varieties of chickpea. The α-galactosidecontent of the most common legumes used for human consumption rangesfrom 0.4 to 9.6%, with some chickpea and pea lines showing very highlevels. Raffinose and stachyose are present in all legume seeds, rangingfrom 0.1 to 2.5% and 0.2 to 5.2%, respectively, the highest levels ofstachyose being found in chickpea and pea. Ciceritol is found only inchickpea and lentil and ranges from 0.4 to 3.1%. Verbascose is present invariable amounts according to species and varieties, with some varieties oflegumes having no, or only traces of, verbascose, while others, such as lentiland chickpea, have quite high amounts (4.2–4.5%). Starch is the maincarbohydrate present in bean, chickpea, lentil, pea and faba bean, thecontent varying between species and between varieties. Wrinkled peas(see Chapter 7) have the lowest starch content and on average, bean andchickpea have the highest starch content. The content of DF for the mostcommon grain legumes used for human consumption is very high, rangingfrom 11 to 27.5%. The soluble component is abundant in bean and fababean, while the highest insoluble DF was found in pea.

3.2.2 The content of carbohydrates in grain legumes used for animalnutrition

Grain legumes utilized in Europe for animal nutrition vary widely in termsof the content and composition of sugars, ranging from about 5% in fababean to about 13% in yellow lupin (Table 3.4). Total soluble sugars containonly a small amount of monosaccharides and from 1 to 3% of sucrose.Soluble sugars consist mostly of α-galactosides. The lowest content of α-galactosides was found in faba bean seeds (less than 2.5%), an intermediate

Nutrition 67




level in pea (about 5%), and the highest level in lupin (up to 12%). Theseeds of white lupin are characterized by having very small amountsof verbascose, their main sugar being stachyose. The highest amounts ofverbascose were found in the seeds of yellow and blue lupin, pea andfaba bean (25, 33 and 50%, respectively) as a proportion of the totalα-galactosides.

Starch is the main carbohydrate present in pea and faba bean, whichare used commonly for animal nutrition. Only in lupin seeds is thestarch content below 1% DM. Abreu and Bruno-Soares (1998) analysed thechemical composition of nine legume species and found the followingstarch contents: 45.3% for pea, 40.0% for faba bean, 0.8% for narrow leafedlupin and 0.7% for yellow lupin. The average starch content in the seedsof 36 different lines of feed peas analysed by Bastianelli et al. (1998) was49.2%, while the average for six lines of wrinkled pea was only 29.4%.

Apart from soluble sugars and starch, grain legume seeds are an impor-tant source of dietary fibre in the diets of animals. The data presentedin Table 3.5 indicate that legumes utilized in animal feed differ in thecontent as well as the composition of DF. The highest amounts of DF occurin the seeds of Lupinus angustifolius (39.2%) and the main NSP componentsin this species are galactose and glucose. A lower proportion of DF is foundin the seeds of Lupinus luteus, where glucose is the main component.



Sucrose Raffinose Stachyose Verbascose

Totalα-galacto-

sides

Solublecarbohy-

drates Starch

Peaa 1.8 0.8 2.5 1.7 4.9 6.8 47.5Faba beanb 2.5 0.2 0.9 1.4 2.4 4.9 41.3White

lupinc2.8 0.7 6.6 0.5 7.9 10.7 ND

Yellowlupind

1.1 2.2 6.9 2.8 11.9 13.0 ND

Narrow leaflupine

1.4 1.4 5.2 2.0 8.6 10.0 ND

Chickpeaf 3.5 0.6 1.9 ND 8.2 11.7 52.6Lentilg 1.9 0.3 1.7 0.4 4.7 6.6 46.2

aFrejnagel et al. (1997); Gdala and Buraczewska (1997) (mean of 19 varieties).bGdala and Buraczewska (1997a,b); Zdu4czyk et al. (1997) (mean of five varieties).cZdu4czyk et al. (1996) (mean of three varieties).dZdu4czyk et al. (1994).eTrugo et al. (1988).fFrias et al. (1998).gFrias et al. (1994) (mean of 16 varieties).ND, not detected.

Table 3.4. Content of soluble carbohydrates (SC) in seeds of different legumespecies (as a %).



Considerably smaller amounts of DF were noted in faba bean and peaseeds, 23 and 18%, respectively.

3.3 Physiological Effect of Grain Legume Carbohydrates inAnimal Nutrition

3.3.1 Consumption of grain legume carbohydrates in feed

The grain legume carbohydrate content in animal diets differs according tothe species and age of animals, and in the amounts of particular grainlegumes introduced into their diets. The proportion of grain legumes indiets of monogastric animals is rather limited for many reasons, butespecially because of antinutritional factors. In practice, grain legumes areused occasionally as the sole high-protein component of pig diets. In theindustrialized parts of the European Union, the mean levels for theincorporation of peas into pig, poultry and cattle diets are 20, 10 and 25%,respectively (Bourdillon, 1998).

Grain legumes provide animal diets with small amounts of mono- anddisaccharides, and with higher amounts of α-galactosides. They are mostoften used as a substitute for soybean meal, which also contains significantlevels of α-galactosides. Toasted soybean meal contains about 5% DM ofα-galactosides (Seve et al., 1989; Coon et al., 1990). Substituting soybeanmeal with meal from grain legumes brings about an increase in theα-galactosides content in animal diets for two main reasons. Firstly, muchmore grain legume meal than soybean meal is needed to obtain the sameconcentration of crude protein in the diet and secondly, some grain

Nutrition 69


NSP Pea Faba bean Lupinus luteus Lupinus angustifolius

Rhamnose 0.9 0.8 0.5 0.8Fructose 0.4 0.1 0.3 0.3Arabinose 19.4 16.4 12.6 10.7Xylose 7.3 13.0 11.3 8.3Mannose 0.9 0.7 1.9 1.6Galactose 8.6 18.3 16.5 34.9Glucose 47.6 45.3 89.0 31.5Uronic acid 15.0 14.6 12.3 12.0Total NSP 172.4 208.6 309.0 365.0DFa 179.2 229.6 325.0 392.0

aNSP + acid detergent lignin (ADL).

Table 3.5. The content of dietary fibre (DF) and composition of non-starchpolysaccharides (NSP) in seeds of different legume species (all data as g kg−1 of drymatter) (Gdala and Braczewska, 1997a; Gdala et al., 1997b,c).



legumes, in particular lupins, have a much higher α-galactoside contentthan soybean.

Table 3.6 presents examples of how the α-galactoside content changesaccording to the proportion of soybean meal and different substitutes inthe diet. In diets with the standard content of soybean meal (16%), the con-tent of α-galactosides is about 8 g kg−1. Only the diet containing faba beanhas a lower amount of α-galactosides. Substituting soybean meal with peaand lupin causes an increase in the α-galactoside content from about 8 to16–20 g kg−1, while substituting about 50% of the soybean meal with seedsof pea and lupin still gives an α-galactoside content in the diets of growingpigs that is higher than the amount found in the standard soybean diet.

Other dietary components also contain α-galactosides. According toCarré et al. (1984), the content of α-galactosides in diets for poultry usuallyranges from 0.5 to 3%, with the main sources being, in decreasing order,soybean meal (6%), peas (5%), faba beans (4%), rapeseed meal (3%) andsunflower meal (2%) of the dry matter.

In animal diets, pea and faba bean meal is a source of legume starch. Inthe case of total substitution of soybean meal with pea meal, legume starchcomposes about 15% of the diet. When soybean meal is partially substitutedwith the pea and faba bean meal, the proportion of legume starch in thediet amounts to 10 and 6%, respectively.

Unlike the seeds of pea and faba bean, lupin seeds contain highamounts of NSP, which constitute 27–35% of the seed dry matter inL. luteus and 35–42% in Lupinus albus (Gdala and Buraczewska, 1997). Forthis reason, diets rich in lupin seeds contain higher amounts of NSP thandiets comprising pea, faba bean and soybean meal. Bioavailability of starchand non-starch polysaccharides from legume seeds can have a significantinfluence, therefore, on the utilization of energy from the diet.



Content in feeda

(g kg−1)Share in dietb

(%)GAL content in diet

(g kg−1)

CP GAL A B A B

Soybean meal 440 49.0 16 6–8 7.8 –Pea 214 49.4 33 20 16.4 13.1Faba bean 259 24.2 27 15 6.5 7.1Yellow lupin 375 104.8 19 10 19.9 14.0Narrow leaf lupin 328 75.5 21 10 15.8 12.4White lupin 333 78.9 21 10 16.6 12.9

aData for soybean meal according to Seve et al. (1989) and Coon et al. (1990).bGrain legumes share in diet: A – total substitution of soybean protein, B – partial(about 50%) substitution of soybean protein.

Table 3.6. Content of α-galactosides (GAL) in diets for pigs with the share ofdifferent components rich in crude protein (CP).



3.3.2 Effect of mono- and disaccharides in animal nutrition

The monosaccharides and the main disaccharide, sucrose, are normallyfully absorbed in the small intestine. In the case of pigs (Canibe and BachKnudsen, 1997; Gdala and Buraczewska, 1997a,b) and poultry (Carré et al.,1995), the apparent ideal digestibility of sucrose from a diet containing peais nearly 99%. Sucrose is readily hydrolysed by sucrase activity locatedon the brush border membrane of enterocytes in the gut and the releasedglucose and fructose are fully absorbed. The mono- and disaccharides fromgrain legumes are not a significant source of feed energy, because they arepresent in only small amounts.

3.3.3 Effect of oligosaccharides in animal nutrition

The first information about antinutritional effect of α-galactosides wasnoted by Kuriyama and Mendel (1917). They reported that test meals of3 or 5 g of raffinose fed to fasting rats resulted in severe diarrhoea withevidence of raffinose residues in the faeces. More recently it has beenshown that the intestinal mucosa of monogastric animals and humans lacksthe α-galactosidase enzyme required to cleave α(1→6) linkages (Gitzelmanand Auricchio, 1965). Oligosaccharides of the raffinose family, whichcontain α(1→6) linkages between α-galactose units and α-galactose andsucrose, therefore, cannot be hydrolysed endogenously and can beclassified as non-digestible carbohydrates. It is generally accepted thatthese oligosaccharides pass undigested into the lower gut of the animal,where they are metabolized by gas-producing bacteria (Rackis, 1975).

Carré et al. (1991) found an apparent α-galactoside digestibility of82–87% in chicken, suggesting extensive microbial fermentation in thelower gastrointestinal tract of the birds. In addition, it has been found thatthe apparent digestibility of α-galactosides in broiler chicken was 86.7%,compared with 99% for adult cockerels (Carré et al., 1995). This indicatesthat the bacterial degradation of α-galactosides in the digestive tract ofadult cockerels is higher than in the digestive tract of young chickens.Also, in the case of pigs, α-galactosides undergo intensive microbialfermentation, especially in the large intestine (Krause et al., 1994). Sincethe net efficiency of digestible energy utilization via hindgut fermentationis 70% of that of the glucose absorbed in the upper intestine (Müller et al.,1989), the net energy value of legume seeds, which contain high amountof α-galactosides, is low. For this reason, the considerable differencein apparent digestibility of α-galactosides between chicken and adultcockerels (86.7 and 99.0%, respectively) results only in 8.7% higher energysupplied by α-galactosides for the adult birds (Carré et al., 1995).

More recent research has suggested that the assumption that oligosac-charides are not digested in the stomach and small intestine may need to be

Nutrition 71




reconsidered. In a study where piglets were fitted with a cannula at theterminal ileum, it was found that 39% of the raffinose oligosaccharidesdisappeared from the stomach and small intestine 3 h after feeding andreached 86–90% at the terminal ileum (Gdala et al., 1997b). This washigher than an earlier report where 75% of the raffinose oligosaccharideswas found to disappear when feeding piglets with pea (Aumaitre et al.,1992). This relatively high amount of digestion in the upper intestine ismost likely due to endogenous plant and microbial α-galactosidases (Gdalaet al., 1997a).

Studies of many authors (e.g. Brenes et al., 1992; Veldman et al.,1993 and Gdala et al., 1997a) have shown that intestinal digestion ofα-galactosides can be increased by supplementation of diets with exoge-nous α-galactosidase (Table 3.7). It has been shown that the addition ofpectinase and α-galactosidase to broiler chicken diets tends to improvegrowth, the apparent metabolizable energy increasing from 12.13 to12.55 MJ kg−1 (P = 0.06) (Igbasan et al., 1997). In contrast, Daveby et al.(1998) reported that supplementation with α-galactosidase significantlyincreased the cumulative feed intake of the milled diets obtained inthe case of chicken, without any apparent effect on the digestibility of theraffinose oligosaccharides. Supplementation of diets with exogenousα-galactosidase does not eliminate other negative effects of α-galactosidespresence in the diet, especially when the content of these sugars is high.Veldeman et al. (1993) reported that the increase in fermentable substratein the lower part of the digestive tract might lead to disturbances ofthe existing microbial balance, increasing the chance of diarrhoea. Theaddition of α-galactosidase (7.1 U g−1) to experimental diets containing2.75% of α-galactosides could not overcome these problems. A highcontent of raffinose in the diet (> 6.7%) results in osmotic catharsis,



α-Galactosidase supplementation

Ileal digestibility (%) − + References

Raffinose 70.2 97.4 Gdala et al. (1997b)Stachyose 88.8 99.5Verbascose 74.2 98.0 (26.7 g GAL kg−1)Raffinose 40.7 95.7 Gdala et al. (1997b)Stachyose 60.1 87.2Verbascose 77.8 86.3 (37 g GAL kg−1)Raffinose (whole lupin) 31.9 75.3Stachyose (whole lupin) 10.4 37.3 Brenes et al. (1992)Raffinose (dehulled lupin) 41.6 86.0Stachyose (dehulled lupin) 23.2 65.0

Table 3.7. Ileal digestibility of α-galactosides (GAL) in pigs fed on a diet, without(−) or with (+) α-galactosidase supplementation.



which causes a portion of the raffinose to be lost before it can be hydrolysedby microbes (Wagner et al., 1976). It has been shown that rapid intestinaltransit of digesta with a high content of α-galactosides (5.3% DM) resultsin a decrease of 20% in true metabolizable energy, compared with a dietcontaining only 1% DM of α-galactosides (Coon et al., 1990). Removal ofoligosaccharides from soybean meal with an 80% ethanol extractionresulted in a lower acidic caecal content and a longer (about 50%) transittime for the diet.

Wiggins (1984) reported that the raffinose family of oligosaccharides(RFO) effect the absorption of nutrients by changing the osmotic pressurein the small intestine. A high content of α-galactosides, therefore, will resultin a reduction in the absorption capacity of the small intestine (Zdunczyket al., 1999). The inclusion of an oligosaccharide extract (4 or 8%) fromlupin seeds to perfusion fluid, i.e. the amount present in a 24-h diet,strongly depressed the intestinal absorption of glucose, methionine andwater, as measured in situ using the perfusion technique (Table 3.8). It ispossible that this affect may be due to other lupin seed componentsextracted together with the oligosaccharides. A high content of fructo-oligosaccharides in perfusion fluid, however, did not depress theabsorption of nutrients from the intestine of rats.

In the experiments presented above, considerably higher amounts ofoligosaccharides than those occurring in practical feeding of animals wereapplied. In the practical feeding of pigs, the content of α-galactosides doesnot exceed 2% of the DM and it is known that a low α-galactoside contentin the diet can significantly decrease their negative effects. In pig diets,where more realistic proportions of α-galactoside were included as soybeanmeal (1.21% of α-galactosides), or water-extracted soybean meal (0.16% ofα-galactosides), there were no differences in the growth performance, feedefficiency, nitrogen digestibility and retention (Seve et al., 1989). Leske et al.(1993), however, confirmed that increasing the amount of raffinose (above0.45%) in diets of leghorn roosters decreased the true metabolizableenergy and DM digestibility. There is also evidence that the α-galactosidesfound in soybean can have a negative effect on protein utilization. Theprotein efficiency ratios determined for chickens fed diets containing

Nutrition 73


Controlfluid

FOS−4%

FOS−8%

OS−4%

OS−8%

Glucose 63.7 74.7 72.2 24.4 14.3Methionine 44.2 37.2 25.8 17.2 18.7Water 6.8 8.7 6.6 0.7 1.2

Table 3.8. Absorption of nutrients (mg rat−1 h−1) from perfusion fluid supple-mented with fructo-oligosaccharides (FOS) or oligosaccharides (OS) from lupinseeds, administered to rat small intestine (Zdu4czyk et al., 1999).



soybean meal, or ethanol-extracted soybean meal, was 2.29 and 2.92,respectively (Leske et al., 1995).

It is evident, therefore, that to improve the nutritional quality of grainlegumes for non-ruminant animals, the level of the raffinose series oligo-saccharides should be reduced, either by plant breeding, the extraction ofseeds, or by using microbial α-galactosidase.

3.3.4 Effect of starch in animal nutrition

Starch is cleaved in the duodenal cavity by secreted pancreatic α-amylase togive a disaccharide (maltose), a trisaccharide (maltotriose) and branchedα-dextrin (Gray, 1991). These final oligosaccharides are further hydrolysedby the complementary action of three integral brush border enzymesat the intestinal surface, glucoamylase (maltase–glucoamylase, amylo-glucosidase), sucrase (maltase–sucrase) and α-dextrinase (isomaltase). Glu-cose, the final product of starch digestibility, is transported via the portalblood to the liver and, subsequently, to the general circulatory system.

Starch is the primary energy source in diets that contain cereals andgrain legumes. There is good evidence that for monogastric animals, thedigestibility of legume starch is lower than that of cereal starch. The resultsof assessing starch bioavailability in the upper gastrointestinal tract ofcolectomized rats, indicated that there are highly significant differences forstarch digestibility between legume and cereal starch. It was reported that15.2% of pea starch was recovered in the ileal digesta of rats compared with0.2% of rice starch (Hildebrandt and Marlett, 1991). It was found that theileal digestibility of legume starch is about 90%, whereas the digestibility ofstarch derived from cereals is nearly 100%. In addition, the digestibilityof isolated starch is higher than that of starch within milled seeds (94.4%versus 84–92%, Table 3.9). Maize and wheat starches were digested betterin the distal ileum by chick (97.8 and 97.6%, respectively) than pea starchpea (94.4%).

The preparation of semi-purified starches for chicken feed has shownthat it is not the physical entrapment of starches within the plant cell wallsthat limits their digestion, but rather the nature of the starches per se (Yusteet al., 1991). In general, legume starch contains between 30 and 40%



Source of starch Apparent digestibility (%) References

Pea seeds 84–92 Carré et al. (1987, 1991)Pea starch 94.4 Yuste et al. (1991)Wheat starch 97.6 Yuste et al. (1991)Maize starch 97.8 Yuste et al. (1991)

Starch was extracted from seeds according to the methods of Faulkes et al. (1989).

Table 3.9. Ileal digestibility of starch in young chicks.



amylose and 60–70% amylopectin compared with cereal starches, which, ingeneral, have 20–25% amylose and 75–80% amylopectin. It has been foundthat the high amylose/amylopectin ratio correlates with the lower digest-ibility of legume starch. It has been suggested that the difference in thedigestibility rate between high amylose and high amylopectin starchescould be due to the larger surface area of amylopectin, which may make itmore available for amylolytic attack (Thorne et al., 1983). This could be oneexplanation for the difference in the digestibility of starch from peas withthe rr genotype, which are characterized by a high amylose content andhave a digestibility of 75.2% compared with 94.4% for normal peas (Carréet al., 1998). It can also be suggested that the lower rate of grain legumestarch digestion may be due to the differences in starch granule structurebetween legumes and cereals (see Chapter 4). Grain legumes also containhigh levels of antinutrients (e.g. enzyme inhibitor, tannins, lectins, andphytic acid) compared with cereals and these compounds will often formcomplexes with nutrients such as starch, which can reduce its digestibility(Thorne et al., 1983; Jenkins et al., 1987).

The digestibility of pea starch is higher than faba bean starch, bothdeclining as the percentage of legume seeds increases in the diet (Table3.10). Coefficients of starch digestibility for different faba bean varietieshave been shown to range from 63.8 to 85.8% (Lacassagne et al., 1988,1991) and for raw pea seeds from 80.9 to 92.2% (Longstaff and McNab,1987; Conan and Carré, 1989; Carré et al., 1991).

Another possible cause of the low digestibility of legume starch (below80%) may be insufficient crushing of the seeds, which could reducethe accessibility of starch particles to digesting enzymes (Carré et al., 1991;Lacassagne et al., 1991). Coarse particles (> 0.5 mm) of excrement havebeen shown to contain the major part (73%) of undigested starch (Carréet al., 1991). Grinding seeds to a small particle size (mean 0.5 or 0.16 mm)increased the starch digestibility coefficients of two faba bean varieties, fedto adult cockerels, from 70.3 to 90.2% and from 63.8 to 80.4%, respectively(Lacassagne et al., 1991). Likewise, in a study conducted on chickens, thedigestibility of starch from different dehulled pea meal fractions was 95.7

Nutrition 75


Source of starchShare in diet

(%)Apparent

digestibility References

Pea 45.1 94.4–99.1 Bengala Freire et al. (1991)Pea 66.1 88.9–92.9 Canibe and Bach Knudsen (1997)Pea 73.7–83.1 85.2–87.3 Gdala and Buraczewska (1997a,b)Faba bean 30.1 97.9 Van der Poel et al. (1992)Faba bean 30.1 96.3 – 98.1 Jansman et al. (1993)Faba bean 62.1 81.5 – 86.4 Gdala and Buraczewska (1997a,b)

Table 3.10. Ileal digestibility of starch in pigs.



and 84.4% for fine (< 100 µm) and coarse (> 100 µm) particle fractions,respectively (Carré et al., 1998).

The nutritional value of diets rich in pea and faba bean starch can beimproved through heating or autoclaving (Longstaff and McNab, 1987;Conan and Carré, 1989). Pelleting has been shown to increase starchdigestibility from 84% to over 95% (Carré et al., 1991). Since starchdigestibility correlates well (r 2 = 0.80) with the true metabolizable energyobtained from pea (Longstaff and MacNab, 1987), it is apparent thatpelleting will increase the metabolizable energy of diets (Table 3.11).Pelleting is the usual process in the feed industry and is very useful forpoultry feed, because chickens have a reduced feed intake when theirdiet is finely ground. Similarly, extrusion has a beneficial effect on thenutritional value of diets containing grain legumes. Extrusion increasesthe in vitro rate of hydrolysis of starch by pancreatic amylase, and stimulatesthe activity of amylase, chymotrypsin and carboxypeptidase A in thepancreatic tissue. As a consequence, the apparent digestibility of starch inthe ileum has been to increase from 94.4 to 99.1% (Bengala Freire et al.,1991).

3.3.5 Effect of non-starch polysaccharides (NSP) in animal nutrition

In general, legume seeds are characterized by having a relatively high levelof structural polysaccharides, mainly comprising cell wall material. As aproportion of the total carbohydrate content of the seed, these compoundsconstitute on average from 73 to 84% in different lupin species, 27% infaba beans, and 25% in peas (Gdala, 1998).

Most NSP are degraded mainly in the hindgut, where they undergomicrobial fermentation providing energy for the animals (Van Engelhardet al., 1989). The energy derived from this hindgut fermentation is about70% of that produced by enzymatic digestion in the small intestine (Mülleret al., 1989; Jorgensen et al., 1996).

Very few studies have reported high digestibility of pea NSP in thedigestive tract of pigs (Goodlad and Mathers, 1990; Canibe and BachKnudsen, 1997). There are reports that the ileal digestibility of NSP of



Meal (n = 25) Pellets (n = 11)

Digestibility of protein (%) 78.4 85.5Digestibility of starch (%) 90.5 98.5Metabolizable energy (kcal kg−1 DM) 2870.5 3150.5

DM, dry matter.

Table 3.11. Nutritional value of meal and pelleted diets with pea seeds(Peyronnet et al., 1996).



legume seeds depends on the age of the pigs and on the species of legumeseed, and ranges from 12.1 to 39.9% (Table 3.12; Gdala et al., 1997a,b).

The differences in digestibility coefficients presented in Table 3.12result from differences in the grain legume species for the level and compo-sition of the NSP. In pea and faba bean seeds the monosaccharide residuesof glucose, arabinose and uronic acids dominate the NSP fraction (Gdalaand Buraczewska, 1997a). Galactose and glucose are the main componentsof lupin seed NSP (in total 60–66%), while uronic acids, arabinose andxylose have intermediate levels (Gdala and Buraczewska, 1997a). Most of thearabinose in pea and faba bean is present as arabinose-containing pectinsubstances in the cell walls of the cotyledons (Selvendran, 1984). Pectins aremore rapidly and extensively digested in the large intestine compared withcellulose, arabinoxylans and xylan polysaccharides (Gdala et al., 1997b).

Pigs are better at utilizing the energy derived from seeds containing ahigh level of NSP. Poultry are able to digest only the water-soluble fractionof NSP, while the water-insoluble fraction remains virtually undigested(Carré et al., 1998). The NSP digestibility for pea diets is about 5.9%in adult cockerels and only 2.8% in chickens (Carré et al., 1995). Theapparent ileal digestibility of DF of milled, or crushed, dehulled peas bycockerels and chickens was 15 and 8%, respectively (Daveby et al., 1998).

Generally, NSP are the main constituents of the DF fraction in grainlegumes and a high content of DF in diets has a negative effect on nutrientdigestibility in animals (Freire et al., 1997). The insoluble DF fractiondecreases intestinal transit time, increases faecal bulk, delays glucoseabsorption and slows starch hydrolysis. The water-soluble fraction of DFincreases the viscosity of the digest in the small intestine, depressing nutri-ent absorption (Low, 1985). Dietary fibre can be a negative factor, there-fore, that dilutes the energy content and decreases the nutrient availabilityfor animals. The soluble DF usually comprises about one-third of the totalDF in cereals, whereas in pea it is about 25% and about 32% in lupin (BachKnudsen, 1997). It has been demonstrated that a 1% increase of crude fibre

Nutrition 77


NSP residues Peaa Faba beanb Yellow lupin Narrow leaf lupin

Arabinose 36.3 43.7 39.2 23.6Xylose 26.3 59.7 −8.0 −6.9Mannose – – 86.0 99.8Galactose 44.8 61.7 42.1 25.4Glucose 4.7 32.8 0.3 26.5Uronic acid 35.8 25.9 25.9 26.5Total NSP 39.9 38.8 14.4 12.1

aAverage for two white flowered varieties.bAverage for two coloured flowered varieties.

Table 3.12. Ileal digestibility of non-starch polysaccharides (NSP) in young pigs(Gdala and Buraczewska, 1997a,b; Gdala et al., 1997a,b).



in the diet diminished the digestibility of gross energy by 1.3% and theutilization of metabolizable energy by 0.9% in pigs (Just et al., 1983).

Many authors have reported that dehulling seeds results in a lower DFcontent and a higher nutritional value. The crude protein content ofdehulled white lupin seeds has been shown to increase by 20% and thecrude fibre content to decrease by 67% (Flis et al., 1997). As a consequenceof changes in the proportion of nutrients, the nutritional value of dehulledseeds of L. albus and L. angustifolius for pigs was increased by 5–10 and 25%,respectively (Fernandez and Batterham, 1995; Flis et al., 1997). Dehullinglupin seeds has been shown also to increase the digestibility of energy inchickens by 18% (Brenes et al., 1993). In addition, enzyme supplementsadded to legume seed diets for chickens also have a positive effect on birdperformance (Brenes et al., 1993). In the case of pigs, enzyme supple-mentation of diets is less effective compared with poultry because of moreintensive fermentation of sugars in the hindgut (Bedford et al., 1992).

3.3.6 Effect of grain legume carbohydrates in ruminant nutrition

There is limited information on the physiological effect of legume seedcarbohydrates in ruminant animals, although it is assumed that they arewell utilized. Information on the digestible energy and gas production byrams fed with grain legumes is presented in Table 3.13.

Differences in the content and composition of carbohydrates are notreflected in clear differences in the amount of digestible energy derivedfrom seeds of different legume species. For example, the level of digestibleenergy in pea seeds, which contain a high level of starch and relatively lowlevels of α-galactosides and NSP, was similar to that found in lupin seeds,which contain very little starch and have large amounts of α-galactosidesand NSP.

The main disadvantage in the use of grain legumes for ruminantsseems to be the high nitrogen degradability in the rumen leading to poor



Totalsugarsa Starch WICWb

Digestibility of energy Gasproduction(ml g−1 DM)(%) (MJ kg−1)

Pea 3.7 45.3 17.1 89.7 16.5 146.7Faba bean 1.9 40.0 20.1 90.5 16.3 112.0Yellow lupin 4.1 0.7 35.3 84.5 16.9 95.9Narrow leaf lupin 4.5 0.8 38.0 82.0 16.5 104.6

aExpressed as saccharase (% dry matter, DM).bWICW – water-insoluble cell wall components.

Table 3.13. Carbohydrate content, the digestibility of energy and gas productionfor different legume seeds (Abreu and Bruno Soares, 1998).



nitrogen values, which make grain legumes uncompetitive with otherprotein sources (Gatel and Champ, 1998). The problem of ruminal degrad-ability of grain legume protein has been the subject of many recent reports(Makkar et al., 1997; Poncet et al., 1998). There is a suggestion that heattreatments, e.g. autoclaving or extrusion, may be useful for improving thenitrogen value of lupin or pea protein for ruminants (Le Guen et al., 1997).

3.4 Physiological Effect of Grain Legume Carbohydrates inHuman Nutrition

3.4.1 Nutritional classification of grain legume carbohydrates

According to their role in plants, carbohydrates can be separated into threegroups, the mono- and disaccharides are a source of energy for growth,the oligosaccharides and starch are storage carbohydrate and the non-cellulosic polysaccharides, pectins, hemicellulose and cellulose comprisethe structural components of the cell walls. From a human nutrition pointof view, carbohydrates can be classified into two groups, available carbo-hydrates, which are enzymatically digested in the small intestine, andunavailable carbohydrates, which are fermented by microflora in the largeintestine (Table 3.14).

The available carbohydrates comprise the mono- and disaccharidesand starch, while the unavailable carbohydrates contain the oligosaccha-rides and the structural components. The mono- and disaccharides arealmost completely digested in the small intestine. Sucrose (a disaccharide)is hydrolysed to its constituent monosaccharides by the sucrase enzymes onthe erythrocyte surface membrane. The digestion of starch starts in themouth, by the enzyme amylase secreted in the saliva, and is continued in

Nutrition 79


Role in the plant Type of saccharide Site of digestionProduct ofdigestion

Physiologicalclassification

Source of energy Mono- anddisaccharides

Small intestine(enzymatic)

Mono- anddisaccharides

Availablecarbohydrates

Storagepolysaccharides

Starch; amyloseand amylopectin

Small intestine(enzymatic)

Mono- anddisaccharides

Availablecarbohydrates

Storageoligosaccharides

α-Galactosides Large intestine(microbial)

Short chain fattyacids: acetate,propionate,butyrate

Unavailablecarbohydrates

Structuralcomponents ofplant cell walls

Non-cellulosicpolysaccharides:pectins, hemi-cellulose,cellulose

Large intestine(microbial)

Carbon dioxide,hydrogenmethane

Unavailablecarbohydrates

Table 3.14. Classification of legume saccharides (Southgate, 1992).



the upper small intestine by enzymes secreted by the pancreas. As withmonogastric animals (see Section 3.3.4), starch can be readily digested inthe human gastrointestinal tract. A part of the consumed starch, especiallyfound in products subjected to hydrothermal processing, is not digested inthe small intestine and passes to the large intestine as ‘resistant starch’,which will be discussed later.

The absence of α-galactosidases in mammalian intestinal mucosa,which cleave the α(1→6) galactose linkage present in raffinose and otherα-galactosides (Gitzelman and Auricchio, 1965), results in these com-pounds passing into the large intestine. These oligosaccharides are thenbroken down to monosaccharides by bacterial enzymes, with the produc-tion of hydrogen and methane gas. Recent research shows that about 30%of the oligosaccharides (raffinose and stachyose) in diets are degraded inthe stomach and the small intestine. The assumption that oligosaccharidesare not digested in the stomach and the small intestine, therefore, must bereconsidered (Sandberg et al., 1993). Despite this apparent partial degrada-tion in the stomach and small intestine, α-galactosides are still includedamongst the unavailable carbohydrates, since they are not absorbed inthe small intestine (Wiggins, 1984). In healthy subjects less than 1% ofthe oligosaccharides are absorbed and when injected directly into thebloodstream they are almost completely recovered (Wheeler et al., 1978).

The other major components of the unavailable carbohydrate groupare the structural components of the plant cell walls, often collectivelytermed DF and described earlier. An additional component of this group isresistant starch. Resistant starch was not recognized until 1982 (Englystet al., 1982). Prior to this time the prevailing concept was that starch wascompletely digested and absorbed. The nutritional properties of starch infoods are to a large extent related to its availability for digestion and/orabsorption in the gastrointestinal tract (Björck and Asp, 1994). From thispoint of view, starch can be classified into three basic groups: rapidlydigestible starch (RDS), slowly digestible starch (SDS) and resistant starch(RS) (Table 3.15).

The digestibility varies according to the plant species and often within aspecies, and relates to the chemical and physical structure of the starch



Starch type SourceDigestibility in smallintestine

Rapidly digestible starch Freshly cooked starchy foods RapidSlowly digestible starch Most raw cereals Slow – but completeResistant starch Partially milled grain and seeds;

raw potato and banana; cooledcooked potato, corn flakes andbread

Resistant

Table 3.15. In vitro nutritional classification of starch (Englyst et al., 1992).



granules. Digestibility also depends on the methods used for preparingfood prior to consumption. Isolated raw starch of pea is almost completelydigested in the small intestine of rats (Table 3.16; Berggren et al., 1995).During technological treatment (heating, freezing, etc.), the physicalstructure of the starch is degraded and, with time, becomes restructured, orretrograded, in a non-digestible form (see Chapter 4). This retrogradedpart of starch and the starch that is not digested in the small intestine forother reasons, for example too little time for digestive enzymes to act onstarch granules, passes to the large intestine as RS. RS can, therefore, bedefined as ‘the sum of starch and products of starch degradation notabsorbed in the small intestine of healthy humans’ (Asp, 1992).

Relatively few studies have tried to quantify the amount of RS inthe small intestine of healthy humans. Table 3.16 presents the content ofRS in legume products, estimated by using either direct (in vivo) methods,including the analysis of starch in ileal effluents from ileostomy patients(Jenkins et al., 1987; Schweizer et al., 1990; Steinhart et al., 1992; Muir andO’Dea, 1993), ileal incubation experiments (Noah et al., 1998), balance

Nutrition 81


Legume productsMethods of RSdetermination

RScontent References

Raw pea starch Weight experiment on ratsa 1.0 Berggren et al. (1995)Pre-cooked lentil Weight experiment on ratsa 11.0 Tovar et al. (1992)Pre-cooked red bean Weight experiment on ratsa 8.0 Tovar et al. (1992)Canned pea Colectomized rats 15.2 Hildebrandt and

Marlett (1991)Canned peab Weight experiment on ratsa

and three methods in vitro27.8 Björck and Sijeström

(1992)Boiled red lentil In vivo, in ileostomists 13.8 Steinhart et al. (1992)

In vivo, in ileostomists 13.6 Jenkins et al. (1987)Boiled white beans In vivo, intubation experi-

ments in healthy subjects16.5 Noah et al. (1998)

Autoclaved whitebeans

In vivo, in ileostomists 5.7 Muir and O’Dea (1993)

Autoclaved whitebeans

In vivo, in ileostomists 20.9 Schweizer et al. (1990)

Boiled lentil In vitro digestibility ofstarch

16.5 Cummings and Englyst(1995)

Boiled red lentil In vitro based on chewing 23.1 Äkerberg et al. (1998)Boiled white beans In vitro based on chewing 16.7 Äkerberg et al. (1998)Autoclaved white

beansIn vitro based on chewing 13.8 Äkerberg et al. (1998)

aRats treated with antibiotics to suppress hindgut fermentation.bPea with low content of starch – 19% dry matter (DM).

Table 3.16. The content of resistant starch (RS) in legume products (g (100 g)−1 ofstarch).



experiments in rats with suppressed hindgut microflora (Tovar et al., 1992;Berggren et al., 1995), analysis of the ileal excreta in colectomized rats(Hildebrandt and Marlett, 1991), or indirect(in vitro) methods (Cummingsand Englyst, 1995; Åkerberg et al., 1998). At present, results of these studiesare too fragmentary to allow the quantity of RS for starches of differentgrain legumes to be estimated satisfactorily. Gray (1991), however, hassuggested that in processed legume seed the content of RS amounts to10.3% of total starch. The results of other studies, presented in Table 3.16,indicate that the RS content may be closer to 15% of total starch. A highercontent of RS was noted in grain legumes processed differently in theItalian diet (Brighenti et al., 1998); dried, canned and frozen seeds ofbeans, peas, lentils and chickpeas containing 11.6, 12.4, 11.4 and 10.9% RS,respectively. In this study, the average RS content in grain legumes wasabout 11.6% compared with 3.2 and 5.7% for cereals and potatoes. As aproportion of total starch, the RS content of grain legumes in this study wasabout 20% (Brighenti et al., 1998).

3.4.2 Consumption of grain legume carbohydrates in food

Immature seeds, dry seeds and processed seeds are all used for food pur-poses. Most grain legumes, after simple processing (sprouting, cooking),are consumed as vegetables, salads, soups, mashed seeds and cooked seeds.The annual consumption of grain legume carbohydrates can be estimatedfrom the consumption and chemical composition of legumes. Examples ofthe average consumption of carbohydrates in grain legumes are presentedin Table 3.17.

From these data the average daily consumption in 1996, ofα-galactosides, digested starch, RS and DF can be calculated and shownto be very low (0.26, 2.89, 0.51 and 1.33 g, respectively). A similar intake ofdigested starch and resistant starch from dried legumes (2.3 and 0.5 g,respectively) has been found in relation to the Italian population(Brighenti et al., 1998). Also, the daily intake of starch and RS in fresh,frozen and canned grain legumes was 1.8 and 0.6 g, respectively (Brighentiet al., 1998).

The calculated amounts given in Table 3.17 do not take into accountthe considerable losses that occur during food preparation. For physio-logical reasons, decreasing the α-galactoside content during preparationof the seeds for consumption is very important, as will be discussed later.The intake of α-galactosides in legume dishes in the Czech Republic andin neighbouring countries are presented in Table 3.18, after taking intoconsideration the losses during the preparation of legume dishes.

Considering the amount of starch in pea seeds (c. 45%), the RS contentin one portion of pea soup can range from 1.3 to 1.9 g. In one portionof mashed pea (75–105 g), the content of total starch and RS ranges from





32.0 to 44.9 g and 4.8 to 6.7 g, respectively. This is a significant amountconsidering that the daily consumption of RS in Western diets ranges fromabout 4 g (Dysseler and Hoffem, 1994), to as much as 20–30 g day−1

(Cummings and Englyst, 1989). The daily intake of total starch and RS inthe Italian population diets reaches 21.4 and 8.5 g, respectively (Brighentiet al., 1998). Other studies, however, have reported considerably lowervalues for RS in the diet of European countries, ranging from 3.2 g day−1

in Norway to 5.7 g day−1 in Spain (Dysseler and Hoffem, 1994). Cummingset al. (1992) calculated a similar amount of NSP ingested daily (8–18 g) inWestern diets. The information presented in Table 3.17 reveals that the

Nutrition 83


Beans Pea Lentil Chickpea Total

Annual consumption of legumes(kg per person)

1.14 0.67 0.43 0.19 2.43

Average content of (g kg−1)α-galactosidesa 36.8 49.4 28.4 47.0 39.6Starcha 549.3 475.0 461.8 526.0 511.5Resistant starch 82.4 72.3 69.3 78.9 77.0Dietary fibre b193.3b b187.9c b214.0d b245.5e 199.5

Annual consumption of (g)α-galactosides 42.0 33.1 12.3 8.9 96.3Starch 626.2 318.3 198.6 99.9 1243.0Resistant starch 93.9 48.4 29.8 15.0 187.1Dietary fibre 220.4 125.9 92.0 46.6 484.9

Resistant starch is 15% of the total starch.aMean content, see Table 3.3.bMean content, see Table 3.4.cMean of 15 cultivars (Zdu4czyk et al., 1997).dSavage (1988).eSidduraju et al. (1998).

Table 3.17. Annual consumption of α-galactosides, starch, resistant starch anddietary fibre in legumes, in Europe (data for 1996).

DishDry legumes

(g)Original GAL

(g)Losses on

cooking (%)Final intake of

GAL (g)

Pea soup 20–30 1.5–2.3 10 1.3–2.1Lentil soup 13–20 0.7–1.0 10 0.6–0.9Bean soup 20–30 0.6–0.9 10 0.5–0.8Mashed peas 75–105 5.6–7.9 70 1.7–2.4Cooked lentils 75–112 4.0–6.0 70 1.2–1.8Cooked beans 75–110 2.5–4.0 70 0.7–1.2

Table 3.18. Estimated intake of α-galactosides (GAL) in legume dishes (g per dish)(Pokorny and Dostalova, unpublished).



daily intake of unavailable carbohydrates from grain legumes reaches2.2 g day−1, including 12% for α-galactosides, 24.4% for RS and 63.1%for DF.

3.4.3 Physiological effect of available carbohydrates from grain legumes

Relatively few studies have tried to quantify the digestibility of starch fromlegumes in the small intestine of humans (Wolever et al., 1986; Schweizeret al., 1990; Bothman et al., 1995). Numerous studies conducted on mono-gastric animals, however, have revealed that starch is easily digested in theupper intestinal tract, however, its digestibility coefficient is lower than thatof cereal starch (see Section 3.3.4). This low digestibility of starch togetherwith the low content of mono- and disaccharides and high content ofunavailable carbohydrates (α-galactosides, RS and NSP), make legumeseeds desirable component of human diets.

It is apparent that the seeds from grain legumes are characterized bytheir relatively low glycaemic index (Table 3.19), which on average is lessthan half that of white and wholemeal bread. It is beneficial, therefore, toinclude grain legumes in diets for people with insulin-dependent diabetes,an illness that is often found in elderly inhabitants of industrializedcountries (Wolever and Brand-Miller, 1995).



Food type GI

Cereal productsBread, white 69Bread, wholemeal 72Rice, white 66Rice, brown 66Spaghetti, white 50Corn flakes 80

Root vegetablesCarrots 92Potato, new 70Potato, instant 80

LegumesBeans, navy 31Beans, kidney 29Beans, soya 15Peas 33Lentils 29

Table 3.19. Glycaemic index (GI) offoods in normal subjects (Gray, 1991).



When grain legumes are used in diets as substitutes for animal products(in the case of vegetarians) they are believed to act in two ways. Firstly, todecrease the consumption of saturated fats and secondly to increasethe content of unavailable carbohydrates in the diet, thus reducing theincidence of digestive tract cancers. A positive association with proteinand fat (r = 0.60 and 0.62, respectively) and a weak negative associationwith NSP (r = −0.23) have been shown in a study of food consumption in12 countries (Cassidy et al., 1994). A much stronger (r = −0.70) inversecorrelation was found, however, between colorectal cancer incidence andstarch intake.

3.4.4 Physiological effect of unavailable carbohydrates from grainlegumes

Within the group of unavailable carbohydrates, the highest proportioncomprises the NSP, or ‘true dietary fibre’ (Burn et al., 1998). Apart fromdecreasing the bioavailability of many mineral components (Torre et al.,1991), DF in the diet provides many advantages. Increased plant fibreconsumption is known to reduce blood lipid levels and is of particularinterest, therefore, in the prevention and treatment of cardiovasculardiseases (Mazur et al., 1990). It appears that DF may influence cholesterolmetabolism in at least four ways (Wolever, 1995; Vanhoof and De Schrijver,1997; Vahouny et al., 1988): (i) it may increase faecal bile acid excretion,resulting in increased cholesterol flux to bile acid synthesis with lesscholesterol being available for lipoprotein synthetic pathways; (ii) it mayalter the absorption of fat and cholesterol, either by binding bile acids or byincreasing small intestine viscosity; (iii) it may reduce post-prandial insulinresponses and, consequently, regulate cholesterol synthesis; and (iv) itresults in the formation of short chain fatty acids, especially propionic acid,during fibre fermentation in the colon, which may decrease cholesterol inthe liver. Faecal output is highly correlated with DF intake and inverselycorrelated with the time taken for materials to pass through the alimentarytract (‘transit time’). Thus, stools formed when the diet is rich in fibreare softer and more voluminous and pass more rapidly through thegut, than when the diet contains little fibre (Gurr and Asp, 1994). Theeffects of DF on the potential to reduce cancer risk in the large intestineare: dilution of carcinogens (via water-holding capacity); provision ofsurface for absorption of carcinogens; faster transit time, so less contacttime; altered bile salt metabolism; and as a consequence of fermentation,lower pH, production of butyrate, altered microbial metabolism and lowerammonia levels in the gut.

When NSP from pea were included in the diet of rats, a model forhuman nutrition, they were found to be associated with increased volatilefatty acid and 3-hydroxy butyrate concentrations in portal and heart blood

Nutrition 85




(Goodlad and Mathers, 1990). Butyrate is considered to be a protectiveagent against colon cancer (Cummings and MacFarlane, 1991).

Earlier studies have equated the term DF with unavailable (complex)carbohydrates, which are the sum of NSP and RS (British NutritionFoundation, 1990). In comparison with DF, this would make starch aquantitatively important source of non-digestible carbohydrates. In fact, ithas been claimed that RS, not DF, is the major substrate for fermentation inthe human colon (Cummings and MacFarlane, 1991). During fermenta-tion of RS, short-chain fatty acids (such as acetic, propionic and butyric) areformed (Björck et al., 1987). Introducing 15–45% cooked haricot beans(Phaseolus vulgaris) into rat diets caused an increase in the absorptionof acetate (from 3.8 to 19.8 mmol day−1), propionate (from 1.2 to 7.5) andbutyrate (from 0.5 to 2.1 mmol day−1), from the large bowel (Key andMathers, 1993). It has been suggested that RS yields a larger proportion ofbutyric acid than DF (Silvester et al., 1995). In in vitro batch cultures, 29% ofbutyrate can be produced from RS, compared with 2–8% from NSP sources(Englyst and Cummings, 1987). An increased amount of butyric acid,which is the preferred energy source for colonocytes, can play an immuno-logical role in relation to the development of colon disease. Butyrate has aprotective effect in the rat model (McIntyre et al., 1993), while in humansubjects, RS has been shown to reduce mucosal proliferation, the level ofsecondary bile acids and the mutagenicity of faecal water (Maunster et al.,1994). In transformed cells, terminal differentiation is induced, resulting inprogrammed cell death or apoptosis (Hague et al., 1993).

Oligosaccharides have been classified with other non-digestible compo-nents (DF, RS) of the diet and collectively referred to as ‘fibre’, because ofthe similarities in response to intestinal enzymes. Oligosaccharides areknown to be fermented mainly by beneficial intestinal microflora (Ferket,1991), which may explain the difference in animal responses observedwhen oligosaccharides, rather than NSP, are included in the diet. Theyreach the colon and are quickly fermented by colonic bacteria, producing,in particular, gas and short-chain fatty acids. The gases are mainly carbondioxide, hydrogen, and methane and are traditionally associated with theflatulence problems that are often linked with the consumption of pulses.Flatulence is poorly tolerated by Western populations who, in addition,might have a greater visceral sensitivity than populations more accustomedto this type of food (Gatel and Champ, 1998).

The intake of grain legumes in northwestern and central EasternEurope is rather low (about 2 kg per person per year), which, theoretically,should not cause digestive troubles if consumption is uniform duringthe year. They are usually consumed, however, only once or twice a week assoup (about 50 g of dry grains per portion), or as a vegetable consumedwith meat, sausages or eggs (about 100 g of dry grains per portion). Suchhigh amounts may cause problems such as flatulence and sometimesdiarrhoea. It would be better to consume small amounts more often and





perhaps to add small amounts of legumes (5–10% of the whole portion) toother conventional dishes (see Table 3.20). Such small amounts would notcontain more than 0.1–0.3 g of α-galactosides per portion, which is notlikely to cause digestive problems.

Nutrition 87


DishLegumesadded

Amount ofcooked

legumes (%)

Sensory acceptancea (%)

Withoutlegumes

Withlegumes

Frankfurter soup Beans 6.6 77 80Tomato soup Soybeans 9.1 80 78Cabbage soup Lentils 7.6 79 63Salad with tunny Beans 9.3 71 78Serbian salad Chickpeas 6.2 81 88Serbian salad Beans 8.1 79 88Vegetable salad Lentils 5.4 70 85Mashed potatoes Peas 5.4 52 71Mashed potatoes Peas 9.3 74 74Potato salad Soybeans 6.2 59 65

aSensory acceptance scale: 0% = unacceptable, 100% = fully acceptable.Values (with legumes) in bold type represent a significant difference (P = 0.95) fromthe corresponding without legumes dish.

Table 3.20. Consumer acceptance of a selection of dishes with added legumes(Dostalova et al., 1999a).



ProcessingB. Czukor et al.4

4ProcessingEditor: Bálint Czukor

Contributors: Tatiana Bogracheva, ZsuzsannaCserhalmi, Bálint Czukor, Jozef Fornal, IldikóSchuster-Gajzágó, Erzsébet T. Kovács, GrazynaLewandowicz and Maria Soral-Smietana

Life is one long process of getting tired.Notebooks, chap. 1 (1912)

Samuel Butler (1835–1902), English novelist and sometimes sheep farmer

4.1 Native Starch

4.1.1 Isolation

Starchy legume seeds are rich in protein, starch and dietetic fibre, all ofwhich are very valuable for food and non-food applications (Salunkhe et al.,1989). For this reason the processing of legume seeds includes the separa-tion and production of these components. Studies that have led to thedevelopment of industrial processing of grain legume seeds have beencarried out mainly on pea (Pisum sativum) and faba bean (Vicia faba). Theprocesses that are generally used are dry processing (air classification)and wet processing. In general, the dry processing procedure producesprotein-rich and starch-rich products, while the wet processing procedureproduces purified protein, starch and dietetic fibre fractions. There areseveral advantages of the dry process: the construction of pilot plants isrelatively simple, the process does not produce waste water and changes inthe structure and functional properties of the components are minimized.Seed processing, which includes water extraction, is more complicated butthe higher purity of the products produced allows a wider range of applica-tions. In addition, the wet process is necessary for scientific research on





starches, which demands a very high purity of starch. The basic principlesof these two processes are described in more detail below.

Dry processingIt has been found that the air classification process can be carried outmore successfully with grain legumes where starch is the main storageproduct rather than oil. Among the starchy grain legumes, air classificationhas been carried out on pea (P. sativum), faba bean (V. faba), mung bean(Vigna radiata), green lentil (Lens culinaris), navy bean (Phaseolus vulgaris),baby lima bean (Phaseolus lunatus) and cowpea (Vigna unguiculata). Fordetailed information, see Reichert and Youngs (1978), Bramsnaes et al.(1979), Talyer et al. (1981), Reichert (1982), Sosulski et al. (1985), Clottet al. (1986) and Sosulski and McCurdy (1987). The first stage of thisprocess includes fine milling of the seeds. Flours prepared from starch-richseeds contain two distinct populations of particles, which differ in both sizeand density and can be separated in a current of air. The starches anddietetic fibres are concentrated mostly in the light, fine fraction and theproteins and lipids in the heavy, coarse one. Repeating the air classifyingprocess can increase the purity of the fractions, however it decreases therecovery of the products. Starch fractions with protein impurities as low as2.5% can be produced; however, the recovery of the starch fraction isonly about 40% (Reichert and Youngs, 1978). Following air classification ofpea meal it has been found that the protein-rich fraction contains mainlystorage proteins, while the starch-rich fraction contains other functionalproteins, which adhere to the surface of the starch granules. The process ofair classification is illustrated in Fig. 4.1, and the influence of the number ofsteps on the purification and recovery of the starch fraction from peas inFig. 4.2. Detailed information on the processing of pea and faba bean by airclassification has been described in a number of reports (Vose et al., 1976;Reichert and Youngs, 1978; Bramsnaes et al., 1979; Vose, 1980; Talyer et al.,1981; Reichert, 1982; Tyler and Panchuk, 1982; Wright et al., 1984; Clottet al., 1986; Clott and Walker, 1987; Uzzan, 1988; Salunkhe et al., 1989).

Wet processingWhen legume seeds are processed for food applications the hulls areremoved because it has been reported that they can contain antinutritionalcompounds that can be released during the extraction process (Sosulskiand McCurdy, 1987; Uzzan, 1988). The dehulled seeds are then pin-milled.It has been found (Gueguen, 1983) that an average flour particle size of100–150 µm is most suitable for further separation of the components.The next step of the process is protein extraction, which is carried out atalkaline pH.

For round-seeded peas, pH 9–10 is most commonly used for extraction,while for wrinkle-seeded (those lines containing the r gene) peas it isusually higher (Schoch and Maywald, 1968; Vose et al., 1976; Colonna et al.,

90 B. Czukor et al.




Processing 91


Fig. 4.1. Dry process of starch extraction (Colonna et al., 1981).

Fig. 4.2. The influence of the number of purification steps on the recovery of peastarch. Protein content, n; yield of starch, l.



1981; Sosulski and McCurdy, 1987; van der Poel et al., 1989; Hoover andSosulski, 1991; Wiege et al., 1995; Salunkhe et al., 1989). In a more recentlydeveloped method (Bogracheva et al., 1995; Davydova et al., 1995), thisprocedure was carried out at a lower pH (8.5), which is much better forfood applications, because stronger alkaline conditions may result in theappearance of antinutritional components in the protein products(Gueguen, 1983; Salunkhe et al., 1989). In the case of the r wrinkled-seededpeas, some applications require a better separation of starch from the othercomponents and this is achieved by high-pressure disintegration (Meuseret al., 1995).

The protein extract, which also contains soluble carbohydrates andemulsified lipids, is separated from the insoluble fraction and the proteinsare isolated from this extract by acid precipitation or by ultra filtration.Protein fractions obtained from such procedures are commonly calledprotein isolates. The wet protein isolates are then dried. In industry this isusually carried out using spread dryers at temperatures of more than100°C. The drying process is relatively quick and so the functional proper-ties of the proteins are not affected significantly. Freeze-drying, however, ismore appropriate when proteins are isolated for scientific purposes.

The insoluble fraction, which is left after separation of the proteinextract, includes starch, cell wall material, insoluble proteins and theremaining lipids. The basis for the further separation of these componentsdepends on differences in their swelling properties. Starch granules haverestricted swelling at room temperatures (Blanshard, 1987; Zobel andStephen, 1995), whereas the swelling capacity of cell wall material is muchgreater. The swelling properties give rise to a difference in size betweenthe starch granules and the cell wall particles. The insoluble fraction isdispersed in a large amount of water and screened through a series of sieveswith pore diameters between 30 and 300 µm (Schoch and Maywald, 1968;Colonna et al., 1981). The liquid passing through the sieves (termed starchmilk) is mainly a dispersion of starch granules, while the material trappedby the sieves contains mainly cell wall material. The smaller the diameter ofthe sieve pores, the smaller the particles of cell walls that are separated fromthe starch milk and the lower the level of impurities. Starch granules arenot uniform in size, the size distribution being dependent on the source ofthe starch (Davydova et al., 1995). The minimum diameter of the sieve to beused is determined by the size distribution of the starch granules. For exam-ple, 50–60 µm sieves are usually used for producing starch from pea andfaba bean (Colonna et al., 1981). The starch produced by such a methodcontains 0.04–0.40% protein and about 0.1–1.0% of lipid as impurities(Elliasson, 1988; Davydova et al., 1995).

In industry the starches are dried by spread drying machines, which arespecially constructed for this purpose. Starches produced for scientificpurposes, however, usually have an additional wash with water, alkaline orsalt solutions, or with organic solvents such as ethanol or acetone, to reduce

92 B. Czukor et al.




the level of impurities (Schoch and Maywald, 1968; Davydova et al., 1995).The starches are then often dried in the open air, in some cases followinga final wash with ethanol, or acetone, to speed up the drying process(Davydova et al., 1995).

Although the methods described above result in satisfactorily purifiedstarches, it should be noted that the use of organic solvents might partiallydisturb starch granular structure, which in turn may affect the properties. Adiagrammatic representation of wet seed processing is shown in Fig. 4.3.

4.1.2 Granular structure

It is generally believed that starch granules are composed mainly oftwo types of glucose polymer; amylose, which is essentially a linear chain

Processing 93


Fig. 4.3. Wet processof starch extraction.



molecule, and amylopectin, which is highly branched (see chapter 2,Chemistry). It is well known that in different starches these two moleculesmay differ in their degree of polymerization and, with regard to amylo-pectin, in the arrangement and degree of polymerization of the branches.Amylose and amylopectin molecules are arranged in granules (Fig. 4.4A),which are complex structures consisting of crystalline and amorphousareas. It is a common point of view that the short chains in the amylopectinmolecules are organized into double helices, some of which then form crys-talline lamellae, or crystallites (French, 1984; Blanshard, 1987; Manners,1989). The regions of starch granules containing these structures arereferred to as the ordered parts, while the remaining regions are called thedisordered or amorphous parts. The amorphous parts of the starch granuleare believed to consist of amylose and long chains from amylopectin(French, 1984). There is evidence that the crystalline and amorphousmaterial form alternate layers in the starch granule (French, 1984;Blanshard, 1987).

The presence of crystallites causes starch granules to be birefringentand this can be studied using light microscopy with cross polarizers(Fig. 4.4B). The interference pattern observed takes the form of a Maltesecross, which indicates that there is an orderly arrangement of the crystallineareas within the granule. The use of a specific plate (the so-called λ-plate, orred 1 compensator; Patzelt, 1974; Morris and Miles, 1994) in conjunctionwith the cross polarizers makes granules appear as blue and yellow sectors,indicating that starches are biaxial crystalline polymers (Patzelt, 1974).

94 B. Czukor et al.


Fig. 4.4. Starch granules from wild-type pea seeds: (A) Using normal lightmicroscopy. Opposite page: (B) viewed using polarized light, prior to gelatinizationin 0.6 M KCl solution; (C) viewed using polarized light, after heating to the meltingpoint for B-type crystallites (66°C) in 0.6 M KCl solution.



If the crystallites were ordered differently with respect to the plane ofpolarization – either towards, or perpendicular to it – then this would giverise to different colours (French, 1984; Morris and Miles, 1994).

Two types of crystallite, or polymorph, structure, A and B, have beenidentified in starch granules, which can be distinguished by the packingdensity of the double helices; A-type polymorphs being more denselypacked than B-type (Fig. 4.5; Sarko and Wu, 1978; Imberty and Perez,1988a; Imberty et al., 1988b; Perez et al., 1996; Wang et al., 1998). Starchesfrom different plant species may have A-, B-, or both A- and B-types of poly-morph (Blanshard, 1987; Wang et al., 1998), the resulting starches being

Processing 95


Fig. 4.4. Continued.



termed A-, B- or C-type, respectively. A-type starches are found in cereals(e.g. maize, wheat and rice), B-type in tubers (e.g. potato) and C-type,containing both A- and B-type polymorphs, in legumes (e.g. pea and fababean).

In the case of C-type starches the arrangement of the two crystal typeswithin the granules will affect the properties of the starch. For example, ifC-type starches are a mixture of granules which contain either A or B poly-morphs, then the properties would be intermediate between those of A-and B-type starches. If, on the other hand, each granule contains both A-and B-type polymorphs, it is likely that the properties of the resulting starchwould be unique and different from those of A- and B-type starches. In thiscase the properties of the starch will depend on the arrangements of A andB polymorphs within the granules.

To understand starch granular structure it is necessary to determinethe proportions of ordered and disordered parts in the granule, the type ofcrystallinity, the proportions of A and B polymorphs (in the case of C-typestarches), the size and arrangement of the crystallites and the properties ofthe amorphous parts of the granule. A range of techniques has been usedfor studying these physical characteristics, in particular, wide-angle X-raypowder diffraction, differential scanning calorimetry and various light andelectron microscopy methods (Donovan, 1979; Yamaguchi et al., 1979;French, 1984; Biliaderis et al., 1986; Meuser et al., 1995). More recently,progress in quantifying the ordered structures within starch granules hasbeen achieved using X-ray diffraction and NMR methods (Gidley andBocick, 1985; Gidley and Robinson, 1990; Cairns et al., 1997; Bograchevaet al., 1998).

With regard to studying the granular structure of legume starches,most progress has been achieved by using starches from peas. Starch

96 B. Czukor et al.


Fig. 4.5. Differences in packing density of A- and B-type polymorphs.



granules from a round seeded pea line have been shown to contain bothA- and B-types of polymorph, the B polymorphs being in the centre and theA polymorphs at the periphery of each granule (Bogracheva et al., 1998).Since B-type polymorphs melt at a lower temperature than A-type, it ispossible to degrade the B polymorphs from the centre of the granules,leaving only the A polymorphs intact at the periphery. This process can becarried out on a microscope stage and observed using polarized light(Fig. 4.4B and C; Bogracheva et al., 1998). In addition, granules from thismaterial have 63% double helices (Bogracheva et al., 1998), 33% of whichare arranged in crystallites at a moisture content of about 20% (Cairns et al.,1997; Bogracheva et al., 1998). A method for determining the polymorphcomposition of C-type starch has been developed recently (Cairns et al.,1997). This method is based on calculations from X-ray diffractionpatterns of the crystalline portions of the starch, using characteristic peaksassociated with either A- or B-type polymorphs (Davydova et al., 1995;Cairns et al., 1997). Using this method the proportions of A and Bpolymorphs in round-seeded peas have been found to be c. 56 and 44%,respectively.

Analysis of starch from a range of pea mutants (see Chapter 6,Breeding and Agronomy) has shown that it is possible to geneticallymanipulate the physical structure of the starch granules (Table 4.l;Bogracheva et al., 1995, 1997, 1999; Davydova et al., 1995) It was found thatgenes that affect the supply of substrate during starch synthesis (rb, rug3and rug4) affect the total crystallinity and possibly the content of the Apolymorphs in the granules. On the other hand, genes that directlyaffect the synthesis of starch polymers (r, rug5 and lam) increase the Bpolymorph content but have little effect on the total crystallinity of thegranules (Table 4.1)

Processing 97


Genotype

Amylosecontenta

(% starch)

CrystallinityTp

(°C)∆T(°C)

∆H(J g−1)total % %B %A B/A

Wild-type 35 20 45 59 0.8 61.8 13.4 10.8rug4 33 23 39 57 0.7 65.4 12.5 9.8rb 23 27 43 58 0.7 66.1 9.0 12.6rug3 12 17 37 63 0.6 70.0 9.4 7.5lam 8 22 69 29 2.4 58.6 8.4 6.8rug5 43 20 52 45 1.2 49.0–57.0 30.0 5.1r 65 19 73 b0b ∞ 52.5–60.0 34.0 2.4

aData from Bogracheva et al. (1997).bA polymorphs not detected.

Table 4.1. The characteristics of the granular structure of starch in pea mutants(Bogracheva et al., 1999).



4.1.3 Functional properties

The applications of starch are determined by their functional properties.For example, an important functional property is pasting, which is thedevelopment of high viscosity after heating of starch–water suspensions.This property is exploited in different foods as well as in non-food usessuch as adhesives. Another important functional property is the capabilityto create gels. This property is also used in different foods and in non-foodapplications such as thermoplastics. Legume starches, in particular thosefrom pea and faba bean, have been studied mostly in relation to theirpasting behaviour.

Heating starch in the presence of water results in disruption of theordered structures within the granules. This process can be studied usingdifferential scanning calorimetry (DSC). Using this method it has beenshown that the disruption of ordered structures in starch granules is anendothermic process, which is often called an order–disorder transition(Donovan, 1979; French, 1984; Blanshard, 1987; Cooke and Gidley, 1992;Zobel and Stephen, 1995). The nature of this transition is strongly depend-ent on the amount of water present during heating (Donovan, 1979;Biliaderis et al., 1986), giving two possible mechanisms for the disturbanceof the ordered structures in the granule. These two mechanisms are usuallycalled melting and gelatinization (see Fig. 4.6). Melting occurs in low mois-ture conditions, when there is no free water in the system. Gelatinizationoccurs when there is an excess of free water in the system (Evans andHaisman, 1982; Blanshard, 1987). Both of these processes occur whenstarches are heated in intermediate conditions of moisture (Donovan,1979; Elliasson, 1980; Biliaderis et al., 1986). The DSC curve describing thegelatinization process shows sharp changes in the absorption of heat, whichare normally described as changes in enthalpy (∆H).

When starch from round-seeded peas is heated in excess of watercontaining a low salt concentration, the A and B polymorphs within thegranules melt independently, giving a double peak of transition in heatcapacity. The transition peak for the B polymorphs is at a lower tempera-ture than that for the A polymorphs (Fig. 4.7; Bogracheva et al., 1994, 1995,1998). Differences were found between the starches from the pea mutants(described in the Breeding chapter and earlier in this chapter) when theywere heated in excess water and the granular disruption followed usinga DSC (Bogracheva et al., 1999). Starches from the rb, rug3, rug4 and lammutants exhibited narrow endothermic peaks that were similar to starchfrom the wild-type. The peaks differed, however, in peak temperature (T p)and peak width (∆T). Starches from the r and rug5 mutants, however, hadvery wide transitions, which were very different to those observed in starchfrom the wild-type and from the other mutants (Table 4.1) Gelatinizationof starches in water is an important factor contributing to starch functional-ity and is widely exploited in industry.

98 B. Czukor et al.




Processing 99


Fig. 4.6. Heating starches in different water contents.

Fig. 4.7. DASM-4differential scanningcalorimetry (DSC)thermogram of peastarch in excess 0.6 MKCl.



When starch suspensions are heated in water, the crystalline structureof the granules is disrupted at a particular temperature, followed by theirintensive swelling and partial solubilization. This results in an increasein viscosity of the starch suspensions. The solubilization and swelling ofstarches from pea and faba bean occur at lower temperatures than cerealstarches (maize and wheat), but at slightly higher temperatures, thanpotato starches (Doublier, 1987; Davydova et al., 1995). In addition, theextent to which legume starches are solubilized is slightly higher than forother starches, although there is a suggestion that they swell less than cerealstarches (Doublier, 1987).

The pasting properties of starches are commonly studied using aBrabender viscograph or a rapid visco-analyser (RVA; Leach et al., 1959;Doublier, 1987). The measurements of viscosity using these two devices aremade during continuous stirring of the starch suspension. It is common touse a heating–cooling cycle during this process, programmed such thatthe starch suspension is heated to 95°C, maintained at this temperaturefor 30 min and then cooled to 50°C (Fig. 4.8). During this treatment, maizeand potato starches give a peak of viscosity during the heating phase. In thecase of potato starch, this peak is especially large. Such behaviour is notdesirable for industry and reduces the number of applications for thesestarches in their native form. The starches from pea and faba bean,however, do not show a peak of viscosity during heating (Schoch andMaywald, 1968; Stute, 1990a,b; Davydova et al., 1995). In addition, the finalviscosity, that these legume starches develop after cooling, is significantlyhigher than that for wheat starch, for example. Such behaviour indicatesthat, in relation to pasting behaviour, legume starches are superior to thosefrom potato and cereals.

The explanation for the stable behaviour of legume starches duringheating lies in their unique crystalline structure described earlier. The

100 B. Czukor et al.


Fig. 4.8. RVA viscograms of starch suspensions. Starch from: (1) r mutant pea;(2) maize; (3) rb mutant pea; (4) wild-type pea; (5) potato.



disruption of crystallinity in pea starch granules begins from the centre,where the B polymorphs are arranged. The disruption of the B polymorphsis accompanied by swelling of the disrupted area. The disruption of the Apolymorphs occurs at a higher temperature and is then followed by furtherswelling of the granules. The swelling of pea starch granules, therefore,occurs much more slowly than the swelling of granules from cereals andpotato, which have only one type of polymorph. The slow development ofviscosity of pea starches can be related to this disruption and swellingbehaviour of the granules. Such rheological behaviour of legume starchesmay widen their applications as thickening agents for industry.

4.2 Modified Starch

According to the ISO standard No. 1227–1979 modified starch is ‘starchwith one or more of its physical or chemical properties modified’. Morespecifically, the term modified starch may refer to a chemically modifiedstarch. Physical or chemical properties of starch can be changed by physicalprocesses, chemical reactions or by biotechnological modifications. Thevarious physical and chemical methods for modifying starch are presentedin Fig. 4.9.

Processing 101


Fig. 4.9. Starch modification.



4.2.1 Physical methods

SteamingA simple method for the physical modification of starch within cells is totreat the legume seed with saturated steam (Kozlowska et al., 1989). Shortperiods of this treatment are used during the production of proteinconcentrates and isolates from faba bean seeds to improve the sensoryproperties of these preparations. The structural changes that take placeduring this type of processing can alter the cellular arrangement of proteinand starch within the seed storage tissue (A and B). After this treatmentthe isolated starch shows intense amylose leakage and marked granuledeformation during heating in water at high temperatures. The steamingof seeds also causes changes in the pasting and gel-forming properties. Thereduced swelling of starch granules from steamed seeds gives rise to pasteswith a lower viscosity and a higher temperature is required for amylosemigration from the granule (Fig. 4.10). Starch isolated from untreatedseeds forms a more rigid and more elastic gel compared with starch fromsteamed seeds. The internal changes within the granule do not markedlyinfluence their appearance when viewed in the scanning electron micro-scope (Kaczymska et al., 1994). Only the denaturation of protein bodies isvisible, the starch granule surface remaining very smooth and unchanged.

The starch properties of pea starches can vary between varieties, thestarch pastes giving different rheological behaviour when heated andcooled. Starch from pea seeds with hard to cook (HTC)-defect was charac-terized by having an increased viscosity of paste during heating as well as ahigher value for the G′ modulus on cooling, indicating a high rigidity ofstarch gels (Fornal et al., 1995).



Fig. 4.10. Viscosity patternof raw (untreated) andsteamed faba bean starches.



AnnealingAnother type of heat treatment used to modify starch is annealing (Hooverand Manuel, 1994; Jacobs et al., 1995). This entails maintaining the starchat temperatures lower than the melting temperature (50–75°C) in excesswater. This process results in a decrease in the potential and extent ofamylose leaching. It is accompanied by an increase in the gelatinizationtransition temperature, the enthalpy of gelatinization and by a decrease inthe gelatinization temperature range. It has been suggested that theseeffects are due to interactions between amylose and the outer branches ofamylopectin, to an increase in double helices and to closer packing of thecrystallities within the granule (Hoover and Manuel, 1994). Annealing alsoresults in an increase in α-amylase hydrolysis, which may be due to a realign-ment of starch chains in the amorphous regions of the granules (Minagawaet al., 1987).

Gamma irradiationAs well as being used in food production, gamma irradiation also canmodify the chemical and physico-chemical properties of starch. Irradiationhas been shown to induce carbonyl derivatives (Raffi et al., 1981a), thedevelopment of acidity (Raffi et al., 1981b) and hydrogen peroxide (Raffiet al., 1981c) in haricot beans. The effect on starches from maize, manioc,wheat, potato and rice is to increase the reducing power, the water-solubledextrin content and the Brabender viscosity values (Raffi et al., 1981d).

ExtrusionA very promising method for physically modifying starch is extrusion(Smietana et al., 1996) and high pressure treatment (Kudla and Tomasik,1992). Extrusion gives products that are free from foreign substances,that can be used for children and in the development of non-allergenicformulas and functional foods. High pressure treatment results in somere-polymerization of dextrin formed during compression and in the order-ing of starch granule structure into more crystal-like matter. To date, bothof these methods have been used for modifying potato starch but, as yet,have not been used to modify legume starches.

In the extrusion processing of cereal and legume seeds, as such or inblends, the transformation of starch and protein determines the propertiesof the final product (Schukla, 1996). Dietary fibre, although less affectedduring extrusion, can also have a significant effect on textural properties.

The thermodynamic effects during extrusion break hydrogen bondsin starches, gelatinizing, or even dextrinizing, them in the process. Therequired energy input is often a function of starch granule size, the extentand type of crystallinity and the purity of the starch extract. Low-proteinand high-amylose starches require high inputs of energy to undergo starchgelatinization. In the case of proteins, the secondary and/or tertiary

Processing 103




structures undergo transformation, resulting in denaturation, associationand coagulation involving reduction or oxidation. Furthermore, starches,proteins, and fibre can be hydrolysed to a varying degree during theextrusion process, which will modify the rheology of the transformed melts.

4.2.2 Chemical methods

The most popular methods for modifying starches are based on the use ofchemicals.

Chemical modification can be carried out on three starch states: (i)in the solid state, where dry starch is moistened with chemicals in a watersolution, air-dried and finally roasted at a temperature of over 100°C; (ii) insuspension, where the starch is dispersed in water, the chemical reaction isthen carried out in water medium, the suspension is then filtered, washedand air-dried; (iii) as a paste, where the starch is gelatinized with chemicalsin small amounts of water, the paste is stirred and when the reaction iscompleted, the starch is air-dried. The chemicals used for modificationreact with the free 2, 3 and 6 hydroxyl groups of the glucose units within thestarch.

The most common chemical modification processes are: oxidationusing sodium hypochlorite, hydrogen peroxide, persulphates or potassiumpermanganate; esterification using acetic anhydride, vinyl acetate, ortho-phosphoric acid, sodium or potassium orthophosphate or sodium tripoly-phosphate, sodium trimetaphosphate, phosphorus oxychloride or urea;etherification using ethylene oxide, propylene oxide, monochloroaceticacid or quarternary amines.

The most important chemically modified starches from an industrialpoint of view are the starch-esters and starch-ethers.

To date, legume starches have not been modified on a commercialscale for non-food applications. There have been laboratory investigations,however, on chemically modifying legume starches and acetylated starch,hydroxypropyl starch, cross-linked starch, cationic and grafted starcheshave been produced.

AcetylationThe only major study on the acetylation of legume starches has beencarried out on a range of bean (P. vulgaris) varieties, using acetic anhydride(Hoover and Sosulski, 1985a,b; Vasanthan et al., 1995). X-ray diffraction ofthe modified starches indicated that the acetyl groups mainly entered theamorphous regions of the starch granule. The result of this process wasstarches with a decrease in hydrolysis, gelatinization temperatures, enthalpyof gelatinization, syneresis and in the extent of the viscosity increase duringthe holding period at 95°C in a viscosity analyses (Fig. 4.11 and Table 4.2).There was also an increase in viscosity in the amylose exudation at 95°C. No





apparent differences in the external morphology of native and acetylatedstarches could be seen under the scanning electron microscope.

The gradual slow rise in viscosity during the holding period at 95°C,and the increased stability during low temperature storage observed withacetylated legume starches, makes acetylation an acceptable method formodifying legume starches for the food industry.

PhosphorylationPhosphorylation of faba bean starch, using a mixture of mono- and disod-ium phosphates at 160°C, resulted in a significant increase of swelling(Soral-Smietana, 1995). Faba bean mono-starch phosphates can bindcomponents from solution or suspension into a homogeneous mass, which

Processing 105


Fig. 4.11. Pasting characteristics of native (normal) and acetylated (chemicallymodified) starches from beans (Hoover et al., 1985a,b).

Starch source Degree of substitution*

Extent of syneresis (%)

4°C −15°C

Pinto bean 0.050 18.2 6.80.050 13.0 5.8

Navy bean 0.050 20.2 7.90.055 12.0 6.2

Black bean 0.050 30.1 15.30.053 11.7 10.40.095 8.9 9.0

*No. of acetyl groups per glucose unit.

Table 4.2. Syneresis of native and acetylated starch gels after storage at two differ-ent temperatures for 7 days (Hoover and Sosulki, 1985a,b).



is of great importance in the preparation of instant products. This modifiedlegume starch can act as a stabilizer of emulsions and can also act as a bufferin fruit produce (Soral-Smietana, 1995).

Cross-linkingCross-linking refined starches from lentil, faba bean and pea with phos-phorus oxychloride decreases water binding capacity, swelling power,α-amylase digestibility and viscosity at 95°C in the amylograph, butincreases the degree of set-back (Table 4.3; Hoover and Sosulski, 1986).Cross-linking occurs mainly in the amorphous regions of the starch granuleand hinders amylose exudation. The stable hot paste viscosities ofcross-linked starches would be of value where low pH and high temperatureare employed, during pressure cooking or sterilization, while the lowdegree of set-back of pea starch should improve the freeze–thaw stabilityand textural quality of frozen foods.

HydroxypropylationThis chemical modification is based mainly on the addition of propyleneoxide to starch moistened with water containing sodium sulphate, themixture being heated and stirred for 24 h at 40°C (Hoover et al., 1988; Kimet al., 1992). The main effect of this modification on native starch granulesis to produce starch with higher molar substitution (0.12) and a higher



Time of incubation (h)

Starch source 1 2 3 4 5 6 7

NativeLentil 3.4 6.4 10.0 17 21 28 28Faba bean 2.1 5.8 9.9 16 20 26 27Field pea 1.9 5.3 9.2 12 19 23 23

Native (gelatinized)Lentil 42.8 57.8 61.8 66 72 79 79Faba bean 32.8 51.8 60.8 65 70 72 72Field pea 30.8 45.8 55.8 62 68 68 70

Cross-linked (ungelatinized)Lentil 3.2 6.0 10.0 16 20 27 27Faba bean 2.0 5.5 9.4 15 19 25 26Field pea 1.8 5.0 8.8 12 18 22 22

Cross-linked (gelatinized)Lentil 38.8 54.8 58.8 63 69 76 76Faba bean 29.8 49.8 58.8 63 68 70 70Field pea 27.8 42.8 52.8 58 64 66 68

Table 4.3. Hydrolysis of native and cross-linked legume starches by pancreaticα-amylase (as % of total conversion to glucose units).



susceptibility to α-amylase attack, whereas a lower substitution results ina reduction in hydrolysis (Fig. 4.12; Hoover and Vasanthan, 1994).

Hydroxypropylation of pea starch, with an amylose content of about34%, with propylene oxide and sodium hydroxide resulted in a decrease inenthalpy of gelatinization, in gelatinization peak temperature, in pastingtemperature and syneresis, and an increase in paste viscosity at 95° and50°C (Hoover et al., 1988).

The low gelatinization temperatures, high water-holding capacity andlow retrogradation rates observed in these chemically modified peastarches would make them suitable for use in the paper and food industries.

CationizationWater-miscible solvents such as ethanol, 2-propanol and methanol are usedas the reaction medium for cationization of pea starches (Kweon et al.,1996). Cationization that results in a degree of substitution of 0.02–0.05reduces the pasting and gelatinization temperatures, increases the peakviscosity and set-back on cooling and eliminates synersis after storage at 4°and −15°C (Yook et al., 1994).

The principal effects of cationization are to promote rapid granuledispersion at low pasting temperatures and to give a molecular dispersionof amylose and amylopectin on heating to 95°C. On cooling, the gelstructures are firm and the cationic groups control the realignment ofstarch during low-temperature storage.

Investigations on grafting green gram, pigeon pea and garden peastarches showed that the graft yield of the reaction is less than for cerealand root starches (Patel et al., 1986). The graft yield in a gelatinized system,however, is almost independent of starch source. The use of starch graftcopolymers as absorbents depends on their competitiveness on price com-pared with full synthetic absorbents produced from partially cross-linkedacryl copolymers. (NB. Graft copolymers are specific copolymers, that areobtained in reactions between macromolecular substances and a substanceof low molecular weight.)

Processing 107


Fig. 4.12. The effectsof degree of molarsubstitution (MS) ofhydroxypropyl groupson rates of hydrolysisof field pea starchduring incubation withα-amylase (Hooveret al., 1994b).



4.2.3 Biotechnological methods

Many modified starches used in food products are the result of germinatingor fermenting legume seed in vivo, or hydrolysing starches in vitro, usingamylolytic enzymes (Bhat et al., 1983; Revilla et al., 1986a,b; Rodriguez et al.,1988; Abia et al., 1993; Frias et al., 1998).

HydrolysisThe first step in enzymic hydrolysis is ‘endocorrosion’, which begins atthe centre of the starch granule. The granule then degrades sequentiallyresulting in compartmentalization and subsequent fragmentation. Thesurface becomes porous and the granule is then divided into numerouspolyhedral forms of various size (0.4–10 µm). This degradation process hasbeen reported for starch from lentil (Revilla and Tarango, 1986a,b) andchickpea (Rodriguez et al., 1988). In general, the susceptibility of starchgranules to modification by amylases depends on the physical structure ofthe granules, the amylose content, the degree of polymerization and on thepresence of non-reducing ends on the granule surface (Bhat et al., 1983;Madhusudhan and Taranathan, 1995).

GerminationGermination induces the release of hydrolytic enzymes, which producechanges in the physical properties and functionality of seed components.Starch extracted from faba bean, chickpea and kidney bean before andafter germination was significantly more digestible than those fromungerminated samples (Table 4.4; Shekib, 1994). Cooking the isolatedstarches from both the ungerminated and germinated samples furtherincreased their digestibility.



Treatment Faba bean Kidney bean Chickpea Corn starch

Isolated starchA 33.2 32.3 39.5B 65.1 62.1 67.2C 47.2 44.7 49.4D 84.3 82.1 90.3

Cooked corn starch – – – 90.3

A, starch from ungerminated seeds; B, cooked starch from ungerminated seeds;C, starch from germinated seeds, D, cooked starch from germinated seeds.Cooking, the samples were steamed for 15 min at 115°C.

Table 4.4. In vitro starch digestibility (as a %) of ungerminated and germinatedisolated legume starches in various forms (Shekib, 1994).



FermentationFermentation can be taken into consideration as a possible method formodifying starch properties for food use, for example puddings. Thisprocess has only limited effects on granule swelling and no apparenteffect in solubility. Marked changes, however, were found in the apparentviscosity of cold pastes and in the intrinsic viscosity (Abia et al., 1993; Ncheet al., 1994; Yadav and Khetarpaul, 1994; Matthews, 1999).

Yadav and Khetarpaul (1994) produced wadi from black-gram dhal(Phaseolus mungo) and examined the in vitro digestibility of starch, phyticacid and polyphenols (Table 4.5). When black-gram dhal wadies werefermented at 25, 30 or 35°C for 12 and 18 h, the improvement in starchdigestibility ranged from 57% to more than 88% over the control value.

4.3 Food Application of Native and Modified LegumeStarches

Native and modified legume starches can be used in the following applica-tions (Blenford, 1994):

• preparation of gels (e.g. puddings) that can be prepared with about50% less starch in comparison to corn starch;

• production of extruded products and instant starches that can beproduced without the significant loss in viscosity that occurs with otherstarches;

• production of roll-dried starches, fruit and vegetable flakes that have apulpy texture after rehydration and a considerable stability at cookingtemperatures;

• production of pulpy products via freeze–thaw processing that keeptheir pulpy texture even after prolonged cooking;

Processing 109


Temperature(°C)

Fermentationtime (h)

Starch digestibility(mg maltose released g−1 meal)

Protein digestibility(%)

Control 0 35.7 53.025 12 56.1 68.1

18 56.9 70.030 12 60.6 71.9

18 61.2 73.835 12 66.6 77.3

18 67.2 79.3

Table 4.5. Effect of temperature and fermentation time on in vitro starch and pro-tein digestibility of wadies prepared from black-gram dhal (Yadav and Khetarpaul,1994).



• production of roll-dried instant starches with cold swelling gellingproperties that can be used as such in formulations of various instantdesserts with a flake-like texture.

There are some patents describing new products based on modification of,among others, legume starch (mostly pea starch). A novel starch-basedtexturizing agent has been produced from high amylose starch (> 40% ofeither corn, barley or pea), by dissolving the starch in water under acidicconditions, while agitating at an elevated temperature and pressure,followed by retrogradation at low temperature and spray drying. The func-tion of texturizing agents is to provide several fat-like attributes such asstructure, viscosity, smoothness and opacity to reduce and/or essentiallyreplace the fat content in foods. In addition, the texturizing agent can beused in full fat foods as a stabilizer. Foods containing the novel texturizingagent include mayonnaise, stoppable and pourable salad dressings,yoghurt, cottage cheese, sour cream, cream cheese, peanut butter, frostingcheesecake, mousse and several sauces (Mallee, 1995; Mallee et al., 1996).

Another application is in the preparation of foods with a reduced lipidcontent. In this case the lipid portion in the food is replaced by an aqueousdispersion made from non-gelling, pre-gelatinized starch (high-amylosestarch, for example pea) derivatives such as dextrin, converted starches andhydroxypropyl starch (Capitani et al., 1996). A reduced fat groundnutbutter product, comprising fine-milled groundnuts in continuous oil phaseand 5–50% native starch (pea or garbanzo bean) has been produced(Finocchiaro, 1996). Finally, the starches can be used as an opacifyingagent. High-amylose starch has been pre-gelatinized under aqueous condi-tions in the form of a complex in which the opacifier (titanium dioxide)has been stabilized or entrapped. This product is recommended for low-fatand fat-free foods and beverages that need to be opacified (coffee creamer,cottage cheese dressing, nutritional beverages, mayonnaise, sour cream, icecream, yoghurt, etc. (Dunn et al., 1996; Dunn and Finocchiaro, 1997).

Further food applications of modified legume starches are possiblebut it depends both on more detailed knowledge of their propertiesand consumer acceptation of this source of starch in traditional or newlydeveloped products.

4.4 Effect of Processing on Starch and Other Carbohydratesin Foods

4.4.1 Resistant starch formation

The most common form of resistant starch (RS) in the diet and the mostimportant from a technological point of view is retrograded starch (RS III),





because it forms as a result of food processing (Escarpa et al., 1996;Soral-Smietana et al., 1998). Despite the extensive work that has been car-ried out on this subject, the correlation between starch structure and resis-tance of starch to amylolysis is still poorly understood.

The formation of RS III is influenced by several factors, theseinclude the conditions of the starch solubilization and retrogradationprocesses (for example, temperature and pressure of the autoclavingprocess and the number of autoclaving cooling cycles), the presence oflipids or sugars and the amylose content in the starch (Siljeström et al.,1989; Sievert and Pameranz, 1989, 1990; Sievert et al., 1991; Czuchajowskaet al., 1991; Eerlingen et al., 1993a,b, 1994a,b). There is a strong positivecorrelation between the amylose content and the level of RS III in cerealsand other grains. The USA has supported plant breeding programmesto produce new plant hybrids with low amylopectin and high amylose(c. 95%) starch (Gordon et al., 1997). This low-amylopectin starch has ahigher gelatinization temperature, lower swelling power in hot waterand is more resistant to enzyme and acid digestion compared with starchcontaining 70% amylose. Many legume starches have a high amylosecontent compared with cereal and tuber starches, ranging from about 30to 70% of the starch (Swinkels, 1985; Blenford, 1994; Soral-Smietana andDziuba, 1995).

The manipulation of RS III content and other nutritional properties ofstarchy foods has been pointed out as a challenge to the food industry(Tovar et al., 1992a,b; Björck and Asp, 1994). In this context, steam-cookingcould provide new ways to increase the present limited industrial utilizationof grain legumes (Sosulski et al., 1989; Tovar et al., 1992a,b). The relation-ship between the total starch content, the proportion of readily availablestarch and RS III after prolonged steam and short dry heat treatment, hasbeen reported (Tovar and Melito, 1996). Also, it has been shown thatduring the steam-heating of intact beans, the interaction between amyloseand protein, and possibly other seed constituents, may also modify thetendency for the polysaccharides to recrystallize (Cerletti et al., 1993).These indigestible transglycosidated starches and other types of modifiedstarches, will probably add in vivo to the unchanged apparent resistantstarch values (Asp and Björck, 1992).

Investigations have been carried out on digestion and large bowelfermentation using rats fed on raw and cooked peas (P. sativum), or whole-meal bread with different levels of cooked haricot beans (P. vulgaris)added. These studies have shown that the resistant starch content increasedin peas after processing and increased progressively in bread withincreasing amounts of haricot beans (Goodlad and Mathers, 1992; Key andMathers, 1993).

Processing 111




4.4.2 Content, composition and digestibility

SoakingGrain legumes are rarely eaten in a raw state and are usually cooked orprocessed first.

Perhaps the simplest method for processing legume seeds is to soakthem in water. This process can reduce the level of reducing and non-reducing sugars 16–40% (Jood et al., 1988). There is an increase, however,in in vitro starch digestibility of 17–23% after a 12-h soaking (Bishnoiand Khetarpaul, 1993). This enhancement of starch digestibility may beattributed to the loss of antinutritional factors such as phytic acid andpolyphenols, which inhibit the activity of α-amylase (Deshpande andCheryan, 1984). Conversley, it has been suggested that prolonged soakingof intact peas may allow the mobilization of phenolics, which are knownto interfere with starch digestion from the seed coat to the cotyledons(Deshpande and Salunkhe, 1982).

Soaking in water and NaHCO3 solution also significantly reducesthe levels of stachyose, verbascose and raffinose. The reduction is usuallyhigher in NaHCO3 solutions than in water and can account for 46–100% ofthe α-galactoside content (Jood et al., 1985; Vijayakumari et al., 1996). Only1–10% of these losses can be explained by leaching into the soakingsolution, the remainder being due to hydrolysis by α-galactosidase releasedby the imbibed seed (Vidal-Valverde et al., 1992a).

In general, soaking is not used by itself, but in combination withgermination, cooking or autoclaving.

High and low pressure cookingCooking and pressure cooking are perhaps the most effective methods forincreasing starch digestibility. The effect of these treatments on differentnutritional components of legumes is very similar, but generally the effectof pressure cooking is more intensive (Jood et al., 1985). Ordinary cookingcan increase starch digestibility by 40–200%, while with pressure cookingthe increase may be 200–400% (Jood et al., 1988; Bishnoi and Khetarpaul,1993; Rani et al., 1996).

The content of reducing and non-reducing sugars and the totalstarch content decreases significantly during cooking. In the case of thenon-reducing sugar content, the losses may be 20–40% (Abdel-Gaward,1993) during normal cooking and significantly higher during autoclaving(Jood et al., 1985).

Cooking also can reduce the α-galactoside content by between 20and 100% (Trugo et al., 1990; Vidal-Valverde et al., 1992a; Abdel-Gaward,1993; Attia et al., 1994; Vijayakumari et al., 1996). There are, however, somereports of increases in the oligosaccharide content after cooking (Rao andBelavady, 1978; Revilleza et al., 1990). For example, boiling mature rawseeds of hyacinth bean in relatively low bean:water ratios resulted in a net





decrease of sucrose after 90 min, while the level of raffinose, stachyose andverbascose increased (Revilleza et al., 1990). This increase could be attrib-uted to hydrolysis of oligosaccharides bound to proteins or other macro-molecules, or to the hydrolysis of high molecular weight polysaccharides.

Steam-heated legumes are rich in resistant starch, while high pressuresteaming and dry pressure cooking decreases the total and the availablestarch, and stabilizes or decreases the resistant starch level (Tovar andMelito, 1996; Periago et al., 1997). There is an increase in the total non-starch polysaccharide content during cooking, the content of soluble non-starch polysaccharides generally increasing and the insoluble non-starchpolysaccharide content decreasing (Periago et al., 1997).

ExtrusionDepending on the extrusion conditions (temperature, moisture content,screw speed), the loss of total sugar and α-galactoside content can be about30% and 50–60%, respectively, while the starch digestibility can increasesignificantly (Tuan and Phillips, 1991; Borejszo and Khan, 1992). Extrusioncooking marginally decreases the total content of dietary fibre of peas at168°C, but the content of resistant starch increases significantly from about1.5% to about 3.3% of the total starch (Berghofer and Horn, 1994).

Dry roastingAt high temperatures, dry roasting results in the complete reduction of theoligosaccharide content in the hyacinth bean after 2 min, although thelevels of sucrose and the oligosaccharides were higher after a roastingtime of less than 0.5 min (Revilleza et al., 1990). The effect after 2 min wasprobably due to a non-enzymatic browning reaction, oxidation of sugars orto pyrolysis (Fig. 4.13). Contrary to the effect of other processes, frying androasting considerably reduces starch digestibility of legumes (Kelkar et al.,1996). Frying significantly reduces the sucrose content of legume seeds,probably because of the composition of the frying medium (Jood et al.,1985).

Freeze-dryingThere is no apparent effect of freeze-drying on the sugar, starch and pectincontents of green beans (Oruna-Concha et al., 1996), although there was aslight decrease in the digestibility of starch in the tepary bean (Abbas et al.,1987).

MicrowavingRepeated microwave treatments decrease the total dietary fibre content ofgreen bean, primarily because of losses in the soluble dietary fibre content(Svanberg et al., 1997).

Processing 113




GerminationGermination is one of the best known methods for decreasing theα-galactoside content and increasing the starch digestibility of legumes.During germination there are significant decreases in the total solublecarbohydrate content and in the total starch content, and increases inthe reducing and non-reducing sugar content and in the in vitro starchdigestibility. In addition, this process totally eliminates the α-galactosides(raffinose, stachyose, verbascose) (Jood et al., 1988; Revilleza et al., 1990;Trugo et al., 1990; Vidal-Valverde et al., 1992a; Vidal-Valverde and Frias, 1992;Bishnoi and Khetarpaul, 1993; Shekib, 1994; Urooj and Puttaraj, 1994;Urbano et al., 1995; Kelkar et al., 1996). The extent of the changes is mainlydetermined by the germination conditions (Nnanna and Philips, 1988).

Germination significantly increases the resistant starch contentresulting in an increase in the total dietary fibre content, the solublefraction decreasing and the insoluble fraction increasing significantly(Veena et al., 1995).

FermentationNatural fermentation enhances protein digestibility and eliminates par-tially or completely the antinutritional factors. The effect of fermentationon starch digestibility has been studied in Bengal gram, cowpea and greengram (Urooj and Puttaray, 1994). The seeds were soaked in water for 3 h,drained, ground and allowed to ferment for 4 h at room temperature. Afterfermentation the meal was steam-cooked for 10 min. The fermentationtreatment reduced the total soluble carbohydrate, the starch and the



Fig. 4.13. The effect of dry roasting on the soluble sugars of hyacinth bean(Lablab purpureus; Revilleza et al., 1990).



dietary fibre content. The fermented meal was digested significantlyfaster than the meal from untreated seeds. This may be due to a loss inthe structural integrity of the starch granules, a change in the nature ofthe interaction between starch and fibre and because of the inactivationof some antinutrients (e.g. α-amylase inhibitors, lectins, phytic acid).

Natural fermentation is a widely accepted, simple and inexpensiveprocessing method for the reduction or elimination of oligosaccharides.Zamora and Fields (1979) studied natural fermentation of cowpea andchickpea and identified the microorganisms involved. They found thatraffinose was eliminated in fermented cowpea and chickpea, that stachyosecontent was decreased in fermented chickpea and eliminated in fermentedcowpea. The reduction or elimination of these compounds was most likelydue to the ability of lactic organisms to utilize oligosaccharides for theirmetabolism. The lactic organisms in cowpea and chickpea were Lacto-bacillus casei, Lactobacillus leichmanni, Lactobacillus plantarum, Pediococcuspentosaceus and Pediococcus acidilactici; Lactobacillus helveticus was found onlyin chickpea. Odunfa (1983) studied the changes in oligosaccharide contentof locust bean during iru preparation. The fermenting organisms werevarious subspecies of Bacillus subtilis.

After 4 days natural fermentation of lentil, the α-galactosides andsucrose in the fermented product could not be detected, the cellulose andhemicellulose content decreased and the lignin increased (Vidal-Valverdeet al., 1993). It was established that during the preparation of suspensionsthe initial concentration of the lentil flour–water suspension had an impor-tant influence on the level of α-galactoside content (Frias et al., 1996c).

Mital et al. (1975) tested a number of lactic cultures for α-galactosidaseactivity. They found that the enzyme is constitutive in Lactobacillus bucheri,Lactobacillus brevis, Lactobacillus cellobiosis, Lactobacillus fermentum and Lacto-bacillus salivarius subsp. salivarius and could be induced in L. plantarum.The fermentation of soya milk with lactic cultures reduces the raffinose andstachyose content in different ways, depending on the Lactobacillus strain(Mital et al., 1979). Lactobacillus fermenti completely utilized raffinose andstachyose by 12 and 25 h respectively, a mixed culture of L. fermenti andStreptococcus thermophilus was less effective, while L. cellobiosis utilized onlyraffinose after 20 h fermentation. Soya milk fermentation with L. plantarumreduced the stachyose content and was less effective on raffinose reduction.

Two enzymes are required to completely hydrolyse oligosaccharides.α-Galactosidase (EC 3.2.1.22) is necessary to hydrolyse the α(1→6) linkagesand invertase (EC 3.2.1.26) hydrolyses the sucrose moiety. Many micro-organisms are able to produce the α-galactosidase enzyme. The enzyme hasbeen reported in brewer’s yeast, in some strains of bacteria (Actinomycetalesand Streptomycetes strains) and moulds. Also, α-galactosidase has been foundin the fruiting bodies of various mushrooms and puffballs and in higherplants (Suzuki et al., 1966). These different α-galactosidases differ for pHand temperature optimum and for substrate specificity (Suzuki et al., 1966).

Processing 115




Variation in seed-derived α-galactosidases has been found between P.vulgaris (Becker et al., 1974) and soybean (Kim et al., 1973; Angel et al., 1988).

Isolated α-galactosidase could be used to reduce or eliminate the flatus-inducing factors in edible legume seeds. It is already used to eliminateraffinose during beet-sugar processing, to improve the crystallization ofsucrose and to increase the sucrose yield. Sugimoto and Buren (1970)purified α-galactosidase enzyme and added it to soya milk, where it effectedthe complete hydrolysis of the oligosaccharides after 2 h incubation.

4.5 Legume Seeds as a Source of Raw Materials

A wide range of starches can be found in pea (see Breeding and Agronomychapter) with amylose contents ranging from near zero to about 70%.Suitable processing methods for the extraction of starches on an industrialscale from peas, however, has only recently become possible and todate has only been applied to conventional round-seeded varieties. Starchfrom round-seeded peas has an amylose content of 30–35%, compared withamylose contents of 20–28% in conventional starches from other species.The main industrial sources of pea starch are Provital Industry SA, Belgium,and Woodstone Food Company, Canada, Provital being the main supplierof raw materials from legumes within Europe.

With regard to the main carbohydrate based products, Provitalproduces (Nastar R) native pea starch, (Nastar R Instant) pre-gelatinizedpea starch and (Swelite R) texture improver.

Nastar R is a native starch with high gel strength. The gelatinizationcapacity and viscosity profile of this native starch are at a similar level ofperformance to that found in certain modified starches (cross-linked). Thestarch shows an excellent stability to high temperatures, to shearing and tovariations in pH and it promotes the formation of film and sliceable gels. Indry products it improves the crispness.

Nastar R Instant is a pre-gelatinized version of Nastar R and is ideal foruse in cold processes. The high gelling capacity of Nastar R is retained withNastar R Instant and an efficient dispersion in water with strong agitationwill rapidly develop a firm and sliceable gel.

Swelite R texture improver is a fat-free functional ingredient composedof fibre and starch. Its dual composition combines the technologicalproperties required from a functional ingredient with the dietetic qualitiesexpected from dietary fibre. The high water capacity, fat binding andtexturing effect of Swelite R gives food preparations an excellent stabilityfor industrial manufacturing and storage, as well as a desirable texture.

These products have demonstrated the usefulness of grain legumes,in this case pea, as a source of raw materials. Given the breadth of geneticvariation now available, there is wide scope for this to progress evenfurther.





Seed Physiology and BiochemistryJ. Górecki et al.5

5Seed Physiology andBiochemistry

Editor: Ryszard J. Górecki

Contributors: Gabriel Fordoñski, Ryszard J.Górecki, Horia Halmajan, Marcin Horbowicz,Rupert G. Jones, and Leslaw B. Lahuta

If you can look into the seeds of time,And say which grain will grow and which not,Speak then to me, who neither beg nor fearYour favours nor your hate.

Macbeth, act 1, sc. 3, l. 58 (1606)William Shakespeare (1564–1616), English playwright

5.1 The Legume Seed

5.1.1 Seed components

The legume seed can be separated into three major tissues – testa, endo-sperm and embryo – which in turn can be further divided into the twocotyledons and the embryonic axis (Fig. 5.1). The testa (Latin for ‘shell’),seed coat or hull is a maternal tissue that surrounds the embryo andattaches the seed to the pea pod via the stalk-like funicle. During earlydevelopment, the testa, acts as a nurturing tissue, distributing nutrientsfrom the mother plant to the developing embryo. Early in development thisis by diffusion via the endosperm and later on by direct contact with theembryo. Sucrose, the main form of imported carbohydrate in legume seeds(Pate et al., 1974; Fellows et al., 1978; Patrick and McDonald, 1980; Schmittet al., 1984), is unloaded from the vascular bundles within the testa. Itis then thought to be transported symplastically through the seed coat(Patrick and Offler, 1995). Its import into the embryo is regulated by





the action of the plasmodesmata (Stitt, 1996), through a possible turgorhomeostat system in the exporting cells (Patrick and Offler, 1995). Oncethe sucrose has been taken up by the cells of the embryo, it is hydrolysed byeither sucrose synthase to fructose and UDP-glucose or, to a lesser extent,by alkaline invertase to fructose and glucose.

Later in development, as the embryo matures and the seed beginsto lose water, the layers of the testa become compressed and accumulatecompounds such as lignin, suberin, cutin, callose and tannins. In matureseeds the testa is a dead tissue, the lignins making it almost impervious towater. In the dry seed, the testa provides a mechanical barrier, therefore,against abiotic and biotic processes that would otherwise damage or killthe embryo within. There is a small opening, the micropyle, that allowsthe diffusion of gases into the otherwise impermeable testa, so that thequiescent or dormant embryo can still respire. The tannins, that are oftenfound in the testa, act as antinutritional compounds and colourants,deterring predatory animals and making the seed less conspicuous.

The endosperm is a triploid tissue consisting of two maternal sets andone paternal set of genetic material. In the early stages of seed developmentit acts as an intermediary in the transfer of carbohydrates from the testa tothe developing embryo. In pea, the unloading of carbohydrates into theendosperm from the testa is believed to be a passive process aided by theactions of a porin-like transporter (DeJong et al., 1996). In developingsoybean cotyledons, however, sucrose causes a membrane depolarization(Lichner and Spanswick, 1981) with a sucrose-specific carrier that isenergetically distinct from the hexose transport systems (Lin et al., 1984). In

118 J. Górecki et al.


Fig. 5.1. Stages of embryo development in pea.



Vicia faba, the process appears to be aided by a transporter molecule(Bouché-Pillon et al., 1994; Wang et al., 1995), although 50% of the carbo-hydrate is still apparently transported passively (Patrick and Offler, 1995).Turgor pressure also influences the efflux of solutes from the testa to theendosperm and later the embryo, with the rate of utilization of the sucrosewithin the cotyledons being the regulatory factor (Patrick and Offler, 1995).

The endosperm may not persist into full seed maturity, as is the casefor temperate grain legumes, the seeds, therefore, being termed exendo-spermous. For example, in pea seeds the endosperm rapidly declines as theembryo increases in size until it is almost entirely absorbed. Nutrients arethen transferred from the testa to the embryo by specialized transfer cellsfound on the inside of the testa.

The largest component of the seed is the embryo, which is the resultof the fusion of one female and one male gamete and represents thesporophyte of the new generation. The embryo itself consists of thecotyledons, which make up the largest component, and the embryonic axis,comprising the root and shoot axes. At first, the embryo is supported inthe nutritive liquid endosperm, but as it develops and its size increases, iteventually fills the embryo sac within the testa, displacing the endosperm.As the embryo progresses to maturity, a number of key developmentalchanges occur, resulting in the deposition of storage components,including the carbohydrates that form the basis of this book.

5.1.2 Seed development

The allocation and partitioning of carbon within legume seeds presents acomplicated picture, with distinct phases when the seed undergoes celldivision, expansion and the laying down of storage products (reviewed byZamski, 1995). Once a plant has flowered, the partitioning of photo-assimilates is altered (Pate, 1984). The carbon that is used for the develop-ment of the seed is principally derived from the photosynthetic activity ofthe plant at the time of seed filling and very little is derived from storedcarbohydrates (Fellows et al., 1978; Pate, 1984; Bewley and Black, 1985),although there may be some limited redistribution of carbon from stemsand pods (Thorne, 1979). In cowpea, up to 77% of the photoassimilatetranslocated to the seeds is used for the accumulation of dry matter (Pate,1984), whereas in lupin, with its thick fibrous pod, only 50% is converted toseed dry matter. This makes seed filling sensitive to biotic and abiotic stressduring and, particularly, after flowering. As seed development proceedsthere are often a number of aborted seeds, which has been attributed to theplant initially producing too many flowers and thus seeds. The supply ofphotoassimilates to many of the seeds is then withdrawn and these seedsabort, ensuring the vigour of a smaller number of seeds in a self-thinningexercise (Wardlaw, 1990, and references therein).

Seed Physiology and Biochemistry 119




Systems to differentiate developmental stages have been producedfor some legume species, such as soybean (Fehr et al., 1971; Table 5.1), pea(Knott, 1987) and faba bean (Knott, 1990). In the case of pea and fababean, however, the key encompasses the whole of plant development, with-out concentrating specifically on seed development. The system for soy-bean was designed to aid insurance loss assessment against damage by hailstorms, rather than for developmental studies. For this reason adaptationsof the soybean key have been made, to better describe the events duringembryo development, leading to the introduction of substages (Spaeth andSinclair, 1984; Dornbos and McDonald, 1986; Lowell and Kuo, 1989).Studies using growth stage and days after anthesis have been comparedas determinants of development (Bewley and Black, 1978). These studiesfavoured the growth stage method as being more consistent when compar-ing lines and plants grown in field conditions. When comparing differentspecies, however, growth stage analysis can be misleading as different stagesare described by different methods. For research into biochemical ormolecular changes in development, growth stages are inappropriate as onestage can encompass a multitude of developmental events.

Many different carbohydrates are deposited within grain legumeseeds during development and these may be metabolic intermediates orend products for storage and other uses. In lima bean, three stages of seeddevelopment have been analysed, half the full size, full size and dry mature(Meredith et al., 1988). The carbohydrates analysed were fructose, sucrose,raffinose, stachyose, verbascose and starch. A reduction in the proportionof fructose and sucrose and an increase in raffinose, stachyose andverbascose was found in successive developmental stages. In general, starchaccumulation occurred before the first growth stage.



Stage number Reproductive stage description

R1 One flower at any nodeR2 Flower at node immediately below the uppermost node with a

completely unrolled leafR3 Pod 0.5 cm long at one of the four uppermost nodes with a

completely unrolled leafR4 Pod 2 cm long at one of the four uppermost nodes with a

completely unrolled leafR5 Beans beginning to develop (can be felt when pod is squeezed)R5.5 At one of the four uppermost nodes with a completely unrolled leafR6 Pod containing full size green beans at one of the four uppermost

nodes with a completely unrolled leafR6.5 Beginning maturityR7 Pods yellowing, 50% of leaves yellow, physiological maturityR8 95% of pods brown, harvest maturity

Table 5.1. Stage of development descriptions for soybean (adapted from Fehret al., 1971, and Lowell and Kuo, 1989).



A modified growth stage system was used in soybean to determine whenoligosaccharides accumulated in the developing seed. As with lima bean, anincrease in these compounds was found during successive developmentalstages (Lowell and Kuo, 1989).

In oil-rich legumes, such as soybean, starch can be present at levels ofup to 10% of the seed dry weight early in development. By full maturity,however, the starch level declines to about 1% and is replaced byaccumulating oil reserves (Yazdi-Samadi et al., 1977; Adams et al., 1980).The amount of soluble carbohydrates increases in the developing soybean(Adams et al., 1980).

With regard to seed development studies, pea has often been used as amodel system for seeds in general and grain legume seeds in particular (seereview by Wang and Hedley, 1993). There are many reports in the literaturerelating to developmental patterns of seed development in pea (e.g. Bissonand Jones, 1932; MacKee et al., 1955; Carr and Skene, 1961; Flinn and Pate,1968; Burrows and Carr, 1970). These papers have generally referred to dif-ferent pea genotypes grown in a range of different environments and withlittle appreciation of the problems of seed to seed variation within samples.This has made a general interpretation of pea seed development difficult.

One of the first studies to use controlled environments and to reduceseed to seed variation was carried out by Eeuwens and Schwabe (1975)using the pea variety Alaska. They were able to define a developmental pat-tern for seeds of this variety that followed a double-sigmoid curve separatedby a lag phase. Using this study and many others from the literature, Pate(1975) was able to develop a general scheme for pea seed development thatrelated all of the cardinal events, including the synthesis of starch, to thegrowth pattern of the seed.

Hedley and Ambrose (1980) reported the first detailed analysis ofa range of pea genotypes, grown in the same controlled environmentconditions. They studied three conventional round-seeded lines plusthree wrinkled-seeded lines containing the r mutation (see Chapter 7), allof the lines differing for seed size. From this study, a general pattern forpea seed development was defined that related the growth of the testa andendosperm to the development of the embryo. This showed three rapidphases of seed growth separated by two lag phases, the first correspondingto a rapid decline in the growth of the testa and endosperm and the secondwhen the testa made contact with embryo following the absorption of theendosperm.

These studies on pea have formed the basis of our understanding of therelationship between the growth and development of the pea seed and thesynthesis of the seed storage products, including the carbohydrates and aseries of papers have now been published in this area (Hedley and Smith,1985; Hedley et al., 1986; Ambrose et al., 1987; Corke et al., 1987; Wang et al.,1987a,b; Cook et al., 1988; Corke et al., 1990a,b; Hauxwell et al., 1990; Wanget al., 1990; Yang et al., 1990; Macleod et al., 1991; Hedley et al., 1994; Rochat





et al., 1995a,b; Hedley et al., 1996; Lloyd et al., 1996a,b; Casey et al., 1998;Craig et al., 1998; Harrison et al., 1998; Craig et al., 1999). Many of thesemore recent studies have been able to utilize the range of starch mutantsdescribed in Chapter 7. They have also provided information for studyingthe growth and development of seeds from other grain legume species, inparticular lentil (Bakhsh et al., 1991, 1992; Frias et al., 1994b, 1996d).

5.2 The Accumulation and Biosynthesis of Carbohydrates

5.2.1 Accumulation of soluble carbohydrates

Mature legume seeds can contain high levels of soluble carbohydrate.For example, in soybean this can be in excess of 14 and 28% of the drymatter for the axis (Table 5.2a) and cotyledons (Table 5.2b), respectively(Horbowicz and Obendorf, 1994). The soluble carbohydrates found withinlegume seeds comprise a number of compounds, including di- and oligo-saccharides (Table 5.3).



Sucrose Raffinose Stachyose Verbascose Galactocyclitols Total

(a) AxesSoybeanb 111.3 26.3 131.4 trace 1.7 288.6Peac 69.7 19.6 65.5 45.6 5.7 200.5Yellow lupina 20.3 23.9 199.6 59.5 5.8 248.0Faba beanb 57.0 15.0 65.3 99.4 0.3 243.6Lentilb 39.2 5.4 92.4 33.7 10.8 236.5Pigeon peab 61.5 13.4 35.7 90.1 4.8 237.1Cowpeab 45.3 12.2 110.0 11.2 0.9 184.7Chickpeab 36.1 22.9 35.1 1.8 22.2 171.5

(b) CotyledonsSoybeanb 76.1 11.8 43.5 trace 2.6 141.0Peac 26.6 4.9 13.7 34.8 2.4 80.1Yellow lupina 7.7 8.4 48.3 23.6 4.3 110.0Faba beanb 19.3 3.4 11.1 52.5 0.0 88.4Lentilb 11.5 2.4 25.4 18.0 33.0 85.2Pigeon peab 18.8 9.4 16.5 34.6 11.4 95.4Cowpeab 15.2 3.3 55.2 10.8 2.3 87.7Chickpeab 20.6 4.3 26.7 1.0 34.5 94.2

aGórecki et al. (1997).bHorbowicz and Obendorf (1994).cLahuta (unpublished).

Table 5.2. Soluble carbohydrates in the axes and cotyledons of different grainlegume species (mg g−1 dry matter).





Sucr

ose

Raf

finos

eSt

achy

ose

Ver

basc

ose

Tota

lRFO

RFO

+su

cros

e

Pea

cv.L

ittle

Mar

vel

62.3

11.6

32.3

19.1

63.0

125.

3So

ybea

ncv

.Will

iam

s82

72.7

12.6

43.4

trac

e56

.012

0.2

cv.A

mso

y71

64.2

11.6

41.0

trac

e52

.612

5.3

Cow

pea

25.9

3.7

46.4

3.6

53.7

79.6

Mun

gbe

ancv

.Ber

ken

13.9

3.9

16.7

26.6

47.2

61.1

Lim

abe

ancv

.For

dhoo

k36

.06.

930

.3tr

ace

37.2

73.2

Com

mon

bean

cv.T

opC

rop

19.4

2.5

34.3

trac

e36

.856

.2cv

.Blu

eLa

ke29

.02.

228

.1tr

ace

30.3

59.3

red

kidn

eybe

an21

.53.

131

.6tr

ace

34.7

56.2

Pole

bean

cv.K

entu

cky

Won

der

26.7

4.3

26.2

trac

e30

.557

.2

Bro

adbe

an20

.72.

310

.711

.424

.445

.1G

roun

dnut

cv.F

loru

nner

81.0

3.3

9.9

trac

e13

.294

.2

Tabl

e5.

3.D

istr

ibut

ion

ofso

lubl

eca

rboh

ydra

tes

indr

yse

eds

inva

riou

sle

gum

es(m

gg−1

defa

tted

mea

l)(K

uoet

al.,

1988

).



MonosaccharidesThe monosaccharides found in legume seeds are often involved as transi-tory intermediates in the synthesis of higher polymers of carbohydrates.They are also often found in the phosphorylated form. Free mono-saccharides are most readily detected during early seed fill and are rapidlyutilized as development continues. Often monosaccharides, such asfructose, glucose and galactose, are found only in trace amounts in matureseeds (Horbowicz et al., 1995; Sun and Leopold, 1995). In soybean, how-ever, the amounts can be relatively high early in development (Yazdi-Samadi et al., 1977), while in mature lucerne seeds no monosaccharideshave been detected (Horbowicz et al., 1995). It has been suggested that it isadvantageous to the plant if the concentrations of the monosaccharides arereduced. This is because the reducing nature of these compounds has beenimplicated in the Maillard reaction, which causes oxidative stress throughthe formation of free radicals, particularly after the seed has germinated(Sun and Leopold, 1995). Once a seed has germinated the amounts ofgalactose still remain almost undetectable, despite the removal of thegalactose moieties from oligosaccharides. This is due to the high levels ofgalactokinase converting any free galactose to a safer phosphorylated formduring germination (Dey, 1985).

DisaccharidesThe most abundant disaccharide found in seeds is sucrose, which isthe principal translocated photoassimilate (Lin et al., 1984; Patrick andMcDonald, 1980; Lichner and Spanswick, 1981; Schmitt et al., 1984;reviewed in Patrick and Offler, 1995). Early in the development of peaseeds it can reach high levels. As the seed develops, however, the sucrosecontent falls as it is utilized for dry matter accumulation, so that by the timeof maximum fresh weight of a pea seed it consists of around 8%, or 20 mgper whole seed (Harrison, 1996).

OligosaccharidesThe most commonly occurring oligosaccharides found in plants are thosebased upon α-galactosyl derivatives of sucrose (reviewed in Dey, 1990).These compounds are almost ubiquitous throughout the plant kingdomand rank second only to sucrose as the most abundant soluble carbo-hydrates. These oligosaccharides can comprise from 2 to 13% of legumeseed dry weight and they are believed to play an important role in seedstorability and in protecting the seed from desiccation stress (Obendorf,1997; Sun, 1997).

The galactosyl moiety of these compounds can be linked to either thefructose or the glucose moieties of sucrose. Depending upon the bondingof the molecules to each other, they will form different families of oligo-saccharides (Table 5.4).





5.2.2 Biosynthesis of soluble carbohydrates

Raffinose family of oligosaccharides (RFO)The synthesis of α-D-galactosyl derivatives of sucrose begins with theproduction of galactinol from UDP-galactose and myo-inositol catalysed bythe enzyme galactinol synthase (GS, EC 2.4.1.123) (Fig. 5.2, equation 3).

The UDP-galactose utilized is derived from the conversion of UDP-glucose by the enzyme UDP-glucose-4′-epimerase.

The galactinol acts as a galactosyl donor and it is thought thatgalactinol has no other role within the plant other than the synthesis ofoligosaccharides (Saravitz et al., 1987), although there are suggestions thatit may be implicated in the synthesis of the galactomannans (Reid, 1985).Sucrose accepts the galactosyl moiety from galactinol to form raffinose withthe regeneration of myo-inositol through the actions of raffinose synthase(RS, EC 2.4.1.82; Fig. 5.2, equation 4). Raffinose synthase can catalyse twotypes of reaction, synthetic and exchange. The synthetic reaction combinessucrose and galactinol to form raffinose and myo-inositol. In the exchangereaction, a galactosyl moiety is transferred between sucrose and raffinose.If the sucrose molecule is radiolabelled, after the exchange reaction,raffinose is radiolabelled. Castillo et al. (1990) reported that in soybeanseeds the synthesis reaction levels off 15 days after flowering, while theexchange reaction increases until day 60. It was suggested that the enzymeeither contains two sites, or that there were two separate enzymes. Theactivity of galactinol synthase, which is thought to be the main rate limitingstep for RFO synthesis (Lowell and Kuo, 1989), increases as the seedbegins to dry off (Saravitz et al., 1987; Castillo et al., 1990). This enzyme hasbeen purified from kidney bean cotyledons and zucchini leaves (Kerr et al.,1993; Liu et al., 1995), which in the future may allow cellular and tissuelocalization and manipulation.

Raffinose can then be used as the substrate to produce the nextoligosaccharide in the RFO, stachyose, by the addition of an α-D-galactosylmoiety from galactinol to the C-6 of the non-reducing α-D-galactose moiety.This reaction is catalysed by stachyose synthase (STS, EC 2.4.1.67;Gaudreault and Webb, 1981; Fig. 5.2, equation 6). In addition to the



α-D-Galactosyl linkage with Name of molecule

C-2 of D-glucose UmbelliferoseC-3 of D-glucose No nameC-6 of D-glucose RaffinoseC-1 of D-fructose No nameC-3 of D-fructose No nameC-6 of D-fructose Planteose

Table 5.4. Mono-O-α-D-galactosyl–sucrose derivatives (Dey, 1990).



genuine STS reaction, the enzyme is able to produce galactosylononitol(Peterbauer and Richter, 1998; Fig, 5.2, equation 7), galactosyl pinitol Aand ciceritol (Hoch et al., 1999; Fig. 5.2, equations 8 and 9). Galactosylo-nonitol and galactosyl pinitol A could also substitute for galactinol in thesynthesis of stachyose from raffinose (Peterbaur and Richter, 1998; Hochet al., 1999; Fig. 5.2, equations 10 and 11). STS possibly synthesizes alsoverbascose from galactinol and stachyose (Tanner et al., 1967; Fig. 5.2,equation 12). High oligomers in the RFO series may be synthesized bya galactinol-independent galactosyltransferase activity (Bachmann andKeller, 1995; Fig. 5.2, equation 13).

Other cyclitol families are also derived from myo-inositol (Loewus andDickinson, 1982) including the galactopinitols A and B series (which aregalactosyl derivatives of D-pinitol), fagopyritol B series, galactosyononitol



Fig. 5.2. Biochemical pathway for some of the major α-galactosides and cyclitols.



and scyllo-inositol (Obendorf, 1997). These compounds have been found inmany legumes and in some cases at relatively high levels, for example,ciceritol (digalactopinitol A) is present in chickpea, lupin, lentil, soybean,kidney bean and lucerne (Obendorf, 1997).

With the synthesis of the galactosyl oligomers, two molecules ofgalactinol can combine to form a higher homologue in the galactinolseries, digalatosyl myo-inositol, with the regeneration of myo-inositol (Peteket al., 1966; Fig. 5.2, equation 5).

Due to the pivotal role of myo-inositol in the synthesis of these variouscompounds, it is found at relatively high levels in young developing seeds,with only a slight reduction through development.

Cyclitols and galactosyl cyclitolsThe trivial term of cyclitols refers to polyhydroxylcycloalkanes and theirderivatives and the term includes the compounds known as the inositols.They are highly soluble, stable and relatively inert within the cell (Loewusand Loewus, 1980). There is a large amount of research interest in thesecompounds due to their importance in cellular metabolism (see reviews byLoewus, 1990; Obendorf, 1997). The inositols are cyclohexanehexols andpossess one hydroxyl group on each of three or more ring atoms. There arenine enantiomers of inositol determined by the position of the hydroxylgroup and, of these, six have been found in plant tissues (Loewus, 1990).

Myo-inositol is synthesized from α-D-glucose-6-phosphate through theactions of myo-inositol-1-phosphate synthase (EC 5.5.1.4) through severalpartial reactions involving the reduction and then subsequent oxidation ofthe co-factor NAD+ (Eisenberg, 1967; Barnett et al., 1973; Chen andEisenberg, 1975; Loewus and Loewus, 1983; Loewus, 1990; RayChaudhuriet al., 1997) and 1-L-myo-inositol phosphatase (EC 3.1.3.25; Fig 5.2,equations 1 and 2).

The synthesized myo-inositol can also be utilized within a numberof synthetic pathways, including those leading to the RFO, inositolphosphates, phosphoinositides, cell wall polysaccharides and bound auxin(Loewus and Loewus, 1983; RayChaudhuri et al., 1997).

There are three steps concerned in the transformation of D-glucoseinto myo-inositol (Fig. 5.3). Initially D-glucose is converted to D-glucose6-phosphate by the hexokinase and then the intermediate is transferredinto L-myo-inositol 1-phosphate, which is finally dephosphorylated to myo-inositol (Hoffmann-Ostenhof and Pittner, 1982). The most interesting stepis the cyclization of D-glucose 6-phosphate to L-myo-inositol 1-phosphate. Itwas found that one enzyme is responsible for the reaction, originally calledcyclase or cycloaldolase, but was finally given the name 1-phosphatesynthase (EC 5.5.1.4). This enzyme has been isolated from various sources,animals, higher plants and yeasts. The enzyme proteins from these sourcesvary in their chemical and physical properties, including their metalcontent, molecular mass and specific activity, but all contain bound NAD+





(or require this coenzyme) for their action (Hoffmann-Ostenhof andPittner, 1982).

Myo-inositol is the primary source for the biosynthesis of many cyclitols(Fig. 5.4). D-Pinitol is formed by two main stages, methylation of myo-inositol and epimerization of the obtained methyl ether (Obendorf, 1997).There exists in nature two pathways for transformation of myo-inositol. Inthe first, myo-inositol is methylated initially by O-methyl transferase intosequoyitol (Scholda et al., 1964). Sequoyitol is then converted to D-pinitolby a two-step epimerization by a NAD+-specific dehydrogenase (EC1.1.1.143) to form 5-O-methyl-D-myo-1-inosose and this intermediate is thenmodified by an NADP+-specific D-pinitol dehydrogenase (EC 1.1.1.142) toD-pinitol (Kremlicka and Hoffmann-Ostenhof, 1966).

In the second pathway, myo-inositol is methylated to D-ononitol(Vernon et al., 1993). The conversion of D-ononitol to D-pinitol may involvea D-ononitol 1-dehydrogenase with 4-O-methyl-D-myo-1-inosose (Obendorf,1997). D-Ononitol is a favoured intermediate for the formation of D-pinitolin Simmondsia chinensis, Ononis spinosa, Medicago sativa and Trifoliumincarnatum (Dittrich and Brandl, 1987).

It is believed that in higher plants D-chiro-inositol is formed fromD-pinitol by demethylation, but an enzyme for this biosynthesis has notbeen characterized (Obendorf, 1997).

5.2.3 Accumulation of starch

The role of starch in the plant has been reviewed by Morrison and Karkalas(1990) and in seeds by Sivak and Preiss (1995). The role of starch in peaseeds has been reviewed by Smith and Denyer (1992) and the biosynthesis



Fig. 5.3. The pathway from D-glucose to myo-inositol.



of starch in pea by Martin and Smith (1995). The structural properties ofpea starch granules have been reviewed by Wang et al. (1998). Due to theinsoluble nature of starch, it has a negligible osmotic pressure, making itideal as the principle carbon store of seeds in non-oil seed legumes. In apea seed it can comprise around 50% of the dry weight (Smith and Denyer,1992).

In most starch-storing plant organs, for example, cereal endosperms,the starch-synthesizing plastids (amyloplasts) are derived from relativelyundifferentiated plastids (leucoplasts). These plastids lack chlorophyll and



Fig. 5.4. Proposed pathways of synthesis of the main cyclitols and methyl-cyclitols present in legume seeds.



have very little internal membrane (Journet and Douce, 1985). In contrast,the starch-storing plastids of developing pea embryos (and probably othergrain legumes, which accumulate starch, as a main storage product) arederived directly from chloroplasts and retain chloroplast-like characteris-tics throughout their development. Developing pea embryos containchloroplasts (located primarily on the outer edge of cotyledons), whichstore little or no starch (Smith et al., 1990).

The starch accumulation in developing faba bean seeds has beenreviewed by Weber et al. (1995b, 1997). During the cell-division phase of thefaba bean embryo (15–18 days after flowering; DAF) starch is mainly foundin two layers of the seed coat, in the hypodermal and chlorenchymal cellsand in the outer parenchymal cells. When storage-product synthesis beginsin the cotyledon (about 25 DAF), starch is deposited as single granules inthe cells of the adaxial region of cotyledons. Later, starch depositionspreads from the adaxial cells to the periphery. During this process thequantity and size of the starch granules increases in the cells of the adaxialregion and new granules appear in the abaxial cells. During the cell-elonga-tion phase (about 30–35 DAF), starch is also present in the axis cells. At nodevelopmental stage is starch found in the palisade cell layers of the seedcoat, the peripheral cells of the abaxial zone, or in the provascular andcalyprogenous cells.

Developing seeds of pea, faba bean and common bean accumulatestarch up to full seed maturity (Meredith et al., 1988; Lahuta et al., 1995).In seeds, which accumulate oil as reserve material (e.g. soybean), starchsynthesis occurs in the cotyledon growth phase (about 35–40 DAF) anddecreases to trace amounts during seed maturation (Monma et al., 1991).

5.2.4 Biochemistry of starch

Native starch is composed of two types of polysaccharide chains, amyloseand amylopectin (see Chemistry Chapter 2), which are formed into gran-ules found in the amyloplast. Amylose is essentially composed of linearα(1→4)-linked glucose units and has a molecular weight between 5 × 105

and 106 (Wang et al., 1998). Along the length of the α(1→4) chain there areoccasional secondary α(1→6) branches. Amylopectin is also composed ofα(1→4)-linked glucan units; the molecular weight, however, is usually inthe millions and it has substantially more α(1→6) branches than amylose,being in the region of 2–4% (Hizukuri and Takagi, 1984; Takeda et al.,1984; 1986).

The first committed step in the starch biosynthetic pathway is theconversion of glucose-1-phosphate to ADP-glucose through the actionsof ADP-glucose pyrophosphorylase (EC 2.7.7.23) in the amyloplast (seeFig 7.1 in Chapter 7, and Chapter 6). The substrate glucose-1-phosphate isderived from glucose-6-phosphate, which is imported into the amyloplast





via a glucose-6-phosphate/Pi transporter (Hill and Smith, 1991; Harrisonet al., 1998).

Glucose-1-phosphate + ATP → ADP-glucose + PPi

This reaction is effectively irreversible due to an efficient pyrophosphory-lase, found in non-photosynthetic plastids, removing the pyrophosphate(PPi; Gross and ap Rees, 1986). The resultant ADP-glucose can then beutilized as the substrate for starch synthesis. The glucose moiety is trans-ferred to the non-reducing end of an α(1→4) glucan chain by the enzymestarch synthase (EC 2.4.1.21). The α(1→6) branches are formed throughthe actions of starch branching enzyme (EC 2.4.1.28), which cleaves shortα(1→4) glucan chains of approximately 20 units and re-attaches them viaan α(1→6) linkage to the original chain or an adjacent chain (Morrisonand Karkalas, 1990). The number of branches along an α(1→4) chain isapproximately one per 20 glucan units.

5.3 Physiological Role of Carbohydrates in Legume Seeds

5.3.1 During seed development

Zygotic seedsSeeds of most plant species exhibit the ability to withstand desiccation, inmany cases achieving water contents of less than 5–10% on a fresh weightbasis. These seeds are termed orthodox and can be stored for many yearsunder dry conditions. Orthodox seeds, which include those from legumes,are not capable of withstanding desiccation at early stages of development.The ability to tolerate desiccation is acquired during later stages of seeddevelopment and is lost after germination. It is believed that the acquisitionof desiccation tolerance is developmentally controlled (Galau et al., 1991;Bewley and Oliver, 1992; Kermode, 1997). Galau and co-workers (1991)suggest that desiccation is acquired before maturation drying at the ‘postabscission stage’, when the vascular connection between the seed coat andthe parent plant is lost.

Acquisition of desiccation tolerance in maturing seeds involves severalcomponents including the accumulation of a special group of proteins(Blackman et al., 1991; Galau et al., 1991; Bewley and Black, 1994; Vertucciand Farrant, 1995), non-reducing sugars and/or galactosyl cyclitols (Kosterand Leopold, 1988; Horbowicz and Obendorf, 1994; Obendorf, 1997), freeradical scavenging systems (Senaratna et al., 1985; Koster and Leopold,1988; Lowell and Kuo, 1989; Hendry, 1993; Leprince et al., 1993; Finch-Savage et al., 1994) and abscisic acid (Bartels et al., 1988; Anandarajahand McKersie, 1990; Blackman et al., 1991; Vertucci and Farrant, 1995).The role of sugars and proteins in seed desiccation tolerance has beenextensively studied. With regard to proteins, in desiccation tolerant seed a





set of heat-stable hydrophilic proteins accumulate during late seed matura-tion. These proteins are so called late-embryogenesis-abundant, or LEA,proteins and their synthesis is controlled by ABA at the transcriptional level(Williamson and Quatrano, 1988; LePrince et al., 1990, 1993). LEA proteinsare believed to protect macromolecular structures and membranes(Blackman et al., 1991, 1995). It has also been shown that another classof stress proteins, heat shock proteins, are synthesized during seeddevelopment (Vierling, 1991). Heat shock proteins presumably stabilizeprotein conformation during tissue dehydration. Recent studies usingseeds from several species have indicated that the presence of proteinsalone is not sufficient to confer complete tolerance to desiccation.

A variety of orthodox seeds, including legumes, accumulate solublecarbohydrates mostly as sucrose and RFO. Soluble sugars account for17.2–28.8% of the dry mass in embryonic axis tissues for soybean andchickpea, respectively, and are present at 2–10 times lower concentrationsin cotyledons than in axes. There are differences in the quantities of RFOmembers in seeds from different species. For example, soybean seedsaccumulate mostly stachyose and raffinose, but only small quantities ofverbascose, whereas, verbascose is the major α-galactoside in pea and fababean seeds. In lupin seeds, the amount of stachyose is much higher thanraffinose and verbascose (Górecki et al., 1997).

Generally, legume seeds accumulate sucrose early in development,while during maturation and desiccation they accumulate raffinose,stachyose and verbascose (Dornbos and McDonald, Jr, 1986; Saravitz et al.,1987; Kuo et al., 1988; Lowell and Kuo, 1989; Horbowicz and Obendorf,1994; Lahuta et al., 1995; Frias et al., 1996b; Górecki et al., 1996, 1997).Blackman et al. (1991, 1992) found that during seed development, soybeanseeds naturally develop desiccation tolerance and that this correlates withthe loss of green colour in embryonic axis tissues, the accumulation of LEAproteins, the breakdown of starch and the accumulation of raffinoseand stachyose in the axis tissues. Immature soybean seeds are capable ofgermination, but do not tolerate rapid desiccation. When these immatureseeds are slowly dried, they develop desiccation tolerance and accumulateamounts of raffinose and stachyose in their embryonic axes that are aboutthree times higher than the values reported when axes mature naturally.When incubated at 100% relative humidity, soybean seeds do not developdesiccation tolerance and do not accumulate stachyose (Blackman et al.,1992). Taken together, these studies provide evidence for the hypothesisthat the RFO are important in conferring tolerance to the stress ofdesiccation.

Yellow lupin seeds retain desiccation tolerance through all stages ofmaturation after 30 days from flowering (Górecki et al., 1997). The acquisi-tion of desiccation tolerance and the ability to germinate is accompaniedby the accumulation of substantial quantities of sucrose and RFO, withstachyose being the predominant soluble carbohydrate (Fig. 5.5). As





maturation proceeds, the mass ratio of sucrose to α-galactosides changesfrom about 7.0 to almost 0.1 at full maturity. Other studies indicate that theRFO function in a similar way in pea, faba bean (Lahuta et al., 1995) andlentil (Piotrowicz-Cieslak et al., 1995) seeds, as in soybean and lupin seeds.

In addition to sucrose and the RFO, seeds of several species accumulategalactosyl cyclitols and small amounts of free cyclitols (Table 5.2a and b).In lupin seeds, D-pinitol, D-chiro-inositol and myo-inositol form four differentseries of galactosyl oligomers (Górecki et al., 1996): the galactinol A seriesincludes D-pinitol, galactopinitol A, ciceritol and trigalactopinitol A; thegalactopinitol B series includes D-pinitol and galactopinitol B; thefagopyritol B series includes D-chiro-inositol, fagopyritol B1 and fagopyritolB2, and the galactinol series includes myo-inositol, galactinol, anddigalactosyl myo-inositol. The accumulation of galactosyl cyclitols in lupin



Fig. 5.5. Contents of soluble sugars, cyclitols and galactosyl cyclitols in the axis ofmaturing yellow lupin seeds (Górecki et al., 1997).



seeds coincides with an increase in the RFO and the acquisition of desicca-tion tolerance, while in pea only the RFO accumulate (Fig. 5.6; Góreckiet al., 1997). Galactopinitol A, galactopinitol B and fagopyritol B1 accumu-late in the embryonic axis tissues of developing soybean seeds in associationwith desiccation tolerance and in parallel with stachyose accumulation(Obendorf, 1997; Obendorf et al., 1998). Immature soybean seeds accumu-late galactopinitols during slow drying (Obendorf et al., 1996). Galacto-pinitol A, galactopinitol B and fagopyritol B1 accumulate in parallel withstachyose in axis and cotyledon tissues during in vitro growth of embryos.Evidence, therefore, supports the suggestion that galactosyl cyclitols inseeds may enhance the physiological role of α-galactosides.

In some seeds that have very low RFO levels, galactosyl cyclitols mayreplace the role of the α-galactosides in the acquisition of desiccationtolerance. Desiccation-tolerant buckwheat seeds, for example, accumulate



Fig. 5.6. Yellow lupin andpea desiccation tolerance ofradicle (as a %) during seeddevelopment and germina-tion. The backgroundshadow indicates thepresence of raffinose familyoligosaccharides (RFO) andgalactosyl cyclitols (GAL-C)(Górecki et al., 1997, 1999;Lahuta et al., 1998).



fagopyritol (galacto-chiro-inositol) as a major soluble carbohydrate inaddition to sucrose (Obendorf et al., 1993). Also, in lucerne somaticembryos galactosyl cyclitols have been proposed to have an importantrole in desiccation tolerance (Horbowicz et al., 1995). Other seeds ofLeguminosae including lentil, kidney bean, castor bean, chickpea, pigeonpea, cowpea and subterranean clover have galactosyl cyclitols in addition tosoluble sugars (Kuo, 1992; Horbowicz and Obendorf, 1994; Obendorf,1997), but their role in desiccation tolerance has not been elucidated.For example, ciceritol is prominent in lentil seed (Frias et al., 1993) andgalactinol in castor bean (Kuo, 1992).

Somatic embryosOligosaccharides and galactosyl cyclitols seem to play an important role inthe acquisition of desiccation tolerance of somatic embryos. These are usedas synthetic or artificial seeds for the propagation of high-value plants, or asa plant breeding tool in the development of new cultivars (see Chapter 6).Somatic embryos have been obtained for more than 150 species ofimportant agricultural crops including legumes, cereals and grasses, andare genetically identical to the donor plant. Lucerne somatic embryosdeposit storage proteins and carbohydrates and acquire desiccation toler-ance (Lai and McKersie, 1994). Morphologically, however, lucerne somaticembryos do not have fully developed cotyledons and lack an endospermand testa. Also, instead of galactomannan being the endospermic carbohy-drate reserve, somatic embryos contain starch, sucrose and raffinose (Laiand McKersie, 1994; Horbowicz et al., 1995).

Unlike zygotic seeds, somatic embryos have elevated levels of sucroseand do not accumulate D-pinitol or its galactosyl derivatives (galactopinitolA, galactopinitol B, ciceritol or trigalactopinitol A). Lower levels ofstachyose accumulate during the maturation of somatic embryos. Duringdesiccation, however, stachyose increases in somatic embryos to levelssimilar to those found in mature seeds. The decrease in sucrose concentra-tion and the increase in stachyose during drying results in a decline inthe sucrose : oligosaccharide ratio. Also, reducing sugars decrease duringdesiccation of somatic embryos (Horbowicz et al., 1995). Except for the lackof pinitol and galactosyl pinitols, changes in soluble carbohydrates duringthe maturation and desiccation of lucerne somatic embryos are similar tozygotic seeds and are associated with desiccation tolerance.

Mechanism of desiccation toleranceHow soluble carbohydrates confer seed desiccation tolerance has beenthe subject of numerous recent publications (Hoekstra et al., 1989; Bruniand Leopold, 1991; Horbowicz and Obendorf, 1994; Koster et al., 1994;LePrince and Waltres-Vertucci, 1995; Vertucci and Farrant, 1995; Buitinket al., 1996; Crowe et al., 1996; Sun et al., 1996). The stabilization of mem-branes appears to be the main role of these compounds in conveying





desiccation tolerance in seed and pollen. Water replacement and glassformation hypotheses present the most interesting models as to how theymay protect cellular constituents. The first hypothesis suggests that carbo-hydrate hydroxyl groups substitute for water and provide the requiredhydrophilic interaction to stabilize membranes and proteins (Bryant andWolfe, 1992; Vertucci and Farrant, 1995). In many orthodox seeds, includ-ing legumes, a high sucrose content may provide hydrogen bondingrequired to prevent lipid phase transitions during drying (Leopoldand Vertucci, 1986; Crowe et al., 1987; Caffrey et al., 1988). Sucrose alone,however, is not sufficient for desiccation tolerance, when measured as theability to germinate after drying (Brenac et al., 1997; Obendorf, 1997).

Pure sucrose solutions when concentrated tend to crystallize, but theaddition of raffinose prevents this crystallization process (Caffrey et al.,1988; Koster, 1991). Thus, the second hypothesis assumes that the presenceof raffinose, stachyose and/or galactosyl cyclitols in seeds inhibit crystalliza-tion of sucrose during drying and enhance the formation of a stable glassystate (Koster, 1991; Sun and Leopold, 1993; Leopold et al., 1994; Vertucciand Farrant, 1995; Koster and Leopold, 1998). Aqueous glasses have beendetected in dried seed tissues (Williams and Leopold, 1989; Vertucci, 1990;Bruni and Leopold, 1991). Whereas this mechanism may apply to dryorthodox seeds, desiccation intolerant recalcitrant seeds die at a waterconcentration much higher than that required for the formation of theglass state. Soluble carbohydrates appear to be required, therefore, butalone are not sufficient to impose desiccation tolerance (Obendorf, 1997).

It can be suggested that oligosaccharides and galactosyl cyclitols areimportant for desiccation tolerance because they reduce the level of mono-saccharides, such as glucose, fructose and galactose, used as substrates fortheir synthesis (Koster and Leopold, 1988; LePrince et al., 1993). Loweringthe monosugar content results in a reduction of easily available respiratorysubstrates and, therefore, may inhibit metabolic events, especially respira-tion, which is a source of free radicals prior to drying (Vertucci and Farrant,1995). Conversely, low levels of monosaccharides may limit Maillard’sreactions, which are destructive to proteins (Wettlaufer and Leopold,1991).

Finally, sucrose, the RFO and galactosyl cyclitols may act as scavengersof free radicals, which are especially destructive when desiccation-sensitivetissues are dried (Hendry et al., 1992; LePrince et al., 1993; Vertucci andFarrant, 1995).

5.3.2 During temperature stress

It is known that the composition of the RFO in leaves is altered bytemperature (Chatterton et al., 1990; Bachmann et al., 1994). There havebeen very few studies, however, relating the effect of temperature during





plant maturation to the content and composition of the RFO and othersoluble carbohydrates in seeds. It has been shown that white lupin seedsmatured at 28°C accumulate only 53–70% as much dry matter as seedsmatured at 13°C (Górecki et al., 1996). These changes were accompaniedby only minor changes in the RFO. Pinitol and the galactose-containingpinitols, however, were more than doubled by seed maturation at 28°C, but,collectively, these compounds make up less than 10% of the total solublecarbohydrates. The effect of maturation temperature on the compositionof soluble carbohydrates in yellow lupin seeds has also been studied(Górecki et al., 1996). In this species, seeds matured at 18°C had morethan twice the amount of stachyose and verbascose compared with seedsmatured at 25°C. From these limited experiments it can be suggested thatthe RFO and galactosyl cyclitols may play some role in temperature stressresponse on maturing seeds.

5.3.3 During seed storage

In general, seeds attain their maximum viability and vigour after the finalstage of maturation and then, during storage, they gradually deteriorateuntil death. The decline of seed quality during storage is expressed firstly asa decrease in the growth rate of germinating embryonic axes (vigour) andsecondly as a loss of the ability to germinate (viability). Seed quality lossduring storage is associated with increased membrane permeability andmany distinct biochemical changes. These include lipid peroxidation,chromosome aberration and damage to DNA, changes in RNA and proteinsynthesis, reduction in respiration and changes in enzymes and reservesubstances (Bewley and Black, 1985).

It has been observed that the content of soluble carbohydrates declineswith increased storage duration (Taufel et al., 1960; Yaklich, 1985; Petruzelliand Tarano, 1989; Kataki et al., 1997; Zalewski and Lahuta, 1998). Similarly,there is a positive correlation between the decline in RFO content and thereduction of seed longevity (Bernal-Lugo and Leopold, 1992). The deple-tion of soluble sugars may result in the limited availability of respiratorysubstates for germination (Edje and Burris, 1970). Other possibilities arethat a depletion in the oligosaccharide content may reduce the protectiveeffects of sugars on the structural integrity of membranes, or may reducethe ability of the seed to maintain a glassy state, resulting in a non-crystalline liquid state of high viscosity (Bruni and Leopold, 1991).

In dry legume seeds, the soluble carbohydrates comprise mainlysucrose, together with different quantities of oligosaccharides, in particularraffinose, stachyose and verbascose. Sucrose is exceptionally effective inprotecting membrane integrity in dry systems (Crowe and Crowe, 1986) aswell as being one of the best vitrifying sugars (Green and Angell, 1986). Asmentioned above (see Section 5.3.3), raffinose and other oligosaccharides





are believed to enhance the protective effects of sucrose by preventingcrystallization. It has been suggested that the RFO, therefore, have animportant role in conferring both desiccation tolerance and seedstorability. According to Horbowicz and Obendorf (1994), seed storabilitydepends on the ratio of sucrose to oligosaccharides (the sum of raffinose,stachyose and verbascose). Seeds of species with a sucrose : oligosaccharideratio of < 1.0 have a storability half-viability period > 10 years, whereas those> 1.0 have a storability half-viability period < 10 years.

Steadman et al. (1996) studied the sugar composition of 46 tissues fromseeds of 18 species, representing three seed storage categories: orthodox,intermediate and recalcitrant. The sucrosyl-oligosaccharides, raffinose andstachyose, were observed to be lower in recalcitrant seeds compared withorthodox seeds. In general, orthodox and recalcitrant seeds had tissueswith sucrosyl-oligosaccharide : sucrose mass ratios of > 1 : 7 and 1 : 12,respectively.

The results from these studies in combination with data in theliterature (e.g. Lin and Huang, 1994) show that the ratio of sucrose tooligosaccharides in seed tissues may provide useful information on theseed storage category.

5.3.4 During germination

During seed germination the resumption of metabolism commenceswithin minutes of the introduction of water to the dry seed. The embryopasses from a dry, quiescent state into a metabolically active phase. This isaccompanied by intensive mobilization of storage reserves, a rapid increasein respiration, initiation of nucleic acid and protein synthesis, and by cellelongation and cell division.

Degradation of starchReserve starch is deposited in amyloplasts within the embryonic axis andcotyledon cells. During seed maturation the membranes of the amyloplastsappear to disintegrate, exposing the starch granules directly to the cyto-plasm of the cells (Harris, 1976; Halmer, 1985). Starch breakdown inlegume cotyledons commences shortly after imbibition, but the rate ofhydrolysis differs between species and varieties. The spatial pattern ofstarch degradation within tissues also varies (Ziegler, 1995). In Phaseoluscotyledons this process progresses from the central region, in peas fromthe outer cotyledon face inwards and in mung bean from the inner faceoutwards (Bewley and Black, 1978). Surface pores on the starch granulesmay facilitate the selective penetration of degrading enzymes, sincegranules appear to be broken down primarily from within (Harris, 1976).Since starch granules are effectively insoluble, breakdown occurs in threephases (Preiss and Levi, 1980). Firstly, the granules are reduced to large





maltodextrins, the maltodextrins are then degraded to glucose andglucose-1-phosphate by debranching and degradation enzymes. Finally, theproducts are metabolized and exported from the site of polysaccharidestorage.

The entire pathway of starch degradation has long been attributedto various combinations of activities of endo- and exo-amylases, starchdebranching enzymes, starch phosphorylase and α-glucosidase.

α-Amylases (EC 3.2.1.1; endoamylase) are the only enzymes that havebeen demonstrated to attack starch granules directly. It has been proposed,therefore, that these enzymes regulate starch granule breakdown (Chang,1982; Steup et al., 1983). It is possible that α-amylase is associated with thestarch granule (Dunn, 1974), or with plastid membranes. Saeed and Duke(1990), however, showed that in pea tissues with a reduced chlorophyllconcentration (e.g. petals, stems, senescent leaves), α-amylases are locatedmainly in the apoplast. The hydrolysis of amylose by α-amylase is biphasic(Preiss and Levi, 1980). Amylose is initially subjected to rapid fragmenta-tion into large maltodextrin chains, which later are hydrolysed more slowly.The hydrolysis of amylopectin by α-amylase is hindered, however, by thepreserve of the α(1→6) branch points (see Chapter 2).

In pea cotyledons from cv. Progress No. 9, amylase activity has beenshown to increase for at least the first 10 days of germination at 21°C.Under these conditions starch is hydrolysed at a rate of 5.3 mg day−1 perseed. Using gelatinized starch as a substrate, it has been shown that there isnearly 600 times more α-amylase activity than is necessary for the in vivorate of starch hydrolysis (Monerri et al., 1986). The enzyme is a 43.5 kDamonomer with pI 4.5 and pH activity optima of 5.5–6.5. When amylose isthe substrate glucose and maltose are the major end products, the enzymecannot attack maltodextrins with degrees of polymerization below that ofmaltotetraose (Beers and Duke, 1990). The development of α-amylase inlegume cotyledons is regulated by endogenous phytohormones, probablyby auxin (Hirasawa, 1989) and cytokinin (Locker and Ilan, 1975) andperhaps is controlled from the embryonic axis (Morohashi et al., 1989).

β-Amylase (EC 3.2.1.2; exoamylase) hydrolyses maltosyl residues fromamylose starting at the non-reducing end. In leguminous plants with starchas the main substrate, β-amylases are the main starch-degrading enzymes(Swein and Dekker, 1966). In germinating soybean seeds, where oil is themain substrate, however, β-amylase is not important in sugar metabolism(Adams et al., 1981). In pea (Chapman et al., 1972) and faba bean (Ziegler,1988), β-amylase is located outside the chloroplasts. In pea seeds, iso-enzymes of β-amylase are relatively stable at lower pH values; at pH 3.5 theisoenzymes retaining over 70% of their initial activity (Zimniak-Przybylska,1992). The β-amylase from pea epicotyl is an approximate 55–57 kDamonomer with a pI of 4.35 and a pH optimum of 6.0. The enzyme is alsounable to hydrolyse native starch grains from pea and glucans smaller thanmaltotetraose (Lizotte et al., 1990).





α-Glucosidases (EC 3.2.1.20) are exo-type carbohydrolases catalysingthe hydrolysis, or transfer, of the terminal α-D-glucosyl residues of α-D-glucosidically linked derivatives. Examples of substrates for hydrolysisinclude maltose, maltotriose, isomaltose, gelatinized starch and nativestarch grains (Sun and Henson, 1990). The ability of some sugar beetα-D-glucosidases to hydrolyse gelatinized starch has led to the suggestionthat the in vivo substrates for α-D-glucosidases may include not only malt-ose, released by the action of α-amylase and β-amylase, but starch as well(Yamasaki and Konno, 1989). Three isoforms of α-D-glucosidases have beenextracted from pea seedlings (Beers et al., 1990), of which two were mostactive under acid conditions and appeared to be apoplastic and the third,most active at about pH 7.0, was identified as a chloroplastic enzyme. In peachloroplasts (Sun et al., 1995) and developing soybean seeds (Monma et al.,1991), α-D-glucosidase is involved in transitory starch degradation. Theenzyme from pea chloroplasts is a homodimer with maximal activity at pH7.0 and maximal stability at 6.5. These properties are compatible with thediurnal oscillations of the chloroplastic stromal pH and of transitory starchaccumulation (Sun et al., 1995).

Debranching enzymes (R-enzymes) are required to hydrolyse the(1→6)-α-glucosidic bonds that constitute the branching points in amylo-pectin and remain in the limit dextrins after amylolytic or phosphorolyticattack (Beck and Ziegler, 1989). Starch phosphorylase releases glucose-1-phosphate as a product, which can be readily further metabolized. Thecotyledons of pea and soybean possess two forms of phosphorylase thatexhibit different substrate specificity. One form is most active against smallmalto-oligosaccharides and corresponds to a leaf plastid enzyme, whereasthe other can better attack larger branched substrates and resembles aleaf cytosolic isoform (Ziegler, 1995). The glucose-1-phosphate produced,following cotyledon starch degradation, is presumably converted to sucrosein the cytoplasm and in this form can be transported to the tissues of thegrowing embryonic axis. The products of amylolysis (glucose and maltose)are also probably utilized in a similar way (Bewley and Black, 1985).

Degradation of the raffinose family of oligosaccharides (RFO)The degradation of the RFO in germinating legume seeds begins duringseed imbibition and proceeds more intensively in the embryonic axis thanin the cotyledons (see also Chapter 4). The RFO in axes are lost during thefirst two days of imbibition of soybean, pea and lupin seeds, whereas, incotyledons RFO hydrolysis is prolonged for 4–6 days (Koster and Leopold,1988; Górecki and Obendorf, 1997; Górecki et al., 1997; Lahuta et al., 1998).Verbascose, stachyose and raffinose are degraded progressively, while thelevel of monosaccharides increases gradually as germination progresses.

The hydrolysis of α(1→6) glycoside bonds between galactosidemoieties of raffinose-type oligosaccharides, cell wall polysaccharides andstorage glycoproteins is catalysed by α-D-galactosidase (EC 3.2.1.22).





Mature seeds usually contain isoforms of this enzyme that differ in activityand molecular mass (Pridham and Dey, 1974).

In developing seeds, the activity of α-D-galactosidase increases duringthe period of intensive RFO synthesis, reaching its highest level at fullmaturity. This increase may result from the structural transformation ofisoenzymes, which leads to changes in their specific activities (Pridham andDey, 1974). In the maturing embryo, the synthesis of the oligosaccharidesand α-D-galactosidase probably occur in different cellular compartments.In pea cotyledons, α-D-galactosidase has been localized in cell vacuoles thatare storing the lectin precursors (Harley and Beavers, 1989). In the cells ofsoybean cotyledons, α-D-galactosidase occurs in cisterns of the Golgiapparatus and it may be deposited in protein bodies (Hermann andShannon, 1985). A similar observation has been made in faba bean (Dattaet al., 1985). A direct role has been established for α-D-galactosidase andα-D-mannosidase in the hydrolysis of glycoproteins and storage galactosidesin the cotyledons of germinating narrow-leafed lupin (Plant, 1984). In yel-low lupin seeds the activity of α-D-galactosidase increases only at the begin-ning of germination (Login et al., 1995), when the accumulated RFO andgalactosyl cyclitols undergo complete decomposition (Górecki et al., 1997).

While the role of α-D-galactosidase in the hydrolysis of saccharides andglycoproteins in germinating seeds is understandable, the role, if any, ofthis enzyme in non-germinating (stored) seeds remains to be elucidated.The seeds of various species appear to use the RFO as part of their storagematerial and it has been observed that the oligosaccharide contentdecreases with increased storage duration (Taufel et al., 1960; Yaklich, 1985;Bernal-Lugo and Leopold, 1992; Horbowicz and Obendorf, 1994).Similarly, there is a positive correlation between the decline in RFOcontent and the depression of seed longevity.

The loss of desiccation toleranceDuring germination seeds undergo a transition from a desiccation tolerantto a desiccation-intolerant state. Generally, after radicle protrusion seedsrapidly lose desiccation tolerance. Koster and Leopold (1998) studied therelationship between soluble sugar content and the loss of desiccationtolerance in the axes of germinating pea, soybean and corn seeds. The lossof desiccation tolerance during imbibition was monitored by measuringthe ability of seeds to germinate after desiccation, following various periodsof pre-imbibition and by measuring the rate of electrolyte leakage fromdry and rehydrated axes. The soluble carbohydrate contents of axesthroughout the transition from desiccation tolerance to intolerance wereanalysed. The results showed that sucrose and the larger oligosaccharideswere consistently present during the tolerant stage and that desiccationtolerance disappeared as the oligosaccharides were lost.

The relationship between the loss of seedling desiccation tolerance andthe content of soluble carbohydrates in legume seeds has also been studied





by Górecki et al. (1997) and Górecki and Obendorf (1997). In these studiesit was found that radicle tissues are often more sensitive to desiccation thanhypocotyls. Pea root tissues lost desiccation tolerance during the first 36 hof germination, while 80% of epicotyls survived slow drying treatmentand 40% survived fast drying treatment of seedlings, for up to 96 h afterimbibition (Fig. 5.7). During desiccation, sucrose levels increased five-to tenfold in root and hypocotyl tissues and even more in epicotyls.Glucose and fructose increased during germination and remained elevatedafter drying. These changes in saccharides reflected the mobilization of



Fig. 5.7. Pea seedling germination (A) and length (B) of axis, radicle and epicotylas a function of hours after imbibition. Desiccation tolerance (C, E) and length(D, F) of radicle and epicotyl at 6 days after rehydration of (C, E) fast-dried or (D, F)slow-dried seedlings as a function of seedlings as a function of seedling age whendried (Górecki and Obendorf, 1997).



oligosaccharides. Drying resulted in a small increase of raffinose in hypo-cotyls and radicles, but this was not enough to protect seedlings from desic-cation damage (Górecki and Obendorf, 1997).

In contrast to pea, soybean and yellow lupin seedlings lost desiccationtolerance within 36 h during germination. This change in desiccationtolerance was associated with the loss of raffinose, stachyose and galactosylcyclitols and an increase in reducing sugars and free cyclitols (Górecki et al.,1997, Górecki and Obendorf, 1997). It can be suggested, therefore, thatsucrose, the RFO and galactosyl cyclitols are not prerequisite for desicca-tion tolerance of pea, soybean and lupin seedlings. On the other hand, theaccumulation of reducing sugars, mainly glucose and fructose, during seedgermination could have a deleterious effect on the seedling by inducingdesiccation injury (Hoekstra et al., 1994).





BiotechnologyN. Kuchuk et al.6

6BiotechnologyEditor: Nickolay Kuchuk

Contributors: Miroslav Griga, GeorginaKosturkova, Nickolay Kuchuk and MladenkaIlieva-Stoilova

And he gave it for his opinion, that whoever could make two ears of cornor two blades of grass to grow upon a spot of ground where only onegrew before, would deserve better of mankind, and do more essentialservice to his country than the whole race of politicians put together.

Gulliver’s Travels, ‘A Voyage to Brobdingnag’, ch. 7 (1726)Jonathan Swift (1667–1745), Anglo-Irish poet and satirist

6.1 Introduction

This chapter concentrates on the biotechnological techniques developedin the fields of plant cell tissue culture and genetic engineering.

The cultivation methods for explants and single cells (protoplasts) andfor plant regeneration in vitro are described as basic approaches that allowvaluable genotypes to be propagated as well as to produce fertile plantsfrom somatic cells. In vitro somaclonal variation and cell selection isconsidered as a new source of diversity for plant breeding. Methods forgenetic transformation and for the production of transgenic grain legumesare summarized to give an idea about ‘the state of art’ of this technology.Hopefully, this information will promote a better understanding of thecurrent opportunities and future prospects of plant biotechnology, as wellas the possibilities for the future manipulation of carbohydrate metabolism,content and composition in grain legume seeds.





6.2 In vitro Cultures and Plant Regeneration of GrainLegumes

6.2.1 Introduction to in vitro culture

The ability of plant cells to express their entire genetic information andregenerate whole plants (i.e. totipotence), is the basis of their developmentin controlled in vitro conditions and to the establishment of biotechnologi-cal techniques and methods for genetic manipulation.

In vitro plant cells from excised tissues (explants) might undergo de-differentiation and re-differentiation following several developmental path-ways, depending on the culture environment. Unorganized cell divisionand growth can be stimulated, leading to the formation of callus andsuspension cultures. Morphogenesis can be induced in meristem, callus,suspension and protoplast cultures, leading to the formation of somaticembryos, organs (shoots and roots) and a whole plant.

Undifferentiated cell growth in callus or suspension cultures can beadequate for the purposes of some biotechnology processes (like secondarymetabolite production, and some investigations in physiology, genetics,cytology, biochemistry, etc.; see Section 6.6). Regeneration of plants, how-ever, is essential for the application of recent advances in biotechnology,especially those concerning genetic engineering for plant improvement.Realization of regeneration capacity in vitro depends on knowledge ofthe requirements for stimulation of the morphogenic response (Halperin,1986). Unfortunately, information on genetic, epigenetic and physiologicalstatus of the explant is still limited and in practice the general approach isto find out the most appropriate chemical or physical stimuli to provoketotipotency of the cell. Up to now, this process has been mainly empiricaland the formulation of strict rules and general protocols has not beenpossible.

The establishment of in vitro cultures and the induction of morpho-genesis in grain legumes has proved more difficult in comparison withmost Solanaceae and Brassicaceae species. For a long time recalcitrancein regeneration has been the largest obstacle for genetic manipulation.Several important observations have led to the development of efficientregeneration systems. These have focused on the role of the genotype,the explant, the application of relatively high auxin concentration forinduction of somatic embryogenesis and the use of powerful cytokininsfor multiple shoot proliferation. Definite success has been achieved in theregeneration of soybean and pea and this success has been immediatelyapplied to genetic manipulation (Barwale and Widholm, 1990; Griga andNovák, 1990; Christou, 1992; see Sections 6.2 and 6.4). The developmentof efficient in vitro methods and plant regeneration protocols for otherlarge-seeded legumes has made significant progress in the last two decadesand organogenesis/somatic embryogenesis has been reported at least

146 N. Kuchuk et al.




Biotechnology 147


Species References

(A) Development through direct and indirect embryogenesisArachis hypogeae Gill and Saxena (1992); Eapen and George (1993a); Baker

et al. (1995); Chengalrayan et al. (1997); Murch and Saxena(1997)

Cicer arietinum Sagara et al. (1993); Barna and Wakhlu (1993, 1995); Islam(1994); Suhasini et al. (1994); Eapen and George (1994);Dinehskumar et al. (1995); Murthy et al. (1996)

Glycine max Barwale and Widholm (1990); Parrott et al. (1992);Komatsuda (1992); Griga et al. (1992); Griga (1993);Trijatmiko and Harjosudarmo (1996); Gai and Guo (1997);Rajasekaran and Pellow (1997)

Lupinus albus Rybczynski and Podyma (1993a)Phaseolus vulgaris Malik and Saxena (1992a); Xu and Yang (1993)Pisum sativum Kysely et al. (1987); Kysely and Jacobsen (1990); Tetu et al.

(1990); Stejskal and Griga (1992); Flandre andSangwan-Norreel (1995); Loiseau (1995); Griga and Slama(1997); Jarkova et al. (1998)

Vicia faba Taha and Francis (1990); Griga et al. (1992); Xu-Zheguyaoand Yang Caiyum (1993); Griga and Klenoticova (1994)

(B) Development through direct and indirect organogenesisA. hypogeae McKently et al. (1990); Eapen and George (1993b); Kanyand

et al. (1994); Venkatachalam and Jayabalan (1997);Ponsamuel et al. (1998)

C. arietinum Altaf and Ahmad (1990); Malik and Saxena (1992b); Islamet al. (1993); Barna and Wakhlu (1994); Fernandez-Romeroet al. (1995); Murthy et al. (1996); George and Eapen (1997)

G. max Barwale and Widholm (1990); Parrott et al. (1992), Shettyet al. (1992); Nawracal et al. (1996); Kaneda et al. (1997)

Lens culinaris Malik and Saxena (1992); Warkentin and McHughen (1993);Halbach et al. (1998)

L. albus Sator (1990); Harzic et al. (1998)P. vulgaris Franklin et al. (1991); Malik and Saxena (1991, 1992c);

Mohamed et al. (1993); Zhang et al. (1997)P. sativum Kallak and Koiveer (1990); Tetu et al. (1990); Nielsen et al.

(1991); Malik and Saxena (1992); Ozcan et al. (1992);Sanago et al. (1996); Kosturkova et al. (1997); Jarkova et al.(1998); Ocatt et al. (1998)

V. faba Ramsay and Middlefell-Williams (1992); Griga andKlenoticova (1994); Fernandez-Romero et al. (1998)

(C) Multiple shoot formation from pre-existing meristems in the explantA. hypogeae Heatley and Smith (1996); Venkatachalam and Jayabalan

(1997)

Continued

Table 6.1. Morphogenic responses of grain legumes cultured in vitro.



in one genotype/cultivar of all economically important legume crops(Christou, 1997; Griga, 1999).

At present, more than 20 research groups are working on the in vitroculture of at least 30 species of grain legumes (Parrott et al., 1992). The invitro procedures for these species, were developed during a period of morethan 20 years to satisfy different goals and the extent to which they havebeen characterized is variable. Also, they follow the historical developmentof in vitro culture methods, utilizing the achievements of the day. Althougha perfect system cannot be offered, the available information gives thepossibility to choose the most appropriate protocol for a particularinvestigation, to adapt a scheme or to successfully develop a new one.

6.2.2 Plant regeneration systems

Different responses and developmental pathways have been observedin vitro in pea and other grain legumes (Table 6.1), depending on internaland external plant factors. Regeneration of plants occurs via organogenesisand/or embryogenesis, either directly from the excised tissue, or indirectlyafter formation of callus. Distinguishing between these processes is impor-tant for making the right choice of scheme to be applied. In organogenesis,a group of cells forms bud and root primordia with subsequent develop-ment into a leafy vegetative shoot and root, respectively. In somatic embryo-genesis, a new individual with a bipolar structure (i.e. a rudimentaryplant with a root/shoot axis) arises from a single cell (Brown, 1986).Regeneration of plants can be obtained by shoot proliferation and bymicropropagation from pre-existing meristems. This chapter will focus onthe development of regeneration systems in pea (Pisum sativum) as themost extensively grown grain legume in Europe.



Species References

C. arietinum Altaf and Ahmad (1990); Malik and Saxena (1992b); Brandtand Hess (1994); Polisetty et al. (1997); Santangelo et al.(1997)

G. max Parrott et al. (1992); Sharma and Kothari (1994); Kanedaet al. (1997)

L. culinaris Malik and Saxena (1992b); Polanco and Ruiz (1997)L. albus Sator (1990); Rybczynski and Podyma (1993b)P. vulgaris Mohamed et al. (1992); Herselman and Mienie (1995)P. sativum Jackson and Hobbs (1990); Kosturkova et al. (1997)V. faba Mohamed et al. (1992)

Table 6.1. Continued



6.2.3 Pioneering studies on pea regeneration

In the early 1970s, Gamborg et al. (1974) and Kartha et al. (1974) firstreported shoot regeneration from callus tissue and from apical meristem,respectively. Initial attempts to induce organogenesis in callus culturesstudied the response of different plant tissues, genotypes and media com-position. Gamborg et al. (1974) first induced shoot formation in callus frommacerated apical meristems grown on media containing 1 µM naphthaleneacetic acid (NAA) and 0.2–5.0 µM 6-benzyl amino purine (BA), the latterbeing important for vigorous shoot formation. Shoots were formed de novoexogenously on the callus. While most of the calli produced one or moreshoots, root formation was poor and did not occur regularly.

Malmberg (1979) used epicotyl sections to obtain callus on MSmedia (Murashige and Skoog, 1962) supplemented with BA and NAA. Heobserved that organogenic ability was genotype dependent (six out of 16lines responded) and decreased with prolonged culture. Root formationwas induced by NAA but, as previously reported, it was not satisfactory.

Atanassov and Mehandjiev (1979) observed that the developmentalstage of the explant influenced the efficiency of callogenesis, the organo-genic response being observed only for 20–22-day-old embryos. Budformation was stimulated by 0.5 mg l−1 BA and could be maintainedfor eight subcultures, but rooting of the regenerants was problematic.Cytological analysis showed that 13% of the newly formed shoots hadmixoploid cells at the vegetative apex.

An effective system of de novo regeneration from callus derived fromimmature leaflets (0.9–1.8 mm) was developed by Mroginski and Kartha(1981). The combination of BAP (10 µM) and NAA (10 µM) was the bestfor callus induction and subsequent shoot regeneration.

Hussey and Gunn (1984) succeeded in obtaining vigorously growncallus with superficial meristems, using plumules that continuously regen-erated shoots over a period of nearly 3 years. Callogenesis was induced onMS medium supplemented with 1 mg l−1 BA and 4–8 mg l−1 indole-3-aceticacid (IAA), and was maintained by reducing indole-3-butyric acid (IBA) to0.25 mg l−1. The in vitro response differed between genotypes and only two(cvs. Puget and Upton) out of five varieties maintained regenerative callus.During the first year of callus growth the plants were diploid and mostlymorphologically normal. Many of the shoots regenerated after 2 years,however, showed considerable morphological variation and difficulties inrooting.

Natali and Cavalini (1987a,b, 1989) examined some factors affectingrhizogenesis in cultures obtained from macerated vegetative apices orimmature embryos. The highest frequency of rooting was achieved athalf strength MS medium supplemented with 2 mg l−1 IBA. Difficultiesin rooting seemed to affect only shoots regenerated from callus, but notformed from meristematic tissues. Grafting of regenerated plantlets on

Biotechnology 149




to rootstocks of the same cultivar was used to overcome these rootingdifficulties.

6.2.4 Regeneration via somatic embryogenesis

The establishment of systems for the induction of somatic embryoformation from callus (indirect embryogenesis) or explant tissue (directembryogenesis) have led to new advances in pea regeneration. Kysely et al.(1987) reported the first regeneration of a whole plant via indirect somaticembryogenesis from immature zygotic embryos and from the youngestnode of shoot apex segments. A key factor for the induction of somaticembryos was the presence of an auxin (picloram or 2,4-dichlorophenoxyacetic acid; 2,4-D). Initially the embryogenic response and rate were low, nomore than 15% with two somatic embryos per responding zygotic embryo,but this was improved after specific factors affecting efficiency were defined(Kysely and Jacobsen, 1990). For the best results embryogenic callus,exclusively originating from embryonic axis tissue, developed somaticembryos on MS media supplemented with 0.2 and 4.0 µM picloram or4.0 µM 2,4-D. In embryogenic cultures from shoot apices 5.0 µM BAstimulated both the development of young somatic embryos and theappearance of new ones. Somatic embryogenesis depended on embryosize (optimal 3–6 mm) and genotype, embryogenic response varying from2 to 31% among the five genotypes tested. It was influenced also by theauxin concentration. The authors concluded that an optimal embryoinduction medium, with regard to auxin type and concentration, has to bedetermined empirically for each pea genotype. The same laboratory(Lehminger-Mertens and Jacobsen, 1989b) first reported pea somaticembryogenesis from protoplast-derived callus.

Stejskal and Griga (1992) used 46 lines of Pisum to study the effect ofgenotype on somatic embryogenesis from immature zygotic embryos.Only one genotype (line HM-6) exhibited good embryogenic competencereaching a mean frequency of embryogenic explants of 14.6%, with oneto six somatic embryos per explant (induction medium with 2,4-D). Inaddition, these authors supported earlier work by Kysely et al. (1987)regarding the development and germination of somatic embryos followingtheir transfer into a medium containing cytokinins (0.15 µM BA, 0.15 µMkinetin, 0.15 µM zeatin) and an auxin (0.15 µM NAA).

In most of the previous studies somatic embryogenesis was indirect,involving an intermediate callus phase. Tetu et al. (1990) first observed incertain genotypes the formation of somatic embryos and buds directly fromthe cotyledonary surface of immature zygotic embryos (3–6 mm in size).Embryogenesis was achieved when explants were cultured for 4–5 weeksin darkness on MS medium containing 43 µM NAA enriched with 15 µMthiamin hydrochloride, 40 µM nicotinic acid and 60 µM arginine. Somatic





embryos developed into plantlets when transferred to germinationmedium containing MS salts, 0.01 M KNO3, 15 µM IBA and 2.2 µM BA.Rooting was possible on MS medium supplemented with 16 µM NAA and13.3 µM BA. An embryogenic response was observed in six out of ninegenotypes and varied from 2 to 48%.

Further data about direct pea somatic embryogenesis from immaturecotyledons or shoot apical meristems were reported by Nadolska-Orczyket al. (1994), Loiseau et al. (1995) and Griga (1998). Recently, the mostefficient and quick protocol for pea somatic embryogenesis has been shownto be by direct regeneration from shoot apical meristems. This was success-fully tested using more than 50 garden pea and field pea genotypes as wellas in wild pea forms (Griga, 1998; Griga, unpublished data). For moredetails connected with pea somatic embryogenesis see Griga (1998, 1999).

6.2.5 Regeneration via organogenesis and multiple shoot formation

There are two organogenic pathways, multiple bud development frompre-existing meristems and de novo shoot bud regeneration after de-differentiation of existing cells. Griga et al. (1986) used a combinationof BA (10 µM) and NAA (0.1 µM) to induce abundant multiple shootformation from shoot apices axillary buds of the first normal leaf, andaxillary buds of the first and second primary scale leaves. Tetu et al. (1990)observed axillary bud initiation from the main meristem and de novo shootbud regeneration from cotyledons of whole zygotic immature embryos.The processes depended on BA (13.3 µM), to induce axillary budformation, and the addition of NAA (16 µM) for axillary shoot formation.When 2,3,5-triiodobenzoic acid (TIBA) (0.2 µM) was added to the NAA–BA-supplemented MS medium bud proliferation was increased, both fromapical meristematic areas and from cotyledons. The results indicated thatthe above-described morphogenesis in pea depends on three major factors:the explant size, the cultivar/genotype and the nutritive media.

Direct organogenesis has been promoted in meristems from shoot tips,axillary buds of primary scale leaves and axillary buds from cotyledons(Kallak and Koiveer, 1990); 80–90% of the explants initiated growth, butfurther development and normal morphology were poor. Shoot regenera-tion was most effective in cotyledonary axillary meristems.

Cotyledonary nodes were suitable explants for Jackson and Hobbs(1990) to develop a scheme for rapid multiple shoot production, whichthey suggested could be applied to a wide range of important pea varieties.For efficient multiple shoot production it was essential to culture the cotyle-donary node explants on MS medium containing 1 mg l−1 BA, to removethe axillary bud region and to remove the initially developed shoot. Thisgave 100% response and up to ten buds per explant in all genotypes. Afterremoval of shoots bigger than 1 cm, the remaining tissue could maintain

Biotechnology 151




organogenesis for several months on MS basal medium with B5 vitamins,supplemented with various concentrations of BA or kinetin. The bestcompromise between the quality and number of buds was achieved using1–5 mg l−1 BA. Rhizogenesis was induced at a frequency of 90% using NAA(0.186 mg l−1).

Nielsen et al. (1991) studied the effect of different auxins by applyingsequential auxin–cytokinin treatment on hypocotyl discs. Explants wereplaced for 2 days on basal K3 medium supplemented with 10 mg l−1 IAA.Transfer to a medium containing 5 mg l−1 zeatin resulted in shoot forma-tion with a frequency of more than 50% in all five cultivars tested. Doublingthe IAA concentration from 5 to 10 mg l−1 increased shooting by a factor oftwo. The effect of IAA and NAA were similar, while 2,4-D treated explantsproduced callus and no shoots. This observation confirmed the persistenceof 2,4-D and supported the assumption that added auxin has to be meta-bolizable to allow shoot formation.

Ozcan et al. (1992) studied factors affecting organogenesis via callus(indirect organogenesis) using explants from various organs at differentdevelopmental stages. Root, epicotyl and shoot tips formed only callus,while 25% of the leaflet explants regenerated shoots at a low frequency. Incontrast, they found rapid and prolific shoot development from immaturecotyledons, following an initial callus growth, on MS medium containing0.5 mg l−1 BA and 4 mg l−1 NAA. The orientation of the cotyledonaryexplants to the medium surface appeared important. The highest regenera-tion frequency was achieved when the distal end was placed on to the agar,suggesting a polar phenomenon affecting morphogenesis. In addition,parts of the cotyledon had different regeneration potential, with the high-est being for sections proximal to the embryonic axis. The developmentalstage also had a crucial effect, with most shoots being produced byfully developed green cotyledons, prior to the shift to yellow maturity.Adventitious shoots developed in a range of media supplemented with BA(0.25, 1, 2 and 4 mg l−1) and NAA (0.25, 1 and 8 mg l−1) or IBA (0.25, 1, 4and 8 mg l−1), with NAA being superior to IBA. BA alone also stimulatedshoot development, but in this case a few shoots could become dominant,inhibiting the elongation of the others. Elongation was stimulated byAgNO3. The authors consider this system to be very suitable for somaclonalvariation and transformation experiments.

The role of phytoregulators is another important regeneration factor.A novel procedure has been developed to initiate shoot regeneration fromintact seedlings produced from mature seeds germinated on a mediumcontaining cytokinin or cytokinin-like substances (kinetin, zeatin and TDZ;Malik and Saxena, 1992c). TDZ, a substituted phenylurea with cytokinin-like activity, commonly used as a cotton defoliant, was found to be mosteffective. Pea seedlings exhibited a unique pattern of shoot formation,which was accomplished in two distinct phases. Multiple shoots developedwithin a week from the nodal and basal regions of the primary epicotyl, in a





medium containing 5–50 µM TDZ. When these seedlings were exposed fora prolonged time (3–4 weeks) to the same medium, numerous shootsemerged de novo from the base, or from the upper part, of multiple shoots.Bohmer et al. (1995) developed a protocol for high frequency shootinduction and plant regeneration from protoplast derived pea callus, usingTDZ as the key factor in the system.

6.2.6 Recent studies to produce more efficient, fast and reliable systemsfor regeneration

Recently, efforts have been focused on optimizing the systems andextending the knowledge of factors effecting regeneration in grainlegumes. Using TDZ, Sanago et al. (1996) developed a simple and rapidregeneration procedure. An average of up to 20 shoots formed from eachhypocotyl explant cultured on MS medium supplemented with 0.5 or1.0 µM TDZ. Shoots (0.5–1.0 cm), detached from the parental tissue, werecultured on MS basal medium with B5 vitamins and 3.0 µM GA3 to facilitateelongation. Formation of roots was high (50–60%) on medium containingeither 2.0 µM NAA or 1.0–2.0 µM IBA, and seeds were harvested fromregenerated plants after only 9–11 weeks.

Ochatt et al. (1998), however, reported that with TDZ, or zeatin, largenumbers of buds were produced that were miniaturized, hyperhydric andhad impaired rootability, plus reduced flowering and fruiting. To studylater effects of growth regulators, these authors used hypocotyl segments,without pre-existing meristems, to induce embryogenesis and organogene-sis on modified MS media supplemented with different phytohormones.2,4-D and picloram induced somatic embryos up to the cotyledonarystage, but they mostly lacked a root or shoot meristem and were too weakto germinate. Callogenesis was more reliable than embryogenesis. Thebest regeneration responses were obtained using 3 or 5 mg l−1 BA and0.01–0.5 mg l−1 NAA, harvesting shoots several times from each explant.Rooting, flowering and fruiting were also better using this hormonalregime compared with other phytoregulators. The authors claim that thiswas the first report where hormones used for bud regeneration and rootingcould be correlated with the subsequent flowering and fruiting of theregenerants.

Stimulation of organogenesis has been observed for other cytokinin-like substances not used traditionally in in vitro cultures (Kosturkovaand Tineva, 1998). Looking for more powerful phytoregulators, however,is only one of the approaches for optimizing conditions for efficientregeneration. Screening for appropriate genotypes able to realize theirmorphogenic potential is another option. By comparing two differentsystems, a more pronounced genotypic effect was observed when indirect,rather than direct, organogenesis was promoted (Kosturkova et al., 1997),

Biotechnology 153




suggesting the involvement of both epigenetic and culture conditions inthe realization of morphogenic potential.

A 100% regeneration efficiency for seven out of ten genotypes testedwas achieved from immature embryonic axes cultured on modified MSmedium containing 10 µM BA and 1 µM NAA (Kosturkova et al., 1997).When the alternative scheme of callus induction with 0.2 µM 2,4-D wasused, only three genotypes responded with bud formation from all explantstransferred to media supplemented with 5 µM BA. Genotypic differencesoccurred in bud initiation and multiple shoot formation of cultures main-tained for several months on media containing 0.5 µM BA and 0.25 µMNAA. Such a prolonged culture with vigorous shoot formation is suitable tostudy the effect of biotic and abiotic stress resistance and to perform cellselection in vitro.

Considerable variation for the frequency of callus formation andfor the onset of regeneration was observed by Jarkova et al. (1998). Among26 genotypes tested, 12 had a morphogenic response from immaturecotyledons on one or both media for embryogenesis or organogenesis.The majority of the explants were characterized by 100% callus formation.Significant differences in embryogenic potential were observed between allsamples. Also, there was wide variation between the samples for the onsettime of morphogenesis, which appears to be very important for normalshoot regeneration and to avoid abnormalities, especially cytogenicinstability (i.e. geotropic reaction, albinism, reduced leaves and internodalinterval).

6.2.7 Factors effecting regeneration

It is obvious that the development of pea in vitro is not a uni-directionalprocess and that development can be manipulated to induce callogenesis,organogenesis or embryogenesis. These processes, however, are influencedby various factors. Recently, considerable information has been obtainedabout the factors affecting the processes of morphogenesis in grainlegumes, which has contributed a great deal to recent success. The mostimportant factors are the explant, growth regulators and genotype.

ExplantAmong the various tissues used as initial material for in vitro cultures itseems that whole or parts of (cotyledons or embryonic axes) immaturezygotic embryos are preferable as embryogenic and organogenic explants.Cotyledonary nodes from seedlings and meristematic tissues are suitablematerial for the induction of adventitious bud formation and micro-propagation, while young leaflets, epicotyl and hypocotyl have been usedless. The developmental stage of the explants is very important as it can





determine the pattern of response, the most efficient being immatureembryos before full maturation and cotyledons of 3–6 mm in size.

Growth regulatorsAuxins and cytokinins, or substances with a similar structure generallyregulate in vitro development and plant regeneration. Choosing the rightgrowth regulators and the correct concentration seems to depend on theexplant, its developmental stage and on the genotype, leading to a varietyof regeneration protocols. Some general observations, however, can bemade. The presence of a high concentration of auxin is essential forsomatic embryogenesis, but the type of auxin can differ: 2,4-D and piclorambeing cited as superior; NAA is less efficient for embryo induction, butis necessary for embryo conversion; NAA and IAA, depending on the con-centration, can induce callogenesis or rhizogenesis. BA alone, or in combi-nation with an auxin, has been the most commonly used cytokinin forinduction of organogenesis and shoot proliferation. Zeatin is very efficientin shoot induction, but TDZ seems to be superior as well as being efficientin the embryo-conversion process (Griga, 1998).

GenotypeRecalcitrance in grain legumes could be the result of a long history ofinbreeding and selection, leading to a reduction in genetic variability.Screening a large number of genotypes could help to discover those witha better response to organogenesis and/or embryogenesis. A correlationbetween embryogenic and organogenic capacity in different respondingcultivars, however, is not always observed. With regard to rooting frequency,the data are also contradictory. There are reports, however, that theseprocesses may be under genetic control (Althers et al., 1993; Bencheikh andGallais, 1996).

6.2.8 Advantages of the different developmental pathways for in vitromanipulation

The range of regeneration systems available allows the most appropriatesystem to be chosen for a particular purpose. Meristem cultures can be usedfor the preservation of germplasm, the production of virus-free plantsand for micropropagation. Somatic embryos can be used for artificial seedproduction. Since somatic embryos are produced from a single cell theywould be the preferred target for genetic manipulation, but embryogeniccultures are more difficult to obtain and sustain. Organogenic cultures are,therefore, more important for somaclonal variation, in vitro selection andtransformation. With regard to Agrobacterium transformation, it has beensuggested (Parrot et al., 1992) that organogenesis de novo is necessary, while

Biotechnology 155




for particle bombardment, transformation proliferation from meristemsalso can be used.

6.3 Isolated Protoplasts from Grain Legumes

6.3.1 Introduction to protoplast cultures

DefinitionThe term protoplast refers to all components of a plant cell excluding thecell wall. The plant cell wall consists of three primary components, cellulose(25–50%), hemicellulose (average 50%) and pectin substances (about5%). Cocking (1960) first used hydrolytic enzymes for digesting the cellwall of tomato root tips to release plant protoplasts. This method allows thequick isolation of an indefinite number of uniform plant protoplasts fromany type of plant tissue from any plant species.

General procedures for the isolation and cultivation of plant protoplastsTo isolate protoplasts the tissue is incubated with digestive enzymes(cellulases, hemicellulases and pectinases) for 1–16 h. Protoplasts arewashed and resuspended, at an appropriate density (103–106 protoplastsml−1), in liquid or on solidified culture medium. They can regenerate a newcell wall within 24–48 h, undergo their first mitotic division between thesecond and tenth day of culture and then form colonies that grow intocallus tissue. This callus can generate plants by the induction of embryo-genesis or organogenesis. More rarely, somatic embryos can be obtaineddirectly from protoplasts or from protoplast-derived colonies. Each stepconsists of several parts, all of which seem to be important and crucial forthe successful protocol (Evans and Bravo, 1984; Eriksson, 1985; Power andChapman, 1985; Binding, 1986).

The complexity of the work makes protoplast cultures more difficult tohandle than meristematic, callus and cell suspension cultures, but theabsence of a hard cellulose cell wall gives plant protoplasts some advantagescompared with the other in vitro cultures (Fowke and Wang, 1992;Paszkowski et al., 1992; Kosturkova, 1993).

Advantages of isolated plant protoplastsThese can be listed as follows:

• The plasmalemma is accessible, allowing unique studies of membranetransport, cell wall biosynthesis, cell growth and differentiation, andother processes in cell biology.

• Each protoplast serves as a single organism and so a population ofseveral million plant cells can be manipulated, allowing events thatoccur at very low frequency (10−6–10−7) to be monitored.





• The regenerated plant has a single-cell origin. This is important formutagenesis and selection in vitro, since the regeneration of chimericplants is assumed to be less likely.

• Protoplasts can be fused from plants belonging to different speciesand other taxonomic units, giving rise to somatic hybrids and cybridscombining various nuclei and cytoplasmic genetic material.

• They allow the isolation and transfer of organelles and singlechromosomes from one cell to another, achieving new combinations ofmitochondria, chloroplasts, vacuoles and nuclei.

• They can undertake direct DNA uptake, which allows the rapid detec-tion of gene expression and genetic transformation in cases whereother methods like Agrobacterium or particle bombardment are notapplicable.

6.3.2 Protoplast cultures from leguminous species

Interest towards protoplasts from leguminous species dates from the early1970s. Most investigations were carried out on soybean and pea, which arethe most important grain legume species. Some of the achievementsare presented in Table 6.2. Grain legumes proved to be recalcitrant,however, which has made success in regenerating plants from protoplastsdifficult. Different sources for producing protoplasts (cell suspension,leaf mesophyll, hypocotyl, epicotyl, etc.) and various culture conditions

Biotechnology 157


In vitro response References

PeaCell division Landgren (1976); Jia (1982)Callogenesis Constabel et al. (1973); Gamborg et al. (1975); von Arnold

and Eriksson (1976): Kuchuk (1989)Embryogenesis,organogenesis

Puonti-Kaerlas and Eriksson (1988); Lehminger-Mertens andJacobsen (1989a); Ochatt et al. (1998)

Plant regeneration Lehminger-Mertens and Jacobsen (1989b); Boehmer et al.(1995); Sanago et al. (1996), Ochatt et al. (1998)

SoybeanCell division Kao et al. (1970); Gamborg et al. (1983); Lu et al. (1983);

Tricoli et al. (1986); Hammat and Davey (1988)Callogenesis Zieg and Outka (1980); Xu et al. (1982); Oelck et al. (1983);

Kuchuck (1989)Plant regeneration Myers et al. (1989); Guo (1991); Dhir et al. (1991, 1992); Lu

et al. (1993)

Table 6.2. Some achievements in the development of isolated pea (Pisumsativum) and soybean (Glycine max) protoplasts to regeneration of whole plants.



(composition of the media, osmotic, phytoregulators, agarose type, etc.)have been examined. Until 1988, callus cultures were obtained fromprotoplasts, but the regeneration of plants was not achieved. Successfulshoot formation from pea protoplasts was first reported by Puonti-Kaerlasand Eriksson (1988; Table 6.2) and the first regeneration of plants byLehminger-Mertens and Jacobsen (1989b; Table 6.2). At about this time,soybean also was regenerated (Dhir et al., 1991; Table 6.2), althoughplants from wild Glycine species had been regenerated several years earlier(Hammat et al., 1987).

6.3.3 Application of grain legumes protoplasts to the study ofcarbohydrates

Plant protoplasts lack cell walls and are, therefore, a good experimental sys-tem for various types of study, such as genetics (using somatic hybridizationand genetic transformation), physiology, cytology, biochemistry and otherfields of biological science. In particular, they are an excellent experi-mental system for basic studies of cell wall regeneration, cell division,membrane fusion, membrane transport, virology, endocytosis and transferof organelles (Fowke and Constabel, 1985; Fowke and Wang, 1992). Thesections below cover the role of carbohydrates in different cell processesof grain legume protoplasts and present possibilities on how protoplastsystems may be exploited as tools for carbohydrate research.

The role of the carbohydrates in protoplast mediaCarbohydrates are essential for protoplast isolation and for maintainingtheir life functions. During the removal and regeneration of the cell wall,its pressure must be replaced by the osmotic pressure of the isolation andculture media, by adding various sugars or sugar alcohols. The type andconcentration of the osmoticum influences protoplast viability, regenera-tion of the cell wall and division. The most frequently used osmotica aremannitol and sorbitol, which are relatively metabolically inert. Sucroseand glucose are also utilized in the early stages of culture. In many systems,additional carbohydrates such as cellobiose, ribose, xylose and arabinosecan be beneficial (Evans and Bravo, 1984; Eriksson, 1985). The mainte-nance of optimal osmotic conditions is closely related to the stability,viability and future development of the protoplasts and highlights therole of carbohydrates in metabolic processes.

von Arnold and Eriksson (1977) observed that mesophyll pea proto-plasts cultured in media free of sucrose, formed poor cell walls and couldnot divide. Xylose, arabinose and glucose, at a concentration of 1 mM, hadfavourable effects on growth, while ribose and galactose did not influenceit. The minimum osmotic pressure was about 500 mOsm, higher values





causing poor wall formation and lower budding of protoplasts. Onlymannitol and sorbitol, however, could function as osmotic stabilizers insuch high concentrations (0.25 M), sucrose and glucose in concentrationshigher than 0.15 M being harmful to the protoplasts (von Arnold andEriksson, 1977).

Protoplast development was closely related to the type and concen-tration of the carbohydrate in the protoplast culture media (Lehminger-Mertens and Jacobsen, 1989a,b). The viability of protoplasts isolated fromshoot tips did not differ dramatically when either mannitol or sucrose wasused as the osmoticum, but if glucose was used then 100% lethalityoccurred within a few hours. In other experiments (Puonti-Kaerlas andEriksson, 1988) using mesophyll protoplasts, however, 0.4 M glucose wassuccessful for maintaining osmolarity in the initial protoplast medium.Whenever liquid media, instead of agarose bead cultures, were used(Lehminger-Mertens and Jacobsen, 1989a), an increasing sugar concen-tration from 3 to 7% and a decrease of pH values to 3.9 were detectedwithin 3 days, these changes correlating with a dramatic decrease in celldivision. The source and concentration of carbohydrate are also essentialfor the induction of embryogenesis in protoplast cultures of pea(Lehminger-Mertens and Jacobsen, 1989b). For the induction process ofprotoplast-derived calli, mannitol could not be substituted by sucrose.Embryogenesis occurred when the mannitol used as an osmoticum had adefined level, 4% but not 3%.

The role of carbohydrates in cell wall synthesisPlant protoplasts offer considerable opportunities for studying the synthe-sis, secretion and assembly of the primary cell wall, as well as the role of thecell wall during development. Hanke and Northcote (1974) examined cellwall formation and found that during the first 20 h of wall regeneration 14Cglucose was predominantly incorporated into protein, starch and celluloseand small amounts were incorporated into an acidic pectin. Klein et al.(1981) observed the synthesis of a broad spectrum of polysaccharide poly-mers during the first 3 h of wall regeneration. Radioactivity was detectedin newly synthesized cellulose within minutes after the protoplasts weretransferred to a wall regeneration medium containing 14C glucose. Thisprocess coincided with the appearance of fibrils on the surface of the proto-plasts. In addition to cellulose, other polysaccharide-containing polymerswere also synthesized. Uridine diphosphate 14C glucose and guanosinediphosphate 14C glucose did not serve as effective substrates for cellulosesynthesis if protoplasts were able to utilize glucose for this process.

In intact cells, polysaccharides and proteins are tightly covalentlylinked. Within the structure of the regenerating protoplast cell wall, how-ever, such linkages are less apparent. Williamson et al. (1976) studied thedistribution of carbohydrate residues on the plasma membrane of soybean

Biotechnology 159




protoplasts and observed that carbohydrate sites were evenly distributed,but mobile within the plane of the membrane. Chemically and physicallyuniform cellulose contrasts with the complex heterogenous mixture ofcovalently linked polysaccharides and proteins that make up the remainderof the wall (Willison, 1985).

Pharnisi et al. (1993) observed that regeneration of cell walls on thesurface of soybean protoplasts was accompanied by the release of smoothvesicles from the cytoplasm to the outside of the plasma membrane.Possibly, the vesicles carried wall materials that were gradually deposited. Acomplex cell wall layer, with a fine cellulose microfibril structure, wasformed and clearly seen on the third day of culture. Nuclear division andsubsequent cytokinesis occurred about 1 day after culture, suggesting thatcell wall formation starts earlier than cell division and then both processesproceed concurrently.

The role of carbohydrates in protoplast divisionIsolated protoplasts are an extremely valuable experimental system forinvestigating the plant cytoskeleton during cell division and the relatedmorphogenic role of the wall, during subsequent development of theprotoplast derived cell.

Fowke et al. (1974) compared cell division between cultured soybeancells and their protoplast derivatives. They found a more rapid formation ofthe phragmoplast and the development of a thicker cell plate in proto-plasts, indicating a slight modification in cross wall formation during cyto-kinesis. Synchronized soybean protoplast cultures permitted the detailedexamination of preprophase bands, which relate to the morphogenicpotential of the protoplasts (Wang et al., 1989; Fowke and Wang, 1992).

Divisions occurring within the first 24 h of Vicia hajastana protoplastculture showed considerable abnormalities in the formation of micro-tubule spindles, phragmoplast, incomplete cross wall and aberrant chromo-some segregation, which were reflected in further development of theprotoplast-derived cells. The importance of a regenerated cell wall fornormal mitotic processes is quite well illustrated by mesophyll protoplastsof lucerne, which are much slower at initiating division and, therefore,have time to form a proper new cell wall. This results in less mitotic abnor-malities (Simmonds, 1992) and leads to normal growth and morphogenesisin protoplast cultures of this species.

Protoplast division can be influenced by starch accumulation in thecell. It was suggested that in pea protoplasts from hypocotyl and primaryroots, large amounts of starch were inhibitory (Landgren, 1981). In potatotuber protoplasts, division began 1 week after their culture, when most ofthe starch grains had been metabolized (Jones et al., 1989). Assuming thathighly meristematic tissues with less starch content were a more suitablesource for protoplast isolation and development, Lehminger-Mertensand Jacobsen (1989a) examined various explants from germinating pea





seedlings (leaf, shoot tip, epicotyl, hypocotyl and root tip) and observed theaccumulation of starch after 1 week. Starch accumulation was not observedin protoplasts derived from shoot tips or from the first lateral shootsoriginating from the cultured embryonic axis minus the cotyledons. Inthese cultures, particularly homogenous meristematic cells and sustainedprotoplast division was achieved.

In contrast to previous reports, Gram et al. (1996), studying starch accu-mulation in relation to the frequency of cell division and regeneration inpea protoplasts, suggested that starch accumulation precedes the divisionof pea protoplasts. They found that the starch content increased rapidlyduring the first 3 days of culture, prior to the onset of division, resulting in a4.2-fold increase in the intracellular starch area and a threefold increase(from 27 to 80%) in the number of protoplasts containing starch. Mitosiswas observed after the fourth day and the number of protoplasts under-going division increased in a stepwise manner, preceded by further starchaccumulation. Since dividing protoplasts were initially 33–66% smaller andcontained 8–42% less starch than non-dividing protoplasts, the dividingprotoplasts contained relatively more starch (6–12%) than non-dividingprotoplasts on a per unit volume basis. Interestingly, the starch levelreached before the onset of the first mitosis was comparable to the levelfound in actively dividing micro-calli, suggesting a requirement of certainlevels of starch accumulation for the induction of pea protoplast division.It can be suggested that there may be an optimum starch content forprotoplast division and that levels below or above this threshold may beinhibitory for mitosis.

Sugar transport through plasmalemmaTransport of solutes through the plasma membrane and the tonoplastmembrane are important cellular activities. For many studies it is desirableto work with relatively homogeneous population of individual cells thatare not organized into tissue. Sugar transport affects the partitioning ofassimilates between the source and the sink regions of a plant thatdetermine crop yield. In developing soybean seeds sucrose is unloadedfrom seed coat phloem into the apoplast prior to its accumulation by thedeveloping seeds. Mechanisms of sugar transport have been analysed usingprotoplasts isolated from developing soybean cotyledons (Lin et al., 1984;Schmitt et al., 1984; Lin, 1985a,b).

Compared with intact cotyledons, isolated protoplasts offer distinctadvantages, such as the absence of bulk diffusion and tissue penetrationbarriers, the accessibility of cell membranes for challenging with sugaranalogues and the prevention of oligosaccharide hydrolysis due to hydro-lases associated with the cell wall. Sucrose and hexose uptake into proto-plasts has shown that, during rapid seed growth, the plasmalemma ofcotyledons contains a sucrose-specific carrier, which is energetically andkinetically distinct from the system(s) involved in hexose transport. Sucrose

Biotechnology 161




uptake by protoplasts is composed of three different mechanisms, a satu-rated, carrier-mediated, energy-dependent mechanism, a carrier-mediated,but non-saturated, or linear mechanism and simple diffusion (Lin et al.,1984). Other studies have focused on proton, sulphydryl reagent andtemperature sensitivities of sucrose uptake kinetics and the operation ofmultiple sucrose uptake mechanisms (Schmitt et al., 1984). The addition ofsucrose to protoplast media causes a specific and transient depolarizationof the membrane potential, acidification of the intracellular pH andalkalization of the external medium. Such results suggest that a proton/sucrose co-transport system is also involved in non-diffusive linear sucroseuptake (Lin, 1985a,b).

6.4 Somaclonal Variation in Grain Legumes

6.4.1 Introduction

There are two basic applications of cultured plant cells that exploit theirtotipotence (ability to express the entire genetic information and regener-ate plants). Firstly, to clone the cells and produce plants that are identicaland, secondly, to change/manipulate the genome and create novel types ofplants. The former application is to maintain valuable genotypic traits andthe latter application is to improve or obtain new characters. Using somaticcell techniques, plants can be genetically manipulated using somaclonalvariation, in vitro mutagenesis and somatic hybridization/cybridization andtransformation by the introduction of foreign genes. Changes in genomesgenerally appear at a low frequency and a large population of an organism,therefore, is necessary for manipulation. In this respect, in vitro culture(callus culture, cell suspension or isolated protoplasts) has the advantage ofa very large number of individuals in a small space, compared with the largegrowing area and intensive labour required to treat equivalent numbers ofplants.

During the early stages of the development of tissue culture methods,in vitro cultured cells were believed to be uniform and similar to the initialmaterial. Regenerants obtained through embryogenesis or organogenesisin vitro as a result of asexual reproduction, therefore, should be pheno-typically and genotypically identical to the donor plant. In the early 1970s,however, there were reports of morphological and cytological changes intobacco plants grown from in vitro cultures (Zagorska et al., 1974). Later,there were similar reports of variability in tissue culture-derived plants. Thisphenomenon was given the name of ‘somaclonal variation’ by Larkin andScowcroft (1981) and is generally found in plants regenerated from callustissue, cell suspensions or isolated protoplasts and, to a lesser extent, inmeristem or shoot tip cultures.





6.4.2 Factors causing variation

The isolation of cells from the integrity of the whole plant and thereorganization of genome function during the establishment of in vitroculture often cause fundamental destabilization of the genetic andepigenetic status. The content and composition of culture media affectchromosomal and cell cycle instability (Gould, 1986). For example, plantgrowth regulators (auxins and cytokinins), that are essential for the de-differentiation and re-differentiation of the cells in vitro, often act as agentsfor genomic changes. Also, inorganic (e.g. phosphates and nitrates) andorganic (e.g. carbohydrates) compounds within the medium contributeto cell cycle abnormalities. Even physical factors, such as temperature, thelight regime and the viscosity and osmolarity of culture media, are known toaffect the cell division cycle of plant cells in culture. The culture phase andthe rate of subculture are also of importance, since prolonged cultivationcan cause more changes in the nuclear and cytoplasmic genome. Selectionof one cell type, however, can occur during subculture, resulting in lessdiversity being observed in such cultures when they are maintained for aprolonged period (Zagorska, 1995). Pre-existing genetic differences, likethe ploidy level of the initial material, the explant origin and the geno-type can be another source of variation. The regeneration pathway, viaorganogenesis or embryogenesis, is another factor causing or eliminatingthe appearance of somaclones. Somatic embryogenesis, as a mechanism ofplant formation from a single cell, was postulated to give ‘free of variability’regenerants (Vasil, 1986). Nevertheless, recent evidence is presentedbelow on variant plants that have been regenerated via somaticembryogenesis.

6.4.3 Mechanisms of somaclonal variation

Somaclonal variation is a complex phenomenon which results from amultiplicity of cellular and genetic mechanisms (Karp, 1993). Generallythe changes that occur have a genetic character and can be inherited,but epigenetic (cannot be transmitted to the progeny) variations arealso observed (Gould, 1986). The most common genetic changes arepolyploidy, aneuploidy, chromosome aberrations, point mutations andalteration in DNA copy number. There may also be alterations in mito-chondrial and chloroplast genomes. These genetic changes, resulting indisturbances in cell cycle and DNA replication, are observed mainly incultures where disorganized growth and a prolonged culture phase areinvolved. In contrast to these genetic effects, epigenetic variation cannotbe transmitted through a sexual cell cycle. Nevertheless, the inducedphenotype modifications can be a valuable source of plant diversity. A

Biotechnology 163




better understanding of the mechanisms and factors of determining varia-tion will contribute to maximizing or minimizing variability as required.Eliminating potential problems of variation in transgenic plants is stillimportant.

6.4.4 Potential and disadvantages of somaclonal variation

Perhaps the greatest benefit of somaclonal variation is in plant improve-ment, by creating additional genetic variability in agronomically usefulcultivars, without the need of sexual hybridization (Lorz and Brown, 1986).It is of primary importance in vegetatively propagated species and inmany horticultural and woody species, for which variation is not readilyavailable. Genomic variation is the first step in the selection of plantsfor crop improvement and the absence of such variation can be a limitingfactor in breeding programmes. It is assumed that somaclonal variationis less important in seed crops, where variation can be created inthe gene pool by reconstruction of genes after crossing. The applicationof tissue culture-derived variability, however, can be useful for suchspecies by combining in vitro culture with in vitro selection (Scowcroft et al.,1987).

By applying selective pressure, variants with a desired character canbe isolated at an early stage of cell development, avoiding regenerationand testing of a great number of useless somaclones. The availability ofrapid screening procedures for useful traits makes somaclonal variationa powerful tool for plant improvement. For example, resistance, or highertolerance, to biotic and abiotic stresses can be achieved by includingselective agents such as pathotoxins, fungal filtrates, herbicides, saltsand heavy metals in the culture media. One of the greatest advantagesof somaclonal variation, however, is for selection of those traits that canbe selected only under in vitro conditions. For example, includingamino acid analogues in the culture media can result in the selection ofover-producers of amino acids. Somaclonal variation is also a pool forcharacters for which there is no adequately defined in vitro response,such as yield, seed protein/oil quality, photosynthetic efficiency, etc. Theidentification and availability of effective plant screening protocols for thistype of trait will contribute much to the wider application of somaclonalvariation.

Somaclones may appear as a negative fact in those cases where clonaluniformity is required (e.g. horticulture, forestry, genetic transformation;Scowcroft et al., 1987). It is likely, however, that the greatest disadvantageof this phenomenon is its unpredictable nature, the same in vitro culture,for example, can generate different types and frequency of somaclonalvariation (Zagorska, 1995).





6.4.5 Variation in grain legumes at the cell and tissue culture level in vitro

Before the term somaclonal variation was introduced into the literature(Larkin and Scowcroft, 1981), a relatively large amount of evidence wasavailable that plant tissues in culture exhibit a broad spectrum of cytologi-cal and karyological changes and abnormalities. This fact also includedgrain legume species. Since the main aim of this part of the chapter is toreview heritable somaclonal variation at the plant level, typical examples ofvariation on cell and tissue culture level in vitro are only mentioned briefly(Table 6.3). Within this table, various altered cell and callus linesare characterized; however, with the absence of plant regeneration, theheritable nature of these changes is subject to speculation.

Cytological instability in vitroSince 1960, the majority of evidence of tissue culture induced variationin grain legumes has involved cytological instability and changes. In calluscultures and cell suspensions of P. sativum with prolonged subcultures,there is a strong tendency towards spontaneous polyploidization, rangingfrom 3n to 32n or higher (Van’t Hof and McMillan, 1969; Frolova andShamina, 1974; Mikhailov and Bessonova, 1975; Knosche and Gunther,1980; Knosche, 1981). Endoreduplication, induced hypothetically bygrowth regulators (cytokinins, IAA, 2,4-D), leads not only to polyploidyand nuclear DNA content increase (Libbenga and Torrea, 1973), but alsoto polytene chromosome formation (Marks and Davies, 1979; Thermanand Murashige, 1984). In addition to polyploidy, a number of aneuploidcells, chromosomal aberrations and karyological abnormalities have beenreported (Kallak and Yarvekylg, 1971, 1976, 1977a,b; Frolova and Shamina,1974; Mikhailov and Bessonova, 1975; Ghosh and Sharma, 1979; Nataliand Cavalini, 1987a). Haploid cells have been found in pollen-derived peacallus (Gupta, 1975) and in callus derived from somatic, diploid, pea tissues(Kunakh et al., 1984; Natali and Cavalini, 1987b). A detailed review aboutcytogenetics of pea callus and cell cultures has been published by Griga andNovák (1990).

Cytological instability in callus and suspension cultures of Vicia fabawas first reported by Venketeswaran (1963) and Venketeswaran and Spiess(1963, 1964). In the 1970s, cultures of faba bean were frequently used as amodel for cytogenetic studies. Observations of all types of euploidy, fromhaploid cells to highly polyploid cells up to 32 n or 64 C, aneuploid cellsand many mitotic abnormalities including endoreduplication, were reported(Frolova and Shamina, 1974, 1978; Yamane, 1975; Shamina and Butenko,1976; Cionini et al., 1978; Papet et al., 1978; Roper, 1979; D’Amato et al.,1980; Hesemann, 1980; Jelaska et al., 1981; Ogura, 1982; Frolova, 1986;Taha and Francis, 1990). A detailed review on the cytogenetics of faba bean

Biotechnology 165






Spec

ies

Type

ofin

vitr

ocu

lture

Trai

tR

espo

nse

(val

ue/d

escr

iptio

n)R

efer

ence

s

Ara

chis

hypo

gaea

(pea

nut,

grou

ndnu

t)A

nthe

r-de

rive

dca

llus

Chr

omos

ome

num

ber

Mix

oplo

idca

llus

(hap

loid

tooc

topl

oid

cells

)B

ajaj

etal

.(19

81)

Caj

anus

caja

nA

nthe

r-de

rive

dca

llus

Chr

omos

ome

num

ber

Hap

loid

,dip

loid

and

aneu

ploi

dce

llsB

ajaj

etal

.(19

80)

Cic

erar

ietin

umC

allu

s,ce

llsu

spen

sion

Salt

tole

ranc

e(N

aCl)

NaC

l-to

lera

ntce

lllin

esG

osal

and

Baj

aj(1

984)

Cal

lus

Salt

tole

ranc

e(N

aCl)

NaC

l-to

lera

ntce

lllin

esPa

ndey

and

Gan

apat

hy(1

984)

Ant

her-

deri

ved

callu

sC

hrom

osom

enu

mbe

rH

aplo

idto

octo

ploi

dce

llsB

ajaj

and

Gos

al(1

987)

;Gos

alan

dB

ajaj

(198

8)

Gly

cine

max

Cel

lsus

pens

ion

Res

ista

nce

(rea

ctio

n)to

elic

itor

from

Phyt

opth

ora

meg

aspe

rma

var.

soja

e

Acc

umul

atio

nof

aph

ytoa

lexi

ngl

yceo

linin

elic

itor

trea

ted

cells

Ebel

etal

.(19

76)

Cal

lus

Res

ista

nce

(rea

ctio

n)to

P.m

egas

perm

ava

r.so

jae

Bio

assa

yfo

rsc

reen

ing

resi

stan

tor

susc

eptib

lege

noty

pes

invi

tro

cultu

re

Hol

liday

and

Kla

rman

(197

9)

Cal

lus;

exci

sed

coty

ledo

nsR

esis

tanc

e(r

eact

ion)

toso

ybea

nbr

own

stem

rot

(Phi

alop

hora

greg

ata)

cultu

refil

trat

e

Bio

assa

yfo

rsc

reen

ing

resi

stan

tmut

ants

incu

lture

;hi

ghde

gree

ofco

rres

pond

ence

betw

een

reac

tion

intis

sue

cultu

rean

dgl

assh

ouse

test

s

Gra

yet

al.(

1986

);W

illm

otet

al.(

1989

)

Cal

lus

Res

ista

nce

toD

iapo

rthe

phas

eolo

rum

cultu

refil

trat

e;du

alcu

lture

with

the

fung

us

Iden

tific

atio

nof

tole

rant

soyb

ean

culti

vars

base

don

invi

tro

test

Sim

onie

tal.

(199

5)

Tabl

e6.

3.So

mac

lona

lvar

iatio

nin

grai

nle

gum

eson

cell,

tissu

ean

dor

gan

cultu

rele

veli

nvi

tro.



Biotechnology 167


Phas

eolu

sco

ccin

eus

(run

ner

bean

)Su

spen

sor-

deri

ved

callu

sC

hrom

osom

enu

mbe

r,D

NA

cont

ent

Gre

atva

riab

ility

inch

rom

osom

enu

mbe

rB

enni

ciet

al.(

1976

)

Phas

eolu

svu

lgar

isEx

cise

dro

ots,

callu

s,ce

llsu

spen

sion

Res

ista

nce

toPs

eudo

mon

asph

aseo

licol

acu

lture

filtr

ate

Diff

eren

tialg

row

thin

hibi

tion

upto

77%

;inc

reas

ein

abno

rmal

cells

;55-

fold

incr

ease

inor

nith

ine

Baj

ajan

dSa

ettle

r(1

968,

1970

)

Cal

lus

from

diffe

rent

plan

tpar

tsPl

oidy

leve

l(D

NA

cont

ent)

Mix

oplo

idca

lli(2

C–8

Can

dev

enhi

gher

DN

Aco

nten

t)H

addo

nan

dN

orth

cote

(197

6)

Ant

her-

deri

ved

callu

sC

hrom

osom

enu

mbe

rH

aplo

id,d

iplo

idan

dpo

lypl

oid

cells

Pete

rset

al.(

1977

)

Cal

lus

Salt

tole

ranc

e(N

aCl)

Salt-

sens

itive

calli

and

plan

tsSm

ithan

dM

cCom

b(1

981)

Cal

lus

Res

ista

nce

toPs

eudo

mon

assy

ring

aepv

.pha

seol

icol

acu

lture

filtr

ate

Cal

lus

scre

enin

gte

st;p

ositi

veco

rrel

atio

nbe

twee

nre

actio

non

callu

san

dpl

antl

evel

Har

tman

etal

.(19

86)

Cal

lus

Res

ista

nce

toSc

lero

tinia

scle

rotio

rum

cultu

refil

trat

eC

allu

s-w

eigh

tass

ay;

scre

enin

gof

part

ial

phys

iolo

gica

lres

ista

nce

with

indr

ybe

ange

rmpl

asm

Mik

las

etal

.(19

92)

Vig

nara

diat

aC

allu

s,ce

llsu

spen

sion

Seed

ling

root

tip-d

eriv

edC

allu

s

Salt

tole

ranc

e(N

aCl)

Thio

prol

ine

resi

stan

ce;N

aCl

resi

stan

ce

NaC

l-to

lera

ntce

lllin

esFi

vefo

ldin

crea

seen

doge

nous

leve

lsof

free

Prol

ine;

elev

ated

tole

ranc

eto

NaC

l

Gos

alan

dB

ajaj

(198

4)K

umar

and

Shar

ma

(198

9)

Vig

nasi

nens

is(c

owpe

a)C

ells

uspe

nsio

nC

hrom

osom

enu

mbe

rPo

lypl

oidy

(3n–

6n);

mul

tinuc

leat

ece

llsG

hosh

and

Shar

ma

(197

9)

Con

tinue

d





Spec

ies

Type

ofin

vitr

ocu

lture

Trai

tR

espo

nse

(val

ue/d

escr

iptio

n)R

efer

ence

s

Pisu

msa

tivum

Cal

lus

Chr

omos

ome

num

ber;

chro

mos

ome

aber

ratio

ns;

mito

ticab

norm

aliti

es

Poly

ploi

dce

lls(3

n,4n

and

mor

e);m

ultin

ucle

arce

lls;

kary

olog

ical

abno

rmal

ities

Kal

lak

and

Yar

veky

lg(1

968,

1971

,197

6,19

77a,

b)

Cal

lus

Chr

omos

ome

num

ber

Poly

ploi

d(u

pto

12n)

and

aneu

ploi

dce

llsFr

olov

aan

dSh

amin

a(1

974)

Imm

atur

eco

tyle

don-

deri

ved

callu

s

Chr

omos

ome

num

ber;

chro

mos

ome

aber

ratio

nsPo

lypl

oid

cells

(up

to8n

and

mor

e);c

hrom

osom

alab

erra

tions

Mik

hailo

van

dB

esso

nova

(197

5)

Polle

nca

llus

Chr

omos

ome

num

ber

Hap

loid

and

mix

oplo

id(m

ainl

yte

trap

loid

)cel

lpo

pula

tions

Gup

ta(1

975)

Cel

lsus

pens

ion

Chr

omos

ome

num

ber

Mul

tinuc

lear

cells

,ane

uplo

idy

Gho

shan

dSh

arm

a(1

979)

Cal

lus

cultu

reC

hrom

osom

enu

mbe

rH

ighl

ypo

lypl

oid

cells

(32n

orm

ore)

Kno

sche

and

Gun

ther

(198

0);

Kno

sche

(198

1)C

allu

scu

lture

Tole

ranc

eto

the

herb

icid

esPr

opha

man

dPr

oban

il(O

-iso

prop

yl-3

-chl

or-

phen

ylca

rbam

ate)

Cal

liw

ithim

prov

edto

lera

nce

tohe

rbic

ides

Jake

leta

l.(1

990)

Cal

lus,

cell

susp

ensi

onSa

ltto

lera

nce

(NaC

l)N

aCl-

tole

rant

cell

lines

Gos

alan

dB

ajaj

(198

4)C

allu

sC

hrom

osom

enu

mbe

r,ch

rom

osom

eab

erra

tions

Hap

loid

,dip

loid

and

poly

ploi

dce

lls(u

pto

8n)

Kun

akh

etal

.(19

84)

Mer

iste

m-d

eriv

edan

dem

bryo

axis

-der

ived

callu

s;re

gene

rate

dsh

oots

Chr

omos

ome

num

ber

Dip

loid

oran

euso

mat

ic(c

hrom

osom

alm

osai

cs)s

hoot

sN

atal

iand

Cav

allin

i(19

87a,

b)

Mes

ophy

llpr

otop

last

sR

esis

tanc

eto

P.sy

ring

aepv

.pi

siM

odel

syst

emfo

rpe

a–

Pseu

dom

onas

inte

ract

ion

Akp

aan

dA

rche

r(1

994)

Tabl

e6.

3.C

ontin

ued.



Biotechnology 169


Vic

iafa

baSu

spen

sion

Chr

omos

ome

num

ber

Poly

ploi

d(4

n–8n

)and

aneu

ploi

dce

llsV

enke

tesw

aran

(196

3)

Cal

lus

Chr

omos

ome

num

ber

Dip

loid

and

poly

ploi

dce

lls(m

ainl

y4n

)Fr

olov

aan

dSh

amin

a(1

974,

1978

);Sh

amin

aan

dB

uten

ko(1

976)

Cal

lus

Chr

omos

ome

num

ber

Poly

ploi

d(3

n–8n

)and

aneu

ploi

dce

llsY

aman

e(1

975)

Cal

lus

Chr

omos

ome

num

ber

(as

DN

Aco

nten

t)B

inuc

lear

and

mul

tinuc

lear

cells

;dip

loid

and

poly

ploi

dce

lls(u

pto

64C

)

Cio

nini

etal

.(19

78a,

b);

D’A

mat

oet

al.(

1980

)

Cal

lus

Chr

omos

ome

num

ber

Dip

loid

,ane

uplo

idan

dpo

lypl

oid

cells

Papes

etal

.(19

78);

Jela

ska

etal

.(19

81)

Cal

lus

and

cell

susp

ensi

onC

hrom

osom

enu

mbe

rD

iplo

id,p

olyp

loid

and

aneu

ploi

dce

llsR

oper

(197

9)

Ant

her-

deri

ved

callu

sC

hrom

osom

enu

mbe

r(p

loid

yle

velm

easu

red

cyto

phot

omet

rica

llyas

C-v

alue

)

Hap

loid

,dip

loid

and

poly

ploi

dce

lls(m

ore

than

16C

)

Hes

eman

n(1

980)

Cal

lus

Chr

omos

ome

num

ber

Poly

ploi

dan

den

eupl

oid

cells

Ogu

ra(1

982)

Cal

lus

cultu

reTo

lera

nce

toth

ehe

rbic

ides

Prop

ham

and

Prob

anil

(O-i

sopr

opyl

-3-c

hlor

-ph

enyl

carb

amat

e)

Cal

liw

ithim

prov

edto

lera

nce

tohe

rbic

ides

Jake

leta

l.(1

990)



callus and cell cultures has been published by Griga et al. (1986). Variationin ploidy level and other chromosomal changes and aberrations in tissueand cell cultures of grain legume species, other than pea or faba bean, areless frequently documented in the literature (Table 6.3).

The inability to regenerate plants from cytologically altered cells andtissues in many of the above cited reports, may be due to the selectiveadvantage of normal (diploid) cells in the process of organ formation(diplontic selection). The inability of plant regeneration from cytologicallyaltered tissues determines that these changes cannot be practicallyexploited at present and may only serve as a model for theoretical studies.The mechanisms of chromosome variation in plant tissue cultures havebeen reviewed by Ogura (1990).

Variation in tolerance/resistance to biotic and abiotic factors (stresses)The most frequent biotic factors studied in grain legumes at an in vitro levelhave been responses to bacterial and fungal pathogens or their toxins, usu-ally contained in culture filtrates (Table 6.3). Some reports are orientatedtowards the formulation of exact and quick bioassays, for indicating thesensitivity/tolerance/resistance of tested genotypes to particular pathogenand its races (Bajaj and Saettler, 1968, 1970; Ebel et al., 1976; Gray et al.,1986; Hartman et al., 1986; Willmot et al., 1989; Miklas et al., 1992; Akpa andArcher, 1994; Simoni et al., 1995). On the other hand, other reports aredirectly aimed at obtaining tolerant/resistant cell lines and subsequentlyplants that are useful for resistance breeding (Table 6.3). Screeninghas been based on the hypothesis that bacterial and fungal toxins playan important role in host–pathogen interactions and that the response ona cell culture level may positively correlate with the whole plant reaction.Unfortunately, at present, this idea is not sufficiently supported byexperimental data (Buiatti and Ingram, 1991).

Probably the first report on the selection of a grain legume species(Phaseolus vulgaris) callus, challenged with the culture filtrate of a bacte-rium (Pseudomonas phaseolicola) was published by Bajaj and Saettler (1968,1970). The authors observed differential tolerance of various callus linesto the host-specific pathotoxin.

Gray et al. (1986) developed a bioassay for the evaluation of soybeanfor resistance to brown stem rot (Phialophora gregata) and for assessingthe pathogenicity of fungal isolates. This was based on testing calli fromsusceptible and resistant soybean genotypes to fungal culture filtrate ofpathogenic and non-pathogenic isolates of P. gregata. Willmot et al. (1989)found that the in vitro reaction of excised cotyledons and callus to P. gregataculture filtrate correlated positively with the greenhouse assay on intactplants (70–100%). In particular, the cotyledon method allowed soybeanlines, resistant to P. gregata isolates, to be accurately identified.

Hartman et al. (1986) observed a highly significant correlation(r = 0.971) between the response of bean calli to the culture filtrate of





halo blight disease (caused by the bacterium Pseudomonas syringae pv.phaseolicola) and the reaction of whole bean plants inoculated with asuspension of the pathogen. Results suggested that a callus screeningsystem could identify bean cultivars resistant to halo blight.

Miklas et al. (1992) developed a screening method to identify partialphysiological resistance of bean to white mould (Sclerotinia sclerotiorum),based on the treatment of callus with pathogen filtrate. The results fromthis callus-weight assay correlated very well with the field reaction of beangenotypes, declared as field-resistant and field-susceptible.

As an alternative to using pathogen filtrate, Simoni et al. (1995) devel-oped an in vitro test for soybean, based on the dual culture of callus or seedswith a fungal culture of Diaporthe phaseolorum var. caulivora. The inhibitionof fungal growth was analysed as an effect of the calli, or germinating seeds,on the mycelium. Based on this test, tolerant and susceptible soybeancultivars could be identified, in addition the system could be used for directin vitro selection of callus lines with improved resistance to the pathogen.

Various in vitro approaches have been studied in pea, includingmultiple shoot cultures, callus cultures, root cultures, direct somaticembryogenesis and dual cultures. These approaches have been used to for-mulate efficient systems based on culture filtrates, pure toxins or mycelialcultures of pea fungal pathogens, including Fusarium oxysporum, Fusariumsolani, Fusarium poae, Fusarium semitectum, Mycosphaerella pinodes, Rhizoctoniasolani, Trichotheceum roseum (Švábová et al., 1995, 1996, 1998; Švábová andGriga, 1997, 1998a,b). The exact identification of specific toxins presentin culture filtrates is one of the prerequisites for more precise work. Seedsproduced by fertile regenerants, obtained during methodological studies,are now available for correlation tests of plants grown in the greenhouseand field.

Among the abiotic stresses, salt tolerance/resistance (NaCl) has beenstudied only in grain legumes on an in vitro level (Table 6.3). Gosal andBajaj (1984) selected NaCl-tolerant cell lines in suspension cultures of Cicerarietinum, P. sativum and Vigna radiata. The number of salt-resistant colonieswas increased by treating the actively growing cell suspensions with 0.25%ethyl methane sulphonate (EMS). Resistant calli of Cicer and Vigna wereable to regenerate roots, although complete plants were not obtained.Pandey and Ganapathy (1984) selected a NaCl-resistant callus line of C.arietinum, which had a growth rate that was comparable with that of thecontrol, non-selected, callus in non-saline medium. Kumar and Sharma(1989) selected V. radiata callus lines that were resistant to thioproline,an analogue of proline. One of the selected clones exhibited an elevatedtolerance to exogenously applied NaCl, as well as a fivefold increased levelof free proline.

Despite positive evidence of a correlation between salt resistance at thetissue culture and at the whole plant level, the data available for grainlegumes are still contradictory (Gale and Boll, 1978), particularly in various

Biotechnology 171




glycophytic and halophytic salt-resistant plant species (see review by Tal,1990).

6.4.6 Variation in grain legumes at the whole plant level

To date most of the data describing somaclonal variation in grain legumesat a whole plant level have been on pea and soybean, with only a limitedamount of information for faba bean, groundnut and Vigna.

Variation in ploidy level, chromosomal aberrations and DNA contentWithin the grain legumes, evidence about the changes of ploidy, chromo-some number and DNA content in complete plants regenerated from invitro cultures only exists for pea (Table 6.4). Kunakh et al. (1984) obtaineddiploid and tetraploid regenerants via organogenesis from pea calli ofvarious ploidy (1n–8n). The ploidy level of callus tissue was affected by theorigin of the primary explant (leaf, shoot or root) and the composition ofthe culture medium, but not by the genotype. In contrast, regenerationability was determined by genotype. Natali and Cavallini (1987a,b)obtained, via organogenesis, diploid and aneusomatic (chromosomalmosaics) pea plantlets from calli derived from macerated shoot apices andembryo axes. As aneusomaty was reduced during plantlet development, theauthors suggested that there may be a selective advantage of diploid overaneuploid cells (diplontic selection). In these early studies no mention wasmade of the fertility of regenerants obtained, or any genetic study of theirseed progenies. Kysely et al. (1987) obtained diploid and tetraploid R0 peaplants (regeneration via somatic embryogenesis) from calli derived fromimmature embryos and shoot apices. All of the tetraploids originated fromthe shoot apex cultures. In contrast to the above mentioned reports, novariation in chromosome number was found in the root tips of R1 plants(seed progenies from immature leaflet calli organogenic regenerants) ofpea (Rubluo et al., 1984). All of the analysed plants had a normal diploidnumber of chromosomes. A possible reason for this may be the eliminationof all cells, other than diploid cells, during the formation of reproductivestructures and seed development on R0 plants. Ahmed et al. (1987)analysed root tips of R0 regenerants of pea, formed directly from shootapical meristems. The regenerants contained a majority of diploid cells(over 80%), plus a low frequency of cells with 10, 12, 21 or 28 chromo-somes. A similar situation was found, however, in root tips of controlplants germinated from seeds. The authors concluded that pea plantsregenerated from meristems might be considered cytologically normal andgenetically stable.

Cecchini et al. (1992) studied cryptic gene alterations, such as amplifi-cations or loss of nuclear DNA, using diploid plants of two pea cultivars,regenerated from meristem-derived calli. Cytogenetic analyses showed a





significant reduction in the nuclear DNA content of regenerants from cv.Dolce Provenza, while the DNA content remained stable in regenerantsfrom line 5075. The DNA reduction included changes in specificsubfamilies of medium repetitive sequences, while highly repetitivesequences remained invariant. In addition, the DNA of cv. Dolce Provenzawas hypermethylated, while modifications in DNA methylation were notdetected from line 5075. The extended hypermethylation of the genome inregenerated plants could be a rapid mechanism for silencing potentiallethal genes during stress conditions. It was concluded that DNA variationsrelated to culture and regeneration stress are dependent, at least in part,on the genotype (5075 more stable than Dolce Provenza). DNA contentvariability, however, was found in line 5075 plants regenerated from callimaintained in culture for a long incubation period. This supports thehypothesis that genetic stability of regenerants is also a function of thelength of time that such cultures are maintained as a callus.

Biochemical and molecular changes (total proteins, isozymes, DNAfingerprints)Rubluo et al. (1984) found no variation in isoenzyme spectra (esterase,glutamate dehydrogenase, 6-phosphogluconate dehydrogenase and leucinamino peptidase) of seed progeny (R1) from pea regenerants, obtained bycallus mediated de novo organogenesis. Amberger et al. (1992) observedvariant isozyme patterns in two independent soybean tissue culture-derivedlines (regenerated via somatic embryogenesis). In the cv. BSR 101, amutation of the Aco2-b (aconitase) gene, to give a null allele, was detected.This mutation had not been previously observed in soybean. In cv. Jilin 3, achlorophyll-deficient plant was identified that also lacked two mito-chondrial malate-dehydrogenase (Mdh null) isozyme bands. These twomutant phenotypes, chlorophyll-deficient and Mdh null, were found toco-segregate. According to the authors, the recovery of two isozymevariants, from the progeny of 185 soybean plants regenerated from somaticembryogenesis, indicates the feasibility of selection for molecular variants.

Griga and Stejskal (1994) found minor changes in the seed storageprotein spectra of seed progenies (R3) from meristem-derived pea plants.Regenerated plants from a 9-year micropropagated shoot culture of peacv. Bohatýr had a high proportion of sterile individuals and various leafmorphological alterations. Differences between these regenerants andcontrol plants were shown in the spectra for leaf peroxidase, esterase, acidphosphatase and seed storage proteins.

Morphological and physiological traits, yield charactersThe first and the most detailed somaclonal variation study within the grainlegumes, which included five seed generations, was performed in pea byGostimiskij et al. (1985) and Ezhova et al. (1989). Variation was observedbetween plants regenerated from long-term callus, derived from macerated

Biotechnology 173






Spec

ies

Type

ofin

vitr

ocu

lture

Plan

treg

ener

atio

nvi

aTr

ait

Res

pons

e(v

alue

/des

crip

tion)

Ref

eren

ces

Gly

cine

max

Imm

atur

ezy

gotic

embr

yo-d

eriv

edca

llus

Som

atic

embr

yoge

nesi

s,or

gano

gene

sis

Qua

litat

ive

and

quan

titat

ive

trai

ts

Alb

inot

icch

imae

ras

(R0)

,ch

loro

phyl

ldef

icie

ncy,

abno

rmal

leaf

mor

phol

ogy,

dwar

fpla

ntha

bit(

R1–

R3)

Bar

wal

ean

dW

idho

lm(1

987)

Cot

yled

onar

yno

dePr

olife

ratio

nof

shoo

tm

eris

tem

san

dde

novo

shoo

ts(o

rgan

ogen

esis

)

Qua

litat

ive

and

quan

titat

ive

trai

ts

Plan

thei

ght,

ster

ility

(R0,

R1,

R2)

Gra

ybos

chet

al.(

1987

)

Imm

atur

eem

bryo

Som

atic

embr

yoge

nesi

sPl

ant

mor

phol

ogy,

lipid

com

posi

tion

Incr

ease

dva

riat

ion

inR

1

(mai

nly

leaf

mor

phol

ogy;

lipid

com

posi

tion)

;not

inhe

rite

dto

R2

Hild

ebra

ndet

al.(

1989

)

Cot

yled

onar

yno

de-

and

epic

otyl

-der

ived

callu

s

Org

anog

enes

isQ

ualit

ativ

etr

aits

Lanc

eola

tele

aves

,lea

fand

pod

vari

egat

ion,

dete

rmin

ate

grow

thha

bit(

R0,

R1,

R2)

Frey

tag

etal

.(19

89)

Cot

yled

onar

yno

de-

and

epic

otyl

-der

ived

callu

s

Org

anog

enes

isA

traz

ine

tole

ranc

eA

traz

ine

tole

rant

plan

ts(R

0,R

1,R

2)W

rath

eran

dFr

eyta

g(1

991)

Imm

atur

eco

tyle

dons

Dir

ects

omat

icem

bryo

gene

sis

Qua

litat

ive

trai

tsC

ompl

ete

and

part

ial

ster

ility

,wri

nkle

dan

dcu

rled

leav

es,c

hlor

ophy

llde

ficie

ncy,

redu

ced

plan

the

ight

,det

erm

inat

egr

owth

habi

t,va

riat

ion

inm

alat

ede

hydr

ogen

ase,

acon

itase

and

diap

hora

se(R

0–R

3)

Am

berg

eret

al.(

1992

)

Tabl

e6.

4.So

mac

lona

lvar

iatio

nin

grai

nle

gum

eson

the

who

lepl

antl

evel

(reg

ener

ants

and

thei

rse

edpr

ogen

ies)

.



Biotechnology 175


Unk

now

nU

nkno

wn

Res

ista

nce

toSe

ptor

iagl

ycin

escu

lture

filtr

ate

Res

ista

ntre

gene

rant

s(n

otpr

oven

)So

nget

al.(

1994

)

Embr

yoge

nic

susp

ensi

onSo

mat

icem

bryo

gene

sis

Res

ista

nce

toFu

sari

umso

lani

cultu

refil

trat

e

Impr

oved

resi

stan

ceto

path

ogen

(R1–

R3)

Jinet

al.(

1996

)

Pisu

msa

tivum

Roo

t,le

afan

dst

empr

imar

yan

dlo

ng-t

erm

callu

s

Shoo

torg

anog

enes

isC

hrom

osom

enu

mbe

rV

ario

uspl

oidy

ofca

llus;

tetr

aplo

idpl

ants

(R0)

Kun

akh

etal

.(19

84)

Shoo

tapi

cal

mer

iste

mPr

olife

ratio

nof

shoo

tm

eris

tem

sC

hrom

osom

enu

mbe

r;ch

rom

osom

eab

erra

tions

Var

iatio

nin

chro

mos

ome

num

ber;

chro

mos

ome

mor

phol

ogy;

anap

hase

abno

rmal

ities

Ahm

edet

al.(

1987

)

Imm

atur

ezy

gotic

embr

yo-d

eriv

edca

llus

Shoo

torg

anog

enes

isC

hrom

osom

enu

mbe

rD

iplo

idan

dan

euso

mat

ic(c

hrom

osom

alm

osai

cs)

plan

tlets

(R0)

Nat

alia

ndC

aval

lini

(198

7a)

Imm

atur

ezy

gotic

embr

yo-

and

shoo

tap

ex-d

eriv

edca

llus

Som

atic

embr

yoge

nesi

sC

hrom

osom

enu

mbe

rTe

trap

loid

plan

ts(R

0)K

ysel

yet

al.(

1987

)

Shoo

tape

x-de

rive

dlo

ng-t

erm

callu

sSh

ooto

rgan

ogen

esis

Qua

litat

ive

and

quan

titat

ive

trai

ts

Leaf

mut

atio

nsw

axy

and

chlo

rotic

a;m

ore

vigo

rous

habi

t;da

rkgr

een

leav

es;

oblo

ngle

afle

ts;f

irst

flow

erpo

sitio

n(R

1–R

5)

Gos

timsk

ijet

al.(

1985

);Ez

hova

etal

.(19

89)

Stem

and

leaf

-der

ived

callu

sSh

ooto

rgan

ogen

esis

Qua

litat

ive

trai

tsA

ntho

cyan

inab

senc

e;le

afty

pe;p

lant

habi

t(R

0–R

1)Lu

tova

and

Zab

elin

a(1

988)

Prim

ary

and

long

-ter

mca

llus

Shoo

torg

anog

enes

isSe

edpr

otei

nsan

dam

ino

acid

com

posi

tion

Incr

ease

dle

gum

in/v

icili

nra

tio;a

ltere

dam

ino

acid

bala

nce

(R1)

Mik

hailo

va-K

rum

ova

etal

.(19

91)

Con

tinue

d





Spec

ies

Type

ofin

vitr

ocu

lture

Plan

treg

ener

atio

nvi

aTr

ait

Res

pons

e(v

alue

/des

crip

tion)

Ref

eren

ces

Shoo

tape

x-de

rive

dca

llus

Shoo

torg

anog

enes

isN

ucle

arD

NA

vari

atio

nG

enot

ype-

depe

nden

t,tis

sue

cultu

rein

duce

dD

NA

cont

entv

aria

tion

inre

gene

rate

dpl

ants

(R0)

Cec

chin

ieta

l.(1

992)

Imm

atur

ezy

gotic

embr

yo-d

eriv

edca

llus,

youn

gle

afle

t-de

rive

dca

llus

Som

atic

embr

yoge

nesi

s,or

gano

gene

sis

Qua

litat

ive

and

quan

titat

ive

trai

ts

Sign

ifica

ntly

alte

red

leaf

and

flow

erm

orph

olog

y;st

erili

ty;l

etha

lity

(R0)

;pod

san

dse

eds

per

plan

t;cr

ude

prot

ein

cont

ent(

R1–

R4)

Stej

skal

and

Gri

ga(1

992)

;Gri

gaet

al.

(199

5);G

riga

and

Léta

l(1

995)

Ara

chis

hypo

gaea

Cot

yled

onca

llus

Unk

now

nR

esis

tanc

eto

Cer

cosp

orid

ium

pers

onat

umcu

lture

filtr

ate

Enha

nced

resi

stan

ceof

R2

plan

tsV

enka

tach

alam

etal

.(1

998)

Phas

eolu

svu

lgar

isSh

ootm

eris

tem

sM

eris

tem

prol

ifera

tion

Res

ista

nce

toph

aseo

loto

xin

Res

ista

ntre

gene

rant

s(n

otpr

oven

)G

anto

ttiet

al.(

1985

)

Vic

iafa

ba(S

hoot

apic

al)

mer

iste

mcu

lture

infe

cted

with

myc

elia

lsus

pens

ion

Mer

iste

mpr

olife

ratio

n/m

ultip

lesh

ootf

orm

atio

n

Res

ista

nce

toB

otry

tisci

nere

a,Ph

ytop

htho

ram

egas

perm

aan

dR

hizo

cton

iaso

lani

Cor

rela

tion

betw

een

low

tom

ediu

mph

ytoa

lexi

nle

vel

inre

gene

rant

san

dfu

ngal

resi

stan

ce(R

0)

Thyn

net

al.(

1989

)

Vig

nara

diat

a‘D

e-em

bryo

nate

d’co

tyle

dons

De

novo

orga

noge

nesi

sQ

ualit

ativ

etr

aits

Chl

orop

hyll

and

mor

phol

ogic

alm

utat

ions

(R0,

R1,

R2)

Mat

hew

set

al.(

1986

)

Tabl

e6.

4.C

ontin

ued.



tissues (shoot-apex, epicotyl, internode, leaf). Changes included bothqualitative (e.g. chlorophyll defects, absence of waxy layer on leaves) andphysiological/quantitative traits (e.g. plant habit, colour and morphologyof leaves, yield parameters) and were stably inherited to seed progeny (T1

to T5 generations analysed). The most frequent changes were: a mutationin the chi (chlorotica) gene (recessive nuclear mutation); the absence of awaxy layer on leaves (recessive nuclear mutation); a more robust habitcompared with the original variety; dark green instead of the light greenleaves found in the original variety; elongate compared with oval-shapedleaves and an earlier or later onset of flowering. Cytological data of calluscells indicated that the phenotypic variability of regenerants was notconnected with large reconstructions of the karyotype. Also, it is unlikelythat stability for a trait, such as flowering period (controlled by four genes),can be the result of a single gene mutation. It is suggested by these authorsand others (Cecchini et al., 1992) that some mechanisms determininggenetic variability are characteristic for cells of in vitro-cultured plants, e.g.amplification of some genome segments and their transposition.

Lutova and Zabelina (1988) analysed R0 and R1 plants obtained byorganogenesis from callus derived from internode and leaf segments.Three qualitative changes were recorded within the R0 regenerants, whichwere inherited in the R1 generation, the presence/absence of anthocyanin,leaf structure and plant habit.

Stejskal and Griga (1992) found, within the R0 regenerants of pea,obtained by somatic embryogenesis from immature zygotic embryos, aplant with a dramatically altered habit (leaflet shape, one pair of leaflets,abnormal flower morphology, reduced flower stalk, shortened internodes,stipules without dentation and with tendrils). The plant exhibited achimaeric character and was completely sterile. The transfer back to in vitroculture did not result in the isolation of a stable mutant (Griga, 2000).The same authors (Griga et al., 1995; Griga and Létal, 1995) comparedsomaclones obtained by somatic embryogenesis and by organogenesis,together with their seed progenies (R1 to R4 generation). Mainly morpho-logical changes were recorded (altered leaflets, tendrils, fasciations), whenevaluating qualitative traits in plants from both tissue systems. Allplants exhibiting such phenomena were chimaeric, and the alteredtraits occurred randomly or were lost in later generations. Analysis of 12quantitative traits (e.g. plant morphology, yield parameters, seed proteincontent) showed that somaclones produced via organogenesis exhibitmore variation compared with those produced via embryogenesis.

An extensive literature exists about soybean somaclonal variation atthe whole plant level. Barwale and Widholm (1987) evaluated plantsregenerated from embryogenic and organogenic cultures of nine soybeangenotypes and found extensive variation in qualitative traits. Threelethal sectorial albinos were seen in the primary regenerants (R0). Variantsobserved in later selfed generations included twin seeds, multiple shoots,

Biotechnology 177




dwarfs, abnormal leaf morphology, abnormal leaflet number, wrinkledleaves, chlorophyll deficiency, partial sterility and complete sterility. Thefrequency of mutations ranged from 0 to 4% in R0 plants, as determinedby studies of corresponding R1, R2, R3 and R4 families. No significantdifferences were seen in the frequency of mutations for embryogeniccompared with organogenic culture-derived plants. Chlorophyll deficiency,sterility and wrinkled leaves, traits that are controlled by single recessivenuclear genes, were stably inherited over two or three generations. Othertraits occurred more randomly and were not in all generations. At presentthe genetic basis of this random variation is not known.

Graybosch et al. (1987) studied somaclonal variation in R1 seedprogenies of plants regenerated from soybean cotyledonary nodes (BA-stimulated shoot formation from pre-existing as well as newly formedmeristematic regions of nodal tissue). The following traits were recordedin three cultivars under field conditions, yield, plant height, lodging,leaf shape and colour and maturity. In addition, the following dominantgenetic markers were evaluated: purple flowers, tawny pubescence, blackhilum and brown pods. Variability for yield was observed in two out of 19families, compared with control cultivars. One of the 22 families exceededthe control in height and variability for height was increased amongregenerated families. Recessive mutations for putative sterility characterswere observed in two out of 89 families, but mutations in six marker geneswere not apparent. Negligible variation in qualitative traits and relativelylow variation in quantitative traits, compared with the control, showedthat cotyledonary node culture was not a source of significant somaclonalvariation. An important fact was that many somaclones retained the yieldpotential of the parental cultivars. This result was significant for the use ofthe cotyledonary node technique for the introduction of foreign genes bygenetic transformation.

Freytag et al. (1989) analysed the progeny (R1 to R3) of soybean plants,regenerated from callus cultures (organogenesis) derived from cotyledon-ary nodes and epicotyls. Variant phenotypes were found that had notbeen previously reported from tissue culture, including lanceolate leaves,leaf variegation (chimaeric variegated plants), pod variegation on other-wise normal plants and a change in growth habit from indeterminate todeterminate. All of the above-mentioned traits were inherited throughthree generations, except pod variation, which was inherited through twogenerations, segregation occurring in each generation. No variation wasobserved in control plants derived from normal seeds.

Hildebrand et al. (1989) studied variation in fatty acid composition ofthe seeds and plant morphological traits, in soybean regenerants obtainedvia somatic embryogenesis. The first seed generation (R1) of regenerantsexhibited higher phenotypic variation, compared with normal seed-derivedpopulations. Changes included lateral indentation (lobing) of the firstunifoliate leaf, sectorial loss of chlorophyll and dual apical meristems,





with three cotyledons and three unifoliate leaves. Variation in fatty acidcomposition was also higher in the R1 generation than in control material.Variation in both morphological and seed composition characters, how-ever, was not observed in the following R2 generation, probably because ofunstable epigenetic effects.

Stephens et al. (1991) observed a wrinkled leaf variant in the R2 genera-tion of a soybean line regenerated through organogenesis. Observations ofprogeny from selfed normal and variant derivatives of this line suggestedgenetic instability for this trait. Reciprocal crosses indicated that the mutanttrait was inherited cytoplasmically. The unusual segregation ratios wereattributed to organelle segregation and to cytoplasmic inheritance.

Amberger et al. (1992) regenerated 475 plants of nine soybeancultivars, via direct somatic embryogenesis from immature cotyledons. TheR1, R2 and R3 progeny from the regenerated plants were scored for qualita-tive variation and inheritance of variant phenotypes. These included partialsterility (R0, R1, R2), complete sterility (R0), abnormal leaf morphology (R0,R1, R2, R3) chlorophyll chimaeras (R2, R3), chlorophyll deficiencies (R2,R3), changes in growth habit (R2, R3), yellow edges on cotyledons (R3), nounifoliates (R3), dwarf plants (R2), yellow–green plants (R3) and isozymevariants (R2). Inheritance studies of chlorophyll-deficient, curled-leaf andwrinkled leaf plants confirmed that these traits were genetically controlled.Although none of the variants exhibited any obvious agronomicallyfavourable characteristics, the study resulted in the identification of novelvariants that may prove useful in the dissection of the soybean genome.New variants included a malate dehydrogenase null and an aconitase null(Amberger et al., 1992), curled leaves, lethal chlorophyll deficiencies, nounifoliates and yellow-edged cotyledons. Similarly, Stephens et al. (1991)observed an unusual segregation of wrinkled-leaf mutation that could beconsidered as a cytoplasmically inherited trait.

Mathews et al. (1986) regenerated mung bean (V. radiata) plants fromde-embryonated cotyledons. Considerable variation was observed in theR2 population, 7% of the R1 plant progenies segregating for chlorophyllmutations and another 7% for viable morphological mutations. The chlo-rophyll mutations included chlorina and xantha types, which were lethalunder field conditions. The viable mutations included those with penta-foliate leaves, sterility, and green seed coat and cotyledon colour. None ofthese mutants was found in the control population. The mutation rate per100 R2 plants was 1.8% for the chlorophyll and for the viable mutants.

Variation in tolerance to biotic and abiotic stressThere are only a few reports about the production of fertile regenerants ingrain legume crops, after in vitro selection using toxic culture filtrates(Table 6.4). The absence of reproducible de novo regeneration systems forthe majority of grain legume species initially led researchers to use the onlyavailable system, meristem or shoot tip culture. Gantotti et al. (1985)

Biotechnology 179




obtained regenerants of P. vulgaris resistant to phaseolotoxin, based on theselection of shoot meristems. Thynn et al. (1989) screened V. faba shootmeristem cultures after treatment with spore suspensions of Botrytis cinerea,Phytophthora megasperma and R. solani, based on the accumulation ofphytoalexins (wyeronic acid, wyerol, DH-wyerone, wyerone) in regeneratedplants. Only low to medium concentrations of phytoalexins were found inresistant regenerated plants from seven faba bean cultivars. The resistanceresponse was affected by the total amount and the ratio of individualphytoalexins. Song et al. (1994) obtained resistant regenerants of soybeanafter in vitro selection with a culture filtrate of Septoria glycines.

One of the most advanced studies of somaclonal variation in grainlegumes with respect to pathogen resistance was published by Jin et al.(1996). Embryogenic suspension cultures from four cultivars were treatedfor 1–2 months with a toxic culture filtrate of F. solani, a fungal diseasecausing sudden death syndrome (SDS). Selected suspensions, regeneratedvia somatic embryogenesis and fertile R0 plants, were obtained. R1 andR2 plants were then tested by artificial inoculation with the pathogen, ina controlled environment and in the greenhouse. Various degrees ofresistance were obtained compared with the resistant control variety.Additional studies, covering further seed generations, will be needed todetermine the stability/heritability of the generated resistance.

Venkatachalam et al. (1998) used a culture filtrate of Cercosporiumpersonatum (tikka leaf late spot disease) to repeatedly treat cotyledon calluscultures of groundnut (Arachis hypogaea). Plants were regenerated fromresistant calli that survived three cycles of selection. R2 seed progenies ofregenerated plants exhibited resistance to the pathogen in field conditions.

Wrather and Freytag (1991) selected soybean cotyledonary node plusepicotyl explants, on a medium containing 48 mg l−1 atrazine. Explantssurviving exposure to atrazine (34%) callused and regenerated shoots viaorganogenesis. Selection in vivo with atrazine-treated soil allowed R0, R1

and R2 atrazine-tolerant plants to be obtained. All non-atrazine selectedcontrol plants died when exposed to the same conditions. Atrazine-tolerantR2 plants appeared to be as healthy and vigorous as the control growing inatrazine-free soil. It was suggested that cytoplasmic inheritance (geneslocated on the chloroplast chromosome) might account for the alteredatrazine-tolerant phenotype.

Product quality changes (proteins, carbohydrates, lipids)Hildebrand et al. (1989) studied variation in fatty acids composition of R1

and R2 seed progenies from soybean plants regenerated via somaticembryogenesis. Variation in the R1 generation for fatty acid compositionwas higher, compared with the control population. In addition, someindividuals showed an unusual fatty acid composition. The progeny ofvariant plants, however, were normal and comparable to the control in theR2 generation.





Mikhailova-Krumova et al. (1991) studied seed proteins from R0 peaplants regenerated by organogenesis following primary and long-termcallus culture. Variation was found in a polypeptide legumin with a 37-kDamolecular weight (MW). With an increasing period of callus culture, thetotal amount of seed protein declined and the variation coefficients forlegumin and vicilin content in the total protein increased. Three sampleswere isolated with an increased legumin/vicilin ratio. In many regeneratedplants the amino acid balance was altered, which was mainly due to anincrease in the phenylalanine and a decrease in the methionine content.

When comparing organogenic and embryogenic somaclones ofpea line HM-6, increased variation in crude protein content (% dryweight) was recorded in regenerant progenies, compared with controlpopulations (Griga, unpublished results). The following data wereobtained in the R2 generation: embryogenic somaclones, mean 24.41% andrange 19.95–26.10%; organogenic somaclones, mean 24.04 and range20.59–27.02%; control population 1, mean 22.91 and range 21.94–23.69%;control population 2, mean 23.89 and range 23.09–25.43%. In the R3 gen-eration, a number of somaclones with significantly higher protein content(based on 95% confidence for means) were found within embryogenicsomaclones compared with the control. On the other hand, organogenicsomaclones exhibited more clones with significantly lower protein content.In the R4 generation, the differences between selected somaclones fromboth origins and from the control populations were less dramatic.

6.5 Transformation Methods in Grain Legumes

6.5.1 Introduction

Targeted genetic transformation of crop plants is a powerful recent com-plement to conventional breeding strategies. In the past decade there hasbeen a significant shift in genetic transformation experiments from the useof plant models to agronomically important crops, including grain legumes(Nisbet and Webb, 1990; Christou, 1992, 1997; Atkins and Smith, 1997).Similarly, as in other important crops (oilseed crops, corn, sugar-beet,potato, tomato), the genetic transformation in legumes was primarilydirected towards the incorporation of genes affecting cultivation (e.g.herbicide tolerance and pathogen resistance) and only later considerednutritional and postharvest product quality (Christou, 1997; James, 1997).There are some recent reports, therefore, on protein improvement in grainlegumes by genetic transformation (Falco et al., 1995; Pickardt et al., 1995;Molvig et al., 1997), but, as yet, few results related to carbohydrate quality/quantity improvement, comparable with those reported in potato (Visserand Jacobsen, 1993; Muller-Rober and Kossmann, 1994; Stark et al., 1996)and corn (James, 1997).

Biotechnology 181




The objective of this section is not to review completely all papersdealing with grain legume transformation, but to show, using selectedexamples, the technological progress in this field, as well as the majortrends towards crop improvement. The techniques discussed for grainlegume transformation, while dealing with traits/genes other thancarbohydrate metabolism, form a methodological background for futurecarbohydrate improvement of grain legumes via genetic transformation.

6.5.2 Gene delivery systems used in agronomically important legumes

Several gene delivery systems have been tested experimentally in grainlegume species. These approaches can be divided into gene transfer medi-ated by bacterial vectors (Agrobacterium tumefaciens, Agrobacterium rhizogenes)and direct gene transfer, comprising polyethylene glycol (PEG)-mediatedgene transfer to protoplasts, electroporation-mediated gene transfer toprotoplasts, biolistic plant transformation (particle bombardment), micro-injection into plant tissues, cells and protoplasts, and tissue (meristem)electroporation. The majority of the above-mentioned techniques need invitro culture and a reliable regeneration protocol. More recently, however,there has been a tendency to modify the transformation protocols byavoiding sophisticated tissue culture steps (Brar et al., 1994; Chowrira et al.,1995, 1996; McKently et al., 1995).

To date, some other transformation methods successfully used in othercrops, e.g., silicon fibre-mediated transformation, vacuum-infiltration ofDNA with intact plants and imbibition of dry seeds/embryos with DNA,have not resulted in complete transgenic plants in grain legumes (fordetailed descriptions of approaches mentioned above, see Gelvin andSchilperoort, 1995; Potrykus and Spangenberg, 1995; Galun and Breiman,1997). Of in vitro regeneration protocols studied during grain legumetransformation, de novo shoot organogenesis from primary explants/callus was more frequently used to obtain transformed regenerants, ratherthan somatic embryogenesis (Parrott et al., 1989, 1993; Ellis, 1995).

There are a number of papers reporting transformation ofproptoplasts, cells and calli in grain legumes (Nisbet and Webb, 1990;Christou, 1992; Atkins and Smith, 1997), however, the following text dealspredominantly with protocols that have allowed the successful productionof complete fertile transgenic plants in grain legume crops.

6.5.3 Methods giving positive results – transgenic plants

Agrobacterium-mediated gene transferAgrobacteria (A. tumefaciens, A. rhizogenes) are Gram-negative soil bacteriathat can infect many plant species (mainly dicotyledonous) and can serve as





natural vector systems. The foreign gene is artificially inserted into amodified bacterial plasmid (Ti or Ri) and, via natural infection, deliveredto the plant cell, where the plasmid T-DNA is integrated into the plantgenomic DNA. Once integrated, the genes transferred by Agrobacteriumhave been shown to be meiotically stable. Plant species subjected to trans-formation must be susceptible to the Agrobacterium strain used and mustshow an ability to regenerate in vitro from protoplasts, cells and tissues. Inspecial cases, however, regeneration can be eliminated by direct germina-tion of Agrobacterium-transformed embryo axes in an autoclaved soil mix(McKently et al., 1995). For the efficient selection of transformed cells ortissues, the appropriate selection conditions must be available. These maybe provided either by selectable marker genes (Schrott, 1995), which con-fer the resistance to some antibiotics (neomycin phosphotransferase, nptII;hygromycin phosphotransferase, hpt) and herbicides (phosphinotricinacetyl transferase, bar; 5-enolpyruvylshikimate-3-phosphate synthase, Epsp),or by reporter genes (Herrera-Estrella et al., 1995), which are codingsequences that provide a clear indication that genetic transformation hastaken place, for example, upon expression in the transgenic plant (Galunand Breiman, 1997). Such reporter genes are usually visual, for example,β-glucuronidase (GUS), luciferase and green fluorescent protein (GFP). Arecent improvement in the Agrobacterium-mediated transformation ofembryogenic suspension cultures by sonication has been reported (Trickand Finer, 1998).

Biolistic plant transformation (particle bombardment)Biolistics or biological ballistics, is the process by which biologicalmolecules (DNA, RNA) are accelerated, usually on microcarriers termedmicroprojectiles, by an explosive charge (gun powder or compressed gas),or by a high-voltage electric charge and shot into plant cells (Galun andBreiman, 1997). Plant species subjected to transformation using ballisticsshould have the ability to regenerate in vitro. As in Agrobacterium-mediatedtransformation, however, there have been successful attempts to producetransgenic shoots from bombarded meristems without in vitro culture(Brar et al., 1994). The efficient selection of transformed tissues basedon selectable marker genes and/or reporter genes is also necessary. Theadvantage of this method is that transformation can be carried outindependently of the variety used (Christou, 1995).

Electroporation and microinjectionIn this system DNA is electroporated into nodal meristems (treated buds),or directly injected into ovaries (Chowrira et al., 1995, 1996; Yue et al.,1996). Transgenic plants then can be recovered in the offspring of electro-porated or microinjected individuals. The advantage of these methods isthat they allow the production of transgenic legume plants without theneed for in vitro tissue culture.

Biotechnology 183




6.5.4 Transgenic plants and useful genes/traits transformed into grainlegumes

The number of successfully transformed grain legume species has beengrowing since 1988, when soybean transgenic plants were recovered as thefirst representative of grain legume crops (Hinchee et al., 1988). During thelast decade, there has been progress in the methodology and a shift awayfrom experiments using only marker and reporter genes, towards transfor-mation with agronomically useful genes. In the past few years, transgenicplants have been obtained in almost all economically important species ofgrain legumes (i.e. Phaseolus spp., P. sativum, A. hypogaea, C. arietinum, Vignaspp., Lens culinaris, Lupinus angustifolius L.). Soybean is the most advancedspecies within grain legumes with regard to genetic transformation and canbe considered as one of the general models for the biolistic approach(McCabe et al., 1988; Christou, 1992, 1995). The first reports of successfultransgenic plant production in particular grain legume species are summa-rized in Table 6.5. A list of the most useful genes/traits transformed intograin legumes and expressed on the plant level are given in Table 6.6 anddiscussed in more detail below.

Herbicide tolerance

GLYPHOSATE TOLERANCE Glyphosate (trivial name for α-phosphonomethylglycine, an active ingredient of the herbicide Roundup®) acts as aninhibitor of aromatic amino acid synthesis, by blocking shikimate bio-synthesis, the target enzyme being 5-enolpyruvylshikimate-3-phosphatesynthase (EPSP). Glyphosate is a non-selective herbicide that is not toxicto animals and is rapidly degraded in the soil (Galun and Breiman, 1997;Ondrej et al., 1998). Tolerance to glyphosate may be engineered by theincorporation of three types of transgenes. The first type, encodes for EPSP(from Petunia and Arabidopsis) and results in an overproduction of thetarget enzyme. Glyphosate is, therefore, fully saturated with EPSP but freeEPSP is still available to exhibit sufficient catalytic activity. The secondtype, is a mutated gene encoding a modified EPSP enzyme (mainly fromSalmonella typhimurium, Escherichia coli, Agrobacterium sp.), which is tolerantto glyphosate. The third type encodes glyphosate-oxido-reductase (GOX;e.g. from Achtomobacter), which can metabolize glyphosate. GOX is normallypresent in bacteria, but not in plants.

The mutated EPSP gene from petunia, together with the gene forkanamycin resistance and GUS, have been used for soybean transformationby Hinchee et al. (1988). Using this system, approximately 6% of the shootsregenerated by de novo organogenesis from seedling cotyledons wereproved to be transformed. Padgette et al. (1995) demonstrated that thetransgene for glyphosate tolerance behaved as a single dominant gene andwas stable over several generations of soybean field trials. A very important





finding was that glyphosate treatment of a field tested transgenic soybeanline grown in several locations did not significantly reduce yield (Delannayet al., 1995).

PHOSPHINOTHRICIN (PPT) TOLERANCE Phosphinothricin (syn. gluphosinate;chemically 4-[hydroxy-(methyl) phosphinoyl]-D,L-homoalanin) is ananalogue of glutamine and acts as an herbicide by inhibiting glutaminesynthesis, following an irreversible inactivation of glutamine synthase.It is widely used as a commercial preparation and may be called Basta®,Liberty®, Bialophos®, Herbiace®, Buster®, Finale® or Radicale® (Galunand Breiman, 1997; Ondrej et al., 1998). Two bacterial genes (bar fromStreptomyces hygroscopicus and pat from Streptomyces viridochromogenes) have theability to detoxify PPT by acetylation. In grain legumes, the bar/pat geneshave been used mainly as selectable markers, e.g. in pea transformation(Schroeder et al., 1993; Grant et al., 1995; Bean et al., 1997; Simonenko et al.,1999). In addition to detecting the presence of the bar gene by Southernanalysis and the PAT assay, leaf paint and spray tests have been carried outon transgenic pea plants and their seed progenies.

Schroeder et al. (1993) found complete and partial tolerance of leavesfrom pea transformants treated with a dose equivalent to 10 l ha−1 Basta ®.At this concentration the control (untransformed leaves) becamecompletely necrotic. Fourteen days after spraying whole pea plants witha dose equivalent to 7 l ha−1 Basta®, transgenic plants showed no symptomsof herbicidal damage and grew normally to maturity, whereas the non-transgenic plants were killed. Grant et al. (1995) found that R1 progeny(first seed generation) containing the gene showed variable resistanceto Buster®. From plants that gave a resistant leaf test at the equivalentconcentration of 10 l ha−1 Buster®, to those that showed susceptibilityat 3 l ha−1 Buster® equivalent. According to Bean et al. (1997), pea trans-formants showing no signs of herbicide damage 3 days after spraying with3 mg l−1 Herbiace® could be classed as clonal transformants, whereas thoseshowing varying combinations of green and brown tissue were categorizedas chimaeras.

ATRAZINE TOLERANCE Herbicides of the S-triazine type (atrazine, simazine)inhibit photosynthesis by binding to the chloroplast thylakoid membraneprotein, resulting in electron transport being blocked in photosystem II.The mode of resistance is to change the target site, i.e. the protein encodedby the chloroplast gene psbA (Galun and Breiman, 1997; Ondrej, 1998). Fuet al. (1993) reported field-testing of F4 and F5 soybean plants transformedfor atrazine resistance. Atrazine treatment at the pre-emergence, or at theseedling stage, did not adversely affect the plant yield, which was the same,or even higher, than that of the unsprayed, non-transformed controls.Yue et al. (1996) transformed 24 soybean varieties by direct injection ofDNA, containing the gene for atrazine resistance, through the ovary and

Biotechnology 185






Spec

ies

Gen

e/tr

aiti

ncor

pora

ted

Tran

sfor

mat

ion

met

hod

Ref

eren

ces

Gly

cine

max

5-En

olpy

ruvy

l-sh

ikim

ate-

3-ph

osph

ate

synt

hase

(EPS

P);h

erbi

cide

tole

ranc

e(g

lyph

osat

e)–

Rou

ndup

®A

grob

acte

rium

Hin

chee

etal

.(19

88)

Phos

phin

otri

cin

acet

yltr

ansf

eras

e(P

AT,

bar)

;her

bici

deto

lera

nce

(PPT

)–

Bas

ta®

Bom

bard

men

tC

hris

tou

and

Swai

n(1

990)

Lysi

ne-f

eedb

ack-

inse

nsiti

veba

cter

ialD

HD

PS/d

apA

and

AK

/lysC

;fiv

efol

dly

sine

incr

ease

inth

ese

edB

omba

rdm

ent

Falc

oet

al.(

1995

)

Met

hion

ine-

rich

2Sal

bum

infr

omB

razi

lnut

;hig

hol

eic

acid

;mod

ified

oil;

viru

sre

sist

ance

Agr

obac

teri

umbo

mba

rdm

ent

Jam

es(1

997)

Synt

hetic

cryI

Ac

gene

(B.t.

)–re

sist

ance

toin

sect

larv

ae(fo

ursp

ecie

s)B

omba

rdm

ent

Stew

arte

tal.

(199

6)A

traz

ine

resi

stan

ce(p

sbA

)D

irec

tinj

ectio

nof

DN

Ain

toov

ary

Yue

etal

.(19

96)

Bea

npo

dm

ottle

viru

s(B

PMV

)coa

tpro

tein

gene

–vi

rus

resi

stan

ceA

grob

acte

rium

Die

tal.

(199

6)So

ybea

nm

osai

cvi

rus

(SM

V)c

oatp

rote

inge

ne–

viru

sre

sist

ance

Agr

obac

teri

umX

uet

al.(

1996

)

Phas

eolu

svu

lgar

isC

oatp

rote

infr

omth

ebe

ango

lden

mos

aic

viru

s(B

GM

V)–

viru

sre

sist

ance

;bar

Bom

bard

men

tR

usse

llet

al.(

1993

)

Ant

isen

sese

quen

ceof

AC

1,A

C2,

AC

3an

dB

C1

gene

sfr

ombe

ango

lden

mos

aic

gem

iniv

irus

–vi

rus

resi

stan

ce;m

ethi

onin

e-ri

ch2S

albu

min

from

Bra

ziln

ut

Bom

bard

men

tA

rago

etal

.(19

96)

Tabl

e6.

5.U

sefu

lgen

es/tr

aits

tran

sfor

med

into

grai

nle

gum

es.



Biotechnology 187


Pisu

msa

tivum

α-am

ylas

ein

hibi

tor

from

com

mon

bean

(αA

I-Pv

)–re

sist

ance

toth

ree

spec

ies

ofbr

uchi

dbe

etle

s;ba

r–

phos

phin

othr

icin

resi

stan

ce(B

asta

®,

Bus

ter®

,Her

biac

e®)

Agr

obac

teri

umSh

ade

etal

.(19

94);

Schr

oede

ret

al.(

1995

);G

rant

etal

.(19

95);

Bea

net

al.(

1997

)H

eat-

stab

leam

ylas

efr

omB

acill

uslic

heni

form

is,i

mpr

oved

seed

star

chde

grad

atio

nA

grob

acte

rium

Saal

bach

etal

.(19

97)

Chi

mae

ric

alfa

lfam

osai

cvi

rus

(AM

V)c

oatp

rote

inge

ne–

part

ial

resi

stan

ceto

AM

VA

grob

acte

rium

Gra

ntet

al.(

1998

a,b)

Cic

erar

ietin

umC

rylA

(c)g

ene

from

B.t

huri

ngie

nsis

(res

ista

nce

topo

d-bo

rer

larv

ae)

Bom

bard

men

tK

aret

al.(

1997

)V

icia

narb

onen

sis

Met

hion

ine-

rich

2Sal

bum

infr

omB

razi

lnut

(thre

efol

din

crea

seof

seed

met

hion

ine

cont

ent)

Agr

obac

teri

umPi

ckar

dtet

al.(

1995

)

Hea

t-st

able

amyl

ase

from

Bac

illus

liche

nifo

rmis

,im

prov

edse

edst

arch

degr

adat

ion

Agr

obac

teri

umSa

alba

chet

al.(

1997

)

Yea

stin

vert

ase,

chan

ges

inre

gula

tion

ofca

rboh

ydra

tean

dpr

otei

nm

etab

olis

mdu

ring

coty

ledo

nde

velo

pmen

tA

grob

acte

rium

Web

eret

al.(

1998

)

Lupi

nus

angu

stifo

lius

Sulp

hur-

rich

sunf

low

erse

edal

bum

in(e

nhan

ced

met

hion

ine

leve

lin

the

seed

),ba

r–

phos

phin

othr

icin

resi

stan

ce(B

asta

®)

Agr

obac

teri

umM

olvi

get

al.(

1997

);Pi

geai

reet

al.(

1997

)





Spec

ies

Expl

ant

Tran

sfor

mat

ion

met

hod

Reg

ener

atio

npr

otoc

olR

efer

ence

s

Gly

cine

max

Seed

ling

coty

ledo

nsA

grob

acte

rium

Org

anog

enes

isH

inch

eeet

al.(

1988

)Pi

sum

sativ

umSh

ootc

ultu

res

epic

otyl

sA

grob

acte

rium

Org

anog

enes

isPu

onti-

Kae

rlas

etal

.(19

90)

Phas

eolu

svu

lgar

isEm

bryo

axis

Bom

bard

men

tO

rgan

ogen

esis

Rus

sell

etal

.(19

93)

Ara

chis

hypo

gaea

Embr

yoge

nic

callu

sB

omba

rdm

ent

Som

atic

embr

yoge

nesi

sO

zias

-Aki

nset

al.(

1993

)C

icer

arie

tinum

Seed

-der

ived

embr

yos

Agr

obac

teri

umO

rgan

ogen

esis

Font

ana

etal

.(19

93)

Vic

iana

rbon

ensi

s(n

arbo

nbe

an,F

renc

hve

tch)

Epic

otyl

segm

ents

Agr

obac

teri

umSo

mat

icem

bryo

gene

sis

Pick

ardt

etal

.(19

95)

Lens

culin

aris

Nod

alm

eris

tem

sin

plan

taEl

ectr

opor

atio

nin

vivo

Seed

prod

uctio

nw

ithou

tin

vitr

ocu

lture

Cho

wri

raet

al.(

1995

)

Vig

naun

guic

ulat

aM

atur

eco

tyle

dons

Agr

obac

teri

umO

rgan

ogen

esis

Mut

huku

mar

etal

.(19

96)

Lupi

nus

angu

stifo

lius

Mat

urin

gem

bryo

axis

Agr

obac

teri

umO

rgan

ogen

esis

Pige

aire

etal

.(19

97)

Tabl

e6.

6.Tr

ansg

enic

plan

tsin

grai

nle

gum

es.



confirmed the integration and normal inheritance of the introducedgene. There was no seedling injury when F2 plants of one variety with theintroduced gene were sprayed with atrazine in the field prior to seedlingemergence. Ninety-two per cent of the control (non-transformed) plantswere killed by the same treatment.

Insect resistanceThere are two general strategies for genetically engineered insect resis-tance in plants. Firstly, the incorporation of genes from specific bacteria,which encode proteins that are toxic to insects. Secondly, the incorporationof genes encoding plant-derived inhibitors of protein, or carbohydrate,digestion in insects (Galun and Breiman, 1997).

BACILLUS THURINGIENSIS ENDOTOXINS Upon sporulation, B. thuringiensis (B.t.)strains produce protein crystals containing ∆-endotoxin, which cause thelysis of epithelium cells in the midgut of insect larvae. These toxins areharmless to mammals and birds and exhibit a limited range of toxicity tospecific groups of insects. The latter criterion is used for clarifying B.thuringiensis strains, those producing CryI are toxic to Lepidoptera, CryIII toColeoptera and CryIV to Diptera.

Within the grain legumes, soybean and chickpea have been successfullytransformed with B.t. ∆-endotoxin genes (Parrott et al., 1994; Stewart et al.,1996; Kar et al., 1996). Stewart et al. (1996) transformed soybean somaticembryos using particle bombardment with a synthetic B.t. CryIAc genelinked to a hygromycin-resistance gene. Three transgenic lines wereselected on hygromycin-containing media and grown into fertile plants.When the transgenic plants were tested, they were found to be protectedfrom damage by larvae of four lepidopteran species, corn earworm(Helicoverpa zea), soybean looper (Pseudoplusia includens), tobacco budworm(Heliothis virescens), and velvet bean caterpillar (Anticarsia gemmatalis).Transgenic plants exhibited less than 3% defoliation upon corn earwormattack, compared with 20% for a lepidopteran-resistant breeding line andmore than 40% for susceptible soybean cultivars. A chimaeric, truncatedbacterial CryIA(c) gene construct, with the nptII gene as a selection marker,was inserted into the embryo axis of mature chickpea seed by particle bom-bardment (Kar et al., 1996). Transgenic kanamycin-resistant plants wereobtained through multiple shoot formation and repeated selection of thebombarded explants. An insect feeding assay indicated that the expressionof CryIA(c) gene was inhibitory to damage by larvae of Heliothis armigera.

AMYLASE INHIBITORS Seeds of the common bean (P. vulgaris) are naturallyresistant to bruchid beetles (e.g. cowpea weevil, Callosobruchus maculatus),because of the presence of an α-amylase inhibitor (αAI-Pv), a seed proteinthat is toxic to the larvae. Other legumes (e.g. pea, chickpea, cowpea, Azukibean), however, do not contain this α-amylase inhibitor-derived tolerance.

Biotechnology 189




Shade et al. (1994) and Schroeder et al. (1995) transferred the gene encod-ing αAI-Pv into pea, using Agrobacterium-mediated transformation basedon the co-cultivation of embryonic axis segments and regeneration viaorganogenesis (Schroeder et al., 1993). The α-amylase inhibitor gene wasstably expressed in the transgenic pea seeds up to the T5 seed generation.αAI-Pv accumulated in the pea seeds up to 3% of the soluble protein, a levelthat was higher than that normally found in beans (1–2%). In the T5 seedgeneration, the development of pea weevil (Bruchus pisorum) larvae wasblocked at an early stage. Seed damage was minimal and seed yield was notsignificantly reduced in the transgenic plants. In addition to the resistanceof transgenic pea plants to the bruchid beetles attacking the crop growingin the field (B. pisorum), the transformed peas also showed complete resis-tance to bruchid species that damage stored seeds, in particular cowpeaweevil (C. maculatus) and Azuki bean weevil (Callosobruchus chinensis; Shadeet al., 1994). Although αAI-Pv also inhibits human α-amylase, it is reportedthat cooked peas should not have a negative impact on human energymetabolism.

Dillen et al. (1997) transformed Phaseolus acutifolius with agenomic fragment encoding the P. vulgaris arcelin-5a-protein, using anAgrobacterium-mediated approach. It is believed that this seed storageprotein confers resistance to the insect Zabrotes subfasciatus, a major pestof P. vulgaris. Arcelin-5a was produced at high levels in the seeds and theauthors suggest using of P. acutifolius as a ‘bridging’ species to introducetransgenes into the economically more important species P. vulgaris.

Virus resistanceVirus coat protein-mediated resistance, based on the concept of ‘crossprotection’ and antisense-RNA derived resistance, has been reported forgrain legumes. The concept of cross protection is derived from the fact thatthe infection of a given crop plant with mild strains of viruses and viroidsprevents or reduces the symptoms caused by a subsequent virulent strain.Rather than using the whole virus, however, only part of the viral genome,encoding the coat protein (CP), is integrated into the plant genome. Theantisense RNA (a mirror sequence of an mRNA sequence) may provideviral tolerance by interfering either with the translation of viral mRNAs, orwith the replication of the viral genome (Greenberg and Glick, 1993; Galunand Breiman, 1997).

Di et al. (1996) transformed soybean, using an Agrobacterium-mediatedapproach, with a bean pod mottle virus (BPMV) CP-gene and found that30% of the R2 plants derived from one transgenic line were resistantto BPMV infection. Similarly, Xu et al. (1996) obtained A. rhizogenes trans-formed transgenic soybean plants with the integrated gene for soybeanmosaic virus (SMV) coat protein. Arago et al. (1996) introduced theantisense sequence of AC1, AC2, AC3 and BC1 genes, from the beangolden mosaic geminivirus, to P. vulgaris transgenic plants, using particle





bombardment. Grant et al. (1998a,b) obtained transgenic pea plants withpartial resistance to the alfalfa mosaic virus (AMV), using A. tumefacienstransformation with chimaeric AMV coat protein gene.

Nutritional qualityGalun and Breiman (1997) categorized nutritional quality of crops intofour groups: fatty acids, lipids and oils; carbohydrates; proteins and pig-mentation. Within grain legumes, most reports relate to the improvementof oils and proteins by genetic transformation. Weber et al. (1998), how-ever, has reported changes in carbohydrate metabolism in transgenic Vicianarbonensis.

In proteins, the strategy has concentrated mainly on improvingamino acid composition, in particular, the methionine (Met) and lysine(Lys) content. Saalbach et al. (1994) transformed V. narbonensis, a closerelative of faba bean (V. faba), with the methionine-rich 2S albumin gene ofthe Brazil nut (Bertholletia excelsa). This synthetic gene, controlled by theCaMV 35S promoter, however, was highly expressed only in leaves androots and was hardly detectable in the seeds. Later, the transformationprotocol was improved by fusing the 2S albumin gene with the seed-specificlegumin B4 promoter from V. faba (Pickardt et al., 1995; Saalbach et al.,1995a,b). Transformation was carried out using the Agrobacterium-mediatedapproach together with a regeneration protocol using somatic embryo-genesis from callus. Transformed calli were selected for kanamycinresistance and the induced somatic embryos were screened for GUS activityand cloned by multiple shoot regeneration. Fertile R0, R1 and R2 transgenicplants were obtained and seed-specific gene expression was found intransformants with the legumin B4 promoter/2S albumin gene fusion.Analysis of the R2 plants indicated a Mendelian inheritance of the 2Salbumin gene. Some transformants exhibited a threefold increase in themethionine content of the salt-soluble protein fraction extracted fromseeds. The same gene was introduced, using the biolistic process, intoP. vulgaris and stable transgenic bean plants were generated (Arago et al.,1996). Again the gene was inherited in a Mendelian fashion in most of thetransgenic bean lines.

Falco et al. (1995) increased the lysine content in soybean seeds bycircumventing the normal feedback regulation of two enzymes of the bio-synthetic pathway, aspartokinase (AK) and dihydropicolinic acid synthase(DHDPS). Lysine-feedback-insensitive bacterial DHDPS and AK enzymes,encoded by the Corynebacterium dapA gene and a mutant E.coli lysC gene,respectively, were expressed in transgenic soybean seeds, following trans-formation via particle bombardment and by fusion with the GUS reportergene. The result was a ten- to several hundredfold increase in free lysineand up to a fivefold increase in the total seed lysine content, lysine contrib-uting up to 33% of the total seed amino acid content. The lysine content inR2 and R3 seeds remained at least as high as that observed in the R1 seed,

Biotechnology 191




demonstrating heritability of the trait. Seeds of soybean transformants (R1)with greatly increased lysine levels had a wrinkled shape and germinationwas poor. List et al. (1996) analysed oils from genetically modified soybeans.Compared with common varieties containing 15% saturated fatty acids,genetically modified soybeans yielded oils with 24–40% saturated acids.

Using an Agrobacterium-mediated approach Molvig et al. (1997) stablytransformed narrow-leafed lupin (L. angustifolius) with a chimaeric genespecifying seed-specific expression of a sulphur-rich, sunflower seed albu-min (SSA). The transgenic seeds contained less sulphate and more totalamino acid sulphur than the non-transgenic parent line. A 94% increase inthe methionine content and a 12% reduction in cysteine content wasrecorded. There was no statistically significant change in other amino acids,or in the total nitrogen and total sulphur contents of the seeds. In feedingtrials with rats, the transgenic seeds gave statistically significant increases inlive weight gain, true protein digestibility, biological value and net proteinutilization, compared with wild-type seeds.

Weber et al. (1998) transformed V. narbonensis with the yeast invertasegene under the control of the legumin B4 promoter. Expression ofthe legumin B4-promoter yeast invertase gene in transgenic cotyledonsresulted in a reduction of sucrose, starch and protein contents and anincrease in the hexose level.

6.5.5 Field trials with transgenic grain legume plants and commercializedtransgenic legume crops

During the period 1986–1997, c. 25,000 transgenic crop field trials wereconducted in 45 countries, on more than 60 crops covering 10 traits.Seventy-two per cent of all transgenic crop field trials were conducted in theUSA and Canada followed in descending order by Europe, Latin Americaand Asia, with a few conducted in Africa. The most frequent crops in thesetrials were maize, tomato, soybean, canola, potato and cotton and the mostfrequent traits were herbicide tolerance, insect resistance, product qualityand virus resistance (James, 1997). Recently, a great deal of progress hasbeen achieved in the transformation and in the commercialization of coolseason legume species in Australia (Atkins et al., 1998; Hamblin et al., 1998).Four co-operating laboratories/companies are now able to transformseven legume species (Lupinus angustifolius, Lupinus albus, Lupinus luteus,C. arietinum, P. sativum, V. faba and L. culinaris), almost all of which havebeen field tested (Table 6.7).

The situation with commercialized transgenic crops in 1996 and 1997 isshown in Tables 6.7, 6.8 and 6.9. From the data it is evident that soybean isthe only grain legume representative within recently commercializedtransgenic crops. On the other hand, soybean, in particular herbicide-tolerant varieties, has a leading position in this context. The progress in





commercialization of transgenics in other legume crops (pea, lupin,chickpea and lentil) compared with the most advanced transgenic cropsis illustrated in Table 6.7.

6.5.6 Future prospects

Based on the results of basic research and on the application of DNArecombinant technology in crop improvement in the last 10–15 years, itis evident that plant genetic transformation has obtained a permanentposition in complementing conventional plant breeding. The absence ofgenetic modification of carbohydrates in grain legumes can be explainedlogically by their lower industrial importance compared with carbo-hydrates, in particular starches, from cereals and potato. If the basic

Biotechnology 193


Crop Traits already commercialized Traits in field trials/development

Canola 1. Herbicide tolerance 1. Improved disease resistance2. Hybrid technology 2. Improved disease resistance3. Hybrid technology andherbicide tolerance

Maize 1. Control of corn-borer 1. Control of Asian corn-borer2. Herbicide tolerance 2. Control of corn rootworm3. Insect protected/herbicidetolerance

3. Disease resistance

4. Hybrid technology 4. Higher starch content5. Hybrid/herbicide tolerance 5. Modified starch content

6. High lysine7. Improved protein8. Resistance to storage grain pests9. Apomixis

Soybean 1. Herbicide tolerance 1. Modified oil2. High oleic acid 2. Insect resistance

3. Virus resistancePea None 1. Insect resistance – pea weevil

2. Quality – sunflower albumin3. High methionine protein4. Antifungal genes

Lupin None 1. Insect resistance – pea weevil2. Virus resistance (BYMV)3. Herbicide tolerance4. High methionine protein

Chickpea None 1. Herbicide tolerance2. High seed protein

Lentil None 1. Herbicide tolerance

Table 6.7. Traits already commercialized in field trials, and under developmentfor selected crops, 1997–1998 (James, 1997; Hamblin et al., 1998).



mechanisms of starch biosynthesis are similar in various crops, then successwith the genetic manipulation of the starch synthesis pathway in potato(Stark et al., 1992, 1996; Visser and Jacobsen, 1993; Muller-Rober andKossmann, 1994) may represent a background and a promise for similarwork in grain legumes. The methodological base (gene transfer systems aswell as regeneration systems) for such oriented research is now available inmajor grain legume species.

At least three aspects will surely play a future role in the deeperinvolvement of DNA technology in the improvement of the compositionand quality of grain legume carbohydrates. Firstly, there will need to be adeeper understanding of the metabolic processes involved in carbohydratebiosynthesis and degradation, including the exact identification of genesresponsible for specific metabolic steps. Secondly, there needs to be furtherimprovement of gene transfer methods, including efficient regenerationsystems. Thirdly, there needs to be economic interest in the improvementof carbohydrates in legume crops. Long-term transformation projects willnot be started without the interest of industry in a potentially profit-makingproduct. This last point can be further substantiated by the fact that of



1996 1997Increase

1996/7 ratioCrop Area % Area %

Soybean 0.5 18 5.1 40 10.0Maize 0.3 10 3.2 25 11.0Tobacco 1.0 35 1.6 13 1.6Cotton 0.8 27 1.4 11 1.8Canola 0.1 5 1.2 10 9.5Tomato 0.1 4 0.1 1 2.0Potato < 0.1< < 1< < 0.1< < 1< 3.0Total 2.8 100 12.8 100 4.5

Table 6.8. Global area (millions of hectares) of transgenic crops grown in 1996and 1997, by crop (James, 1997).

1996 1997Increase

1996/7 ratioTrait Area % Area %

Herbicide tolerance 0.6 23.5 6.9 54 10.7Insect resistance 1.1 37.5 4.0 31 3.8Virus resistance 1.1 40.5 1.8 14 1.6Insect and virus resistance – – < 0.1< < 1< –Quality traits < 0.1< < 0.1< 0.1 < 1< 2.0Total 2.8 100.5 12.8 100 4.5

Table 6.9. Global area (millions of hectares) of transgenic crops grown in 1996and 1997, by trait (James, 1997).



22 technology proprietors that approved 48 transgenic crop products forcommercialization in 1997, 20 (90%) were private corporations and onlytwo (10%) public sector organizations (James, 1997).

6.6 The Availability and Possible Manipulation of GenesInvolved in Starch Biosynthesis

6.6.1 Biochemical pathways of starch biosynthesis

The chemistry and physical structure of starches have been discussedelsewhere (see Chapters 2 and 4). Likewise, a more detailed description ofstarch biosynthesis is described in Chapter 5. For the purpose of discussingthe possibilities of using biotechnology to modify starch, however, it is nec-essary to have some basic information on the biochemical process involved.The biosynthetic pathway of starch synthesis involves three main enzymesystems ADP-glucose pyrophosphorylase (ADPGPPase), starch synthases(SS) and starch branching enzymes (SBE; for review see Martin and Smith,1995).

Amylose and amylopectin are synthesized from ADP-glucose, which issynthesized from glucose-1-phosphate and ATP in a reaction that is catalysedby ADPGPPase. The glucose-1-phosphate can be supplied from the Calvincycle, in photosynthetic tissues, or may be imported directly from thecytosol (Tyson and ap Rees, 1988) and/or synthesized from glucose-6-phosphate (Hill and Smith, 1991) in storage tissues. Most of the glucose-6-phosphate in storage tissues is formed from sucrose, which is the maintransported carbohydrate in plants (ap Rees and Morrell, 1990). Incomingsucrose is cleaved by sucrose synthase to form UDP-glucose and fructoseand the UDP-glucose is then converted to glucose-1-phosphate by the actionof UDP-glucose pyrophosphorylase (UGPase). The fructose is phosphory-lated to fructose-6-phosphate by fructokinase and possibly by hexokinase(Renz and Stitt, 1993). The hexose monophosphates are freely intercon-verted by the action of phosphoglucomutase and phosphoglucoisomerase.The carbon then enters the amyloplast at the level of hexose monophos-phates (Viola et al., 1991), where ADPGPPase starts starch biosynthesis.

6.6.2 The availability of genes involved in starch biosynthesis

Plant ADPGPPase is reported to be a tetrameric enzyme that is formedfrom two distinct polypeptides (Copeland and Preiss, 1981). The cDNAclones encoding both subunits have been isolated from wheat (Olive et al.,1989), maize (Bhave et al., 1990), potato (Mueller-Rober et al., 1990),barley, (Villand et al., 1992), arabidopsis (Villand et al., 1993) and fababean (Weber et al., 1995a). This enzyme is allosterically regulated by

Biotechnology 195




3-phosphoglycerate (3-PGA) as an activator and by inorganic phosphate(Pi) as an inhibitor. It seems that this enzyme has a key role in starchbiosynthesis, since the genetic manipulation of ADPGPPase genes clearlydemonstrates the influence of this enzyme on starch yield in transgenicplants. For example, the inhibition of the ADPGPPase gene activity byantisense regulation (Mol et al., 1990) led to a near-complete inhibition ofstarch synthesis. A small (20–50%) decrease in the activity of this gene,however, did not result in a proportional decrease in starch content(Mueller-Roeber et al., 1992). Also, the tuber-specific expression of theglgC-16 gene encoding E. coli ADPGPPase, active in the absence ofallosteric activation, led to a 30% increase of starch content in cv.Russet Burbank potatoes, whereas the over-expression of wild-type E. coliADPGPPase had no significant effect (Stark et al., 1992).

In the next step of starch biosynthesis, the starch synthases (SS) catalysethe synthesis of α(1→4) linkages between the pre-existing glucan chain andthe glucosyl part of ADP-glucose, in the synthesis of both amylose andamylopectin. Starch synthases are present in different isoforms, which arelocated both bound to the starch granule and in the soluble phase ofthe amyloplast. After biochemical analysis of amylose-free (waxy) mutantsfrom several species, it was suggested that the granule-bound SSs (GBSS)synthesize amylose, whereas the soluble SSs synthesize amylopectin.

Amylose-free starch could be obtained, therefore, by the manipulationof gene(s) encoding the granule-bound starch synthase. Expression of anantisense-RNA that inhibits the granule-bound starch synthase has yieldedpotato starch composed only of amylopectin (Visser et al., 1991; Kuiperset al., 1994). Reductions in soluble starch synthase activity of 70–80% by theantisense approach, however, had no measurable effect on the starchcontent, or on the amylose/amylopectin ratio of transgenic potato tubers(Marshall et al., 1996), although a profound change in the morphology ofstarch granules was detected. Transgenic potatoes have been generated, inwhich the activities of both main soluble starch synthases responsible foramylopectin synthesis in the tuber (SSII and SSIII) have been reduced(Edwards et al., 1998). The properties of starch from tubers of these plantshave been compared with those of starches from transgenic plants in whichthe activity of either SSII or SSIII has been reduced. Starches from the threetypes of transgenic plants are qualitatively different from each other andfrom the starch of control plants, with respect to granule morphology,the branch lengths of amylopectin, and the gelatinization behaviour. Theeffects of reducing SSII and SSIII together could not be predicted fromthe effects of reducing these two isoforms individually.

The α(1→6) branches in starch polymers are made by starch-branching enzymes (SBE), which are also present in multiple isoforms. Thegeneric effect of SBE is to hydrolyse an α(1→4) linkage within a chain andthen catalyses the formation of an α(1→6) linkage between the reducingend of the glucan chain and another glucose residue. Two SBE families, A





and B, have been identified in maize (Stinard et al., 1993), rice (Mizunoet al., 1993) and pea (Burton et al., 1995). The proteins of these two familiesare structurally related and are similar to glycogen-branching enzymesfrom E. coli bacteria, which have been used to produce highly branchedstarch in transformed potato (Shewmaker et al., 1994).

In the embryos of peas containing the r mutation (see Chapter 7),the percentage of amylopectin is reduced from about 65% to about 35%.This relative decrease in amylopectin has been associated with decreasedbranching activity and the loss of one branching enzyme isoform (Smith,1988). Antisense experiments inhibiting isoform B activity in potato,however, resulted in no significant modification of starch synthesis, or ofthe amylose : amylopectin ratio (Kossmann et al., 1991; Mueller-Roeber andKossmann, 1994).

Recently, different lines of transgenic potatoes have been generatedwhere the expression of starch biosynthetic genes has been repressed, usingan antisense RNA technique. In this way plants have been obtained thatsynthesize a wide variety of structurally modified starches. These starchesare currently being assessed for their applicability in different industrialprocesses in the food and non-food sectors (Kossmann et al., 1997).

6.6.3 The availability of other genes influencing starch biosynthesis andstarch quality

It remains unclear whether all enzymes and proteins involved in deter-mining starch structure have been characterized. The phosphorylation ofstarch, which occurs to a greater extent in starches derived from vegetativestorage organs like potato tubers and also starches from other sources(Jane et al., 1996), is not associated with the catalytic activity of the starchbiosynthetic enzymes described above. A gene involved in the incorpora-tion of phosphate into starch-like glucans has been cloned and incorpo-rated into transgenic potato plants. This results in the production of starchwith a reduced phosphate content (Lorberth et al., 1998). This reducedphosphate content drastically influences the pasting properties of thestarch. It also results in some secondary effects on the transgenic plants,which have excess starch in their leaves and a reduction in cold sweeteningin their tubers.

Sink strength of storage organs can also contribute to starch accumula-tion, sink strength being defined as the ability of an organ to attractphotoassimilates (Ho, 1988). It is dependent on the transport mechanismand on the physical and biochemical isolation of the transported carbonin the sink tissue. As sucrose is the major transport form of fixed photo-assimilates into the storage organs of major crop plants, sucrose meta-bolism is of particular interest. Two classes of sucrose cleavage enzymes,invertase and sucrose synthase, are present in plants.

Biotechnology 197




Sucrose synthases (Susy, UDP-glucose: D-fructose-glycosyl-transferases)catalyse reversible reactions associated with storage functions, suchas starch synthesis (Heim et al., 1993). On the other hand, invertases(β-fructofuranosidases) catalyse the irreversible cleavage of sucrose intoglucose and fructose. Genes of faba bean (Heim et al., 1993) and potato(Zrenner et al., 1995) sucrose synthase, as well as faba bean (Weber et al.,1995b) and maize invertases (Xu et al., 1996) have been cloned and couldbe used to study the influence of the enzymes on carbohydrate accumula-tion in legume species. The size of potato tubers was increased by over-expression of yeast invertase specifically in the cytosol of tubers. There was a10–15% reduction in the starch content of mature tubers of these plants,however, plus a large increase in the quantity of glucose (Sonnewald et al.,1997). It was thought that starch accumulation could be improved byincreasing the glucose phosphorylating capacity of the invertase-expressingtubers. The additional expression of another transgene encoding aglucokinase from Zymomonas mobilis, in combination with an invertase gene,however, led to a dramatic reduction in starch accumulation and astimulation of glycolysis (Trethewey et al., 1998). Weber et al. (1998)transformed V. narbonensis with the yeast invertase gene, under the controlof the legumin B4-promoter. Expression of the legumin B4-promoter yeastinvertase gene in transgenic cotyledons resulted in a reduction of sucrose,starch and protein contents and an increase in the hexose level.

Pyrophosphate (PPi) and inorganic phosphate (Pi) are inhibitors ofmany biosynthetic pathways leading to carbohydrate accumulation. Totest whether sucrose and starch biosynthesis can be accelerated by removalof PPi, the ppa gene for pyrophosphatase from E. coli was introduced intotobacco and potato under control of the constitutive 35S-promoter. Thisresulted in a small shift of partitioning in favour of sucrose and a reductionin starch content (Sonnewald, 1992).

Although most of the information presented above has been obtainedfor plant species other than grain legumes, the modification of the bio-synthesis and the quality of starch within legume species would be possibleby making use of the cloned genes.

6.7 The Availability and Possible Manipulation of GenesInvolved in a-Galactoside Accumulation and Degradation

6.7.1 Biochemical pathways of a-galactoside biosynthesis

The chemical, biochemical and nutritional aspects of the raffinose oligosac-charides are covered elsewhere (see Chapters 2, 3 and 5). As with the sec-tion on starch, it is necessary to present information on these compoundshere to make it easier to discuss their possible genetic manipulation.





The biosynthesis of the raffinose family of oligosaccharides (RFO) hasbeen fairly well characterized. The committed reaction of raffinose bio-synthesis involves the synthesis of galactinol from UDP-galactose andmyo-inositol. The enzyme that catalyses this reaction is galactinol synthase(GS). The synthesis of raffinose and higher RFO homologues from sucroseis thought to be catalysed by distinct galactosyltransferases (for example,raffinose synthase and stachyose synthase). Studies with many species,however, suggest that GS is the key enzyme controlling the flux of reducedcarbon into the RFO biosynthetic pathway. The GS enzyme has beenpurified and characterized from courgette, the purified enzyme being amonomer of Mr 42,000 with an isoelectric point of 4.1 (Smith et al., 1991).

6.7.2 The availability of genes involved in a-galactoside accumulation anddegradation and their possible manipulation

There are some biotechnological approaches to produce commercialbred lines of grain legume species with a low content of antinutritionalcarbohydrates. One approach is antisense inhibition of genes (Mol et al.,1990) involved in the production of antinutritional carbohydrates. For thispurpose it is essential to clone the key genes of α-galactosides biosynthesis.

There is one patent describing nucleotide sequences of galactinolsynthase (GS) genes from courgette and soybean (US Pat. No. 5,648,210;Kerr et al., 1993) and the isolated nucleic acid fragments that encodesoybean seed and courgette leaf GS have been provided. Chimaeric genes,including these fragments and suitable regulatory genes that are capable oftransforming plants to produce GS at levels higher or lower than that foundin the target plant, also have been provided. Also, there are methods forvarying the content of D-galactose-containing oligosaccharides of sucrose inplants to produce transformed plants and seeds.

Recently, Peterbauer et al. (1999) have reported on the gene cloningand functional expression of stachyose synthase from seeds of adzuki bean(Vigna angularis Ohwi et Ohashi). The complete cDNA sequence comprised3046 nucleotides and included an open reading frame that encoded apolypeptide of 857 amino acid residues. The recombinant protein wasexpressed in a heterologous expression system and the raised product wasimmunologically active with polyclonal antibodies specified for stachyosesynthase.

Another approach can be based on the use of genes encoding enzymesthat degrade α-galactosides, to produce legume plants with low levels ofthe RFO. α-Galactosidases (EC 3.2.1.22) catalyse the hydrolysis of α(1→6)-galactosyl linkages and have been known for a long time, in micro-organisms, plants and animals (Dey and Pridham, 1972). Genes encodinga number of α-galactosidases have been cloned and sequenced. Many of

Biotechnology 199




these genes are present as operons in association with other genes involvedin galactoside utilization. Genes have been isolated from a number ofsources including Bacillus stearothermophillus (Ganter et al., 1988), E. coli(Aslandis et al., 1989), Streptoccoccus mutans (Aduse-Opoku et al., 1991) andPedicoccus pentosaceous (Gonzales and Kunka, 1986).

In processing soybean and cowpea meal, α-galactosidases from varioussources including Bifidiobacterium sp. (Sakai et al., 1987), Aspergillus awamori(Smiley et al., 1976), Aspergillus niger (Somiari and Balogh, 1995) and Lacto-bacillus fermentum (Garro et al., 1996) have been used to remove flatulencefactors. More information concerning the utilization of α-galactosidases inprocessing has been discussed elsewhere (see Chapter 4).

The development of transgenic grain legume plants with seedspecific expression of α-galactosidases could be a tool to overcome theRFO problem. At present, genes encoding seed-specific proteins fromsoybean (Okamura et al., 1986), pea (Ellis et al., 1988) and bean (Green-wood and Grispefls, 1985) have been cloned and their promoters couldbe used to construct chimaeric α-galactosidase genes with seed-specificexpression. The physiological role of the antinutritional carbohydrateswithin the plant, however, is not clear. As discussed earlier (see Chapter 5),there is a hypothesis suggesting that the α-galactosides have an importantrole in seed maturation and in resistance to desiccation. In which case,an α-galactosidase functioning during seed maturation could bedisadvantageous for seed quality. A better strategy could be α-galactosidasesthat could be activated by external factors, preferably after seed harvesting.From this point of view, genes encoding thermostable enzymes fromhyperthermophiles could be of interest for this purpose. One group ofhyperthermophilic bacteria is the genus Thermotoga. To date, Thermotogaspp. are the only known hyperthermophiles capable of growing oncellulose. They produce a multiplicity of hydrolases, which are involvedin the metabolism of various polysaccharide substrates. Recently anα-galactosidase from Thermotoga neapolitana has been used for the hydrolysisof guar (galactomannan) gum (McCutchen et al., 1996). This enzyme hasa temperature optimum close to 100°C, its activity decreasing with lowertemperatures.

It may be possible to transfer the thermostable α-galactosidasechimaeric gene, under the control of a seed-specific promotor, to commer-cial legume breeding lines. Hypothetically, the activity of this enzyme intransgenic seeds would be low at temperate climatic conditions. Theaccumulation of the RFO in transgenic seeds, therefore, could be at asimilar level to the non-transferred control cultivar and, hence, normalseed quality would not be reduced. After harvesting, during seedprocessing the enzyme could be activated by high temperature and couldthen decrease the content of α-galactosides. In addition, this processcould be applied to canned green peas, where it is impossible to remove theRFO by other methods.





6.8 Cell Suspension Culture as a Model for StudyingCarbohydrate Metabolism

6.8.1 Introduction

Plant cell suspension culture provides a defined and controlled system forthe study of cell metabolism. Some advantages of cell suspension culturesfor metabolic studies, compared with the whole plant are: rapid anduniform growth with the possibility of controlling nutritional conditions;ease of extraction and purification of large quantities of metabolicproducts; a more efficient use of labelled compounds and a lack of inter-fering microorganisms. Although the metabolic systems of cell cultures andof whole plants are frequently similar, some quantitative differences dooccur.

The aim of this section is to illustrate how cell suspension cultures canbe used to study the metabolism and biochemistry of plant cell walls. Inparticular, the possibility of using cell suspensions from legumes as a modelsystem for studying their plant cell wall metabolism.

6.8.2 Composition of plant cell walls

The plant cell wall is a highly dynamic structure playing an important rolein growth and development, morphology, cell-to-cell communication andtransport processes (Fry, 1989; Hayashi, 1989; Bowles, 1990; Sakurai, 1991;Takeuchi et al., 1994; Heredia et al., 1995; Roberts, 1996; Ishii, 1997;Driouich and Staehelin, 1997). It enables the plant cell to resist internaland/or external pressures, provides a structural barrier to some moleculesand protects against invasion by insects and by pathogens (Bowles, 1990).The cell cytoplasm obtains its metabolic substrates through the cell walland excretes other substances across it. The cell wall is a complex structure,which contains mainly carbohydrates, proteins, lignins and water as wellas other substances embedded in it, such as cutin, suberin and certaininorganic compounds.

Morphologically, three zones can be differentiated in plant cell walls,the middle lamella, the primary wall and the secondary wall. The middlelamella is the most external of the three zones and acts as a separatingpanel between two cells. It consists almost exclusively of pectic substances.The primary cell wall consists of cellulose microfibrils, complex polysaccha-rides and N- and O-linked glycoproteins. Most plant cells have only a pri-mary wall and the middle lamella, but some specialized cells exist that alsohave the secondary wall. This is a supplementary wall with a predominantlymechanical function.

It has been established (Mauch and Staehelin, 1989; Bolwell, 1993;Penel and Greppin, 1996) that there are many glycoproteins and soluble

Biotechnology 201




proteins with various enzyme activities, located in either the cell wall orthe extracellular space. Some of the enzymes are needed for modifyingthe organization of the macromolecular network, others are involved inprocesses such as defence reactions against pathogens (Bowles, 1990; Coteand Hahn, 1994). The cell wall hydrolysis enzymes (cellulases, pectinasesand a series of glycosidases, such as α- and β-galactosidases, α- andβ-mannosidases, etc.) are involved in processes such as fruit maturation andsoftening, as well as in responses to pathogen exposure and other stressfactors (Fry, 1989; Bowles, 1990). Peroxidases are known to take part in theformation of links between lignin, proteins, hemicellulose and ferulic acid(Bowles, 1990).

6.8.3 Biosynthesis of the cell wall components

The structural components of primary plant cell walls are cellulose micro-fibrils, other complex polysaccharides and N- and O-linked glycoproteins(Heredia et al., 1995). Of these components, only cellulose microfibrils aresynthesized at the cell surface by plasma membrane enzymes. All othertypes of cell matrix molecules are produced by Golgi-based enzyme systems(Driouich et al., 1993; Driouich and Staehelin, 1997) and are transported,via secretory vesicles, to the cell surface. Recent biochemical investigationshave led to the identification and partial characterization of Golgi-localizedglycosyltransferases, involved in the synthesis of xyloglucans, pectic polysac-charides and glucoronoxylans (Camiranel et al., 1987; Gibeat and Carpita,1994).

Immunolabelling experiments, with libraries of anti-polysaccharideantibodies, have been used to outline the spatial organization of polysac-charides (Moore et al., 1991; Zhang and Staehelin, 1992). It is important toremember, however, that all of these studies carried out with antibodiesare directed against specific sugar domains of the polysaccharides and notagainst specific glycosyltransferases. This is because none of these enzymeshas been fully purified to date, although some of them have been identifiedand partially characterized (Driouich and Staehelin, 1997). The isolation ofsuch enzymes and the generation of specific antibodies against them is amajor problem that still has to be resolved.

6.8.4 Oligosaccharides as signals and substrates in the plant cell wall

A few selected oligosaccharides, at very low concentrations, can exert‘signalling’ effects on plant tissues (Fry et al., 1993). Such oligosaccharidesare termed ‘oligosaccharines’ and their discovery has provoked muchresearch (Sakurai, 1991; Fry et al., 1993). There is still an urgent needfor additional knowledge on these oligosaccharides, however, including a





more detailed physiological description of known effects, the definition ofstructure–activity relationships and documentation of the biosynthesis,transport, binding action and turnover of oligosaccharides in the plant (Fryet al., 1993).

Almost all of the investigations of oligosaccharides have utilized in vitropreparations, often made using hard chemical treatments that are unlikelyto be encountered in plants. This raises questions as to whether oligo-saccharides are actually present in vivo, how they are released from largercomplex carbohydrates and how they fulfil their biological functions.

There is evidence suggesting that two biologically active oligo-saccharide fragments can be generated in vivo from plant cell wall poly-saccharides, oligogalacturonides and xyloglucan oligosaccharides. Partialdepolymerization of homogalacturonan generates oligogalacturonides,which, in the presence of Ca2+ (Farmer et al., 1991; Fry et al., 1993), exhibitvarious regulatory effects in plants. These include the elucidation ofdefence responses, the regulation of growth and development (Ryan andFarmer, 1991; Aldington and Fry, 1993; Roberts, 1996), the productionof protease inhibitors (Cote and Hahn, 1994), fruit ripening, flowerformation and the inhibition of auxin action (Fry et al., 1993). Specificoligosaccharides can be produced from xyloglucan by partial digestion withcellulase. The xyloglucan oligosaccharides have shown growth inhibitingeffects (Jork et al., 1984; Farmer et al., 1991; Angur et al., 1992) and havebeen closely associated with cell extension (Cutillas-Iturralde and Lorences,1997). The mechanism of xyloglucan oligosaccharide growth promotion,however, remains poorly understood.

Enzymatic hydrolysis offers the most likely biological mechanism forthe generation of oligosaccharides (Fry et al., 1993). Plants and pathogensare known to produce a number of hydrolytic enzymes that could beinvolved in this process (Schlumbaum et al., 1986; Mauch et al., 1988; Bolet al., 1990; Ioshikava et al., 1990; Ham et al., 1991). The demonstration thatbiologically active oligosaccharides are generated at the plant pathogeninterface by hydrolytic enzymes would provide evidence that these frag-ments do have a role in vivo. The release of such fragments by purifiedenzymes has been demonstrated in the case of glucanases. Several proteinswith β(1→3)-glucanase activity have been purified from soybean and shownto release elicitor active fragments from the mycelial walls of Phytophthorasojae (Ioshikava et al., 1990; Ham et al., 1991). Other hydrolytic enzymes,possibly involved in the generation of oligosaccharides during interactionswith plant pathogens, are pectic-modifying enzymes (Bowles, 1990; Fryet al., 1993; Cote and Hahn, 1994). They exist in uninfected plant tissuesand have the potential to generate biologically active oligogalacturonidesin plants, by de-esterification of pectic methyl esters and cleavage ofhomogalacturonans.

The results from the extensive research on oligosaccharide effects hasled to the conclusion that these compounds are important signal molecules

Biotechnology 203




and they play major roles in plant development processes and plant–pathogen interactions. The structural characterization of these molecules isa major experimental challenge. Knowledge of the structure of oligosac-charides and the biological responses to these signals, now has progressedto the point where detailed studies on their mode of action can beundertaken.

6.8.5 Plant cell suspension cultures – a powerful tool in investigating cellwall metabolism

In spite of the complexity of working with cell walls, knowledge of cell wallpolymer structure has expanded considerably in recent years (Becker et al.,1974; Sakurai, 1991; Mutafschiev et al., 1993; McCann and Roberts, 1994;Heredia et al., 1995). Knowledge of the biosynthesis and the functions ofthe polymers, however, remains limited.

Plant cell suspension culture offers new possibilities for investigatingsome aspects of cell wall metabolism (Bolwell, 1985; Butenko, 1985; Morriset al., 1985; Dwight Camper and McDonald, 1989; Wink, 1994). In this typeof culture, single cells, or clumps of cells, grow and multiply suspendedin liquid media. Plant cell suspension cultures provide a model system forstudying various molecular, physiological and genetical problems, whichcan be manipulated in ways that cannot be applied to whole plants. Cellsuspensions can be cultivated in flasks on shakers or in bioreactors, whichallows control of the nutritional and environmental conditions. As a result,controlled growth and morphological development can be ensured. Cellgrowth is rapid and more uniform than for cells within the plant. Further-more, they offer a possibility for the controlled supply of metabolites, andevery cell in such a suspension has direct access to the external mediumcontaining these metabolites. Plant cell suspensions also offer a standardsystem that allows the results obtained by different research groups to becompared.

It has been established (Wink, 1994) that plant cell suspension is aphysiologically complex system, in which both the cell biomass and theculture medium are included. The culture medium is not only the sourceof all necessary nutrients, but also a functional cell compartment with anumber of different metabolites sequested in it (Olson et al., 1969; VanHuystee and Lobazzewski, 1982; Wink, 1984, 1994; Morris et al., 1985;Konno et al., 1986, 1987, 1989; Kawasaki, 1989; Uchiyama et al., 1993; VanHuystee et al., 1994; Dolaptchiev et al., 1996; Ilieva et al., 1996a,b; Sims et al.,1996). It is well known that suspension cultured plant cells secrete variouspolysaccharides and glycoproteins into the culture medium (Olson et al.,1969; Kawasaki, 1989; Uchiyama et al., 1993; Sims et al., 1996). Thesesecreted substances are thought to be components of the middle lamella,derived from primary cell walls, two elements that are difficult to separate





in the intact plant (Heredia et al., 1995). Investigation of these compoundsshould contribute, therefore, to our understanding of the composition andfunctions of the middle lamella and primary cell walls (Olson et al., 1969;Uchiyama et al., 1993; Wink, 1994; Sims et al., 1996).

High activities of various enzymes, involved in the biosynthesis of thecell wall during growth, can be determined in the culture medium (Olsonet al., 1969; Wink, 1984, 1994; Konno et al., 1989; Dolaptchiev et al., 1996;Ilieva et al., 1996a,b). In addition, plant cells release enzymes into theextracellular space when they are attacked by microbes. The secretion ofchitinase, glucanase and other enzymes has been studied in this context(Schlumbaum et al., 1986; Mauch et al., 1988; Bol et al., 1990; Ioshikava et al.,1990; Ham et al., 1991). Since the suspension cultured cells also can bestressed by factors other than microbe invasion and culture conditions, thesecretion of high amounts of hydrolytic enzymes into the medium may beconsidered a cell response (Wink, 1984, 1994; Messner and Boll, 1993).

It has been established that during the growth and ageing of cellcultures the content of the secreted metabolites changes (Kawasaki, 1986;Goubet and Morvan, 1993; Schaumann et al., 1993; Dolaptchiev et al.,1996). The culture medium acts, therefore, as an external sink, in whichthe turnover of secreted metabolites occurs. Lytic processes have beenobserved in the medium, carried out by secreted enzymes (Wink, 1984;Konno et al., 1989). As a result some of the secreted metabolites aretransformed into inert endproducts or compounds, which fulfil differentfunctions (Wink, 1984; Konno et al., 1986, 1987, 1989).

The rapid and uniform growth of cell cultures can be used to follow thegrowth cycle of the culture and the turnover of cell wall polysaccharides.During growth, changes in the enzyme activities can be recorded in thecells and in the culture medium (Uchiyama et al., 1993; Dolaptchiev et al.,1996; Ilieva et al., 1996a,b). Specific physiological features of the cultureand its dependence on the biosynthetic time course of different metabo-lites can also be studied (Schaumann et al., 1993; Uchiyama et al., 1993; Faiket al., 1995; Dolaptchiev et al., 1996; Ilieva et al., 1996a). Suspension cultureshave been successfully used to study the time course of synthesis andsecretion of different polysaccharides and enzymes. This allows enzymeactivities to be correlated with polysaccharide content and composition(Schaumann et al., 1993; Ilieva et al., 1996a, b). It should be noted thatthe composition of the culture medium markedly affects the cell wallpolysaccharide content, offering a system for studying the influence ofdifferent environments on cell wall physiology. On the other hand, thisinfluence may interfere with or even corrupt the results obtained forheterotrophic suspensions grown on rich media. Recently, Lozovaja et al.(1996) overcame this problem by using photoautotrophic soybean (Glycinemax) cell suspension cultures grown on minimal medium, with CO2 as thesole carbon source. This system was used to investigate cell wall polysaccha-rides and starch biosynthesis and turnover. Photoautotrophic suspension

Biotechnology 205




culture could be a convenient new model, therefore, to study some largelyunknown aspects of plant physiology and biochemistry. This system couldalso allow the relationship between photosynthesis and cell growth to bestudied with regard to cell wall component accumulation, which could leadto strategies for increasing growth and biomass production.

Since the supply of different components is controlled and every cellhas direct access to the external medium, it is possible to study the influ-ence of different added molecules on plant cell metabolism. For example,cell suspension cultures have been used as model systems for studying theherbicide turnover by plant cells (Dwight Camper and McDonald, 1989).Also, cell suspension cultures from legumes have been used to study therole of oligosaccharides in eliciting and inducing enzyme activities (Kohleet al., 1984, 1985; Roffs et al., 1987; Stab and Ebel, 1987; Bruce and West,1989; Davis et al., 1993). Investigations using cell suspension cultures fromlegumes also have contributed to the knowledge of signal transductionpathways activated by oligosaccharides and an understanding of theirbiological significance (Young et al., 1982; Young and Kauss, 1983; Apostolet al., 1989; MacKintosh et al., 1994).

Plant cell wall polysaccharide studies have been hindered by the use ofheterogenous wall extracts (Becker et al., 1974; Takeuchi et al., 1994). Theintact plant tissues contain various types of cells, as well as a mixture ofprimary and secondary cell walls. Suspension cultured cells have beenselected for investigating the structure of plant cell walls, because they cangrow as a fairly homogeneous population with predominantly primary cellwalls. This allows homogeneous preparations of primary cell walls to beobtained and homogeneous polysaccharide fragments to be isolated fromthe cell wall and from the culture medium. These fragments have beenused as substrates for investigating enzyme functions (Olson et al., 1969;Konno et al., 1986, 1987). Some polysaccharide fragments can be isolated ina native form from the culture medium and their structure and functionscan be studied by subsequent model experiments.

Cell suspensions are a very promising system for obtaining pureenzymes. Enzymes involved in the biosynthesis of cell wall polysaccharidescan be isolated from the cell biomass (Olson et al., 1969; Konno et al., 1987;Faik et al., 1995) and some biosynthetic enzymes can be obtained fromthe culture medium (Wink, 1984, 1994; Konno et al., 1989; Le Bansky et al.,1992; Van Huystee et al., 1994; Ilieva et al., 1996c). Once purified, theseenzymes can be used in model experiments together with isolatedpolysaccharide fragments for elucidating their mode of action, their func-tion in cell growth and development and in the turnover of the cell wallpolysaccharides. For example, Kono et al. (1986, 1987, 1989) succeededin isolating some enzymes involved in polysaccharide biosynthesis andperformed model experiments using homogenous preparations of primarycell walls (isolated from carrot cell biomass) and with polysaccharidefragments.





A number of enzymes that have been found in culture media are ofcommercial interest. Wink (1984, 1994) reported that the culture mediumof Lupinus polyphyllus cells is a promising source for the isolation ofdifferent hydrolytic enzymes. Van Huystee et al. (1994) showed thatcultured groundnut cells in suspension cultures are an ideal system forthe isolation of peroxidase, in terms of the ease of purification and yield.Since some of these enzymes are involved in the plants defence againstattack by microorganisms and against other stress factors, their enhancedbiosynthesis and secretion into the medium can be induced by subsequentsignal molecules or stress factors. This presents possibilities for obtainingan enhanced enzyme yield (Wink, 1994; Ilieva et al., 1996c). Cultivation ofNicotiana tabacum 1507 cell suspension in an aqueous two phase systemresulted in an enhanced yield of phosphomonoesterase with a high specificactivity (Ilieva et al., 1996c).

Biotechnology 207




Breeding and AgronomyG. Engqvist et al.7

7Breeding and AgronomyEditor: Goran Engqvist

Contributors: Mike Ambrose, Nikolai Chekalin,Peter Chekrygin, Goran Engqvist, Saima Kalev,Martin Mrskos, Paolo Ranalli and Ion Scurtu

You write with ease, to show your breeding,But easy writing’s vile hard reading.

Clio’s protest (written 1771, published 1819)Richard Brinsley Sheridan (1751–1816), Anglo-Irish playwright

7.1 Current Breeding Goals

The main grain legume within Europe is pea (Pisum sativum) and so themajor breeding programmes are aimed at improving the characteristics ofthis species. It follows, therefore, that a significant part of this chapter willconcentrate on pea, with reference being made to other grain legumeswhere relevant and when information exists.

A major part of dry pea production within European Union countriesis used as a high-protein animal feedstuff. The EU, however, only producesabout a third of its required animal feed protein, the other two-thirds beingimported. In other parts of the world, particularly in developing countries,the major use of dry peas is for direct human consumption, a trend thatis becoming more popular in western countries as more people turn tovegetarian diets for health or for moral reasons.

In order to obtain a significant increase in dried pea cultivation andproduction, it is important that the net return from peas is competitive withthat from other crops grown by farmers. The primary breeding goal, there-fore, is to obtain a high harvested seed yield. This requires, in the firstplace, the development of crops that have good standing ability to improve





the ease of harvesting, which in Western countries is carried out usingcombine harvesters normally used for cereals. Standing ability can beimproved by the incorporation of a stiff stem combined with the ‘semi-leafless’ phenotype, which has well-developed tendrils (Fig. 7.1). The JohnInnes Centre (Norwich, UK) played a vital role in adopting the semi-leaflesscharacter into pea breeding programmes (Snoad, 1974; Davies, 1977;Hedley and Ambrose, 1981). The height of the crop at harvest is a goodand simple measure when evaluating the ease of harvesting.

There are a number of other important agronomic characters. Earli-ness of ripening, which is essential in those countries that have a limitedgrowing season. Plant height, which must not be too short because this willmake combining difficult. Seed size, which must not be too large, or thecost of the seed for sowing will be too high. This is particularly important incountries such as Canada, where the seed yield per unit area is low and theproduction capacity covers a vast acreage. There can be some contradictionwith this character relating to breeding goals, because a larger seed size canincrease yield. Seed colour can be important, with yellow and green beingthe preferred types. It is also important to reduce the proportion of seedthat may be shed during harvesting.

In addition to agronomic characters, seed quality is also important. Inparticular, the protein content should be high, since the main use of peas isas a high protein source for animal and human diets. There has been a ten-dency for the protein content to be lower in new higher-yielding varietiesand it is important that efforts are made to reverse this trend. As peasbecome more important in the human diet, good cooking ability will

210 G. Engqvist et al.


Fig. 7.1. Crop of the ‘semi-leafless’ pea held up by the tendrils intertwining.



become an increased priority for breeders. In developing countries thischaracteristic is of paramount importance because of the energy requiredin preparing grain legumes for human consumption.

Perhaps the major problem with the production of all crops, includinggrain legumes, is susceptibility to diseases. Breeding for increased diseaseresistance is, therefore, an important part of all breeding programmes.Aphanomyces root rot, caused by Aphanomyces euteiches, is one of the mostimportant root pathogens of peas worldwide. Mycosphaerella pinodes, whichcauses leaf, stem and pod spot, and root rot is one of the three fungi in theAscochyta blight disease complex. No strong resistance has been foundagainst these two fungi, although there is resistance to other importantpathogens, for example, Peronospora viciae, Fusarium oxysporum, Erysiphepolygoni and Pseudomonas syringae.

The breeding objectives discussed so far are the traditional ones andthey will remain as the basis for all pea improvement, at least in the nearfuture. Seed quality characters, however, will undoubtedly become moreimportant with time as grain legume crops, and pea in particular, areused more in human diets, also, as these crops become more important aspotential sources of raw material for use in processed food and non-foodapplications. In this respect, manipulating and improving the carbohydratefraction of the seed, which in most grain legumes comprises by far thegreatest proportion of the seed, will become of increasing importance toplant breeders. Part of this chapter, therefore, will describe and discuss theavailability of genetic variation for carbohydrates and possible ways that theseed composition for these compounds can be altered. These discussionswill include information on the inheritance of major carbohydrate compo-nents and the importance of these components to the growth of theplant within the crop environment. Information is also provided on theregistration requirements for new varieties within Europe.

7.2 Breeding Techniques

7.2.1 Pedigree breeding

Traditional field pea breeding techniques often start with making crosses ina greenhouse in the autumn, growing the bulked first filial generation – F1

seeds in a greenhouse in the spring and then growing the F2 in the field thefollowing summer. If a pedigree breeding method is followed, the F2 willusually be planted spaced out and the first selections, based on individualplants, will then be carried out. Pedigree plant selections will be made atthe F3, often during an off-season cultivation at a location in the SouthernHemisphere (if the breeding programme is based in the NorthernHemisphere). The F4 of each pedigree line would then be grown in anobservation plot at the home station location. At this stage a vigorous

Breeding and Agronomy 211




selection for stem stiffness and seed quality would be performed betweenthe lines. The F5 selected lines could then again be grown at an off-seasonlocation in the Southern Hemisphere. At the F5 stage, selection based onagronomic characters would be made.

The selection process at the F5 is often preceded by progeny tests of theF4 generation in the greenhouse for certain diseases and this information isthen used to aid the F5 selection. The first comparative yield trials takeplace at the F6 stage together with the start of elite seed production. Thefirst major comparative yield trial and replicated trials in other countrieswill be carried out at the F7. The official trials can start at the F8 andcontinue through to the F10.

The pedigree method allows an effective and relatively quick searchthrough a population following a cross, although it is rather labourintensive and demanding and, therefore, expensive.

7.2.2 Bulk selection

The main alternative to the pedigree breeding method is the bulk method,in which the F2 is grown as a bulk. The F3 is then grown in plots for yielddetermination at the home station, with two or three replicates of each plotbeing grown depending on the amount of available seed. Part of the F3 seedis sown later in the same season as spaced plants, to facilitate individualplant selection. The F3 yield plots are harvested and weighed. Thosepopulations that show the highest relative yield are identified and singleplant selections are made on the spaced plants from the same population.Preliminary yield estimates of the lines selected in the F3 start in F5.

7.2.3 Deviations from the pedigree and bulk methods

The schedules outlined for the pedigree and bulk selection methods maysometimes be changed. For example, repeat plant selections may be madeto increase homogeneity. Also, when a cross is made to incorporate plantdisease resistance characters, the F2 may be put directly through a selectionprocedure to identify plants with the desired character. If and when thebreeder wishes to search through a large number of populations the selec-tion work needs to be simplified. In this instance the bulked populationscan be grown for several years, delaying the plant selection until the F4 orF5 generation. This has the advantage that the selected plants are morehomozygous and the derived lines more uniform than when selections aremade in the F2. At the same time the bulked populations can be put into comparative yield trials, preferably under severe conditions, and those





populations that show obvious shortcomings can be discarded. In this waythe selection work can be concentrated on the best populations.

According to plant breeding theory on the subject of making predic-tions from a cross, it is stated that when different populations are comparedtheir mean and standard deviation can be used to identify the populationthat is most likely to contain the plants with the desired recombinedcharacteristics. For a population mean to be reliable, however, it should bedetermined as the mean of a sample of individuals from that population. Inreality, the cost of carrying out such a determination is very high and so thisprediction technique is not often used in practical breeding. If a cheap andsimple method for estimating m could be found then there would be abreakthrough in the application of the cross prediction theory. Oneimportant and principle difference between the pedigree and bulkselection methods is that the bulk method as outlined above gives a yieldmeasurement in the F3, from which a rough estimate of the populationmean can be derived. Both the pedigree and bulk breeding methods havebeen relatively successful in creating good pea cultivars.

7.3 Access to Genetic Variation

7.3.1 Germplasm banks

Most countries maintain collections of grain legumes of one sort oranother. The type of collection will vary according to the resourcesavailable and according to the nature of the work that the collection issupporting. Some countries maintain reference collections of material thathas been bred, or has originated, in their country and for which they holdnational responsibility. Other germplasm banks have built up collections ofresearch lines.

The genetic resources community operates through networks thataim towards developing common work plans for the collation of passportdata on collections. The networks also aim to identify gaps where futurecollection work is required and to ensure common evaluation andcharacterization strategies. One of the most successful of these networks isthe European Co-operative Programme for Plant Genetic Resources. Oneof the groups within this programme is focused on grain legumes. Thisgrain legume group has instigated the development of a series of EuropeanCentral Crop databases for European grain legume collections, with differ-ent institutions acting as co-ordinator for particular species (Table 7.1).

As an example, the European Pisum database comprises records ofthe holdings of 22 collections in 20 countries (Table 7.2). Informationon these databases can be obtained through the following web site:www.cgiar.org/ecpgr





7.3.2 Existing variation for the carbohydrates

Information on the existing variation for carbohydrate characters canbe obtained from the centres detailed in Table 7.1. Pea can be used asan example of the range of variation that can be found and analyticalinformation on a representative sample of lines from the collection heldat the John Innes Centre is presented in Table 7.3. This table containsinformation on the carbohydrate composition of 227 round-seeded pealines that were analysed as part of an EU-funded ECLAIR project (AGRE0048), running from 1990 to 1994. The range of lines covered the spectrumof Pisum germplasm available, including representatives from subspecies,landraces and old and new cultivars. The aim of the project was to improvethe nutritional quality of grain legume seeds for use in the animal feedstuffindustry. The data are from seeds multiplied in one season on a single site.The distributions for soluble sugars, starch, neutral detergent fibre (NDF)and acid detergent fibre (ADF) were all normally distributed, but demon-strated a range of values. It is evident, from these analyses that considerable‘natural’ variation can be found within existing Pisum germplasm, all ofwhich is available for incorporation within breeding programmes. Thisgives considerable scope for modifying the carbohydrate composition ofthis species using conventional breeding techniques.

7.3.3 Newly identified genetic variation

Breeders often work without knowledge of the underlying genetic basisto their crossing programmes and selections, since many of their mostimportant characters, such as yield or standing ability, are determined byinteractions between many genes. Some quality characteristics of seeds,however, are known to be controlled by relatively simple genetic systemsand in these cases major genes have been identified.

At present, the best characterized carbohydrate pathway is starch,which has been studied for many years by biochemists. Major advances in



Species Database management centre

Vicia faba INRA, Le Rheu, FranceCicer arenatum National Plant Breeding Station, Elvas, PortugalGlycine max N.I. Vavilov Research Institute of Plant Industry, St Petersburg,

RussiaLens spp. Aegean Agricultural Research Institute, Izmir, TurkeyPisum spp. John Innes Centre, Norwich, UK

Wiatrowo, Wagrowic, PolandPhaseolus spp. Federal Office of Agrobiology, Linz, Austria

Table 7.1. European central crop databases and management centre.





Org

aniz

atio

n/in

stitu

teA

cron

ymC

ount

ryN

umbe

rof

acce

ssio

ns

Aeg

ean

Agr

icul

tura

lRes

earc

hIn

stitu

teA

RA

Turk

ey88

Inst

ituto

delG

erm

opla

sma

BA

RIta

ly42

97In

stitu

tfür

Pfla

nzen

bau

und

Pfla

nzen

zuch

tung

BR

NG

erm

any

1009

Cen

tre

for

Gen

etic

Res

ourc

esC

GN

Net

herl

ands

1008

Dep

artm

ento

fAgr

icul

ture

and

Fish

erie

sfo

rSc

otla

ndEC

SSc

otla

nd19

80Z

entr

alIn

stitu

tfür

Gen

etik

und

Kul

turp

flanz

en-f

orsc

hung

GA

TG

erm

any

2478

Cen

tre

deR

eche

rche

sA

gron

omiq

ues

deL’

Etat

GB

XB

elgi

um34

8In

tern

atio

nalC

entr

efo

rA

gric

ultu

ralR

esea

rch

inth

eD

ryar

eas

ICA

Syri

a33

18In

stitu

toN

acio

nald

eIn

vest

igac

ione

sA

grar

ias

IMD

Spai

n25

6Jo

hnIn

nes

Cen

tre

JIIU

K30

08In

stitu

teof

Plan

tBre

edin

gJO

KFi

nlan

d17

Agr

obio

logi

sche

Inst

itute

LIN

Aus

tria

33N

ordi

cG

ene

Ban

kfo

rA

gric

ultu

rala

ndH

ortic

ultu

ralP

lant

sN

GB

Swed

en30

55Pe

aan

dLe

ntil

Bre

edin

gPr

ogra

mm

eR

AB

Mor

occo

65In

stitu

teof

Plan

tInt

rodu

ctio

nan

dG

enet

icR

esou

rces

SAD

Bul

gari

a15

98R

esea

rch

and

Bre

edin

gIn

stitu

teof

Tech

nica

lCro

psan

dLe

gum

esSU

MC

zech

Rep

ublic

937

Inst

itute

for

Plan

tPro

duct

ion

and

Qua

lific

atio

nTA

PH

unga

ry93

4V

eget

able

Cro

psR

esea

rch

Inst

itute

,Res

earc

hSt

atio

nU

jmaj

orU

JMH

unga

ry74

8Se

rvic

iode

Inve

stig

acio

nA

grar

iaV

AL

Spai

n49

1N

.I.V

avilo

vA

ll-U

nion

Scie

ntifi

cIn

stitu

teof

Plan

tInd

ustr

yV

IRR

ussi

a65

18Is

rael

Gen

eban

kfo

rA

gric

ultu

ralC

rops

,Vol

cani

Cen

tre

VO

LIs

rael

690

Pisu

mG

ene

Ban

kW

TDPo

land

2899

Tabl

e7.

2.M

anag

emen

tcen

tres

with

inEu

rope

for

the

mai

ngr

ain

legu

me

data

base

s.



understanding the synthesis of starch have been made following the identi-fication and development of mutants within the starch biosynthetic path-way. As well as aiding fundamental studies on starch synthesis, such mutantsare also available to breeders for the development of crops where the seedcontains starches with improved nutritional characteristics, or new uses.

Other pathways that lend themselves to genetic characterization, thatcould provide breeders with new sources of variation, are those leading tothe synthesis of soluble carbohydrates, in particular the raffinose family ofoligosaccharides (RFO). Information on the known variation for both theRFO and starch biosynthetic pathways is outlined in the following sections.

StarchTwo mutations, r and rb, affecting the content and composition of starchhave been known for many years and were first identified by their affect onthe shape of the dry mature seed, which in both cases appeared wrinkled.Mendel (1865) used a naturally occurring mutation at the r locus in hisepoch making studies on inheritance. This mutation is the genetic changethat has brought about the development of the vined pea crop, which isharvested when immature and then either quick-frozen, quick-dried orcanned. A second, apparently naturally occurring, mutation at the rb locuswas first genetically characterized by Kooistra (1962), who also determinedthat r and rb were independent loci, even though the presence of recessivegenes at either locus gave rise to wrinkled seeds.

The presence of mutations at either the r or rb locus results in areduction in the starch content of the dry seed to about 35%, comparedwith about 50% found in the non-mutant wild type. In addition to affectingstarch content, the presence of these mutations also affects starch composi-tion. Starch from r mutant seed contains about 70% amylose, while starchfrom the rb mutant contains about 20% amylose, compared with thewild-type level of about 30% amylose (Wang et al., 1998). It is now knownthat the mutation at the r locus is in a gene encoding a starch-branchingenzyme (Fig. 7.2; Bhattachatryya et al., 1990; Martin and Smith, 1995) andresults in a lack of activity of this enzyme during seed development.The mutation at the rb locus reduces the activity of ADP-glucose



Mean Range CV%

Starch 45.9 32.7–54.5 7.75Soluble sugars 5.3 3.9–8.2 11.51NDF 16.7 11.8–26.3 15.18ADF 8.3 5.9–12.7 15.51

CV, coefficient of variation.

Table 7.3. Storage component analysis of 227 lines of round-seeded peas expressed as a percentage of the dry weight.



pyrophosphorylase, another step in the starch biosynthetic pathway (Fig.7.2; Hylton and Smith, 1992).

Until 1987, these mutations at the r and rb loci were the only onesknown to directly affect the content and composition of starch in pea seeds.In 1987 a chemical mutagenesis programme was carried out to producenew mutants affected in pea seed development and composition, in partic-ular starch content and composition (Wang et al., 1990; Wang and Hedley,1991). As a result of this programme, mutations at six loci were identified,five of which resulted in the seed having a wrinkled (rugosus) shape, whilethe sixth (lam) did not appear to significantly affect seed shape. In additionto identifying mutations at specific loci, a range of mutations or alleles wasidentified at each locus (Table 7.4). Two sets of mutants were geneticallycharacterized and found to correspond to either the r or rb locus. In eachcase, however, the effect on starch content and composition of the newmutants widened the available variation, such that the starch and amylose



Fig. 7.2. The pathway of starch biosynthesis in pea (Wang et al., 1998).



contents ranged from 27 to 36% and 60 to 75%, for the r alleles, and from30 to 37% and 23 to 32%, for the rb alleles, respectively (Table 7.4).

The remaining mutants were assigned to four previously unidentifiedloci (rug3, rug4, rug5 and lam). The presence of mutations at the rug3locus resulted in seeds containing only 1–12% starch, depending on theparticular mutant allele, and the starch had relatively low amountsof amylose (Table 7.4). Mutations at this locus decrease the activity ofplastidial phosphoglucomutase (Fig. 7.2; Harrison et al., 1998). The seedsfrom plants containing this mutation are viable even though they possessvery low or no starch and the resulting plants appear to grow normally(Harrison et al., 1998).

Seeds from the rug4 mutants are only mildly wrinkled and the starch andamylose contents are decreased to only 38–43% and 31–33%, respectively(Table 7.4). It is now known that mutations at the rug4 locus result in adramatic reduction in the activity of sucrose synthase (Craig et al., 1999), anenzyme that is outside of the dedicated starch biosynthetic pathway (Fig. 7.2).

Mutations at the rug5 locus result in a reduction in starch content to29–35% and an increase in the proportion of amylose in the starch to43–52% (Table 7.4). Mutants containing the mutations at the rug5 locushave a reduced level of one of the major soluble starch synthases (Fig. 7.2;Craig, 1998).

The sixth group of alleles isolated from the chemical mutagenesisprogramme carried out by Wang et al. (1990) differed from the other fivein that seed shape and starch content were more similar to the wild type(Table 7.4). The composition of the starch, however, was similar to that oflow amylose or ‘waxy’ mutants identified in maize and other species, hencethe gene symbol lam, for low amylose. Mutants containing the lam mutationlack activity for a major granule-bound starch synthase (Denyer et al., 1995).

The presence of all of these new mutants affecting starch gives breedersan opportunity to develop pea lines with seeds containing a range ofstarches or, in the case of the rug3 mutants, seeds with little or no starch.



Loci Number of alleles Starcha Amyloseb

r 10 27–36 60–75rb 8 30–37 23–32rug3 5 1–12 12rug4 3 38–43 31–33rug5 3 29–35 43–52lam 5 39–49 4–10

aStarch is given as a percentage of the dry weight.bAmylose is given as a percentage of the starchon a dry weight basis.

Table 7.4. Pea loci and alleles affecting starchsynthesis and composition (Wang et al., 1998).



The physical properties of the starches produced by these new mutants aredescribed in Chapter 4. Having identified a range of loci affecting starch, itthen becomes possible to combine the genes in different ways to producedouble or perhaps treble mutants that could extend further the availablevariation and potential uses.

Raffinose family of oligosaccharides (RFO)Although variation for the content and composition of the RFO andcyclitols between species is quite well established (see review by Horbowiczand Obendorf, 1994) there have been few reports of variation withinparticular species. A recent screen of lentil (Lens culinaris) germplasm,however, has revealed variation within this species for the total level of RFOand for the levels of individual members of the RFO (Frias et al., 1994b). Inthis study, 16 lentil lines composed of exotic germplasm and cultivatedvarieties were analysed and quantified for the RFO using thin-layerchromatography (TLC) and high performance liquid chromatography(HPLC). Total RFO levels ranged from about 1.8 to about 4.3% of the seeddry weight. The most notable variation within the individual RFO memberswas for verbascose, which ranged from about 1% of the seed dry weightto a level, observed in several of the lines, that could not be detected. Thisvariation was used in a genetic analysis and evidence suggests that the lackof verbascose may be due to the presence of a single recessive gene (Friaset al., 1999). In addition, there is evidence of an inverse relationshipbetween a reduction in the level of verbascose and an increase in the levelof a cyclitol, ciceritol, suggesting a link between these two pathways.

A screen for RFO variation also, has been carried out on 70 pea lines,covering the genetic variation available within the John Innes pea genebank. The screening procedure was similar to that used for lentil. Theinitial screen used TLC and lines that apparently had extreme variation forthe RFO were reanalysed using HPLC, to quantify the observed differences(Jones et al., 1999b). Variation for the total level of the RFO in the selectedlines ranged from about 3.5 to about 7.0% of the seed dry weight. As withthe lentil study, the most extreme variation for individual members of theRFO was for verbascose, which ranged from about 3% to an undetectablelevel. Those lines that lacked verbascose had an increased level ofstachyose, the previous homologue in the RFO pathway.

There is similar evidence of within species variation for RFO inPhaseolus vulgaris (Burbano et al., 1999). In this study, 19 varieties ofbean were screened for RFO content and composition and several lineswere found in which no verbascose could be detected. Once again thepredominant α-galactoside was stachyose.

SaponinsAlthough not present in large quantities in legume seeds, saponins areconsidered to be antinutritional. Genetic variation for saponin type has





been found within soybean seeds and a genetic model linking structure (ofthe sugar chains) and five genes; Sa-1a, Sg-1b, Sg-2, Sg-3 and Sg-4, has beenproposed by Tsukamoto et al. (1993). Variation for soyasapogenol B hasbeen reported in a study of 19 P. vulgaris varieties (Burbano et al., 1999).This now opens up the possibility of breeders selecting lines that have lowamounts of saponins with antinutritional properties and higher amounts ofthose saponins with known health benefits, improving the quality of theirseed.

7.4 Selection Methods

The basis of plant breeding is the selection of individuals with desiredcharacteristics from a gene pool. The gene pool may be natural, as found ingene banks, or created, for example, by mutagenesis or by crossing differ-ent individuals with the hope of finding a new combination of characters.In the case of gene banks, there are often relatively large amounts of seedfor each of the lines and so the selection can be made for seed qualitycharacters using destructive chemical analysis, or using destructive or non-destructive physical techniques (Fig. 7.3a). The selected line then can bemultiplied and released as a variety, or could become part of a breeding



Fig. 7.3. (A) Plant breeding selection methods for chemical component. Selectionfrom gene banks. Opposite page: (B) Selection from F2 populations.





Fig. 7.3. Continued



programme, where some of its characteristics would be incorporated intoa new variety. In the example presented in Fig. 7.3A, the plant would bea typical inbreeding species, such as pea. The scheme would only workfor an out breeding species, such as faba bean (Vicia faba), if the chemicalcharacter being selected was not segregating in the population.

A crossing programme using an inbreeding species, where one parenthas a useful seed quality characteristic, will produce a segregating F2

population consisting of a large number of seeds, all of which could begenetically different (Fig. 7.3B). Destructive analysis of the whole seedin this case cannot be considered and there are basically two strategiesthat can be adopted. In the first strategy, the single seeds are multipliedfor several generations by single-seed descent to produce inbred lines.Destructive physical or chemical techniques can then be used to allowselection for the character to take place, followed by multiplication anddevelopment of the new variety. This strategy, however, is expensive andwill result in the production of large numbers of inbred lines, the majorityof which will be discarded.

The second strategy is to carry out the selection on the single seeds(Fig. 7.3B). This requires a non-destructive method of analysis, whichshould also be rapid because of the large numbers of seeds that would needto be screened. The rapid methods do not need to be too precise, however,since the main objective of the screen will be to select seeds that are high(or low) for a particular chemical component. There are basically two typesof non-destructive analysis: either a portion of the seed is removed foranalysis or the whole seed is used. In the first case, parts of each seed areremoved, which can make use of the drilling technique described by Joneset al. (1995; Fig. 7.4). This material can then be analysed either chemicallyor physically and selections made. If the seed is maintained whole then onlyphysical techniques can be used for analysis (see below) and the selectionsare made using this information. The seeds, selected following either typeof analysis, will then be multiplied for several generations to produceinbred lines. Seeds from these lines can be used to carry out more precisechemical analyses and final selections can then be made, the selected linesbeing multiplied and developed into a new variety.

The approach of screening seeds for chemical characteristics in earlygenerations is very efficient but does require the development of screeningtechniques based on single seeds. The chemical methodology associatedwith these techniques has been discussed in Chapter 2. The non-destructivetechniques based on physical methods are briefly described below.

7.5 Physical Screening Methods

The most common physical selection methods used by breeders are basedon near-infrared (NI) transmission or reflectance. Some studies have also







Fig. 7.4. Miniature drill used for sampling from single seeds. The insert shows ahole bored through a pea seed with the resultant flour sample – 30 mg, more thanenough for chemical or physical analyses. The seed can be grown normally toproduce a plant.



been carried out using mid-infrared (MI), which it has been suggestedcould yield more chemical information than NI. Both methods are basedon the selective absorption and vibration of specific chemical bonds withinmolecules. The fundamental vibrations result from absorption in the MIwhile the second and third overtones occur in the NI.

7.5.1 Near-infrared (NI) spectroscopy

The principle of NI spectrometry is based on the generation of a polychro-matic beam from a tungsten lamp, which is passed through a monochroma-tor to produce a monochromatic beam in the NI region. The beam is thenfocused on the sample and the reflected diffuse energy may be detected asreflectance (R), or the beam may be passed through the sample and theremaining energy detected as transmittance (T). NIR can be used onmilled material, including samples taken from whole seeds, while NIT canbe used to analyse whole seed samples, including single seeds.

The analyses are based on the property of organic molecules to absorbenergy in the < 2.5 µm region of the spectrum, which lies between theinfrared and the visible ranges. Most seed components, including thecarbohydrates, absorb energy in the infrared region, giving a unique finger-print. In a crude sample, for example pea meal, the spectrum obtained willbe the sum of all of the organic constituents. The relationship between theabsorbed energy of a particular component and its concentration inthe mixture will be affected by the overlapping of spectral bands from thedifferent constituents, the particle size and by the temperature of thesample. Using NI to determine precise concentrations of a particular seedcomponent, therefore, is difficult and indirect. If it is to be used for thispurpose it is necessary to develop a predictive model based on multipleregressions of spectral versus chemical data, using samples differing widelyin concentration for the component of interest (Kim and Williams, 1990;Orman and Schumann, 1991; Sinnaeve et al., 1995).

It is more easy to use NI to directly identify changes in the relativeamounts of a component, particularly if it is reduced to very low levelsor removed completely, for example, following genetic changes within acrossing programme. In this way it is possible to rapidly select segregantsfrom a population, which differ from the norm. These selected seeds canthen be subjected to a more precise chemical analysis using part of theseed, or whole seeds can be chemically analysed following inbreeding andmultiplication as discussed earlier.

At least one NIT instrument is available for use by breeders (InfratecGrain Analyser) produced by Foss Tecator, Sweden. This instrument hasbeen developed to cope mainly with batches of seed samples, but a singleseed adapter kit is available that will allow it to be used for the analysis ofsingle seeds, including peas.





7.5.2 Mid-infrared spectroscopy

The MI region of the spectrum (2.5–25 µm) is less familiar to breedersthan NI, although it could in the future have significant advantages.Bands in this region can be specifically assigned to chemical groups, whichholds out the possibility of using it for quantitative analysis of specific seedcomponents with less need for the complex statistical analysis used with NItechniques. The main drawbacks of MI are that the samples are opaque andcontain water, which is a very strong IR absorber. The performance can beimproved using Fourier transformation (FT) methods and a recent paperhas applied the combination of FT with MI to the analysis of pea seeds(Letzelter et al., 1995). The technique described in this paper combinesFT-MI with photo-acoustic detection (PAS) and can be applied to smallamounts of seed material (c. 100 mg), which it is possible to remove froma pea seed while retaining seed viability (Jones et al., 1995). There is also areport of this method being applied to whole maize seeds (Greene et al.,1992), suggesting in the future that the technique could be used to analysethe seeds of grain legumes.

7.6 Some Agronomic Considerations of Carbohydrates

7.6.1 During plant growth and development

The capacity of plants to accumulate carbohydrates is a function of theirphotosynthetic capacity and of the carbon distribution pattern between theplant parts, both of which appear to be genetically determined. Within acrop this capacity also depends on the ability of plants to maintain drymatter accumulation when faced with a variety of abiotic stresses. Theproductivity of plants within crops, therefore, often falls far short of its fullgenetic potential because of such environmental stresses. The allocation ofrecently fixed carbon to export and to storage enables plants to maintaina steady supply of carbohydrates both for development and for therestoration and maintenance of homeostasis during environmental stress.

The regulation of carbon allocation between starch synthesis andsynthetic processes in the chloroplast and cytosol determines the amountof stored assimilate that is available for export or use in the leaf at timesof low or no photosynthesis. Carbon that exits the chloroplast can beused for sucrose synthesis, for respiration or for the synthesis of compoundsthat remain in the leaf. The regulation of two cytosolic enzymes, fructosebisphosphatase and sucrose phosphate synthase, is particularly importantin controlling the flux of carbon to sucrose. Sucrose may be either storedtemporarily in the cytosol and vacuole, or moved to the vicinity of the veinsand enter the sieve elements to be translocated to sinks. At night and dur-ing daytime periods of low photosynthesis, stored sucrose is made available,





or starch is degraded, to support sucrose export. The synthetic, transportand regulatory processes involved in the allocation of assimilates are themeans by which leaves maintain a relatively steady supply of carbon for uselocally or in translocation sinks.

A major environmental stress that is often found within crops is adeficiency of nitrogen. The restriction of leaf canopy development innitrogen-stressed plants consists of two general effects. One is a decrease inthe rate of leaf initiation (Rufty et al., 1984) and the other is a decreasein the development of existing leaves (Rufty et al., 1988). Both effectscontribute to a decreased utilization of carbohydrate in the shoot. Carbohy-drate metabolism in leaves is altered in nitrogen-limited plants, with starchand sucrose levels being elevated relative to controls, even though growth isrestricted. This observation implies firstly that growth is not limited byassimilate availability and secondly that sucrose degradation and possiblyglycolysis are reduced.

A large portion of the carbohydrate utilized in growth reactions in sinktissues is derived from sucrose imported from leaves. It has been proposed(Huber and Akazawa, 1986; Black et al., 1987) that an important pathwayfor sucrose utilization in sink tissues is catalysed by phosphofructokinase,which is activated by fructose-2,6-bisphosphate (F26BP). The sharp declinein the concentration of F26BP in leaves following the imposition ofnitrogen stress, therefore, may cause the accompanying decrease in carbo-hydrate metabolism. Such observations clearly suggest that a decrease insucrose utilization contributes to a decrease in demand for assimilateswithin the shoot and to the reported diversion of carbohydrate to theroot system. Legumes have evolved to live in low nitrogen soils and in thesespecies the diversion of carbohydrates to the root system during nitrogenstress has probably played a major role in the development of the symbioticrelationship with nitrogen-fixing bacteria.

7.6.2 During seed development

The availability of water to crops during the time when the seeds are devel-oping is a major factor determining yields. It is known, for example, thatwater deficits during the time of seed filling in soybean crops decreasesseed size. This may result from a reduction in the supply of assimilates fromthe maternal plant and/or an inhibition of seed metabolism. Experimentshave been carried out to determine whether it is maternal or zygotic factorsthat limit seed growth at times of water deficit (Bernal-Lugo and Leopold,1992). When water was withheld from greenhouse grown soybean plantsduring the linear seed-filling period, leaf water potential decreased rapidly,inhibiting canopy photosynthesis completely within 3 days. Seed dry weightcontinued to increase, however, at or near the control rate. The level oftotal extractable carbohydrates in leaf, stem and pericarp tissue decreased





by 70, 50 and 45% respectively, indicating that reserves were mobilized tosupport seed growth. The content of sucrose in the cotyledons decreasedfrom about 60 to 30 mg g−1 dry weight and the sucrose concentration inthe interfacial apoplast of the cotyledons decreased from about 100 to50 mmol. The rate of sucrose accumulation by excised embryos, measuredin a short-term in vitro assay, however, increased in response to the waterdeficit.

Results from such experiments indicate that both source and sinkactivity in soybean are altered by water deficits to maintain the flux ofassimilates to the developing embryos. This may explain why seed growth ismaintained, albeit for a shorter duration, when soybean crops are exposedto water deficits during the seed filling period.

7.7 European Registration Requirements for New Varieties

7.7.1 Background

Most European countries have specific requirements that new varietiesmust meet the demands of ‘Value for Cultivation and Use’ (VCU) in theregistration process. Generally, a variety has a VCU if, in comparison withother registered varieties in an important growing region, it makes apositive contribution to the growing of the crop, to the use of the crop, orto the products derived from it. The characters and minimum conditionsincluded in the official examination of agricultural varieties should cover:

• yield;• resistance to harmful organisms;• behaviour with respect to factors in the physical environment;• quality characters;• alternativity – for spring and winter form varieties.

The importance of new varieties and their characters vary accordingto the technical facilities of a given country (e.g. farming management,harvesting machines, susceptibility to spill at harvest), the quality of a newvariety (e.g. standing ability) and the corresponding level of plant breedingprogrammes (e.g. resistance to diseases), climatic and soil conditions (e.g.suitability of leafy types vs. semi-leafless varieties in wet and/or aridregions), etc. Those countries that are members of the International Unionfor the Protection of New Varieties of Plants (IUPOV), follow IUPOVguideline recommendations on the conduct of tests, the choice of varietalcharacteristics on which to establish distinctness, uniformity and stability(DUS). For a variety to be distinct, it must be clearly distinguishable by oneor more important characters from any other variety, whose existence is amatter of common knowledge at the time when protection by a grant ofrights is sought. A variety is required to be sufficiently uniform, depending





on its breeding system, to allow accurate description and assessment ofdistinctness and to ensure stability. To measure stability with any degreeof certainty would take at least 3 years and would require tests to be madeon seed from succeeding stages in the multiplication cycle.

7.7.2 Agronomic characters

The variability for pea registration requirements throughout Europe canbe divided into three groups, according to the evaluating and analysingmethods (Table 7.5a–c). Table 7.5a shows the agronomic characters, whichare very extensive and include those that are of most importance forfarmers. Farmers take these into account first when making decisionsabout which variety is to be grown. The foremost character in all Europeancountries (except Estonia) is yield and stability of yield, a new variety being



Agronomic character

Yield � � � � � � � � � � �Earliness of ripening � � � � � � � � �Shortness of straw � � � � � � �Height of crop canopy at harvest � � �Standing ability � � � � � � � � � � �Ease of combining � � � � �One-step combining � �Height of lower pod attachment � �Pod shattering or spill at harvest � � �Resistance to:

Drought � �Alternaria alternata � �Ascochyta pisi/Mycosphaerella pinodes � � � � � � �Black rust � �Colletotrichum � �Downy mildew (Peronospora pisi) � � � � � � �Erysiphe pisi � � � � �Pea enation virus � � � �Pea wilt (race 1) Fusarium sp. � � � � � � � � �

Winter hardening – cold tolerance � �1000 seed weight � � � � � � � � � �

Table 7.5a. The registration requirements of several European countries for newvarieties of pea – agronomic characters.

Cze

chR

epub

licB

ulga

ria

Den

mar

kEs

toni

aFr

ance

Ger

man

yH

unga

ryIta

lyM

oldo

vaPo

land

Rom

ania

Spai

nSw

eden

UK

Ukr

aine





Processing character

Grain colour stability � � � �Fungal diseases � � �Pests � � �Grading 3 mm �Grading 4 mm �Grading 4.5 mm �Grading 5 mm �Grading 5.5 mm �Grading < 6 mm � � �Grading 6–7 mm � � �Grading > 7 mm � � �Grading 8 mm �Grading 9 mm �Seed characteristics:

Shape, colour, surface bleaching �Soakability � �Cookability � � �Seed colour after cooking �Taste �Consistency �Grain cooking uniformity �

Table 7.5b. The registration requirements of several European countries for newvarieties of pea – processing characters.

Cze

chR

epub

licB

ulga

ria

Den

mar

kEs

toni

aFr

ance

Ger

man

yH

unga

ryIta

lyM

oldo

vaPo

land

Rom

ania

Spai

nSw

eden

UK

Ukr

aine

Chemical character

Trypsin inhibitor activity (TIU) � �Amino acid content:

lysine, methionine, cystine, alanine �Protein content of seed � � � � � � � � � �Protein yield from seed � � � �

Table 7.5c. The registration requirements of several European countries for newvarieties of pea – chemical characters.

Cze

chR

epub

licB

ulga

ria

Den

mar

kEs

toni

aFr

ance

Ger

man

yH

unga

ryIta

lyM

oldo

vaPo

land

Rom

ania

Spai

nSw

eden

UK

Ukr

aine



compared to control varieties that are supplied by the appropriate testingauthorities. Thousand seed weight is a character that is very dependent ongrowing conditions and should be taken as a guide to the genetic potentialof the variety. There is a close link between future yield and characters suchas standing ability, height of crop canopy at harvest time in relation toshortness of straw and ease of combining. In other words, a variety couldbe prone to lodging, but mature pods may be held off the ground bythe haulm in such a way that harvesting may be easier than the standingability score may suggest. This character can be particularly importantin a difficult harvest season, especially in leafy types. Nevertheless, thischaracteristic is observed in only four countries, two of which (Ukraineand Bulgaria) still do not utilize semi-leafless varieties with good standingability. Instead they tend to grow tall leafy types that are susceptible tolodging and not easy to combine, and they are interested, therefore,in the height of attachment of the lower pods and shattering or spill atharvest. In Romania, for example, it is always necessary to check varietiesfor their suitablility to one-step combining.

The behaviour of new varieties, with respect to factors in the physicalenvironment mentioned above, is tested in Ukraine through resistance todrought. The typical continental weather in Ukraine gives an advantage totaller leafy types of peas that are able to cover the ground and protectthe crop from the high evaporation that occurs during the hot late springand summer months. Another specific character, common in southerncountries (e.g. Bulgaria, Italy, Spain and France), where farmers preferautumn sowing to spring sowing because of too much activity in the spring,is winter-hardiness or cold resistance of winter pea varieties specially bredfor this purpose, or normal spring varieties sown in autumn.

Diseases can have a major influence on yield. Growers of varietiessusceptible to specific diseases should either ensure that appropriatefungicide is applied to the seed coat prior to drilling or try to avoid landthat is known to produce disease problems. In Table 7.5a, resistance toharmful organisms is distributed equally, which means that all concernedcountries consider these agronomic characters very important. Differencesin observations can originate from differences in races, or diseasedistribution, throughout Europe.

The most common and observed fungus disease is wilt (Fusariumoxysporum f. sp. pisi), which reduces yields and can only be controlledeffectively by genetic resistance. Race 1 is thought to be the commonestform and it is a very persistent, soil-borne disease. The foremost foliardisease complex Ascochyta pisi and M. pinodes is responsible for leaf andpod spot. The latter is seed-borne and may be soil-borne and developsrapidly in wet conditions.

Downy mildew (P. viciae) is favoured by the cool, moist conditionscommon in coastal countries such as the UK, in northern parts of France,Germany and Poland, and in Denmark and Sweden. Once grey mould





(Botrytis cinerea) is established in a crop, it cannot be controlled effectively.Growers in areas of countries where wet weather during the flowering andpod-setting period is more likely to occur, may be able to choose some ofthe more determinate, shorter-strawed varieties with a semi-leafless habit.These plant types produce a more open crop with a drier microclimate.

Powdery mildew (Erysiphe polygoni, Erysiphe pisi) seldom occurs in theabove-mentioned countries because it is usually a disease of late-sownpeas and spreads in hot and dry summers, particularly in France, Germany,Poland and the Czech Republic (i.e. in countries with a continentalclimate). Infected plants are covered in a fine white film and, if thedisease appears early in the season, pods may fail to fill and maturation ofthe crop is delayed.

Other specific diseases may become more important, for exampleAlternaria alternata and black rust in Bulgaria, or Colletotrichum in theUkraine.

Pea enation mosaic virus is seldom noticed before the approach offlowering as it is aphid (Acyrthosiphon pisum) transmitted. Since the weathergreatly influences the migration and reproduction of aphids, it influencesalso the occurrence and severity of this disease.

The number of characters represented in this section is evidence ofthe great emphasis given to them by breeders, seed-merchants, farmers,growers and processors.

7.7.3 Technological characters

The seed market and the food industry strongly appreciate grain colourstability (evaluated by Bulgaria, Czech Republic, Sweden and Ukraine),either green or yellow-seeded varieties being preferred (Table 7.5b). Thetime of harvest is important, therefore, because strong sunshine can lead tothe bleaching of seed colour (tested in Hungary). In addition, seed sampleshave to be free from waste and stain, as well as the effects of fungal diseases(e.g. blemishes, contaminants) and pests (e.g. damaged by pea moth cater-pillar and pea seed beetle). These characters are regarded as important forcanning and packet sales, and meeting these requirements results in higherpayments for such samples.

Some marrowfat varieties may need special agronomic measures toensure high quality produce. Samples which do not meet the technologicalrequirements mentioned above (except colour) may be suitable for themicronizing market. The micronising process produces a high protein feedfor use in certain dried animal feedstuff and pet foods. Another criterionfor marrowfat peas might be large, even-sized seed, which is tested throughgrading (Table 7.5b). It is necessary for a variety to be graded in only onelarge group and not to be divided into more size groups (a high number ofsize groups is analysed in Ukraine).





Peas for human consumption also should be tested for soakability,cookability, grain cooking uniformity, seed colour after cooking, taste(smell) and consistency. It is generally known that peas harvested at lowmoisture contents, or are over-dried, may fail soaking tests.

7.7.4 Chemical characters

Seed protein content is strongly influenced by location and season andvaries only slightly between varieties. Hungary is the only country wherethe content of four major amino acids is relevant for the registrationprocess (Table 7.5c).

Trypsin inhibitors are low molecular weight proteins that can bind to,and inhibit, the hydrolytic activity of pancreatic protease enzymes, leadingto reduced protein digestibility and even pancreatic enlargement in rats.The presence of trypsin inhibitors in grain legume seed has been shown todecrease the nutritional value of the seed proteins. The trypsin inhibitorlevels found in pea seeds are 5–20 times lower than those found insoybeans. Although the importance of the digestibility problems associatedwith trypsin inhibitors in peas is highlighted, this character is officiallyanalysed only by the French and Czech testing authorities (Table 7.5c).





Manipulating Grain Legume CarbohydratesC. Hedley8

8Strategies for ManipulatingGrain Legume Carbohydrates

Editor: Cliff Hedley

. . . the other is a conclusion, shewing from various causes why theexecution has not been equal to what the author promised to himselfand to the public.

Boswell Life, vol. 1, p. 2 (1755)Samuel Johnson (1709–1784), English poet, critic and lexicographer

8.1 The Problems

The main use of grain legume seeds is for human and animal nutrition andso improving the seeds nutritional components is of paramount impor-tance to the development of legume crops. A major problem with improv-ing the nutritional value of grain legume seeds, however, is the possiblenegative impact of such changes, especially on the plant or seed. This is aparticular problem when dealing with so called antinutritional componentswithin the seed. Many inhibitors that affect the digestion of nutrients byanimals or humans are useful within the plant, where they may serve as pro-tective agents against attack by insects or disease-forming microorganisms.They may also have a positive effect on the consuming organism. For exam-ple, some inhibitors are rich in the sulphur-containing amino acids that areoften present in low amounts in legume seeds (Domoney, 1999).

With regard to the carbohydrates, the raffinose family of oligosaccha-rides (RFO) poses such a problem. Although not antinutritional in the truesense of the word, they restrict the use of legume seeds in animal feed and,because they are associated with flatulence, they are often avoided in





the human diet (see Chapter 3). It is likely, however, that removal of thesecompounds by plant breeders would have an adverse affect on the growthand development of the plant and seed. Since the RFO are believed to beinvolved in protection against abiotic stress and may have an important rolein germination (see Chapter 5). As with the protein inhibitors, the RFOalso may have a positive affect in the diet by promoting beneficial changesin the gut flora, in particular increasing the proportion of Bifidobacteria,which have been implicated in protection against colon cancer (seeChapter 3).

With the possible exception of the storage proteins, nutritionists havebeen more interested in the effect of the antinutritional, or non-nutritionalcomponents in legume seeds, rather than the nutritional components. Themain nutritional carbohydrate in most legume seeds is starch. It is knownthat existing legume starches are digested more slowly than those fromother species, in particular from cereals, and that this can have a positiveeffect in reducing the glycaemic index in humans, which is an advantage tothose people with type 2 diabetes (see Chapter 3). The lack of information,in general, on starch digestion and, more specifically, on why legumestarches are digested more slowly, however, creates the problem of notknowing the nutritional consequences of changing the starch content orcomposition in legume seeds.

Also, there is very little information on the effect of manipulatingstarch on the growth and development of the plant or seed. Studies usingpea seed mutants affected in starch content, composition (see Chapter 7)and granular structure (see Chapter 4) have given some information on thepossible consequences of manipulating legume starches. For example, it isknown from using these lines that blocking starch synthesis completelyin peas, by introducing a mutation at the rug3 locus, appears to have aminimal affect on the growth of the plant and on the viability of the seed(Wang et al., 1998). There is also no evidence that changing the chemicalcomposition and the granular structure of pea starches has any adverseaffects on the seed biology. One interesting observation, following theintroduction of mutations directly affecting starch, however, is that theyhave pleiotropic effects on other seed components, in particular, proteins(Casey et al., 1998a,b), lipids (Jones et al., 1995) and soluble carbohydrates(Jones, 2000). For example, introducing recessive alleles at the r and rb lociin pea gives a small percentage increase in the protein content, a largedifference in the protein composition and a large percentage increase inthe lipid content of the seed (Wang and Hedley, 1993).

The third carbohydrate category, after the soluble carbohydratesand starch is the ‘fibre’ fraction, which contains a multitude of solubleand insoluble compounds, generally characterized by nutritionists as non-digestible elements in the diet. Many of the fibre components are lookedon as beneficial within the human diet, mainly from a health point of view(see Chapter 3). Within the plant, most of these compounds are associated

234 C. Hedley




with the cell wall and so reducing or manipulating particular fibrecomponents could have a detrimental effect on the structure of plantcells and the development of the seed (see Chapter 6). Likewise, anygenetic manipulation of plant or seed development that results in an effecton the content or composition of the cell walls could affect the nutritionalusefulness of grain legumes in the diet.

One component of the ‘fibre’ fraction, as recognized by nutritionists,that could be manipulated, however, is ‘resistant starch’, much of whichresults from the processing of native starches in food, prior to being con-sumed in the diet (see Chapter 4). There is little information on why somestarches produce more ‘resistant starch’ when processed than others,although this has often been associated with the amylose content. There issome evidence for this from work using pea mutants with starches that areknown to have different levels of amylose (Skrabanja et al., 1999). As men-tioned earlier, there is no evidence that manipulating starch is detrimentalto the seed (at least in pea) and so producing high-amylose starches wouldnot be a problem from this point of view. There would, however, need to bea balance between the proportion of starch that can be digested, the rate ofstarch digestion and the proportion of the starch that becomes resistant todigestion.

8.2 Strategies for Overcoming the Problems

Before discussing possible ways that grain legume seed can be improvedwith regard to their carbohydrates, one simple fact should be borne inmind. The thing that links all carbohydrates together, and all other organiccomponents within the plant, is that they were all initially derived fromsimple sugars such as sucrose. It is very likely, therefore, that reducingor increasing one carbohydrate component will have an effect on others,either as a direct consequence of a change on partitioning of sugars,or because of an effect on the cellular environment of changing theconcentration of a cellular component.

8.2.1 The soluble carbohydrates

As mentioned above and in Chapter 3, the main problems with the solublecarbohydrates lie with the inability to digest the RFO, with the resultingreduction in nutritional value for animal feed and the problem offlatulence in humans. Any changes in the content and composition of theRFO would also need to take into consideration their role in the plant,mentioned above and in Chapter 5.

The first requirement of any conventional breeding programmedesigned to genetically manipulate plant characteristics, such as the

Manipulating Grain Legume Carbohydrates 235




content and composition of the RFO, is to identify suitable levels of geneticvariation. As discussed in Chapter 7, papers have been published recentlydescribing genetic variation for these compounds in pea, lentil and bean.In particular, lines with very low levels of verbascose have been identified.What is not known at present, however, is the importance of individualRFO components, either to the growth and development of the plant andseed, or as a contributory factor to the nutritional problems in animals orhumans.

These are two areas of research, therefore, that should be undertaken in the short termand preferably within a co-ordinated programme utilizing similar genetic material.

If genetic variation cannot be found within existing germplasm thenit will be necessary to create it, either by modifying existing genes usingmutagenesis, or by using molecular techniques, either to manipulate geneaction by reverse transcription, or by the introduction of novel genes fromother species. The use of mutagenesis is a random technique and does notrequire information on the biochemistry of a particular pathway. On theother hand, the use of techniques based on molecular biology requiresinformation on the biochemistry and may require the isolation of specificproteins controlling steps in a pathway. There is a developing literature onthe biochemistry of the RFO and other associated cyclitols and galactosylcyclitols (see Chapters 2 and 5).

There are still many gaps in our knowledge, however, and very little information aboutspecific steps in the pathways or the links between them.

It is known that legume species differ for the range of these com-pounds produced within the seed and for the presence or absence ofparticular pathways. For example, pea only produces the RFO within itsseeds, while lentil has, in addition, the D-pinitol pathway and can producerelatively high amounts of ciceritol. Pea lines that are verbascose minusaccumulate more stachyose, the previous homologue in the RFO pathway,while lentil lines that are verbascose minus accumulate ciceritol, demon-strating a link between the two pathways in this species.

It is important that studies are carried out to assess the effect of compounds such asciceritol on human and animal nutrition, as well as their possible protective role in theplant.

If ciceritol, for example, has a reduced adverse effect on nutritionwhile maintaining a positive role within the plant, then a strategy formanipulating these compounds can be developed. This could involveblocking the RFO pathway, or parts of the pathway, in species such as lentil,that possess both pathways. Assimilate then being diverted into the D-pinitolpathway. The D-pinitol pathway could be engineered to function in speciessuch as pea, followed by blocking, or partly blocking, the RFO pathway.Much of the biotechnology required to carry out these procedures is inplace, including the transformation systems (see Chapter 6).

236 C. Hedley




The alternative strategy of manipulating the activity of α-galactosidase,to degrade the RFO during storage and prior to inclusion in the diet,should also be considered (see Chapter 6).

8.2.2 Starch

Starch biosynthesis is relatively simple, compared with the complexity ofthe soluble carbohydrate pathways. This simplicity is reflected in the wealthof information in pea on starch biochemistry and the presence of welldefined mutants at most of the steps in the pathway. Unlike the solublecarbohydrates, starch behaves as a relatively inert substance within theplant, with little evidence of major problems following its manipulation.The main problems with starch concern its use as a source of nutritionand as an important fraction of the non-digestible material in food. Thereis little knowledge as to why legume starches are digested more slowly thanthose from cereals, or why legume starches produce high levels of resistantstarch when processed or cooked. To manipulate starches with regard tothese characteristics demands knowledge of the relationship betweenstarch chemical and granular structure and the starch nutritional charac-teristics, on the one hand, and the genetic control of starch chemical andgranular structure, on the other.

The genetic variation required to study these relationships exists in pea. This shouldbe used to determine the effects of specific mutations on starch chemical and granularstructure and the link between these characteristics of starch and starch digestibility inthe native and processed condition.

Although pea can probably be used as a model for other starchstoring grain legumes, it will become important to extend the studies onlegume starches to other species. This will entail either identifying geneticvariation, similar to that now available for pea, in gene banks, creating thevariation using a mutagenesis programme, similar to that carried out inpea, or using information gained from the pea studies to modify specificsteps in starch biosynthesis using transformation.

One or all of these alternatives should be initiated as soon as possible, particularly inthose species that are commonly used for human nutrition (lentil, common bean andchickpea).

8.2.3 Fibre

The complexity of the ‘fibre’ fraction makes it much more difficult todefine strategies for improvement compared with the soluble carbohy-drates and starch. In this case, it is a primary requirement to determinewhich compounds are the most important from a nutritional/health point





of view. It will then be necessary to determine what the consequences to theplant may be if the selected compounds are changed, reduced or increased.This process would require the selection or derivation of variation thatwould serve the dual purpose of helping to answer these fundamentalquestions and providing variation for future breeding programmes. Therole of specific ‘fibre’ components within the plant cell and the productionof sufficient material to utilize for nutritional tests could make use of thecell culture systems described in Chapter 6.

8.3 Conclusions

Defining strategies for improving or manipulating grain legume carbohy-drates will only result in increased scientific and technological activity ifgrain legume crops are considered worth developing from an economicpoint of view. As stated in the introduction to this book, in general theconsumption of grain legumes has been declining for many years in thedeveloped parts of the world. A shrinking market gives rise to decreasedinterest from producers and a reduction in available funding for researchand development. This downward spiral can only be stopped and perhapsreversed by creating new opportunities for grain legume crops and byadding value to the raw materials derived from them.

It is very important to get away from the ‘poor man’s meat’ identity thatgrain legumes have had in the past. The health benefits from consumingmore grain legumes in the diet are well documented and with currentawareness of diet and health issues in western populations this should be agood ‘selling point’. Another current concern of western populations is theeffect that intensive farming is having on the environment. An increasedacreage of grain legumes and reintroducing these species into farmingrotation systems would reduce the chemical inputs required for other cropssuch as cereals. A more sustainable agricultural system would reduce thelevel of fertilizers reaching water supplies and have a positive effect onreducing chemical pollution.

An alternative, or perhaps additional, strategy for increasing the useand production of grain legumes is to consider the seeds as a source of rawmaterials for the processing industry, rather than as an entity to be eaten asa vegetable. The three major constituents of the starchy legumes, protein,starch and fibre, all have useful functional properties that can be readilyutilized in food products. Procedures have already been developed forisolating these three fractions (see Chapter 4) relatively easily, from pea.Considering the two carbohydrate fractions, pea starch has unique pastingproperties, having a stable development and high end-point viscosity,compared with equivalent amounts of cereal and tuber starch. Most legumestarches have good gelling properties, although this is usually accompaniedby a high level of syneresis, which could be a negative property. As

238 C. Hedley




mentioned above and in Chapter 4 the introduction of mutant genesaffecting starch synthesis in pea has resulted in a spectrum of starches withproperties that could and should be utilized within the food processingindustry. Likewise, the insoluble fibre fraction extracted from the seedembryo has excellent water-holding properties that could be utilized withinthe processed meat industry as an alternative to inorganic salts, such asphosphates.

Once the proteins, starch and fibre have been isolated from legumeseeds these materials could also find alternative uses in non-food products.For example, some legume starches, in particular those with high amylosecontents, have properties that make them an excellent raw material forthermoplastics. The current knowledge of starch genetics, chemistry andgranular structure, based on pea, has opened the door to the developmentof an almost infinite selection of starches that could be produced to suit awide range of food and non-food applications.





ReferencesReferencesReferences

References

AACC American Association of Cereal Chemists (1995) Sugars. In: AACC Methods,Vol. 2, Section 80.

Abbas, I.R., Scheerens, J.C. and Berry, J.W. (1987) Tepary bean starch. Part III.In vitro Digestibility. Starch/Stärke 39, 280–284.

Abdel-Gawad, A.S. (1993) Effect of domestic processing on oligosaccharide contenton same dry legume seeds. Food Chemistry 46, 25–31.

Abia, R., Buchanan, C.J., Saura-Calixto, F. and Eastwood, M.A. (1993) Structuralchanges during the retrogradation of legume starches modify in vitro fermenta-tion. Journal of Agricultural Food Chemistry 41 (11), 1856–1863.

Abreu, J.M.F. and Bruno-Soares, A.M. (1998) Chemical composition, organicmatter digestibility and gas production of nine legumes grains. Animal FeedScience and Technology 70, 49–57.

Acevedo, E. and Bressani, R. (1989) Ingestion de fibra dietetica en los paises delistmo controamericano: implicationes nutricionales. Archivos Latinoamericanosde Nutricion 39, 392–404.

Acevedo, E., Velazquez-Coronado, L. and Bressani, R. (1994) Changes in dietaryfibre content and its composition as affested by processing of black beans(Phaseolus vulgaris Tamazulapa veriety). Plant Foods for Human Nutrition 46,139–145.

Adams, C.A., Rinne, R.W. and Fjerstad, M.C. (1980) Starch deposition and carbo-hydrate activities in developing and germinating soya bean seeds. Annals ofBotany 45, 577–582.

Adams, C.A., Broman, T.H. and Rinner, W. (1981) Starch metabolism in develop-ing and germinating soybean seeds is independent of α-amylase activity. Annalsof Botany (London) 48, 433–440.

Adsule, N.R., Lawande, K.M. and Kadam, S.S. (1989) Pea. In: Salunkhe, D.K. andKadam, S.S. (eds) CRC Handbook of World Food Legumes: Nutritional Chemistry,

241

A3867: AMA: Hedley: First Proof:30-Oct-00 References



Processing Technology and Utilization, Vol. I. CRC Press, Boca Raton, Florida,pp. 215–251.

Aduse-Opoku, J., Tao, L., Ferretti, J. and Russell, R. (1991) Biochemical and geneticanalysis of Streptococcus mutans – galactosidase. Journal of General Microbiology 137,757–764.

Ahmed, A.P. and Labavitch, J.M. (1977) A simplified method for accurate determi-nation of cell wall uronide content. Journal of Food Biochemistry 1, 361.

Ahmed, R., Gupta, S.D. and Ghosh, P.D. (1987) The cytological status of plantsregenerated from shoot-meristem culture of Pisum sativum. Plant Breeding 98,306–311.

Äkerberg, A.K.E., Liljeberg, H.G.M., Granfeldt, Y.E., Drews, A.W. and Björck, M.E.(1998) An in vitro method, based on chewing, to predict resistant starchcontent in foods parallel determination of potentially available starch anddietary fibre. Journal of Nutrition 128, 651–660.

Akinyele, I.O. and Akinlosotu, A. (1991) Effect of soaking, dehulling and fermenta-tion on the oligosaccharides and nutrient content of cowpeas (Vignaunguiculata). Food Chemistry 41, 43–53.

Akpa, A.D. and Archer, S.A. (1994) Interaction between Pseudomonas syringae pv.Pisi and isolated pea mesophyll protoplasts. Acta Phytopathology and Entomology(Hungary) 29, 95–104.

Aldington, Z. and Fry, S.C. (1993) Oligosaccharins. Advances in Botany Research 19,1–101.

Altaf, N. and Ahmad, M.S. (1990). Chickpea (Cicer arietinum L.). In: Bajaj, Y.P.S.(ed.) Biotechnology in Agriculture and Forestry, Vol. 10. Legumes and Oilseed Crops 1.Springer-Verlag, Berlin, pp. 100–113.

Althers, S., Stirm, S. and Jacobsen, H.J. (1993) Immunobiochemical analysis of anuclear protein marker for regeneration potential in higher plants. Journal ofPlant Physiology 141, 415–422.

Aman, P. (1979) Carbohydrates in raw and germinated seeds from mung bean andchick pea. Journal of the Science of Food and Agriculture 30, 869–875.

Amberger, A.L., Palmer, R.G. and Shoemaker, R.C. (1992) Analysis of culture-induced variation in soybean. Crop Science 32, 1103–1108.

Ambrose, M.J., Wang, T.L., Cook, S.K. and Hedley, C.L. (1987) An analysis of seeddevelopment in Pisum sativum. IV. Cotyledon cell populations in vivo andin vitro. Journal of Experimental Botany 38, 1909–1920.

Amuti, K.S. and Pollard, C.J. (1977) Soluble carbohydrates of dry and developingseeds. Phytochemistry 16, 529–532.

Anandarajah, K. and McKersie, B.D. (1990) Manipulating the desiccation toleranceand vigor of dry somatic embryos of Medicago sativa L. with sucrose, heat shockand abscisic acid. Plant Cell Reports 9, 451–455.

Andersen, K.E., Buskov, S., Bjergegaard, C., Sorensen, H., Sorensen, J.C. andSörensen, S. (1997) Analyses of dietary fibre associated non-carbohydrateconstituents based on supercritical fluid extraction. In: Abstract European FoodChemistry IX. Interlaken, Switzerland, pp. 209–214.

Anderson, T.A. (1982) Recent trends in carbohydrate consumption. Annual ReviewNutrition 2, 113–132.

Anderson, J.W. and Bridges, S.R. (1988) Dietary fibre content in selected foods.The American Journal of Clinical Nutrition 47, 440–447.

242 References




Andersson, H. (1992) The ileostomy model for the study of carbohydrate digestionand carbohydrate effects on sterol excretion in man. European Journal of ClinicalNutrition 46 (Suppl. 2), 69–76.

Angel, C.R., Sell, J.L. and Zimmerman, D.R. (1988) Autolysis of α-galactosidesof defected soy flakes: influence on nutritive value for chickens. Journal ofAgricultural Food Chemistry 36, 542–546.

Angur, Ch. Lu In, Sakai, K., Ogata, T., Sinay, P., Darvill, A.G. and Albersheim, P.(1992) Further studies of the ability of xyloglucan oligosaccharides to inhibitauxin-stimulated grown. Plant Physiology 99, 180–185.

Anonymous (1994) Analysis of carbohydrates by anion exchange chromatographywith pulsed amperometric detection. Dionex Technical Note 20, Dionex Corpora-tion, Sunnyvale, California.

AOAC Association of Official Analytical Chemists (1984) Sugars and sugarproducts. In: Official Methods of Analysis, Vol. 14, Section 31, 573.

Apostol, I., Heinstein, P.F. and Low, P.S. (1989) Rapid stimulation of an oxidativeburst during elicitation of cultured plant cells. Role in defense and signaltransduction. Plant Physiology 90, 109–116.

Arago, F.J.L., Barros, L.M.G., Brasileiro, A.C.M., Ribeiro, S.G., Smith, F.D., Sanford,J.C., Faria, J.C. and Rech, E.L. (1996) Inheritance of foreign genes in trans-genic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment.Theoretical and Applied Genetics 93, 142–150.

Arcioni, S., Pezzotti, M. and Damiani, F. (1987) In vitro selection of alfalfa plantsresistant to Fusarium oxysporum f. sp. Medicaginis. Theoretical and Applied Genetics74, 700–705.

Arentoft, A.M. and Sorensen, H. (1992) Alpha galactosides and dietary fibresin relation to pea quality: methods of oligosaccharide analyses. In: AEP (ed.)Proceedings of the 1st Conference on Grain Legumes, Angers, France. AEP, Paris,France, pp. 457.

Arentoft, A.M., Michaelsen, S. and Sorensen, H. (1993) Determination of oligosac-charides by capillary zone electrophoresis. Journal of Chromatography A 652,517–524.

Aslandis, C., Schmidt, K. and Schmidt, R. (1989) Nucleotide sequences and operonstructure of plasmid-borne genes mediating uptake and utilization of raffinosein Escherichia coli. Journal of Bacteriology 171, 6753–6763.

Asp, N.G. (1992) Preface. European Journal of Clinical Nutrition 46, 1.Asp, N.G. (1996) Dietary carbohydrates: classification by chemistry and physiology.

Food Chemistry 57 (1), 9–14.Asp, N.G. and Björck, I. (1992) Resistant starch. Trends in Food Science and Technology

3, 111–114.Asp, N.G. and Johansson, C.G. (1984) Dietary fibre analysis. Nutrition Abstracts

Reviews 54, 735–752.Asp, N.G., Johansson, C.-G., Hallmer, H. and Siljeström, M. (1983) Rapid enzymatic

assay of insoluble and soluble dietary fibre. Journal of Agricultural Food Chemistry31, 476–482.

Asp, N.G., van Amelsvoort, J.M.M. and Hautvast, J.G.A.J. (eds) (1995) Proceedingsof the concluding plenary meeting of EURESTA – including the final reports ofthe working groups. European FLAIR Concerted Action No II (COST 911) on:Physiological implications of the consumption of resistant starch in man.Wageningen, The Netherlands, IBSN 90-9008390-1.

References 243




Asp, N.G., van Amelsvoort, J.M.M. and Hautvast, J.G.A.J. (1996) Nutritional implica-tions of resistant starch. Nutrition Research Reviews 9, 1–3.

Atanassov, A.I. and Mehandjiev, A.D. (1979) In vitro induced morphogenesis in peaembryos. Comptes Rendus de l’Academie Bulgare de Sciences 32, 115–118.

Atkins, C.A. and Smith, P.M.C. (1997) Genetic transformation and regenerationof legumes. In: Legocki, A., Bothe, H. and Puhler, A. (eds) Biological Fixation ofNitrogen for Ecology and Sustainable Agriculture. Springer-Verlag, Berlin, Germany,pp. 283–304.

Atkins, C.A., Smith, P.M.C., Gupta, S., Jones, M.G.K. and Caligari, P.D.S. (1998)Genetics, cytology and biotechnology. In: Gladstones, J.S., Atkins, C.A. andHamblin, J. (eds) Lupins as Crop Plants: Biology, Production and Utilization. CABInternational, Wallingford, pp. 67–92.

Attia, R.S., El-Tabey Shehata, A.M., Aman, M.E. and Hamza, M.A. (1994) Effect ofcooking and decortication on the physical properties, the chemical composi-tion and the nutritive value of chickpea (Cicer arietinum L.). Food Chemistry 50,125–131.

Asworth, E.N., Stirm, V.E. and Volenec, J.J. (1993) Seasonal variations in solublesugars and starch within woody stems of Cornus sericea L. Tree Physiology 13,379–388.

Aumaitre, A., Peiniau, J., Bengala Freire, J. and Seve, B. (1992) Sensitivity of theweaned piglet to pea antinutritional factors: effect of technical treatments. In:AEP (ed.) Proceedings of the 1st European Conference on Grain Legumes, Anger. AEP,Paris, pp. 487–488.

Bach Knudsen, K.E. (1997) Carbohydrates and lignin contents of plant materialsused in animal feeding. Animal Feed Science and Technology 67, 319–338.

Bach Knudsen, K.E. and Li, B.W. (1991) Determination of oligosaccharides inprotein-rich feedstuffs by gas-liquid chromatography and high performanceliquid chromatography. Journal of Agricultural and Food Chemistry 39, 689–694.

Bachmann, M. (1993) Metabolism of the raffinose family oligosaccharides inleaves of Ajuga reptans L. Cold induction, sink to source transition andcompartmentation. PhD thesis, University of Zurich, pp. 125.

Bachmann, M. and Keller, F. (1995) Metabolism of the raffinose family oligosaccha-rides in leaves of Ajuga reptans L. Inter- and intracellular compartmentation.Plant Physiology 109, 991–998.

Bachmann, M., Matile, P. and Keller, F. (1994) Metabolism of the raffinose familyoligosaccharides in leaves of Ajuga reptans L. Cold acclimation, translocation,and sink to source transition: discovery of chain elongation enzyme. PlantPhysiology 105, 1335–1345.

Bajaj, Y.P.S. (1985) In vitro induction of genetic variability in ground nut. ProceedingsInternational Workshop Cytogenetics of Arachis. ICRISAT, Patancheru, pp. 165.

Bajaj, Y.P.S. and Dhanju, M.S. (1979) Regeneration of plants from apical meristemtips of some legumes. Current Science 48, 906–907.

Bajaj, Y.P.S. and Gosal, S.S. (1987) Pollen embryogenesis and chromosomalvariation in cultured anthers of chickpea. Chickpea Newsletter 17, 12–13.

Bajaj, Y.P.S. and Gosal, S.S. (1981) Induction of genetic variability in grain-legumesthrough tissue culture. In: Rao, A.N. (ed.) Proceedings COSTED Symposium onTissue Culture of Economically Important Plants, Singapore, pp. 25–41.

244 References




Bajaj, Y.P.S. and Saettler, A.W. (1968) Effect of culture filtrates of Pseudomonasphaseolicola on the growth of excised roots and callus tissue cultures of bean.Phytopathology 58, 1041–1042.

Bajaj, Y.P.S. and Saettler, A.W. (1970) Effect of halo toxin-containing filtrates ofPseudomonas phaseolicola on the growth of bean callus tissue. Phytopathology 70,1065–1067.

Bajaj, Y.P.S., Singh, H. and Gosal, S.S. (1980) Haploid embryogenesis in anthercultures of pigeon-pea (Cajanus cajan). Theoretical and Applied Genetics 58,157–159.

Bajaj, Y.P.S., Ram, A.K., Labana, K.S. and Singh, H. (1981) Regeneration of geneti-cally variable plants from the anther-derived callus of Arachis hypogaea and A.villosa. Plant Science 23, 35–39.

Baker, C.M., Durham, R.E., Burns, J.A., Parrott, W.A. and Wetzstein, H.Y. (1995)High frequency somatic embryogenesis in peanut (Arachis hypogeae L.) usingmature dry seed. Plant Cell Reports 15, 38–42.

Bakhsh, A., Jones, D.A., Lambert, N. and Hedley, C.L. (1991) Genetic variation forstorage product composition in lentil (Lens culinaris). Aspects of Applied Biology(Production and protection of grain legumes) 27, 279–281.

Bakhsh, A., Bird, J., Lambert, N., Bacon, J., Chambers, S., Jones, A., Wang, T.L.and Hedley, C.L. (1992) Quantitative variation for seed storage productcomposition in lentil (Lens culinaris). In: AEP (ed.) Proceedings of the 1st EuropeanConference on Grain Legumes, Angers. AEP, Paris, pp. 153–154.

Barna, K.S. and Wakhlu, A.K. (1993) Somatic embryogenesis and plant regenera-tion from callus cultures of chickpea (Cicer arietinum L.). Plant Cell Reports 12,521–524.

Barna, K.S. and Wakhlu, A.K. (1994) Whole plant regeneration of Cicer arietinumfrom callus via organogenesis. Plant Cell Reports 13, 510–513.

Barna, K.S. and Wakhlu, A.K. (1995) Direct somatic embryogenesis and plantletsregeneration of chickpea. In Vitro Cellular and Developmental Biology – Plant 31,137–139.

Barnett, J.E.G., Rasheed, A. and Corina, D.L. (1973) Partial reactions of D-glucose6-phosphate-1L-myo inositol 1-phosphate cyclase. Biochemical Journal 131,21–30.

Bartels, D., Singh, M. and Salamini, F. (1988) Onset of desiccation tolerance duringdevelopment of the barley embryo. Planta 175, 485–492.

Bartle, K.D. (1993) Introduction to the theory of chromatic separations withreference to gas chromatography. In: Baugh, P.J. (ed.) Gas Chromatography: aPractical Approach. IRL Press, Oxford, pp. 1–14.

Barwale, U.B. and Widholm, J.M. (1987) Somaclonal variation in plants regener-ated from cultures of soybean. Plant Cell Reports 6, 365–368.

Barwale, U.B. and Widholm, J.M. (1990) Soybean: Plant regeneration and soma-clonal variation. In: Bajaj, Y.P.S. (ed.) Biotechnology in Agriculture and Forestry,Vol. 10. Legumes and Oilseed Crops 1. Springer-Verlag, Berlin, pp. 114–133.

Bastianelli, D., Grosjean, F., Peyronnet, C., Duparque, M. and Regnier, J.M. (1998)Feeding value of pea (Pisum sativum L.) 1. Chemical composition of differentcategories of pea. Animal Science 67, 609–619.

Bean, S.J., Gooding, P.S., Mullineaux, P.M. and Davies, D.R. (1997) A simple systemfor pea transformation. Plant Cell Reports 16, 513–519.

References 245




Beck, E. and Ziegler, P. (1989) Biosynthesis and degradation of starch in higherplants. Annual Reviews Plant Physiology and Plant Molecular Biology 40, 95–117.

Becker, G., Kauffman, P., McNeil, M. and Albesheim, P. (1974) The structure ofplant cell wall. VI. A survey of the walls of suspension-cultured monocots. PlantPhysiology 544, 109–115.

Becker, R., Olson, A.C., Frederick, D.P., Kon, S., Gubmann, M.R. and Wagner, J.R.(1974) Conditions for autolysis of alpha-galactosides and phytic acid inCalifornia small white beans. Journal of Food Science 39, 766–769.

Bedford, M.R., Patience, J.F., Classen, H.L. and Inborr, J. (1992) The effect ofdietary fibre enzyme supplementation of rye- and barley-based diets on diges-tion and subsequent performance in weaning pigs. Canadian Journal of AnimalSciences 72, 97–105.

Beers, E.P. and Duke, S.H. (1990) Characterisation of α-amylase from shoots andcotyledons of pea (Pisum sativum L.) seedlings. Plant Physiology 92, 1154–1163.

Beers, E.P., Duke, S.H. and Henson, C.A. (1990) Partial characterisation andsubcellular localization of three α-glucosidases isoforms in pea (Pisum sativumL.) seedlings. Plant Physiology 94, 738–744.

BeMiller, J.N. and Whistler, R.L. (1996) Starch. In: Fennema, O.R. (ed.) FoodChemistry. Marcel Dekker, New York, pp. 191–204.

Bengala Freire, J., Aumaitre, A. and Peiniau, J. (1991) Effect of feeding raw andextruded pea on ileal digestibility, pancreatin enzymes and plasma glucose andinsulin in early weaned pigs. Journal of Animal Physiology and Animal Nutrition65, 154–164.

Benamouzig, R., Mahre, S., Rautureau, J. and Tome, D. (1994) Physiologicalproperties of leguminous fibres. Grain Legumes 7, 18–19.

Bencheikh, M. and Gallais, A. (1996) Somatic embryogenesis in pea (Pisum sativumL. and Pisum arvense L.): dialel analysis and genetic control. Euphytica 90,257–264.

Bennici, A., Cionini, P.G. and D’Amato, F. (1976) Callus formation from thesuspensor of Phaseolus coccineus in hormone-free medium: a cytological andDNA cytophotometric study. Protoplasma 89, 251–261.

Berggren, A., Björck, I.M.E. and Nyman, E.M.G.L. (1995) Short-chain fatty acidcontent in caecum of rats fed various source of starch. Journal of the Science ofFood and Agriculture 68, 241–248.

Berghofer, E. and Horn, T. (1994) Bildung resistenter Stärke bei derHeissextrusion Stärkereicher Rohstoffe. (How to produce resistant starchwhen using starch with raw materials in cooking extrusion). Ernährung/Nutri-tion 18, 341–348.

Bernabé, M., Fenwick, R., Frias, J., Jiménez-Barbero, J., Price, K., Valverde, S. andVidal-Valverde, C. (1993) Determination by NMR spectroscopy of the structureof ciceritol, a pseudo trisaccharide isolated from lentils. Journal of Agriculturaland Food Chemistry 41, 870–872.

Bernal-Lugo, I. and Leopold, A.C. (1992) Changes in soluble carbohydrates duringseed storage. Plant Physiology 98, 1207–1210.

Bernicot, H., Carrouée, B., Lacampagne, J.P., Muel, F. and Schneider, A. (1998)Peas: a recent raw material for animal feed. Grain Legumes 21, 12–23.

Berry, C.S. (1986) Resistant starch: formation and measurement of starch that sur-vives exhaustive digestion with amylolytic enzymes during the determination ofdietary fibre. Journal of Cereal Science 4, 301–314.

246 References




Berry, C.S., Anson, I., Miles, M., Morris, V.J. and Russel, P.L. (1988) Physico-chemical characterisation of resistant starch from wheat. Journal of Cereal Science8, 203–206.

Bewley, D. and Oliver, M.J. (1992) Desiccation tolerance in vegetative plant tissuesand seeds: protein synthesis in relation to desiccation and a potential rolefor protection and repair mechanisms. In: Osmond, C.B., Somero, G. andBolis, C.L. (eds) Water and Life: A Comparative Analysis of Water Relationships at theOrganismic, Cellular and Molecular Levels. Springer-Verlag, Berlin, pp. 141–160.

Bewley, J.D. and Black, M. (1978) Physiology and Biochemistry of Seeds. Development,Germination and Growth, 1st edn. Springer-Verlag, New York.

Bewley, J. D. and Black, M. (1985) Seeds. Physiology of Development and Germination,1st edn. Plenum Press, New York.

Bewley, J.D. and Black, M. (1994) Seeds. Physiology and Development and Germination,2nd edn. Plenum Press, New York.

Bewley, J.D. and Reid, J.S. (1985) Mannans and glucomannans. In: Dey, P.M. andDixon, R.A. (eds) Biochemistry of Storage Carbohydrates in Green Plants. AcademicPress, London, pp. 289–304.

Bhat, U.R., Paramahans, S.V. and Taranathan, R.N. (1983) Scanning electronmicroscopy of enzyme-digested starch granules. Starch 35, 261–265.

Bhattacharyya, M.K., Smith, A.M., Ellis, T.H.N., Hedley, C.L. and Martin, C. (1990)The wrinkled-seed character of peas described by Mendel is caused by atransposon-like insertion in a gene encoding starch branching enzyme. Cell 60,115–122.

Bhatty, R.S. (1990) Cooking quality of lentils: the role of structure and compositionof cell walls. Journal of Agricultural and Food Chemistry 38, 376–383.

Bhave, M.R., Lawrence, S., Barton, C. and Hannah, L.C. (1990) Identification andmolecular characterization of shrunken-2 cDNA clones of maize. Plant Cell 2,581–588.

Biermann, C.J. (1988) Introduction to analysis of carbohydrates by gas-liquidchromatography (GLC). In: Bierman, C.J. and McGinnis, G.D. (ed.) Analysis ofCarbohydrates by GLC and MS. CRC Press, Boca Raton, Florida, pp. 1–17.

Biliaderis, C.G., Page, C.M., Maurice, T.J. and Juliano, B.O. (1986) Thermal charac-terisation of rice starches: a polymeric approach to phase transitions ofgranular starch. Journal of Agricultural Food Chemistry 34, 6–14.

Biliaderis, C.G. and Tonogai, J.R. (1991) Influence of lipids on the thermal andmechanical properties of concentrated starch gels. Journal of Agricultural FoodChemistry 39, 833–840.

Binarova, P., Nedelnik, J., Fellner, M. and Nedbalkova, B. (1990) Selection forresistance to filtrates of Fusarium spp. in embryogenic cell suspension cultureof Medicago sativa L. Plant Cell Tissue and Organ Culture 22, 191–196.

Binder, R.G. and Haddon, W.F. (1984) Analysis of O-methylinositols by gas–liquidchromatography-mass spectrometry. Carbohydrate Research 129, 21–32.

Binding, H. (1986) Regeneration from protoplasts. In: Vasil, I.K. (ed.) Cell Cultureand Somatic Cell Genetics of Plants, Vol. 3. Academic Press, Orlando, Florida,pp. 259–274.

Bingham, S. and Selvendran, R.R. (1983) Workshop report: dietary fibre estima-tion. In: Wallace, G. and Bell, L. (eds) Fibre in Human and Animal Nutrition. TheRoyal Society of New Zealand, pp. 119–121.

References 247




Bishnoi, S. and Khetarpaul, N. (1993) Effect of domestic processing and cookingmethods on in vitro starch digestibility of different pea cultivars (Pisumsativum). Food Chemistry 47, 177–182.

Bisson, C.S. and Jones, H.A. (1932) Changes accompanying fruit development inthe garden pea. Plant Physiology 7, 91–105.

Bitter, R. and Muir, H.M. (1962) A modified uronic acid carbazole reaction. Analyti-cal Biochemistry 4, 330.

Bjergegaard, C., Sörensen, H. and Sörensen, S. (1997a) Dietary fibres and associ-ated compounds in rape seed and biorefined rapeseed products compared toDF in pea. Journal of Animal and Food Sciences 6, 163–184.

Bjergegaard, C., Sörensen, H. and Sörensen, S. (1997b) Dietary fibres – importantparts of high quality food and feeds. Journal of Animal and Food Sciences 6,145–161.

Björck, I., Nyman, M., Pedersen, B., Siljeström, M., Asp, N.G. and Eggum, B.O.(1986) On the digestibility of starch in wheat bread – studies in vitro and in vivo.Journal of Cereal Science 4, 1–11.

Björck, I., Nyman, M., Siljeström, M., Asp, N.G. and Eggum, B.O. (1987) Formationof renzyme resistant starch during autoclaving of wheat starch: studies in vitroand in vivo. Journal of Cereal Science 6, 159–172.

Björck, M.E. and Asp, N.G. (1994) Controlling the nutritional properties of starchin foods – a challenge to the food industry. Trends in Food Sciences and Technology5, 213–218.

Björck, M.E. and Siljeström, M.A. (1992) In-vivo and in-vitro digestibility of starch inautoclaved pea and potato products. Journal of the Science of Food and Agriculture58, 541–553.

Black, C.C., Mustardy, L., Sung, S.S., Kormanik, P.P., Xu, D.P. and Paz, N. (1987)Regulation and roles for alternative pathways of hexose metabolism in plants.Plant Physiology 69, 387–394.

Black, M., Corbineau, F., Grzesik, M., Guy, P. and Come, D. (1996) Carbohydratemetabolism in the developing and maturing wheat embryo in relation to itsdesiccation tolerance. Journal of Experimental Botany 47, 161–169.

Blackman, S.A., Wettlaufer, S.H., Obendorf, R.L. and Leopold, A.C. (1991) Matura-tion proteins associated with desiccation tolerance in soybean. Plant Physiology96, 868–874.

Blackman, S.A., Obendorf, R.L. and Leopold, A.C. (1992) Maturation proteins andsugars in desiccation tolerance of developing soybean seeds. Plant Physiology100, 225–230.

Blackman, S.A., Obendorf, R.L. and Leopold, A.C. (1995) Desiccation tolerance indeveloping soybean seeds: the role of stress proteins. Physiologia Plantarum 93,630–638.

Blakeney, A.B., Harris, P.J., Henry, R.J. and Stone, B.A. (1983) A simple and rapidpreparation of alditol acetates for monosaccharide analysis. CarbohydrateResearch 113, 291.

Blanshard, J.M.V. (1987) Properties and potential. In: Gallard, T. (ed.) Starch. JohnWiley & Sons, New York, pp. 15–54.

Blenford, D. (1994) Pea starches. Unique natural ingredients. International FoodIngredients 6, 27–32.

Blumenkrantz, M. and Asboe-Hansen, G. (1973) New method for quantitativedetermination of uronic acids. Analytical Biochemistry 54, 484.

248 References




Bochicchio, A., Rizzi, E., Balconi C., Vernieri, P. and Vazzana, C. (1994) Sucroseand raffinose contents and acquisition of desiccation tolerance in immaturemaize embryos. Seed Science Research 4, 123–126.

Bogracheva, T.Ya., Leontiev, S.P. and Genin, Ya.V. (1994) The effect of solutes onthe Gelatinisation of smooth pea starch. Carbohydrate Polymers 25, 227.

Bogracheva, T.Ya., Davydova, N.I., Genin, Ya.V. and Hedley, C.L. (1995) Mutantgenes at the r and rb loci affect the structure and physico-chemical properties ofpea seed starches. Journal of Experimental Botany 46, 1905–1913.

Bogracheva, T.Ya., Ring, S., Morris, V., Lloyd, J.R., Wang, T.L. and Hedley, C.L.(1997) The use of mutants to study the structural and functional propertiesof pea starch. In: Richmond, P., Frazier, P.J. and Donald, A.M. (eds) Starch:Structure and Function. Royal Society of Chemistry, Cambridge, pp. 230–237.

Bogracheva, T.Ya., Morris, D.G., Ring, S.G. and Hedley, C.L. (1998) The granularstructure of C-type pea starch and its role in gelatinisation. Biopolymers 45,232–332.

Bogracheva, T.Y., Cairns, P., Noel, T.R., Hulleman, S., Wang, T.L., Morris, V.J.,Ring, S.G. and Hedley, C.L. (1999) The effect of mutant genes at the r, rb, rug3,rug4, rug5 and lam loci on the granular structure and physio-chemical proper-ties of pea seed starch. Carbohydrate Polymers 39, 303–314.

Bohmer, P., Meyer, B. and Jacobsen, H.-J. (1995) Thidiazuron-induced highfrequency of shoot induction and plant regeneration in protoplast derivedpea callus. Plant Cell Reports 15, 26–29.

Bol, J.F., Linthorst, H.J.M. and Cornelissen, B.J.C. (1990) Plant pathogenesisrelated proteins induced by virus infection. Annual Reviews of Phytopathology 28,113–138.

Bolwell, G.P. (1985) Use of tissue cultures for studies of vascular differentiation.In: Dixon, R.A. (ed.) Plant Cell Culture – a Practical Approach. JRL Press, London,pp. 107–125.

Bolwell, P. (1993) Dynamic aspects of the plant extracellular matrix. InternationalReviews of Cytology 146, 261–323.

Borejszo, Z. and Khan, K. (1992) Reduction of flatulence-causing sugars byhigh temperature extrusion of pinto bean high starch fractions. Journal of FoodScience 57, 771–777.

Botham, R.L., Cairns, P., Morris, V.J. and Ring, S.G. (1995a) Physicochemicalcharacterisation of resistant starch in ileostomy effluent. In: Asp, N.G., vanAmelsvoort, J.M.M. and Hautvast, J.G.A.J. (eds) Proceedings of the ConcludingPlenary Meeting of EURESTA – Including the Final Reports of the Working Groups.ATO, Wageningen, pp. 71–72.

Bothman, R.L., Cairns, P., Morris, V.J., Ring, S.G., Englyst, H.N. and Cummings,J.H. (1995b) A physicochemical characterization of chick pea starch resistantto digestion in the human small intestine. Carbohydrate Polymers 26, 85–90.

Bouché-Pillon, S., Fleurat-Lessard, P., Serrano, R. and Bonnemain, J.L. (1994)Asymmetric distribution of the plasma-membrane H+-ATPase in embryos ofVicia faba L. with special reference to transfer cells. Planta 193, 392–397.

Bourdillon, A. (1998) Advantages and constraints of grain legumes for the feedmarket. In: AEP (ed.) Opportunities for High Quality, Health and Added-value Cropsto Meet European Demands. Proceedings 3rd European Conference on Grain Legumes.AEP, Paris, France, p. 5.

References 249




Bowles, D. (1990) Defence-related proteins in higher plants. Annual Reviews ofBiochemistry 59, 873–907.

Bramsnaes, F. and Olsen, H.S. (1979) Development of field pea and faba beanproteins. Journal of American Oil Chemistry’s Society 56, 450–454.

Brandt, E.B. and Hess, D. (1994) In vitro regeneration and propagation of chickpea(Cicer arietinum L.) from meristem tips and cotyledonatry nodes. In Vitro Cellularand Developmental Biology – Plant 30, 75–80.

Brar, G.S., Cohen, B.A., Vick, C.L. and Johnson, G.W. (1994) Recovery of transgenicpeanut (Arachis hypogaea L.) plants from elite cultivars utilizing ACCELLtechnology. The Plant Journal 5, 745–753.

Brenac, P., Horbowicz, M., Downer, M., Dickerman, S.M., Smith, A.M. andObendorf, R.L. (1997) Raffinose accumulation related to desiccation toleranceduring maize (Zea mays L.) seed development and maturation. Journal in PlantPhysiology 150, 481–488.

Brenes, A., Marquardt, R.R., Guenter, W. and Smolinski, B. (1992) Broiler chickperformance, gastrointestinal size, and digestibility of non-starch polisac-charides (NSP) and oligosaccharides as affected by enzyme addition todiets containing whole and dehulled lupin (L. albus). In: AEP (ed.) Proceedingsof the 1st European Conference on Grain Legumes, Angers. AEP, Paris, France,pp. 477–478.

Brenes, A., Marquardt, R.R., Guenter, W. and Rotter, B.A. (1993) Effect of enzymesupplementation on the nutritional value of raw, autoclaved, and dehulledlupins (Lupinus albus) in chicken diets. Poultry Sciences 72, 2281–2293.

Brett, C. and Waldron, K. (1990) Cell wall structure and the skeletal functions of thewall. In: Physiology and Biochemistry of Plant Cell Walls. Unwin Hyman, London,pp. 4–57.

Brighenti, F., Casiraghi, M.C. and Baggio, C. (1998) Resistant starch in the Italiandiet. British Journal of Nutrition 80, 333–341.

British Nutrition Foundation (1990) Complex carbohydrates in foods. In: The Reportof the British Nutrition Foundation’s Task Force. Chapman & Hall, London.

Brown, D.C.W. (1986) Plant regeneration through organogenesis. In: Vasil, I.K.(ed.) Cell Culture and Somatic Cell Genetics in Vitro, Vol. 3. Academic Press, NewYork, pp. 49–65.

Bruce, J.I.A. and West, C.A. (1989) Elicitation in lignic biosynthesis andisoperoxidase activity by pectic fragments in suspension-cultures of bean. PlantPhysiology 91, 889–897.

Bruni, F. and Leopold, A.C. (1991) Glass transition in soybean seed. Relevance toanhydrous biology. Plant Physiology 96, 660–663.

Brunt, K., Sanders, P. and Rozema, T. (1997) The enzymatic starch determination.Rapport 97–06, Nederlands Institut voor Koolhydraat Onderzoek TNO.

Bryant, G. and Wolfe, J. (1992) Can hydration forces induce lateral phase separa-tions in lamellar phases? Cryo-Letters 13, 23–36.

Buiatti, M. and Ingram, D.S. (1991) Phytotoxins as tools in breeding and selectionof disease-resistant plants. Experientia 47, 811–819.

Buitink, J., Walters-Vertucci, C., Hoekstra, F.A. and Leprince, O. (1996). Calorimet-ric properties of dehydrating pollen: analysis of a desiccation-tolerant and anintolerant species. Plant Physiology 111, 235–242.

250 References




Burbano, C., Muzquiz, M., Ayet, G., Cuadrado, C. and Pedrosa, M.M. (1999) Evalua-tion of antinutritional factors of selected varieties of Phaseolus vulgaris. Journal ofthe Science of Food and Agriculture 79, 1468–1472.

Burchfield, H.P. and Storrs, E.E. (1962) Biochemical Applications of Gas Chromatogra-phy. Academic Press, New York, pp. 119–120.

Burn, J., Chapman, P.D., Bishop, D.T. and Mathers, J. (1998) Diet and cancerprevention: the concerted action polyp prevention (CAPP) studies. Proceedingsof the Nutrition Society, 57, 183–186.

Burrows, W.J. and Carr, D.J. (1970) Cytokinin content of pea seeds during theirgrowth and development. Physiologia Plantarum 23, 1064–1070.

Burton, R.A., Bewley, J.D., Smith, A.M., Bhattacharyya, M.K., Tatge, H., Ring, S.,Bull, V., Hamilton, W.D.P. and Martin, C. (1995) Starch branching enzymesbelonging to distinct enzymes families are differentially expressed during peaembryo development. The Plant Journal 7, 3–15.

Buta, J.G. and Galletti, G.C. (1989) FT-IR investigation of lignin components invarious agricultural lignocellulosic by-products. Journal of the Science of Food andAgriculture 49, 37–43.

Butenko, R.G. (1985) Some features of cultured plant cells. In: Butenko, R.G. (ed.)Plant Cell Culture. MIR Publishers, Moscow, pp. 11–34.

Caffrey, M., Fonseca, V. and Leopold, A.C. (1988) Lipid–sugar interactions:relevance to anhydrous biology. Plant Physiology 86, 754–758.

Cairns, P., Bogracheva, T.Ya, Ring, S.G., Hedley, C.L. and Morris, V.J. (1997) Deter-mination of the polymorphic composition of smooth pea starch. CarbohydratePolymers 31, 275–282.

Camiranel, A., Brummell, D.A. and MacLachlan, G.A. (1987) Fucosylation ofxyloglucan: localization of the transferase in dicotisoms in pea stem cells. PlantPhysiology 84, 753–756.

Canibe, N. and Bach Knudsen, K.E. (1997) Digestibility of dried and toasted pea inpigs. 1. Ileal and total tract digestibility of carbohydrates. Animal Feed Science andTechnology 64, 293–310.

Capitani, T.A., Trzasko, P., Zallie, J.P. and Mason, W.R. (1996) Starch based lipidmimetic for foods. US Patent 5512311.

Carr, D.J. and Skene, K.G.M. (1961) Diauxic growth curves of seeds, with specialreference to french beans (Phaseolus vulgaris L.). Australian Journal of BiologicalSciences 14, 1–12.

Carré, B., Prevotei, B. and Leclercq, B. (1984) Cell wall content as a predictor ofmetabolisable energy value of poultry feedingstuffs. British Poultry Science 25,561–572.

Carré, B., Escartin, R., Melcion, J.P., Champ, M., Roux, G. and Leclerco, B. (1987)Effect of pelleting and association with maize or wheat on the nutritive value ofsmooth pea (Pisum sativum) seeds in adult cockerels. British Poultry Science 28,219–229.

Carré, B., Beaufils, E. and Melcion, J.-P. (1991) Evaluation of protein and starchdigestibilities and energy value of pelleted or unpelleted pea seeds from winteror spring cultivars in adult and young chickens. Journal of Agricultural and FoodChemistry 39, 468–472.

Carré, B., Gomez, J. and Chagneau, A.M. (1995) Contribution of oligosaccharideand polysaccharide digestion, and excreta losses of lactic acid and short chain

References 251




fatty acids, to dietary metabolisable energy value in broiler chickens and adultcockerels. British Poultry Science 36, 611–629.

Carré, B., Melcion, J.P., Widiez, J.L. and Biot, P. (1998) Effect of various processes offermentation grinding and storage of peas on the digestibility of pea starch inchickens. Animal Feed Sciences and Technology 71, 19–33.

Carrouee, B. (1995) Grain legumes in the European Union: current status andprospect. In: AEP (ed.) Improving production and utilisation of grain legumes.Proceedings of 2nd European Conference on Grain Legumes. AEP, Paris, France,pp. 2–4.

Casey, R., Domoney, C., Forster, C., Hedley, C., Hitchin, E. and Wang, T. (1998)The effect of modifying carbohydrate metabolism on seed protein geneexpression in peas. Journal of Plant Physiology 152, 636–640.

Casey, R., Forster, C., Hedley, C.L., Hitchin, E. and Wang, T.L. (1998a) The rela-tionship between carbohydrate metabolism and seed protein composition inpeas. In: AEP (ed.) Proceedings of the 3rd European Conference on Grain Legumes:Opportunities for High Quality, Healthy and Added-value Crops to Meet EuropeanDemands. AEP, Paris, France, pp. 84–85.

Casey, R., Domoney, C., Forster, C., Hedley, C.L., Hitchin, E. and Wang, T.L.(1998b) The effect of modifying carbohydrate metabolism on seed proteingene expression in peas. Journal of Plant Physiology 152, 636–640.

Cassidy, A., Bingham, S.A. and Cummings, J.H. (1994) Starch intake and colorectalcancer risk: an international comparison. British Journal of Cancer 69, 937–942.

Castillo, E.M., de Lumen, B.O., Reyes, P.S. and de Lumen, H.Z. (1990) Raffinosesynthase and galactinol synthase in developing seeds and leaves of legumes.Journal of Agricultural and Food Chemistry 38, 351–355.

Cavallini, A. and Natali, L. (1991) Nuclear DNA variability within Pisum sativum(Leguminosae): cytophotometric analyses. Plant Systematics and Evolution 173,179–185.

Cecchini, E., Natali, L., Cavallini, A. and Durante, M. (1992) DNA variations inregenerated plants of pea (Pisum sativum L.). Theoretical and Applied Genetics 84,874–879.

Cegla, G.P. and Bell, K.R. (1977) High pressure liquid chromatography for the anal-ysis of soluble carbohydrates in defatted oilseed flours. Journal of the AmericanOil Chemists Society 54, 150–152.

Cerletti, P., Eynard, L. and Guerrieri, N. (1993) Resistant starch: a negative orpositive entity in foods? Agro Food Industry Hi-Technology 4, 27–29.

Cerning-Beroard, J. and Filiatre-Verel, A. (1976) A comparison of the carbohydratecomposition of legume seeds: horsebeans, peas and lupines. Cereal Chemistry 53,968–978.

Cerning-Beroard, J. and Filiatre-Verel, A. (1979) Etude comparee de la compositionglucidique des graines de pois lisee et ride. Lebesmittel-Wissenschaft undTechnologie 12, 273–280.

Champ, M. (1992) Determination of resistant starch in foods and food products:interlaboratory study. European Journal of Clinical Nutrition 46, 51–62.

Champ, M. (1995) Definition, analysis, physical and chemical characterizationof RS. In: Asp, N.G., van Amelsvoort, J.M.M. and Hautvast, J.G.A.J. (eds) Proceed-ings of the Concluding Plenary Meeting of EURESTA – Including the Final Reports ofthe Working Groups. European FLAIR Concerted Action No. 11(COST 911),Brussels, pp.1–14.

252 References




Champ, M., Brilluet, J.M. and Rouau, X. (1986) Nonstarch polysaccharides ofPhaseolus vulgaris, Lens esculenta, and Cicer arietinum seeds. Journal of Agriculturaland Food Chemistry 34, 326–329.

Chang, C.W. (1982) Enzymic degradation of starch in cotton leaves. Phytochemistry21, 1263–1269.

Chapman, G.W., Pallas, J.E. and Mendicino, J. (1972) The hydrolysis ofmaltodextins by a α-amylase isolated from leaves of Vicia faba. BiochimicaBiophysica Acta 276, 491–507.

Chapman, N.S. (1985) An Introduction to Gas Chromatography. Pye Unicam,Cambridge, p. 20.

Chatterton, N.J., Harrison, P.A., Thornley, W.R. and Bennett, J.H. (1990)Sucrosyloligosaccharides and cool temperature growth in 14 forb species. PlantPhysiology and Biochemistry 28, 167–172.

Chauhan, G.S. and Bains, G.S. (1990) Equilibrium moisture content, BETmonolayer moisture and crispness of extruded rice-legume snacks. Journal ofFood Science and Technology 25, 360–363.

Chavan, J.K., Kadam, S.S. and Salunkhe, D.K. (1989) Chickpea. In: Salunkhe, D.K.and Kadam, S.S. (eds) CRC Handbook of World Food Legumes: Nutritional Chemis-try, Processing Technology and Utilization, Vol. I. CRC Press, Boca Raton, Florida,pp. 247–288.

Chen, C.H.-J. and Eisenberg, F. Jr (1975) myoInosose-2 1-phosphate: an inter-mediate in the myo-inositol 1-phosphate synthase reaction. Journal of BiologicalChemistry 250, 2963–2967.

Chen, W.-J.L. and Anderson, J.W. (1982) Soluble and insoluble fibre in selectedcereal and vegetables. American Journal of Clinical Nutrition 34, 1077–1082.

Chen, Y. and Burris, J.S. (1990) Role of carbohydrates in desiccation tolerance andmembrane behavior in maturing maize seed. Crop Science 30, 971–975.

Chengalrayan, K., Mhaske, V.B. and Hazra, S. (1997) High frequency conversion ofabnormal peanut somatic embryos. Plant Cell Reports 16, 783–786.

Chien, C.T., Lin, T.P., Juo, C.G. and Her, G.R. (1996) Occurrence of a novelgalactopinitol and its changes with other non-reducing sugars during develop-ment of Leucaena leucocephala seeds. Plant and Cell Physiology 37, 539–544.

Chirva, V.J., Kretsu, L.G. and Kintia, P.K. (1970) Triterpene glycosides from plants.I. Chemical characteristics of phaseoloside E. Chimija Prirodnych Soedinenij 93,377–378.

Chowrira, M.G., Akella, V. and Lurquin, P.F. (1995) Electroporation-mediated genetransfer into intact nodal meristems in planta. Generating transgenic plantswithout in vitro tissue culture. Molecular Biotechnology 3, 17–23.

Chowrira, M.G., Akella, V., Fuerst, P.E. and Lurquin, P.F. (1996) Transgenicgrain legumes obtained by in planta electroporation-mediated gene transfer.Molecular Biotechnology 5, 85–96.

Christou, P. (1992) Genetic Engineering and In Vitro Culture of Crop Legumes.Technomic Publishing Company, Lancaster, Pennsylvania, USA.

Christou, P. (1995) Strategies for variety-independent genetic transformation ofimportant cereals, legumes and woody species utilizing particle bombardment.Euphytica 85, 13–27.

Christou, P. (1997) Biotechnology applied to grain legumes. Field Crops Research 53,83–97.

References 253




Christou, P. and Swain, F. (1990) Cotransformation frequencies of foreign genes insoybean cell cultures. Theoretical and Applied Genetics 79, 337–341.

Cionini, P.G., Bennici, A. and D’Amato, F. (1978) Nuclear cytology of callusinduction and development in vitro. I. Callus from Vicia faba cotyledons.Protoplasma 96, 101–112.

Clott, P. and Walker, A.F. (1987) Air classification of flours of three legume species:fractionation of protein. Journal of Science of Food and Agriculture 38, 177–186.

Clott, P., Walker, A.F. and Pike, J. (1986) Air classification of flours of three legumespecies: effect of starch granule size distribution. Journal of Science of Food andAgriculture 37, 173–184.

Cocking, E.C. (1960) A method for the isolation of plant protoplasts and vacuoles.Nature 187, 962–963.

Colonna, P., Gueguen, J. and Mercier, C. (1981) Pilot scales preparation of starchand cell wall material from Pisum sativum and Vicia faba. Sciences des Aliments 1,415–426.

Colonna, P. and Mercier, C. (1984) Macromolecular structure of wrinkled- andsmooth-pea starch components. Carbohydrate Research 126, 233–247.

Conan, L. and Carre, B. (1989) Effect of autoclaving on motabolizable energy valueof smooth pea seed (Pisum sativum) in growing chicks. Animal Feed Science andTechnology 26, 337–345.

Constabel, F., Kirkpatrick, J.W. and Gamborg, O.L. (1973) Callus formation frommesopfyll protoplasts of Pisum sativum. Canadian Journal of Botany 51,2105–2106.

Cook, S.K., Adams, H., Hedley, C.L., Ambrose, M.J. and Wang, T.L. (1988) Ananalysis of seed development in Pisum sativum. VII. Embryo development andprecocious germination in vitro. Plant, Cell, Tissue and Organ Culture 14, 89–101.

Cooke, D. and Gidley, M.J. (1992) Loss of crystallinity and molecular order duringstarch gelatinisation: origin of the enthalpic transition. Carbohydrate Research227, 103–112.

Coon, C.N., Leske, K.L., Akavanichan, O. and Cheng, T.K. (1990) Effect of oligosac-charides-free soybean meal on true metabolizable energy and fibre digestion inadult roosters. Poultry Science 69, 787–793.

Copeland, L. and Preiss, J. (1981) Purification of spinach leaf ADP-glucosepyrophosphorylase. Plant Physiology 68, 996–1001.

Corke, F.M.K., Hedley, C.L., Shaw, P.J. and Wang, T.L. (1987) An analysis of seeddevelopment in Pisum sativum V. Fluorescence triple staining for investigatingcotyledon development. Protoplasma 140, 164–172.

Corke, F.M.K., Hedley, C.L. and Wang, T.L. (1990a) An analysis of seed develop-ment in Pisum sativum L. XI Cellular development and the deposition ofstorage protein in immature embryos grown in vivo and in vitro. Protoplasma155, 127–135.

Corke, F.M.K., Hedley, C.L. and Wang, T.L. (1990b) An analysis of seed develop-ment in Pisum sativum L. XII. In vitro manipulations of embryo developmentusing xenobiotic compounds. Protoplasma 155, 136–143.

Cote, F. and Hahn, M.G. (1994) Oligosaccharins: structures and signal trans-duction. Plant Molecular Biology 26, 1379–1411.

Craig, J., Lloyd, J.R., Tomlinson, K., Barber, L., Edwards, A., Wang, T.L., Martin, C.,Hedley, C.L. and Smith, A.M. (1998) Mutations in the gene encoding starch

254 References




synthase II profoundly alter amylopectin structure in pea embryos. The PlantCell 10, 413–426.

Craig, J., Barratt, P., Tatge, H., Dejardin, A., Handley, L., Gardner, C.D., Barber, L.,Wang, T., Hedley, C.L., Martin, C. and Smith, A.M. (1999) Mutations at therug4 locus alter the carbon and nitrogen metabolism of pea plants through aneffect on sucrose synthase. The Plant Journal 17, 353–362.

Cristofaro, E., Mottli, F. and Whurmann, J.J. (1974) Involvement of raffinose familyof oligosaccharides in flatulence. In: Sepple, H.L. and McNutt, K.W. (eds)Sugars in Nutrition. Academic Press, New York, pp. 313–363.

Crowe, J.H. and Crowe, L.M. (1986) Stabilization of membranes in anhydrobioticorganisms. In: Leopold, A.C. (ed.) Membranes Metabolism and Dry Organisms.Cornell University Press, Ithaca, New York, pp. 188–209.

Crowe, J.H., Crowe, L.M., Carpenter, J.F. and Aurell Wistrom, C. (1987) Stabiliza-tion of dry phospholipid bilayers and proteins by sugars. Biochemical Journal242, 1–10.

Crowe, J.H., Hoekstra, F.A., Nguyen, K.H.N. and Crowe, L.M. (1996) Is vitrificationinvolved in depression of the phase transition temperature in dryphospholipids? Biochemica et Biophysica Acta 1280, 187–196.

Crowe, L.M., Mouradian, R., Crowe, J.H., Jackson, S.A. and Womersley, C. (1984)Effects of carbohydrates on membrane stability at low water activities.Biochimica et Biophysica Acta 769, 141–150.

Cummings, J.H. and Englyst, H.N. (1989) Measurement of starch fermentationin the human large intestine. Canadian Journal of Physiology and Pharmacology69, 121–129.

Cummings, J.H. and Macfarlane, G.T. (1991) The control and consequences ofbacterial fermentation in the human colon. Journal Applied Bacteriology 70,443–459.

Cummings, J.H., Bingham, S.A., Heaton, K.W. and Eastwood, M.A. (1992) Fecalweight, colon cancer risk, and dietary intake of non polysaccharides (dietaryfibre). Gastroenterology 103, 1783–1789.

Cummings, J.H. and Englyst, H.N. (1995) Gastrointestinal effect of carbohydrate.American Journal of Clinical Nutrition 61 (Suppl.), 938S–945S.

Cutillas-Iturralde, A. and Lorences, E.P. (1997) Effect of xyloglucan oligosaccha-rides on growth, viscoelastic properties and long-term extension of pea shoots.Plant Physiology 113, 103–109.

Czuchajowska, Z., Sievert, D. and Pomeranz, Y. (1991) Enzyme-resistant starch. IV.Effects of complexing lipids. Cereal Chemistry 68, 537–542.

Dahmer, M.L., Collins, G.B. and Hildebrand, D.F. (1991) Lipid content and compo-sition of soybean somatic embryos. Crop Science 31, 741–746.

Dahmer, M.L., Hildebrand, D.F. and Collins, G.B. (1992) Comparative proteinaccumulation patterns in soybean somatic and zygotic embryos. In Vitro Cellularand Developmental Biology – Plant 28, 106–114.

D’Amato, F., Bennici, A., Cionini, P.G., Baroncelli, S. and Lupi, M.C. (1980)Nuclear fragmentation followed by mitosis as mechanism for wide chromo-some number variation in tissue cultures: its implications for plant regenera-tion. In: Sala, F., Parisi, B., Cella, R. and Ciferri, O. (eds) Plant Cell Cultures:Results and Perspectives, Developments in Plant Biology, Vol. 5. Elsevier/North-Holland Biomed. Press, Amsterdam, pp. 67–72.

References 255




Damiani, F., Pezzotti, M. and Arcioni, S. (1990) Somaclonal variation in Lotuscorniculatus L. in relation to plant breeding purposes. Euphytica 46, 35–41.

Datta, P.K., Basu, P.S. and Datta, T.K. (1985) Some biologically active proteinconstituents of Vicia faba protein bodies. Journal of Food Science and Technology22, 97–101.

Daveby, Y.D. and Aman, P. (1993) Chemical composition of certain dehulledlegume seeds and their hulls with special reference to carbohydrates. SwedishJournal of Agriculture Research 23, 133–139.

Daveby, Y.A., Razdan, A. and Åman, P. (1998) Effect of particle size and enzymesupplementation of diets based on dehulled peas on the nutritive value forbroiler chickens. Animal Feed Science and Technology 74, 229–239.

Davies, D.R. (1977) Restructuring the pea plant. Science Progress Oxford 64, 201–214.Davis, P., Merida, J., Legendre, L., Low, P.S. and Heinstein, P. (1993) Independent

elicition of the oxidative burst and phytoalexin formation in cultured plantcells. Phytochemistry 32, 607–611.

Davydova, N.I., Leont’ev, S.P., Genin, Y.V., Sasov, A.Y. and Bogracheva, T.Y. (1995)Some physico-chemical properties of smooth pea starches. Carbohydrate Polymers27, 109–115.

DeJong, A., Koerselman-Kooij, J.W., Schuurmans, J.A.M.J. and Borstlap, A.C. (1996)Characterization of the uptake of sucrose and glucose by isolated seed coathalves of developing pea seeds. Evidence that a sugar facilitator with diffusionalkinetics is involved in seed coat unloading. Planta 199, 486–492.

Delannay, X., Bauman, T.T., Beighley, D.H., Buettner, M.J., Coble, H.D., DeFelice,M.S., Derting, C.W., Diedrick, T.J., Griffin, J.L., Hagood, E.S., Hancock, F.G.,Hart, S.E., LaVallee, B.J., Loux, M.M., Lueschen, W.E., Matson, K.W., Moots,C.K., Murdock, E., Nickell, A.D., Owen, M.D.K., Paschal, E.H., Prochaska, L.M.,Raymond, P.J., Reynolds, D.B., Rhodes, W.K., Roeth, F.W., Sprankle, P.L.,Tarochionae, L.J., Tinius, C.N., Walker, R.H., Wax, L.M., Weigelt, H.D. andPadgette, S.R. (1995) Yield evaluation of a glyphosate-tolerant soybean lineafter treatment with glyphosate. Crop Science 35, 1461–1467.

Delcour, J.A. and Eerlingen, R.C. (1996) Analytical implications of the classificationof resistant starch as dietary fibre. Cereal Foods World 41, 85.

Denyer, K., Barber, L.R., Burton, R., Hedley, C.L., Hylton, C.M., Johnson, S.,Marshall, J., Smith, A.M., Tatge, H., Tomlinson, K. and Wang, T.L. (1995) Theisolation and characterization of novel low-amylose mutants of Pisum sativum L.Plant, Cell and Environment 18, 1019–1026.

Deshpande, S.S. and Cheryan, M. (1984) Effect of phytic acid, divalent cations andtheir interactions on α-amylase activity. Journal of Food Science 49, 516–519.

Deshpande, S.S. and Salunkhe, D.K. (1982) Interactions of tannic acid and catechinwith legume starches. Journal of Food Science 47, 2080–2081.

Detherage, W.L., McMasters, M.M. and Rist, C.E. (1955) A partial survey of amylosecontent in starch from domestic and foreign varietes of corn, wheat, andsorghum and from other starch-bearing plants. Transactions of the AmericanAssociation of Cereal Chemistry 13, 31–42.

Dey, P. and Pridham, J. (1972) Biochemistry of galactosidases. Advanced Enzymology36, 91–130.

Dey, P.M. (1985). D-galactose-containing oligosaccharides. In: Dey, P.M. (ed.)Biochemistry of Storage Carbohydrates in Green Plants. Academic Press, London,pp. 53–129.

256 References




Dey, P.M. (1990) Oligosaccharides. In: Dey, P.M. and Harbourne, J.B. (eds) Methodsin Plant Biochemistry, Vol. 2. Carbohydrates. Academic Press, New York, pp.189–218.

Dhir, S.K., Dhir, S. and Widholm, J.M. (1991) Plantlet regeneration from immaturecotyledon protoplasts of soybean (Glycine max L.). Plant Cell Reports 10, 39–43.

Dhir, S.K., Dhir, S. and Widholm J.M. (1992) Regeneration of fertile plants fromprotoplasts of soybean (Glycine max L. Merr): genotypic differences in cultureresponse. Plant Cell Reports 11, 285–289.

Di, R., Purcell, V., Collins, G.B. and Ghabrial, S.A. (1996) Production of transgenicsoybean lines expressing the bean pod mottle virus coat protein precursorgene. Plant Cell Reports 15, 746–750.

Dierick, N. and Decuyper, J. (1998) Effect of exogenous carbohydrase supple-mentation to high pea (Pisum sativum, L.) and soy bean meal diets on digestionand nutrient excretion in growing pigs. In: AEP (ed.) Opportunities for HighQuality, Healthy and Added-Value Crops to Meet European Demands. Proceedings ofthe 3rd European Conference on Grain Legumes, Valladolid, Spain. AEP, Paris,pp. 12–13.

Dillen, W., de Clerq, J., Goossens, A., van Montagu, M. and Angenon, G. (1997)Agrobacterium-mediated transformation of Phaseolus acutifolius A. Gray. Theoreti-cal and Applied Genetics 94, 151–158.

Dinehskumar, V., Kirti, P.B., Sachan, J.K.S. and Chopra, V.L. (1995) Picloraminduced somatic embryogenesis in chickpea (Cicer arietinum L.). Plant Science109, 207–213.

Dittrich, P. and Brandl, A. (1987) Revision of the pathway of D-pinitol formation inLeguminosae. Phytochemistry 26, 1925–1926.

Dolaptchiev, L., Bakalova, A., Vassileva, P., Ilieva, M. and Michneva, M. (1996)Tobacco plant cell cultures with high content of extracellular phospho-hydrolases. Enzyme Microbiology and Technology 19, 384–388.

Domoney, C. (1999) Inhibitors of legume seeds. In: Shewry, P.R. and Casey, R.(eds) Seed Proteins. Kluwer Academic Publishers, Dordrecht, the Netherlands,pp. 635–655.

Donovan, J.W. (1979) Phase transitions of the starch–water system. Biopolymers 18,263–275.

van Doorne, L.E., Marahall, L.E. and Kirkwood, R.C. (1995) Somatic embryogenesisin pea (Pisum sativum L.): effect of explant, genotype and culture conditions.Annals of Applied Biology 126, 169–179.

Dornbos, D.L. Jr. and McDonald, M.B. Jr (1986) Mass and composition of develop-ing soybean seeds at five reproductive growth stages. Crop Science 26, 624–630.

Dostálová, J., Krejcová, A., Réblova, Z. and Pokorný, J. (1999a) Changes ofα-galactosides during soaking and cooking of peas (in Czech). BulletinPotravinárskeho Výskumu 38, 133–140.

Dostálová, J., Zátopková, M., Réblová, Z. and Pokorný, J. (1999b) Changes ofundigestible saccharides during food processing (in Czech). Zborník PríspevkovZjazdu Chemických Spolebnosti 51, 1–2.

Dostálová, J., Borovicková, L. and Pokorný, J. (2000) Modification of recipesfor reduction of daily α-galactoside intake from legumes (in Czech). Výyiva apotraviny – Zpravodaj 55, 18–19.

Doublier, J.L. (1987) A rheological composition of wheat, maize, faba bean andsmooth pea starches. Journal of Cereal Science 5, 247–262.

References 257




Driouich, A. and Staehelin, L.A. (1997) The plant Golgi apparatus: structuralorganization and functional properties. In: Berger, E.G. and Roth, J. (eds) TheGolgi Apparatus. Birkhauser Verlag, Basel, pp. 275–301.

Driouich, A., Faye, L. and Staehelin, L.A. (1993) The plant Golgi apparatus: afactory for complex polysaccharides and glycoproteins. Trends in BiochemicalSciences 18, 210–214.

Dunn, G. (1974) A model for starch breakdown in higher plants. Phytochemistry 13,1341–1346.

Dunn, J.M. and Finocchiaro, E.T. (1997) Starch-based texturising agents andmethods of manufacture. US Patent 564243.

Dunn, J.M., Akliva, A.T. and Finocchiaro, E.T. (1996) Starch-based opacifying agentfor food beverages. US Patent 5571334.

Dwight Camper, N. and McDonald, S.K. (1989) Tissue and cell cultures as modelsystems in herbicide research. Reviews of Weed Science 4, 169–190.

Dysseler, D. and Hoffem, D. (1994) Estimation of resistant starch intake in Europe.In: Asp, N.G., van Amelsvoort, J.M.M. and Hautvast, J.G.A.J. (eds) Proceedings ofthe Concluding Plenary Meeting of EURESTA, April 1994. European Fair-ConcertedAction no. 11 (COST 911). ATO, Wageningen, pp. 84–86.

Dysseler, P. and Hoffem, D. (1995) Ring test 1993–1994 for total and resistant starchdetermination: results and discussion. In: Asp, N.G., van Amelsvoort, J.M.M.and Hautvast, J.G.A.J. (eds) Proceedings of the Concluding Plenary Meeting ofEURESTA – Including the Final Reports of the Working Groups. ATO, Wageningen,pp. 87–94.

Eapen, S. and George, L. (1993a) Somatic embryogenesis in peanut: influence ofgrowth regulators and sugars. Plant Cell Tissue and Organ Culture 35, 151–156.

Eapen, S. and George, L. (1993b) Plant regeneration from leaf disks of peanut andpigeonpea: Influence of benzyladenine, indolacetic acid and indoleaceticacid-aminoacid conjugates. Plant Cell Tissue and Organ Culture 35, 223–227.

Eapen, S. and George, L. (1994) Somatic embryogenesis in Cicer arietinum:influence of genotype and auxins. Biologia Plantarum 36, 343–349.

Ebel, J., Ayers, A.R. and Albersheim, P. (1976) Host-pathogen interactions. XII.Response of suspension-cultured soybean cells to the elicitor isolated fromPhytopthora megasperma var. Sojae, a fungal pathogen of soybean. Plant Physiology57, 775–779.

Edje, O.T. and Burris, J.S. (1970). Physiological and biochemical changes indeteriorating soybean seeds. Proceedings of the Association of Seed Analysis 60,158–166.

Edwards, A., Fulton, D., Hylton, C., Jobling, S.A., Gidley, M., Roessner, U., Martin,C. and Smith, A. (1998) A combined reduction in activity of starch synthases IIand III of potato has novel effects on the starch of tubers. Plant Journal 17,251–261.

Eerlingen, R.C. and Delcour, J.A. (1995) Formation, analysis, structure and proper-ties of type III enzyme resistant starch. Journal of Cereal Science 22, 129–138.

Eerlingen, R.C., Crombez, M. and Delcour, J.A. (1993a) Enzyme resistant starch. I.Quantitative and qualitative influence of incubation time and temperature ofautoclaved starch on resistant starch formation. Cereal Chemistry 70, 339–344.

Eerlingen, R.C., Deceunick, M. and Delcour, J.A. (1993b) Enzyme resistant starch.II. Influence of amylose chain length on resistant starch formation. CerealChemistry 70, 345–350.

258 References




Eerlingen, R.C., Cillen, G. and Delcour, J.A. (1994a) Enzyme resistant starch. IV.Effect of endogenous lipids and added dodecyl sulphate on formation ofresistant starch. Cereal Chemistry 71, 170–177.

Eerlingen, R.C., Jacobs, H. and Delcour, J.A. (1994b) Enzyme resistant starch. V.Effect of retrogradation of waxy maize starch on enzyme susceptibility. CerealChemistry 71, 351–355.

Eeuwens, C.J. and Schwabe, W.W. (1975) Seed and pod wall development in Pisumsativum L. in relation to extracted and applied hormones. Journal of ExperimentalBotany 26, 1–14.

Eisenberg, F. Jr. (1967) D-myo-Inositol 1-phosphate as product of cyclization ofglucose-6-phosphate and substrate for a specific phosphatase in rat testis.Journal of Biological Chemistry 242, 1375–1382.

Elliasson, A.-C. (1980) Effect of water on the gelatinisation of wheat starch.Starch/Stärke 32, 270–272.

Elliasson, A.-C. (1988) Physical and chemical characteristics of legume starches. ISIAtlas of Science: Animal and Plant Sciences, pp. 89–94.

Ellis, D. (1995) Genetic transformation of somatic embryos. In: Bajaj, Y.P.S. (ed.)Biotechnology in Agriculture and Forestry 30, Somatic Embryogenesis and SyntheticSeed 1. Springer-Verlag, Berlin, pp. 207–220.

Ellis, J.R., Shirsat, A.H., Hepher, A., Yarwood, J.N., Gatchause, J.A., Croy, R.R.D. andBoulter, D. (1988) Tissue specific expression of a pea legumin gene in seeds ofNicotiana lumbaginifolia. Plant Molecular Biology 10, 203–214.

Englyst, H.N. and Cummings, J.H. (1985) Digestion of polysaccharides of somecereal foods in the human small intestine. American Journal of Clinical Nutrition42, 778–787.

Englyst, H.N. and Cummings, J.H. (1986) Digestion of the carbohydrates of banana(Musa paradisiaca sapientum) in the human small intestine. American Journal ofClinical Nutrition 44, 42–50.

Englyst, H.N. and Cummings, J.H. (1987) Digestion of polysaccharides of potato inthe small intestine of man. American Journal of Clinical Nutrition 45, 423–431.

Englyst, H.N. and Cummings, J.H. (1988) Improved method for measurement ofdietary fibre as non-starch polysaccharides in plant foods. Journal of Associationof Official Analytical Chemists International 71, 808–814.

Englyst, H.N., Wiggins, H.S. and Cummings, J.H. (1982) Determination of thenon-starch polysaccharides in plant foods by gas–liquid chromatography ofconstituent sugars as alditol acetate. Analyst 107, 307–318.

Englyst, H.N., Kingman, S.M. and Cummings, J.H. (1992) Classification andmeasurement of nutritionally important starch fractions. European Journal ofClinical Nutrition 46 (Suppl. 2) 33S–50S.

Eriksson, T. (1985) Protoplast isolation and culture. In: Fowke, L. and Constable, F.(eds) Plant Protoplasts. CRC Press, Boca Raton, Florida, pp. 1–20.

Escarpa, A., Gonzales, M.C., Manas, M., Garcia-Diz, L. and Saura-Calixto, F. (1996)Resistant starch formation: standardization of a high-pressure autoclaveprocess. Journal of Agricultural Food Chemistry 44, 924–928.

Eskin, N.A.M., Johnsons, S., Vaisey-Genser, M. and McDonald, B.E. (1980) A studyof oligosaccharides in a selected group of legumes. Canadian Institute FoodScience and Technology Journal 13, 40–42.

Evans, D.A. and Bravo, J.E. (1984) Protoplast isolation and culture. In: Evans, D.A.(ed.) Handbook of Plant Cell Cultures, Vol. 1. Globe, UK, pp. 124–176.

References 259




Evans, I.D. and Haisman, D.R. (1982) The effect of solutes on the gelatinizationtemperature range of potato starch. Starch/Stärke 34, 224–231.

Ezhova, T.A., Bagrova, A.M., Hartina, G.A. and Gostimski, S.A. (1989) Study ofinheritance of somaclonal variations in Pisum sativum L. Genetics (Moscow) 25,878–885.

Faik, A., Chambat, G. and Joselean, J.P. (1995) Changes in wall-bound poly-saccharidase activities during the culture cycle of a Rulus fruticosus cellsuspension. International Journal of Biological Macromolecules 17, 381–386.

Faisant, N., Champ, M., Colonna, P. and Buleon, A. (1993a) Structural discrepan-cies in resistant starch obtained in vivo in humans and in vitro. CarbohydratePolymers 21, 205–209.

Faisant, N., Champ, M., Colonna, P. and Buleon, A. (1993b) Structural features ofstarch that escapes digestion in the human small intestine. In: Bioavailability 93.Proceedings of the Conference on Nutritional, Chemical and Food Processing Implicationsof Nutrient Availability, 146–150.

Faisant, N., Champ, M., Colonna, P., Buleon, A., Molis, C., Langkilde, A.M.,Schweizer, T., Fluorie, B. and Galmiche, J.P. (1993c) Structural features ofstarch at the end of the human small intestine. European Journal of ClinicalNutrition 47, 285–296.

Faisant, N., Buleon, A., Colonna, P., Molis, C., Lartigue, S., Galmische, J.P. andChamp, M. (1995a) Digestion of raw banana starch in the small intestine ofhealthy humans: structural features of resistant starch. British Journal of Nutrition73, 11–123.

Faisant, N., Colonna, P., Buleon, A., Bouchet, B., Gallant, D.J. and Champ, M.(1995b) Characteristics of starches escaping human small intestine digestion/absorption. In: Asp, N.G., van Amelsvoort, J.M.M. and Hautvast, J.G.A.J. (eds)Proceedings of the Concluding Plenary Meeting of EURESTA – Including the FinalReports of the Working Groups. ATO, Wageningen, pp. 115–116.

Faisant, N., Gallant, D.J., Bouchet, B. and Champ, M. (1995c) Banana starchbreakdown in the human small intestine studied by electronic microscopy.European Journal of Nutrition 49, 98–104.

Faisant, N., Planchot, F., Kozlowski, F., Pacouret, M.-P., Colonna, P. and Champ, M.(1995d) Resistant starch determination adapted to products containing highlevel of resistant starch. Scientific Alimentation 15, 85–91.

Falco, S.C., Guisa, T., Locke, M., Mauvais, J., Sanders, C., Ward, R.T. and Webber, P.(1995) Transgenic canola and soybean seeds with increased lysine. Bio-Technology (New York) 13, 577–581.

FAO Food Balance Sheets (1996) Statistics Series No. 133. FAO, Rome.FAO Yearbook Production (1996) 49.FAO Yearbook Production (1998) 51.FAO Yearbook Production (2000) 53.Farmer, E.E., Miloshok, T.D., Saxton, M.J. and Ryan, C.A. (1991) Oligosaccharide

signalling in plants. Specificity of oligouronid-enhanced plasma membraneprotein phosphorylation. Journal of Biological Chemistry 266, 3140–3145.

Faulks, R.M. and Bailey, A.L. (1990) Digestion of cooked starches from differentfood sources by procine α-amylase. Food Chemistry 36, 191–203.

Faulks, R.M., Southon, S. and Livesey, G. (1989) Utilisation of α-amylase resistantmaize and pea starch in the rat. British Journal of Nutrition 61, 291–300.

260 References




Feeney, R.E. and Whitaker, J.R. (1982) The Maillard reaction and its prevention. In:Cherry, J.P. (ed.) Food Protein Deterioration: American Chemical Society SymposiumNo. 206. American Chemical Society, Washington, DC, pp. 201–230.

Fehr, W.R., Caviness, C.E., Burmood, D.T. and Pennington, J.S. (1971) Stage ofdevelopment descriptions for soybeans, Glycine max (L.) Merrill. Crop Science 11,929–931.

Fellows, R.J., Egli, D.B. and Legget, J.E. (1978) A pod leakage technique for phloemtranslocation studies in soybean (Glycine max (L.) Merr.). Plant Physiology 62,812–814.

Fenwick, G.R., Price, K.R., Tsukamoto, C. and Okubo, K. (1991) Saponins. In: ToxicSubstances in Crop Plants, pp. 285–327.

Ferket, P.R. (1991) Effect of diet on gut microflora of poultry. Zootecnica Inter-national 7–8, 44–49.

Fernandez, J.A. and Batterham, E.S. (1995) The nutritive value of lupin-seed anddehulled lupin-seed meals as protein sources for growing pigs as evaluated bydifferent technique. Animal Feed Sciences and Technology 53, 279–296.

Fernandez-Romero, D., Moral, R., Gil, J. and Moreno, M.T. (1998) The effect ofTDZ on shoot regeneration from faba bean (Vicia faba). In: AEP (ed.) Opportu-nities for High Quality, Healthy and Added-Value Crops to Meet European Demands.Proceedings 3rd European Conference on Grain Legumes, Valladolid, Spain. AEP,Paris, p. 366.

Fernandez-Romero, D., Moreno, M.T., Cubero, J.I. and Gil, J. (1995). Plantletregeneration of chickpea (C. arietinum L.). In: AEP (ed.) Improving Productionand Utilisation of Grain Legumes. Proceedings 2nd European Conference on GrainLegumes, Copenhagen, Denmark. AEP, Paris, p. 435.

Fidanza, F.A.A., Zuccorini, M.G.P and Corvi, R.R. (1982) Contenuto in fibraalimentare di alcuni alimenti. La revista della Societa Italiana di Scienza dellAlimentazione 11, 3–7.

Finch-Savage, W.E., Hendry, G.A.F. and Atherton, N.M. (1994) Free radical activityand loss of viability during drying of desiccation-sensitive tree seeds. Proceedingsof the Royal Society of Edinburgh Section B (Biological Sciences) 102B, 257–260.

Finocchiaro, E.T. (1996) Starch containing reduced fat peanut butter and methodof manufacture. US Patent 5549923.

Flandre, F. and Sangwan-Norreel, B.S. (1995) Direct somatic embryogegenesis fromaxis of mature pea (Pisum sativum L.). Revue de Cytologie et de Biologie Vegetales leBotaniste 18, 39–60.

Fleming, S.E. (1980) A study of relationships between flatus potential and carbohy-drate distribution in legume seeds. Journal of Food Science 46, 794–798.

Fleming, S.E. (1981) Influence of cooking method on digestibility of legume cerealstarches. Journal of Food Science 47, 1–3.

Flinn, A.M. and Pate, J.S. (1968) Biochemical and physiological changes duringmaturation of fruit of the field pea (Pisum arvense L.). Annals of Botany 32,479–495.

Flis, M., Sobotka, W. and Zdunczyk, Z. (1997) Effect of variety and dehulling onnutritional value of white lupin seeds for growing pigs. Journal of Animal andFeed Sciences 6, 521–531.

Fontana, G., Santini, L., Caretto, S., Frugis, G. and Mariotti, D. (1993) Genetictransformation in the grain legume Cicer arietinum L. (chickpea). Plant CellReports 12, 194–198.

References 261




Ford, C.W. (1982) Accumulation of O-methyl-inositols in water stressed Vignaspecies. Phytochemistry 21, 1149–1151.

Fornal, J., Szmatowicz, B., Parkkonen, T. and Autio, K. (1995) Characteristics of thestarch isolated from pea seeds with various hardness. In: AEP (ed.) ImprovingProduction and Utilisation of Grain Legumes. Proceedings of the 2nd Conference onGrain Legumes, Copenhagen. AEP, Paris, pp. 398–399.

Fowke, L.C. and Constabel, F. (1985) Plant Protoplasts. CRC Press, Boca Raton,Florida, p. 245.

Fowke, L.C. and Wang, H. (1992) Protoplasts as tools in cell biology. PhysiologiaPlantarum 85, 391–395.

Fowke, L.C., Bech-Hansen, C.W., Constabel, F. and Gamborg, O.L. (1974) A com-parative study on the ultrastructure of cultured cells and protoplasts of soybeanduring cell division. Protoplasma 81, 189–203.

Franklin, Ch.I., Trieu, T.N., Gonzales, R.A. and Dixon, R. (1991) Plant regenerationfrom seedling explants of green bean (Phaseolus vulgaris L.) via organogenesis.Plant Cell Tissue and Organ Culture 24, 199–206.

Freire, J.P.B., Peiniau, J., Cunha, L.F., Almeida, J.A.A. and Aumaitre, A. (1997)Comparative effect of pea hulls and fat source on the digestive performanceand the activity of pancreatic and intestinal enzymes in the Large White andAlentejano weaned piglets. EAAP Publication 88, 554.

Frejnagel, S., Zdunczyk, Z. and Krefft, B. (1997) The chemical composition andnutritive value of low- and high-tannin faba bean varieties. Journal of Animal andFeed Sciences 6, 401–412.

French, D. (1984) Chemistry and Technology. In: Whistler, R.L., Bemiller, J.N. andPaschall, E.F. (eds) Starch. Academic Press, San Diego, California, pp. 183–247.

Freytag, A.H., Anand, S.C. and Rao-Arelli, A.P. (1988) Plant regeneration of cystnematode-resistant genotypes of soybeans. Current Science (Bangalore) 57,291–294.

Freytag, A.H., Rao-Arelli, A.P., Anand, S.C., Wrather, J.A. and Owens, L.D. (1989)Somaclonal variation in soybean plants regenerated from tissue culture. PlantCell Reports 8, 199–202.

Frias, J., Hedley, C.L., Price, K.R., Fenwick, G.R. and Vidal-Valverde, C. (1994a)Improved methods of oligosaccharide analysis for genetic studies of legumeseeds. Journal of Liquid Chromatography 17, 2469–2483.

Frias, J. Vidal-Valverde, C., Bakhsh, A., Arthur, A.E. and Hedley, C.L. (1994b) Anassessment of variation for nutritional and non-nutritional carbohydrates inlentil seeds (Lens culinaris). Plant Breeding 113, 170–173.

Frias, J., Prodanov, M., Sierra, I. and Vidal-Valverde, C. (1995) Effect of light oncarbohydrates and hydrosoluble vitamins of lentils during soaking. Journal ofFood Protection 58, 692–695.

Frias, J., Price, K.R., Fenwick, G.R., Hedley, C.L., Sörensen, H. and Vidal-Valverde,C. (1996a) Improved method for the analysis of α-galactosides in pea seeds bycapillary zone electrophoresis. Comparison with high-performance liquidchromatography-triple-pulsed amperometric detection. Journal of Chromato-graphy A 719, 213–219.

Frias, J., Vidal-Valverde, C., Kozlowska, H., Górecki, R., Honke, J. and Hedley, C.L.(1996b) Evolution of soluble carbohydrates during the development of pea,faba bean and lupin seeds. Zeitschrift für Lebensmittel-Unteruchung und-Forschung203, 27–32.

262 References




Frias, J., Vidal-Valverde, C., Kozlowska, H., Tabera, T., Honke, J. and Hedley, C.L.(1996c) Natural fermentation of lentils. Influence of time, flour concentrationand temperature on the kinetics of monosaccharides, disaccharide andalpha-galactosides. Journal of Agricultural Food Chemistry 44, 579–584.

Frias, J., Diaz-Pollan, C., Hedley, C.L. and Vidal-Valverde, C. (1996d) Evolution andkinetics of monosaccharides, disaccharide and alpha-galactosides duringgermination of lentils. Zeitschrift für Lebensmitteln Untersuchungen und Forschungs202, 35–39.

Frias, J., Sotomayor, C., Diaz-Pollan, C., Hedley, C.L., Fenwick, R.G. and Vidal-Valverde, C. (1996e) Influence of milling, concentration and temperatureduring soaking on soluble carbohydrate content and trypsin inhibitor activityin lentils seeds. In: Fenwick, R.G., Hedley, C., Richards, R.L. and Khokhar, S.(eds) Agri-Food Quality. An Interdisciplinary Approach. The Royal Society ofChemistry, Thomas Graham House, Science Park, Milton Road, Cambridge,pp. 284–288.

Frias, J., Fornal, J., Ring, S.G. and Vidal-Valverde, C. (1998) Effect of germinationon physico-chemical properties of lentil starch and its components. Food Scienceand Technology (LWT), in press.

Frias, J., Vidal-Valverde, C., Sotomayor, C., Diaz-Pollan, C. and Urbano, G. (1999)Influence of processing on available carbohydrate content and antinutritionalfactors of chickpeas. European Food Research and Technology, 210, 340–345.

Frias, J., Vidal-Valverede, C., Sotomayor, C., Diaz-Pollan, C. and Urbano, C. (2000)Effect of processing on available and non available carbohydrates content andtrypsin inhibitor activity on chick pea. European Food Research and Technology(in press).

Frokiaer, H., Nielson, D, Sorensen, H., Sorensen, J.C. and Sorensen, S. (1998)Determinations of soyasaponins in grain legumes by enzyme linked immuno-sorbent assay based on monoclonal antibodies. In: AEP (ed.) Proceedings of the3rd European Conference on Grain Legumes, Valladolid. Opportunities for High Quality,healthy and added-value crops to meet European demands. AEP, Paris, pp. 334–335.

Frolova, L. (1986) Cytogenetic characteristics of a cell population of Vicia faba invitro. Biologisches Zentralblatt 105, 105.

Frolova, L.V. and Shamina, Z.B. (1974) Cytogenetic characteristic of tissue cultureof plants from the Leguminaceae family. Cytology and Genetics (Kiev) 8, 413–418.

Frolova, L.V. and Shamina, Z.B. (1978) (in Russian) The kinetics of cell populationof Vicia faba cultured in vitro. Citologija 20, 551–556.

Frühbeck, G., Monreal, I. and Santidrian, S. (1997) Hormonal implications of thehypocholesterolemic effect of intake of field bean (Vicia faba L.) by young menwith hipercholesterolemia. American Journal of Clinical Nutrition 66, 1452–1460.

Fry, S.C. (1988) Wall polymers: Extraction and fractionation. In: Wilkins, M. (ed.)The Growing Plant Cell Wall: Chemical and Metabolic Analysis. Longman Scientificand Technical, Harlow, UK, pp. 49–101.

Fry, S.C. (1989) Cellulases, hemicellulases and auxinstimulated growth: a possiblerelationship. Physiologia Plantarum 75, 532–536.

Fry, S.C. (1994) Chemical analysis of components of the primary cell wall. In:Harris, N. and Oparka, K.J. (eds) Plant Cell Biology: a Practical Approach. OxfordUniversity Press, Oxford, pp. 199–220.

Fry, S.C., Aldington, Z., Hetherington, P.R. and Aitkai, J. (1993) Oligosaccharides assignals and substrates in the plant cell wall. Plant Physiology 103, 1–5.

References 263




Fu, J.H., Li, L.C., Yuan, H.L., Yue, S.X. and Zhu, L.H. (1993) Expression of theatrazine-resistant transgenic soybean plants in field tests. Acta Agronomica Sinica19, 497–500.

Gai, J.Y. and Guo, Z. (1997) Efficient plant regeneration through somatic embryo-genesis from germinated cotyledon of the soybean. Soybean Genetics Newsletter24, 41–44.

Galau, G.A., Jakobsen, K.S. and Hughes, D.W. (1991) The controls of dicots lateembryogenesis and early germination. Physiologia Plantarum 81, 280–288.

Gale, J. and Boll, W.G. (1978) Growth of bean cells in suspension culture in thepresence of NaCl and protein-stabilizing factors. Canadian Journal of Botany 57,777–782.

Gallant, D.J., Bouchet, B., Buleon, A. and Perez, S. (1992) Physical characteristics ofstarch granules and susceptibility to enzymatic degradation. European Journal ofClinical Nutrition, 46 (Suppl. 2), 3–16.

Galun, E. and Breiman, A. (1997) Transgenic Plants. Imperial College Press,London.

Gamborg, O.L., Davies, B.P. and Stahlhut, R.W. (1983) Cell division and differentia-tion in protoplasts from cell cultures of Glycine species and leaf tissue ofsoybean. Plant Cell Reports 2, 213–215.

Gamborg, O.L., Shyluk, J. and Kartha, K.K. (1975) Factors affecting the isolationand callus fromation in protoplasts from the shoot apices of Pisum sativum L.Plant Science 4, 285–292.

Gamborg, O.L., Constabel, F. and Shyluk, J. (1974) Organogenesis in callus fromshoot apices of Pisum sativum. Physiologia Plantarum 30, 125–128.

Ganter, C., Bock, A., Buckel, P. and Mattes, R. (1988) Production of thermostable,recombinant alpha-galactosidase suitable for raffinose elimination from sugarbeet syrup. Journal of Biotechnology 8, 301–310.

Ganter, J.L.M.S., Correa, J., Reicher, F., Heyraud, A. and Rinaudo, M. (1991) Lowmolecular weight carbohydrates from Mimosa scabrella seeds. Plant Physiologyand Biochemistry 29, 139–146.

Gantotti, B.V., Kartha, K.K. and Patil, S.S. (1985) In vitro selection of phaseolotoxinresistant plants using meristem culture of bean (Phaseolus vulgaris). Phyto-pathology 75, 1316–1317.

Garcia-Alonso, A., Goni, I. and Saura-Callixto, F. (1998) In vitro starch availabilityin raw and cooked legumes (lentils, chick peas and beans). In: AEP (ed.) Oppor-tunities for High Quality, Healthy and Added-value Crops to Meet EuropeanDemands.Proceedings of the 3rd European Conference on Grain Legumes, Valladolid,Spain. AEP, Paris, pp. 22–23.

Garcia-Olmedo, R., Diaz, A. and Villanueva, J. (1985) Estudio de la fibra alimentariaen legumbres cocinadas segun recetas tipicas de la cocina espanola. Anales deBromatologia 37, 79–90.

Garleb, K.A., Bourquin, L.D. and Fahey, G.C. (1991) Galactouronate in pecticsubstances from fruits and vegetables: comparison of anion exchange HPLCwith Pulsed amperometric detection to standard colorimetric procedure.Journal of Food Sciences 56, 423.

Garro, M., de Valdez, G.F., Oliver, G. and de Giorgi, G. (1996) Purification of alpha-galactosidase from Lactobacillus ferentum. Journal of Biotechnology 45, 103–109.

Gatel, F. and Champ, M. (1998) Grain legumes in human and animal nutrition – upto date results and question marks. In: AEP (ed.) Opportunities for High Quality,

264 References




Health and Added-value Crops to Meet European Demands. Proceedings of 3rd Euro-pean Conference on Grain Legumes. AEP, Paris, pp. 7–11.

Gaudreault, P.R. and Webb, J.A. (1981) Stachyose synthesis in leaves of Cucurbitapepo. Phytochemistry 20, 2629–2633.

Gdala, J. (1998) Composition, properties, and nutritive value of dietary fibre oflegume seeds. A review. Journal of Animal and Feed Sciences 7, 131–149.

Gdala, J. and Buraczewska, L. (1997) Ileal digestibility of pea and faba beanscarbohydrate in growing pigs. Journal of Animal Feed Sciences 6, 235–245.

Gdala, J., Buraczewska, L., Wasilewko, J. and Grala, W. (1995) Ileal digestibility ofnutrients in pigs fed diets with whole or dehulled faba beans (Vicia faba, L.)supplemented with enzymes. In: AEP (ed.) Improving Production and Utilisationof Grain Legumes. Proceedings of 2nd European Conference on Grain Legumes,Copenhagen, Denmark. AEP, Paris, pp. 264.

Gdala, J., Jansman, A.J.M., Buraczewska, L., Huisman, J. and van Leeuwen, P.(1997a) The influence of α-galactosidase supplementation on the ileal digest-ibility of lupin seed carbohydrates and dietary protein in young pigs. AnimalFeed Sciences and Technology 67, 115–125.

Gdala, J., Johansen, H.N., Bach Knudsen, K.E., Knap, I.H., Wagner, P. andJorgensen, O.B. (1997b) The digestibility of carbohydrates, protein and fatin the small and large intestine of piglets fed non-supplemented and enzymesupplemented diets. Animal Feed Sciences and Technology 65, 15–33.

Gatel, F. and Champ, M. (1998) Grain legumes in human and animal nutrition – upto date results and question marks. Opportunities for High Quality, Health andAdded-value Crops to Meet European Demands. Proceedings of 3rd European Conferenceon Grain Legumes, Valladolid, Spain. AEP, Paris, pp. 7–11.

Gee, M., McComb, E.A. and McCready, R.M. (1958) A method for the characteriza-tion of pectic substances in fruit and sugar-beet marks. Food Research 27, 72.

Gelvin, S.B. and Schilperoort, R.A. (1995) Plant Molecular Biology Manual. Kluwer,Dordrecht.

George, L. and Eapen, S. (1993) Influence of genotype and explant on somaticembryogenesis in peanut. Oleagineux 48, 361–364.

George, L. and Eapen, S. (1997). Influence of cytokinins and explant type on plantregeneration in chickpea (Cicer arietinum L.). Physiology and Molecular Biology ofPlants 3, 129–134.

Gerarde, J. (1636) The Herball; or, Generall Historie of Plantes, new edn. Norton andWhittakers, London, pp. 1219–1221.

Ghosh, P. and Sharma, A.K. (1979) Chromosome analysis in suspension culture ofVigna sinensis var. Black and Pisum sativum L. Caryologia 32, 419–424.

Gibeaut, D.M. and Carpita, N.C. (1994) Biosynthesis of plant cell wall polysaccha-rides. Review FASEB Federation of American Societies for Experimental Biology Journal8, 904–915.

Gidley, M. and Bociek, S.M. (1985) Molecular organisation of starch. A 13CCP/MAS NMR study. Journal of American Chemical Society 107, 7040–7044.

Gidley, M. and Robinson, G. (1990) Techniques for studying interactions betweenpolysaccharides. In: Dey, P.M. (ed.) Methods in Plant Biochemistry, Vol. 2. Carbohy-drates. Academic Press, London.

Gill, R. and Saxena, P.K. (1992) Direct somatic embryogenesis and regeneration ofplants from seedling explants of peanut (Arachis hypogeae): promotive role ofthidiazuron. Canadian Journal of Botany 70, 1186–1192.

References 265




Gitzelman, P. and Auricchio, S. (1965) The handling of soya alpha-galactosides by anormal and galactosemic child. Pediatrics 36, 231–232.

Goldberg, R., Devillers, P., Prat, R., Morvan, C., Michon, V. and Pentoat, C. (1986)Relationship to pectin properties. In: Lewis, N.G. and Paice, M.G. (eds) Controlof Cell Wall Plasticity. American Chemical Society, Washington, DC, pp.313–323.

Goldberg, R., Prat, R. and Morvan, C. (1994) Structural features of water-solublepectins from mung bean hypocotyls. Carbohydrate Polymers 23, 203–210.

Gonzales, C. and Kunka, B. (1986) Evidence for plasmid linkage of raffinose utiliza-tion and associated lactosidase and sucrose hydrolase activity in Pedicoccuspentosaeus. Applied Environmental Microbiology 51, 105–109.

Goodlad, J.S. and Mathers, J.C. (1990) Large bowel fermentation in rats given dietscontaining raw peas (Pisum sativum). British Journal of Nutrition 64, 569–587.

Goodlad, J.S. and Mathers, J.C. (1992) Digestion of complex carbohydrates andlarge bowel fermentation in rats fed on raw and cooked peas (Pisum sativum).British Journal of Nutrition 67, 475–488.

Gordon, D.T., Topp, K., Yong-Cheng, S., Zallie, J. and Jeffcoat, R. (1997) Resistantstarch: physical and physiological properties. In: Yalpani, M. (ed.) New Technol-ogies for Healthy Foods and Nutraceuticals. ATL Press, Shrewsbury, Massachusetts,pp. 157–178.

Górecki, R.J., Brenac, P., Clapham, W.M., Willcott, J.B. and Obendorf, R.L. (1996)Soluble carbohydrates in white lupin seeds matured at 13 and 28°C. Crop Science36, 1277–1282.

Górecki, R.J., Piotrowicz-Cieslak, A.I., Lahuta, L.B. and Obendorf, R.L. (1997)Soluble carbohydrates in desiccation tolerance of yellow lupin seeds duringmaturation and germination. Seed Science Research 7, 107–115.

Górecki, R.J. and Obendorf, R.L. (1997) Galactosyl cyclitols and raffinose familyoligosaccharides in relation to desiccation tolerance of pea and soyabeansseedlings. In: Ellis R.H., Black, M., Murdoch, A.J. and Hong, T.D. (eds)Applied Aspects of Seed Biology: Proceedings of the Fifth International Workshop onSeeds, Reading. Kluwer, Dordrecht, pp. 119–128.

Górecki, R.J., Lahuta, L.B., Jones, D.A. and Hedley, C.L. (1999) Soluble sugarsin maturing pea seeds of different lines in relation to desiccation tolerance. In:Black, M., Bradford, K.J. and Vazquez-Ramos, J. (eds) Seed Biology: Advances andApplications. CAB International, Wallingford, UK, pp. 67–74.

Gosal, S.S. and Bajaj, Y.P.S. (1984) Isolation of sodium chloride-resistant cell lines insome grain-legumes. Indian Journal of Experimental Biology 22, 209–214.

Gosal, S.S. and Bajaj, Y.P.S. (1988) Pollen embryogenesis and chromosomalvariation in anther culture of three food legumes – Cicer arietinum, Pisumsativum and Vigna mungo. Sabrao Journal 20, 51–58.

Gostimski, S.A., Bagrova, A.M. and Ezhova, T.A. (1985) (in Russian) Discoveryand cytogenetic analysis of variation originated during plant regeneration inpea tissue culture. Proceedings of the Academy of Sciences USSR 283, 1007–1011.

Goubet, F. and Morvan, C. (1993) Synthesis of cell wall galactans from flax (Linumusitassinum L.) suspension-cultured cells. Plant Cell Physiology 35, 719–727.

Gould, A.R. (1986) Factors controlling generation of variability in vitro. In: Vasil,I.K. (ed.) Cell Culture and Somatic Cell Genetics in Plants, Vol. 3. Academic Press,New York, pp. 549–599.

266 References




Gram, T., Mattsson, O. and Joersbo, M. (1996) Division frequency of pea proto-plasts in relation to starch accumulation. Plant Cell Tissue and Organ Culture 45,179–183.

Grant, J.E., Cooper, P.A., McAra, A.E. and Frew, T.J. (1995) Transformation of peas(Pisum sativum L.) using immature cotyledons. Plant Cell Reports 15, 254–258.

Grant, J.E., Pither-Joyce, M.D., Fifield, W., Cooper, P.A., Erasmuson, S.K. andTimmerman-Vaughan, G.M. (1998a) Resistance to alfalfa mosaic virus in trans-genic pea (Pisum sativum L.). In: Abstracts IX International Congress Plant TissueCell Culture, Jerusalem, p. 147.

Grant, J.E., Pither-Joyce, M., Fifield, W., Cooper, P.A. and Timmerman-Vaughan, G.(1998b) In: AEP (ed.) Opportunities for High Quality, Healthy and Added-ValueCrops to Meet European Demands. Proceedings of 3rd European Conference on GrainLegumes, Valladolid, Spain. AEP, Paris, pp. 372–373.

Gray, J. (1991) Starches and Sugars. A Comparison of their Metabolism in Man. ILSIEurope, Springer-Verlag, London, pp. 1–21.

Gray, L.E., Guan, Y.Q. and Widholm, J.M. (1986) Reaction of soybean callus toculture filtrates of Phialophora gregata. Plant Science 47, 45–55.

Graybosch, R.A., Edge, M.E. and Delannay, X. (1987) Somaclonal variation insoybean plants regenerated from the cotyledonary node tissue culture system.Crop Science 27, 803–805.

Green, J.L. and Angell, C.A. (1989) Phase relations and vitrification in saccharide–water solutions and the trehalose anomaly. Journal of Physical Chemistry 93,2880–2882.

Green, R.V., Gordon, S.H., Jackson, M.A., Bennett, G.A., McClelland, J.F. and Jones,R.W. (1992) Detection of fungal contamination in corn: potential of FT-IR PASand DRS. Journal of Agricultural and Food Chemistry 40, 883–886.

Greenberg, B.M. and Glick, B.R. (1993) The use of recombinant DNA technologyto produce genetically modified plants. In: Glick, B.R. and Thompson, J.E.(eds) Methods in Plant Molecular Biology and Biotechnology. CRC Press, BocaRaton, Florida, pp. 1–10.

Greenwood, J.S. and Chrispefls, M.J. (1985) Correct targeting of the bean storageprotein phaseolin in the seeds of transformed tobacco. Plant Physiology 79,65–71.

Griga, M. (1993) Some factors affecting somatic embryogenesis efficiency insoybean (Glycine max L. Merr). Biologia Plantarum 35, 179–189.

Griga, M. (1996) Agronomic performance of pea (Pisum sativum L.) somaclonesregenerated from embryogenic and organogenic cultures. In: Extended AbstractsCOST 822 WG, Mechanisms and Markers of Regeneration and Genetic Stability,Turku, Finland, pp. 10–11.

Griga, M. (2000) Morphological alterations in sterile mutant of Pisum sativumobtained via somatic embryogenesis. Biologia Plantarum, 43, 161–165.

Griga, M. and Klenoticova, H. (1994) Plant regeneration in callus cultures of fababean (Vicia faba). Rostlinna Vyroba 40, 697–709.

Griga, M. and Letal, J. (1995) Somaclonal variation study of pea (Pisum sativum L.).In: Recent Advances in Plant Biotechnology, International Conference of the Institute ofPlant Genetics. 95-IPG, Nitra, Slovakia, pp. 130–135.

Griga, M. and Letal, J. (1996) Improved protocol for pea (Pisum sativum L.) somaticembryogenesis and field performance of pea somaclones. In: Abstracts 2ndAsia–Pacific Conference Plant Cell Tissue Culture, Beijing, p. 4.

References 267




Griga, M. and Novak, F.J. (1990) Pea (Pisum sativum L.). In: Bajaj, Y.P.S. (ed.) Bio-technology in Agriculture and Forestry, Vol. 10. Legumes and Oil Seed Crops 1.Springer-Verlag, Berlin, pp. 65–99.

Griga, M. and Slama, J. (1997) Direct pea somatic embryogegenesis and screeningPisum genotypes for embryogenic capacity. In: Mechanisms and Markers ofRegeneration and Genetic Stability. COST Extended Abstracts 3rd Meeting ofCOST 822 Working Group, Gieben, Belgium, pp. 226–227.

Griga, M. and Stejskal, J. (1993) Organogenesis and somatic embryogenesis in vitroin pea (Pisum sativum L., P. arvense L.) and field evaluation of somaclones – apreliminary report. In: Book of Abstracts XXIII Annual ESNA Meeting, Halle,p. 127.

Griga, M. and Stejskal, J. (1994) Micropropagation of pea (Pisum sativum L.) – invitro system and its practical applications. In: Lumsden, P.J., Nicholas, J.R. andDavies, W.J. (eds) Physiology, Growth and Development of Plants in Culture. Kluwer,Dordrecht, pp. 278–283.

Griga, M., Tejklova, E., Novak, F.J. and Kubalakova, M. (1986) In vitro clonalpropagation of Pisum sativum L. Plant Cell Tissue and Organ Culture 6, 95–104.

Griga, M., Stejskal, J. and Klenoticova, H. (1992) Plant regeneration in soybean,pea and faba bean via somatic embryogenesis. In: AEP (ed.) Proceedings of 1stEuropean Conference on Grain Legumes, Angers. AEP, Paris, pp. 107–108.

Griga, M., Stejskal, J. and Beber, K. (1995) Analysis of tissue culture-derivedvariation in pea (Pisum sativum L.) – preliminary results. Euphytica 85, 335–339.

Grosjean, F. and Gatel, F. (1986) Pea for pigs. Pig News Information 7, 443–448.Gross, P. and ap Rees, T. (1986) Alkaline inorganic pyrophosphatase and starch

synthesis in amyloplasts. Planta 167, 140–145.Gruchala, L. and Pomeranz, Y. (1993) Enzyme-resistant starch: studies using

differential scanning calorimetry. Cereal Chemistry 70, 163–170.Gueguen, J. (1983) Legume seed protein extraction, processing, and end product

characteristics. Qualitas Plantarum Plant Food and Human Nutrition 32, 267–303.Gujska, E. and Khan, K. (1991) Functional properties of extrudates from high

starch fractions of navy and pinto beans and corn meat blended with legumehigh protein fractions. Journal of Food Science 56, 431–435.

Guo, H. (1991) Protoplast isolation, culture and differentiation responses insoybean. MSc thesis, Kentucky University, p. 122.

Gupta, S. (1975) Morhogenetic response of haploid callus tissue of Pisum sativum(var. B22). Indian Agriculturist 19, 11–21.

Gurr, M.I. and Asp, N.G. (1994) Dietary Fibre, 2nd edn. ILSI Press, Washington, DC.Haase, N.U. and Kempf, W. (1991) Ein Beitrag zur Biosynthese amylosereicher

Erbsestarken. Starch 43, 2–5.Haase, N.U. and Shi, H.L. (1991) A characterization of faba bean starch (Vicia faba

L.). Starch 43, 205–208.Haddon, L.E. and Northcote, D.H. (1976) The effect of growth conditions and

origin of tissue on the ploidy and morphogenetic potential of tissue cultures ofbean (Phaseolus vulgaris L.). Journal of Experimental Botany 27, 1031–1051.

Hague, A., Manning, A.M., Hanlon, K.A., Huschtscha, L.I., Hart, D. and Paraskeva,C. (1993) Sodium butyrate induces apoptosis in human colonic tumour ceelines in a p53-independent pathway. Implications for the possible role ofdietary fibre in the prevention of large-bowel cancer. International Journal ofCancer 55, 498–505.

268 References

A3867: AMA: Hedley: First Proof:14-Nov-00 References



Halbach, T., Kiesecker, H., Jacobsen, H.J. and DeKathen, A. (1998) Tissue cultureand engineering of lentil (Lens culinaris Medik). In: AEP (ed.) Opportunities forHigh Quality, Healthy and Added-Value Crops to Meet European Demands. Proceedings3rd European Conference on Grain Legumes, Valladolid, AEP, Paris, p. 376.

Hall, J.M. (1989) A review of total dietary fiber methodology. Cereal Foods World 34,526–528.

Halmer, P. (1985) The mobilisation of storage carbohydrates in germinated seeds.Physiologie Vegetale 23, 107–125.

Halperin, W. (1986) Attainment and retention of morphogenetic capacity in vitro.In: Vasil, I.K. (ed.) Cell Culture and Somatic Cell Genetics in Vitro, vol. 3. AcademicPress, New York, pp. 3–48.

Ham, K.S., Kauffmann, S., Albersheim, P. and Darvill, A.G. (1991) Host pathogeninteractions XXXIX. A soybean pathogenesis related phytoalexin elicitor-activeheat-stable fragments from fungal walls. Molecular Plant–Microbe Interactions 44,545–552.

Hamblin, J., Barton, J., Atkins, C., Jones, M., Smith, P., Wylie, S., Schroeder, H.,Molvig, L., Tabe, L. and Higgins, T.J. (1998) The development and status oftransgenic pulses in Australia. In: AEP (ed.) Opportunities for High Quality,Healthy and Added-Value Crops to Meet European Demands. Proceedings 3rd EuropeanConference on Grain Legumes, Valladolid. AEP, Paris, pp. 70–71.

Hammat, N. and Davey, M.R. (1988) Isolation and culture of soybean hypocotylprototplasts. In Vitro Cellular and Developmental Biology 24, 601–604.

Hammat, N., Kim, H-I., Davey, M.R., Nelson, R.S. and Cocking, E.C. (1987) Plantregeneration from cotyledon protoplasts of Glycine canescence and G. cladestina.Plant Science 48, 129–135.

Hammat, N., Ghose, T.K. and Davey, M.R. (1986) Regeneration in legumes. In:Vasil I.K. (ed.) Cell Culture and Somatic Cell Genetics in vitro, Vol. 3. AcademicPress, New York, pp. 67–95.

Hanke, D.E. and Northcote, D.H. (1974) Cell wall formation by soybean callusprotoplasts. Journal of Cell Science 14, 29–50.

Harley, S.M. and Beevers, L. (1989) Coated vesicles are involved in the transport ofstorage proteins during seed development in Pisum sativum L. Plant Physiology91, 674–678.

Harris, N. (1976) Starch grain breakdown in cotyledon cells of germinating mungbean seeds. Planta 129, 271–272.

Harrison, C.J. (1996) The rug-3 locus of pea. PhD thesis, University of East Anglia,Norwich.

Harrison, C.J., Hedley, C.L. and Wang, T.L. (1998) Evidence that the rug3 locus ofpea (Pisum sativum L.) encodes plastidial phosphoglucomutase confirms thatthe imported substrate for starch synthesis in pea amyloplasts is glucose-6-phosphate. The Plant Journal 13, 753–762.

Hartman, C.L., McCoy, T.J. and Knous, T.R. (1984) Selection of alfalfa (Medicagosativa) cell lines and regeneration of plants resistant to the toxin(s) producedby Fusarium oxysporum f. sp. Medicaginis. Plant Science Letters 34, 183–194.

Hartman, C.L., Secor, G.A., Venette, J.R. and Albaugh, D.A. (1986) Response ofbean calli to filtrate from Pseudomonas syringae pv. Phaseolicola and correlationwith whole plant disease reaction. Physiology Molecular Plant Pathology 28,353–358.

References 269




Harzic, N., Guilloteau, A. and Huyghe, C. (1998) In vitro shoot formation of Lupinusalbus from cotyledonary node. In: AEP (ed.) Opportunities for High Quality,Healthy and Added-Value Crops to Meet European Demands. Proceedings 3rd EuropeanConference on Grain Legumes, Valladolid. AEP, Paris, p. 369.

Hauxwell, A.J., Corke, F.M.K., Hedley, C.L. and Wang, T.L. (1990) Storage proteingene expression is localised to regions lacking mitotic activity in developingpea embryos. An analysis of seed development in Pisum sativum XIV. Develop-ment 110, 283–289.

Hayashi, T. (1989) Xyloglucans in the primary cell wall. Annual Review of PlantPhysiology and Plant Molecular Biology 40, 139–168.

Heatley, M.E. and Smith, R.H. (1996) Whole plant regeneration from shoot apex ofArachis hypogaea. In Vitro Cellular and Developmental Biology – Plant 32, 115–118.

Hedley, C.L. and Ambrose, M.J. (1980) An analysis of seed development in Pisumsativum L. Annals of Botany 46, 89–105.

Hedley, C.L. and Ambrose, M.J. (1981) Designing ‘leafless’ plants for improvingyields of dried pea crops. Advances in Agronomy 34, 225–277.

Hedley, C.L. and Smith, C.M. (1985) Genetic variation for pea-seed development.In: Hebblethwaite, P.D., Heath, M.C. and Dawkins, T.C.K. (eds) The Pea Crop:a Basis for Improvement. Butterworths, Sevenoaks, UK, pp. 329–338.

Hedley, C.L., Smith, C., Ambrose, M.J., Cook, S. and Wang, T.L. (1986) An analysisof seed development in Pisum sativum. II. The effect of the r-locus on thegrowth and development of the seed. Annals of Botany 58, 371–379.

Hedley, C.L., Lloyd, J.R., Ambrose, M.J. and Wang, T.L. (1994) An analysis of seeddevelopment in Pisum sativum. XVII. The effect of the rb locus alone and incombination with r on the growth and development of the seed. Annals ofBotany 74, 365–371.

Hedley, C.L., Bogracheva, T.Ya., Lloyd, J.R. and Wang, T.L. (1996) Manipulation ofstarch composition and quality in pea seeds. In: Fenwick, G.R., Hedley, C.L.,Richards, R.L. and Khokhar, S. (eds) Agri-Food Quality, an InterdisciplinaryApproach. The Royal Society of Chemistry, Cambridge, pp. 138–148.

Heim, U., Weber, H., Baumlein, H. and Wobus, U. (1993) A sucrose-synthase geneof Vicia faba. Expression pattern in developing seeds in relation to starchsynthesis and metabolic regulation. Planta 191, 394–401.

Hendry, G.A.F. (1993) Oxygen, free radical processes and seed longevity. SeedScience Research 3, 141–153.

Hendry, G.A.F., Finch-Savage, W.E., Thrope, P.C., Atherton, N.M., Buckland, S.H.,Nilson, K.A. and Seel, W.E. (1992) Free radical processes and loss of seedviability during desiccation in the recalcitrant species Quercus rubur L. NewPhytologist 122, 273–279.

Heredia, A., Jiménez, A. and Guillén, R. (1995) Review. Composition of plant cellwalls. Zeitschrift für Lebensmittel-Unteruchung und-Forschung 200, 24–31.

Hermann, E.M. and Shannon, L.M. (1985) Accumulation and subcellular localisa-tion of alpha-galactosidase-hemagglutinin in developing soybean cotyledons.Plant Physiology 77, 886–890.

Herrera-Estrella, L., Leon, P., Olsson, O. and Teeri, T.H. (1995) Reporter genesfor plants. In: Gelvin, S.B. and Schilperoort, R.A. (eds) Plant Molecular BiologyManual. Kluwer, Dordrecht, pp. 1–32.

270 References




Herselman, L. and Mienie, C.M.S. (1995) The production of disease free dry beanseed through meristem tip culture. Centro International de Agricultura Tropical,Cali, Columbia. Workshop Proceedings. CIAT, SACCAR 188–190.

Hesemann, C.U. (1980) Haploid cells in calli from anther culture of Vicia faba.Zeitschrift für Pflanzenzuchtung 84, 18–22.

Hildebrand, D.F., Adams, T.R., Dahmer, M.L., Williams, E.G. and Collins, G.B.(1989) Analysis of lipid composition and morphological characteristics insoybean regenerants. Plant Cell Reports 7, 701–703.

Hildebrandt, L.A. and Marlett, J.A. (1991) Starch bioavailability in the uppergastrointestinal tract of colectomized rats. Journal of Nutrition 121, 679–686.

Hill, J. (1756) The British Herbal, an History of Plants and Trees, Natives of Britain,Cultivated for Use, or Raised for Beauty. Osborne, J., Shipton, J., Hodges, J.,Newbery, B., Collins, S., Crowder and H. Woodgate, London, 279 pp.

Hill, L.M. and Smith, A.M. (1991) Evidence that glucose-6-phosphate is imported asthe substrate for starch synthesis by plastids of developing pea embryos. Planta185, 91–96.

Hiller, K., Keipert, M. and Linzer, B. (1966) Triterpensaponine. Die Pharmazie 21,713–751.

Hinchee, M.A.W., Connor-Ward, D.V., Newell, C.A., McDonnell, R.E., Sato, S.J.,Gaser, C.S., Fischhoff, D.A., Re, D.B., Fraley, R.T. and Horsch, R.B. (1988)Production of transgenic soybean plants using Agrobacterium-mediated DNAtransfer. Bio-Technology (New York) 6, 915–922.

Hirasawa, E. (1989) Auxin induce α-amylase activity in pea cotyledons. PlantPhysiology 91, 484–486.

Hizukuri, S. and Takagi, T. (1984) Estimation of the molecular weight for amylaseby the low angle laser-light-scattering technique combined with high-performance chromatography. Carbohydrate Research 134, 1–10.

Ho, L.C. (1988) Metabolism and compartmentation of imported sugars in sinkorgans in relation to sink strength. Annual Review of Plant Physiology and PlantMolecular Biology 39, 355–378.

Hoch, G., Peterbauer, T. and Richter, A. (1999) Purification and characterizationof stachyose synthase from lentil (Lens culinaris) seeds: galactopinitol andstachyose synthesis. Archives of Biochemistry and Biophysics 366, 75–81.

Hoekstra, F.A., Crowe, L.M. and Crowe, J.H. (1989) Differential desiccationsensitivity of corn and Pennisetum pollen linked to their sucrose contents. PlantCell and Environment 12, 83–91.

Hoekstra, F.A., Haigh, A.M., Tetteroo, F.A.A. and Roekel, van T. (1994) Changesin soluble sugars in relation to desiccation tolerance in cauliflower seeds. SeedScience Research 4, 143–147.

Hoffmann-Ostenhof, O. and Pittner, F. (1982) Biosynthesis of myo-inositols and itsisomers. Canadian Journal of Chemistry 60, 1863–1871.

Holliday, M.J. and Klarman, W.L. (1979) Expression of disease reaction types insoybeans callus from resistant and susceptible plants. Phytopathology 69, 576.

Honda, S., Nishimura, Takahashi, M.Y., Chiba, H. and Kayehi, K. (1982) A manualmethod for the spectrophotometric determination of reducing carbohydrateswith 2-cyanoacetamide. Analytical Biochemistry 119, 194–199.

Hoover, R. and Sosulski, F. (1985a) Studies on the functional characteristics anddigestibility of starches from Phaseolus vulgaris biotypes. Starch 37, 181–191.

References 271




Hoover, R. and Sosulski, F. (1985b) A comparative study of the effect of acetylationon starches of Phaseolus vulgaris biotypes. Starch 37, 397–404.

Hoover, R. and Sosulski, F. (1986) Effect of cross-linking on functional properties oflegume starches. Starch/Stärke 38, 149–154.

Hoover, R. and Sosulski, F.W. (1991) Composition, structure, functionality andchemical modification of legume starches: a review. Canadian Journal of Physiol-ogy and Pharmacy 69, 79–92.

Hoover, R. and Manuel, H. (1994) The effect of annealing on the physicochemicalproperties of legume starches. In: Fenwick, G.R., Hedley, C., Richards, R.L. andKhokhar, S. (eds) Agricultural Food Quality. An Interdisciplinary Approach. TheRoyal Society of Chemistry, Cambridge, pp. 157–160.

Hoover, R. and Vasanthan, T. (1994) Effect of heat-moisture treatment on thestructure and physicochemical properties of cereal, legume and tuber starches.Carbohydrate Research 252, 33–53.

Hoover, R., Hannouz, D. and Sosulski, F.W. (1988) Effects of hydroxypropylationon thermal properties, starch digestibility and freeze–thaw stability of field pea(Pisum sativum cv Trapper) starch. Starch 40, 383–387.

Hoover, R., Swamidas, G. and Vasanthan, T. (1993) Studies on the physico-chemicalproperties of native, defatted, and heat-moisture treated pigeon pea. Carbohy-drate Research 246, 185–203.

Horbowicz, M. and Obendorf, R.L. (1994) Seed desiccation tolerance andstorability: dependence on flatulence-producing oligosaccharides and cyclitols– review and survey. Seed Science Research 4, 385–405.

Horbowicz, M., Obendorf, R.L., McKersie, B. and Viands, D.R. (1995) Soluble sac-charides and cyclitols in alfalfa (Medicago sativa L.) somatic embryos, leaflets,and mature seeds. Plant Science 109, 191–198.

Horbowicz, M., Brenac, P. and Obendorf, R.L. (1998) Fagopyritol B1, O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol, a galactosyl cyclitol in maturingbuckwheat seeds associated with dessication tolerance. Planta 205, 1–11.

Huber, S.C. and Akazawa, T. (1986) A novel sucrose synthase pathway for sucrosedegradation in cultured sycamore cells. Plant Physiology 81, 1008–1013.

Hudlicky, T. and Cebulak, M. (1993) Cyclitols and their Derivatives: a Handbook ofPhysical, Spectral, and Synthetic Data. VCH Publishers, New York.

Hussey, G. and Gunn, H.V. (1984) Plant production in pea (Pisum sativum L. Cvs.Puget and Upton) from long-term callus with superficial meristems. PlantScience 37, 143–148.

Hylton, C. and Smith, A.M. (1992) The rb mutation of peas causes structural andregulatory changes in ADP glucose pyrophosphorylase from developingembryos. Plant Physiology 99, 1626–1634.

Igbasen, F.A., Guenter, W. and Slominski, B.A. (1997) The effect of pectinase and α-galactosidase supplementation on the nutritive value of peas for broilerchickens. Canadian Journal of Animal Science 77, 537–539.

Ilieva, M., Bacalova, A., Pavlov, A., Michneva, M. and Dolaptchiev, L. (1996a) Pro-duction of phosphomonoesterases by Nicotiana tabacum 1507 in an aqueoustwo-phase system. Biotechnology and Bioengineering 51, 488–493.

Ilieva, M., Kratchanova, M., Pavlova, E., Dimova, Ts. and Pavlov, A. (1996b) Poly-galacturonase and pectinmethylesterase activities during growth of Helinthusannuus 1805 cell suspension. In: Visser, I. and Voragen, A.G.J. (eds) Pectin andPectinases. Elsevier Sciences, Amsterdam, pp. 869–874.

272 References




Ilieva, M., Kratchanova, M., Pavlova, E., Dimova, Ts., Petrova, D., Pavlov, A. andMichneva, M. (1996c) Polygalacturonase and pectinmethylesterase activitiesduring growth of Nicotiana tabacum 1508 cell suspension. In: Proceedings of XVIIIInternational Carbohydrate Symposium, Milan, pp. 21–26.

Imberty, A. and Perez, S. (1988) A revisit to the three dimensional structure ofB-type starch. Biopolymers 27, 1205–1221.

Imberty, A., Chanzy, H. and Perez, S. (1988) The double-helical nature of thecrystalline part of A-starch. Journal of Molecular Biology 201, 365–378.

Ioshikava, M., Takenchi, I. and Horino, O. (1990) A mechanism for ethylene-induced disease resistance in soybean: enhanced synthesis of an elicitor-releasing factor 3-endoglucanase. Physiology and Molecular Plant Pathology 37,367–376.

Ishii, T. (1997) Structure and functions of feruloylated polysaccharides. PlantSciences 127, 111–127.

Islam, R. (1994) Somatic embryogenesis from immature cotyledons of chickpea(Cicer arietinum L.). Pakistan Journal of Botany 26, 197–199.

Islam, R., Farooqui, H. and Riazuddin, S. (1993) In vitro organogenesis of chickpeaand its transformation by Agrobacterim tumefaciens. Plant Tissue Culture 3, 29–34.

Iyer, V., Salunkhe, D.K., Satha, S.K. and Rockland, L.B. (1980) Quick cooking beans(Phaseolus vulgaris L.) II. Phytate, oligosaccharides and antienzymes. QualitiesPlantarum Foods for Human Nutrition 30, 45–52.

Iyengar, A.K. and Kulkarni, P.R. (1977) Oligosaccharide levels of processedlegumes. Animal Feed Sciences and Technology 14, 222–223.

Jackson, J. and Hobbs, S. (1990) Rapid multiple shoot production from cotyledon-ary node explants of pea (Pisum sativum L.). In Vitro Cellular and DevelopmentalBiology – Plant 26, 835–838.

Jacobs, H., Eerlingen, R.C., Clauwert, W. and Delcour, J.A. (1995) Influence ofannealing on the pasting properties of starches from varying botanical sources.Cereal Chemistry 72, 480–487.

Jacobsen, H.J. (1992) Biotechnology applied to grain legumes – current stateand approaches. In: AEP (ed.) Proceedings of 1st European Conference on GrainLegumes, Angers. AEP, Paris, pp. 99–103.

Jakel, L., Knosche, R. and Gunther, G. (1990) Cytological investigations on tisuueculture of Pisum sativum L. and Vicia faba L. cultured on herbicide-containingselection media. Biologisches Zentralblatt 109, 159–167.

James, C. (1997) Global Status of Transgenic Crops in 1997. ISAAA, Ithaca, New York.Jane, J., Kasemsuvan, T., Chen, J.F. and Juliano, B.O. (1996) Phosphorus in rice and

other starches. Cereal Foods World 41, 827–832.Jansman, A.J.M., Huisman, J. and van der Poel, A.F.B. (1993) Ileal and faecal

digestibility of field beans (Vicia faba L.) varying in tannin content. Animal FeedSciences and Technology 42, 83–96.

Jarkova, T.V., Deineko, E.V. and Shumny, V.K. (1998) Screening of pea (Pisumsativum L.) collection effects on the morphogenetic capacity in tissue culture.Plant Tissue Culture and Biothechnology 4, 175–182.

Jelaska, S., Pevalek, B., Papes, D. and Devide, Z. (1981) Developmental aspects oflong-term callus culture of Vicia faba L. Protoplasma 105, 285–292.

Jeltema, M.A. and Zabik, M.E. (1979) Fiber components-quantification andrelationship to cake quality. Journal of Food Science 44, 1732–1735.

References 273




Jeltema, M.A. and Zabik, M.E. (1980) Revised method for quantitative fibrecomponents. Journal of the Science of Food and Agriculture 31, 820–829.

Jenkins, D.J.A., Cuff, D., Wolever, T.M.S., Knowland, D., Thompson, L., Cohen, Z.and Prokipchuk, E. (1987) Digestibility of carbohydrate foods in anileostomate: relationship to dietary fiber, in vitro digestibility, and glycemicresponse. American Journal of Gastroenterology 82, 709–717.

Jesh, P. and Harzburg, B. (1997) Kartoffeln sind ihre Starke. Kartoffelnbau 9/10,348–351.

Jia, S. (1982) Factors affecting the division frequency of pea mesophyll protoplasts.Canadian Journal of Botany 60, 2192–2196.

Jia-Ping, Z., Roth, E.J., Terzaghi, W. and Lark, K.G. (1981) Isolation of sodiumdependent variants from haploid soybean cell culture. Plant Cell Reports 1,48–51.

Jin, H., Hartman, G.L., Huang, Y.H., Nickell, C.D. and Widholm, J.M. (1996)Regeneration of soybean plants from embryogenic suspension cultures treatedwith toxic culture filtrate of Fusarium solani and screening of regenerants forresistance. Phytopathology 86, 714–718.

Johansen, H.N., Glitse, V. and Bach Knudsen, K.E. (1996) Influence of extractionsolvent and temperature on quantitative determination of oligosaccharidesfrom plant materials by high-performance liquid chromatography. Journal ofAgricultural and Food Chemistry 44, 1470–1474.

Johansson, C.-G., Siljeström, M. and Asp, N.G. (1984) Dietary fibre in bread andcorresponding flours – formation of resistant starch during baking. Zeitschriftfür Lebensmittel Untersuchung und Forschung 179, 24–28.

John, M., Roehring, H., Smidt, J., Walden, R. and Schell, J. (1997) Cell signalling byoligosaccharides. Current Trends in Plant Science 2, 111–115.

Jones, D.A., Barber, L.M., Arthur, A.E. and Hedley, C.L. (1995) An analysis of seeddevelopment in Pisum sativum L. XVI. Assessing variation for fatty-acid contentby use of a non-destructive technique for single seed analysis. Plant Breeding114, 81–83.

Jones, D.A., Urban, J. and Copikova, J. (1999a) A micro-analytical method for thedetermination of starch and amylose/amylopectin content in pea seeds.Biologia Plantarum 42, 303–308.

Jones, D.A., Dupont, M.S., Ambrose, M.J., Frias, J. and Hedley, C.L. (1999b) Thediscovery of compositional variation for the raffinose family of oligosaccharidesin pea seeds. Seed Science Research 9, 305–310.

Jones, H., Karp, A. and Jones, M.K. (1989) Isolation, culture, and regeneration ofplants from potato protoplasts. Plant Cell Reports 8, 307–311.

Jones, R.G. (2000) Carbon partitioning into carbohydrates through pea seeddevelopment. PhD thesis, University of East Anglia, UK.

Jood, S., Chauhan, B.M. and Kapoor, A.C. (1988) Contents and digestibility ofcarbohydrates of chickpea and black gram as affected by domestic processingand cooking. Food Chemistry 30, 113–127.

Jood, S., Mehta, U., Singh, R. and Bhat, C.M. (1985) Effect of processing on flatusproducing factors in legumes. Journal of Agricultural and Food Chemistry 33,268–271.

Jorgensen, H., Zhao, X.O. and Eggum, B.O. (1996) The influence of dietary fibreand environmental temperature on the development of the gastrointestinal

274 References




tract, digestibility, degree of fermentation in the hind gut and metabolism inpigs. British Journal of Nutrition 75, 365–378.

Jork, W.C., Darvill, A.G. and Albersheim, P. (1984) Inhibition of 2,4-D stimulatedelongation of pea stem segments by a xyloglucan oligosaccharide. Plant Physiol-ogy 75, 295–297.

Journet, E. and Douce, R. (1985) Enzymic capacities of purified cauliflower budplastids for fat synthesis and carbohydrate metabolism. Plant Physiology 79,458–467.

Just, A., Fernandez, J.A. and Jorgensen, H. (1983) The net energy value of diets forgrowth in pigs in relation to the fermentative processes in the digestive tract andthe site of absorption of the nutrients. Livestock Production of Science 10, 171–186.

Kaczymska, B., Autio, K. and Fornal, J. (1994) Heat induced structural changes infaba bean starch paste: the effect of steaming of faba bean seeds. Food Structure12, 217–224.

Kallak, H. and Koiveer, A. (1990) Induction of morphogenesis in meristems ofdifferent cultivars of Pisum sativum L. Plant Science 67, 221–226.

Kallak, H. and Yarvekylg, L. (1968) (in Russian) On morphological and cytologicaldifferences of pea callus. Cytology and Genetics (Kiev) 2, 408–414.

Kallak, H. and Yarvekylg, L. (1971) On the cytogenetic effects of 2,4-D on pea callusin culture. Acta Biologica Academiae Scientiarum Hungaricae 22, 67–73.

Kallak, H. and Yarvekylg, L. (1976) On nuclear form and division in plant tissueculture. Acta et Commentationes Universitatis Tartuensis 383, 52–63.

Kallak, H. and Yarvekylg, L. (1977a) Nuclear behaviour in callus cells: morphologyand division. Biologia Plantarum 19, 48–52.

Kallak, H. and Yarvekylg, L. (1977b) Changes in chromosome complement inlong-term pea callus cultures. Acta Biologica Academiae Scientiarum Hungaricae28, 183–189.

Kandler, O. and Hopf, H. (1980) Occurrence, metabolism, and function of oligo-saccharides. In: Preiss, J. (ed.) The Biochemistry of Plants, Vol. 3. Carbohydrates:Structure and Function. Academic Press, New York, pp. 221–270.

Kaneda, Y., Tabei, Y., Nishimura, S., Harada, K., Akihama, T. and Kitamura, K.(1997) Combination of thidiazuron and basal media with low salt concentra-tions increases the frequency of shoot organogenesis in soybeans (Glycine maxL. Merr). Plant Cell Reports 17, 8–12.

Kanyand, M., Dessai, A.P. and Prakash, C.S. (1994) Thidiazuron promotes highfrequency regeneration of peanut (Arachis hypogeae) plant in vitro. Plant CellReports 14, 1–5.

Kao, K.N., Keller, W.A. and Miller, R.A. (1970) Cell division in newly formed cellsfrom protoplasts of soybean. Experimental Cell Research 62, 338–340.

Kar, S., Johnson, T.M., Nayak, P. and Sen, S.K. (1996) Efficient transgenic plantregeneration through Agrobacterium-mediated transformation of chickpea(Cicer arietinum L.). Plant Cell Reports 16, 32–37.

Karp, A. (1993) Mechanisms of somaclonal variation. Biotechnology and Biotechnologi-cal Equipment 7, 20–25.

Kartha, K.K. (1984) Culture of shoot meristems: Pea. In: Vasil, I.K. (ed.) Cell Cultureand Somatic Cell Genetics in vitro, Vol. 1. Academic Press, New York, pp. 106–110.

Kartha, K.K., Gamborg, O.L. and Constable, F. (1974) Regeneration of pea (Pisumsativum L.) plants from shoot apical meristems. Zeitschrift für Pflanzenphysiologie72, 172–176.

References 275




Kataki, P.K., Horbowicz, M., Taylor, A.G. and Obendorf, R.L. (1997) Changes insucrose, cyclitols and their galactosyl derivatives with seed ageing. In: Ellis,R.H., Black, M., Murdoch, A.J. and Hong, T.D. (eds) Basic and Applied Aspects ofSeed Biology. Kluwer, Dordrecht, pp. 515–522.

Kawasaki, S. (1989) Extensin secreted into the culture medium by tobacco cells. I.Purification and some properties. Plant Cell Physiology 30, 259–265.

Keijbets, M.J.H. and Pilnik, W. (1974) Some problems in the analysis of pectin inpotato tuber tissue. Potato Research 17, 169.

Kelkar, M., Shastri, P. and Rao, B.Y. (1996) Effect of processing on in vitro carbohy-drate digestibility of cereals and legumes. Journal of Food Science and Technology,India 33, 493–497.

Keller, F. and Ludlow, M.M. (1993) Carbohydrate metabolism in drought stressedleaves of pigeon pea (Cajanus cajan). Journal of Experimental Botany 44,1351–1359.

Kermode, A.R. (1997) Approaches to elucidate the basis of desiccation-tolerance inseeds. Seed Science Research 7, 75–95.

Kerr, P.S. (1993) Soybean products with improved carbohydrate composition andsoybean plants. International Patent Publication Number WO 93/07742,PCT/US/92/08958.

Kerr, P.S., Pearlstein, R.W., Becker-Manley, M.F. and Pierce, J.W. (1993) Nucleotidesequences of galactinol synthase from zucchini and soybean. InternationalPatent Publication Number WO 93/02196, PCT/US92/06057.

Key, F.B. and Mathers, J.C. (1993) Complex carbohydrate and large bowel fermen-tation in rats given wholemeal bread and cooked haricot beans (Phaseolusvulgaris) fed in mixed diets. British Journal of Nutrition 69, 497–509.

Kim, H.-O. and Williams, P.C. (1990) Determination of starch and energy in feedgrains by near-infrared reflectance spectroscopy. Journal of Agricultural and FoodChemistry 38, 682–688.

Kim, H.R., Hermansson, A.-M. and Eriksson, C.E. (1992) Structural characteristicsof hydroxypropyl potato starch granules depending on their molar substitu-tion. Starch 44, 111–116.

Kim, W.J., Smit, C.J.B. and Nakayama, T.O.N. (1973) The removal of oligosaccha-rides from soy beans. Lebensmittel Wissenschaft und Technologie 6, 201–204.

Kitagawa, I., Saito, M. and Taniayama, T. (1984a) Quantitative determination ofsoyasaponins in soybeans of various origins and soybean products by meansof high-performance liquid chromatography. Yakagaku Zasshi 104, 275–279.

Kitagawa, I., Yoshikawa, M., Hayashi, T. and Taniayama, T. (1984b) Characteriza-tion of saponin constituents in soybeans of various origins and quantitativeanalysis of soyasaponins by gas-liquid chromatography. Yakagaku Zasshi 104,162–168.

Klein, A.S., Montezinos, D. and Delmer, D.P. (1981) Cellulose and 1,3-glucansynthesis during the early stages of wall regeneration in soybean protoplasts.Planta 152, 105–114.

Kmita-Glazewska, H. and Kostyra, H. (1998) Chemical composition of solubleand insoluble fractions of dietary fibre isolated from pea seeds. In: AEP (ed.)Opportunities for High Quality, Healthy and Added-Value Crops to Meet EuropeanDemands. Proceedings of 3rd European Conference on Grain Legumes, Valladolid.AEP, Paris, p. 382.

276 References




Knosche, R. Von (1981) Investigations on the spontaneous polyploidizations in atissue culture from Pisum sativum L. II. Connections between mitotic index andfrequencies of restitution mitoses. Biologisches Zentralblatt 100, 55–65.

Knosche, R. Von and Gunther, G. (1980) Investigations on the spontaneouspolyploidizations in a tissue culture from Pisum sativum L. I. The evidence ofrestitution cell cycles. Biologisches Zentralblatt 99, 311–323.

Knott, C.M. (1987) A key for stages of development of the (Pisum sativum). Annals ofApplied Biology 111, 233–244.

Knott, C.M. (1990) A key for stages of development of the faba bean (Vicia faba).Annals of Applied Biology 116, 391–404.

Koch, K.E. (1996) Carbohydrate-modulated gene expression in plants. AnnualReview of Plant Physiology and Plant Molecular Biology 47, 509–540.

Kohle, H., Young, D.H. and Kauss, H. (1984) Physiological changes in suspensioncultured soybean cells elicited by treatment with chitosan. Plant Sciences 33,221–230.

Kohle, H., Jeblik, W., Poten E., Blaschek, W. and Kauss, H. (1985) Chitosan elicitedcallose synthesis in soybean cells as a Ca2+ dependent process. Plant Physiology77, 544–551.

Komatsuda, T. (1992) Research on somatic embryogenesis and plant regenerationin soybean. Bulletin of the National Institute of Agrobiological Resources (Japan) 7,1–78.

Konno, H., Yamasaki, Y. and Katoh, K. (1986) Characteristics of lactiosidase puri-fied from cell suspension cultures of carrot. Physiologia Plantarum 68, 46–52.

Konno, H., Yamasaki, Y. and Katoh, K. (1987) Purification of an arabinofurano-sidase from carrot cell cultures and its involvement in arabinose rich polymerdegradation. Physiologia Plantarum 69, 405–412.

Konno, H., Yamasaki, Y. and Katoh, K. (1989) Extracellular exo-polygalacturonasesecreted from carrot cell cultures. Its purification and involvement in pecticpolymer degradation. Physiologia Plantarum 76, 514–520.

Kooistra, E. (1962) On the differences between smooth and three types of wrinkledpeas. Euphytica 11, 357–373.

Kossmann, J., Visser, R.G.F., Mueller-Roeber, B., Willmitzer, L. and Sonnewald, U.(1991) Cloning and expression analysis of a potato cDNA that encodesbranching enzyme, evidence for co-expression of starch biosynthetic genes.Molecular General Genetics 230, 39–44.

Kossmann, J., Riesmeier, J. and Willmitzer, L. (1997) Understanding and influenc-ing starch biochemistry (poster abstract 123). 5th International Congress of PlantMolecular Biology, Singapore.

Koster, K.L. (1991) Glass formation and dessication tolerance in seeds. PlantPhysiology 96, 302–304.

Koster, K.L. and Leopold, A.C. (1988) Sugars and desiccation tolerance in seeds.Plant Physiology 88, 829–832.

Koster, K.L., Webb, M.S., Bryant, G. and Lynch, D.V. (1994) Interactions betweensoluble sugars and POPC (1 palmitoyl-2-oleoylphosphatidylcholine) duringdehydration: vitrification of sugars alters the phase behavior of thephospholipid. Biochimica et Biophysica Acta 1193, 143–159.

Kosturkova, G.P. (1993) Application of alfalfa protoplast cultures in genetic studies.Biotechnology and Biotechnological Equipment 7(2), 40–43.

References 277




Kosturkova, G.P. (1997) In vitro cultures of leguminous species. Proceedings Inter-national Balkan Botanical Congress. Thessaloniki.

Kosturkova, G.P. and Mehandjiev, A. (1998) In vitro cultures of leguminous species.In: Tsekos, I. and Moustakas, M. (eds) Progress in Botanical Research. Kluwer,Dordrecht, the Netherlands, pp. 529–532.

Kosturkova, G.P. and Tineva, N.A. (1998) Organogenesis stimulation in pea in vitrocultures by o-allylthioureidobenzoic acid. In Vitro Cellular and DevelopmentalBiology – Plant 34, 344–345.

Kosturkova, G.P., Mehandjiev, A., Dobreva, I. and Tsvetkova, V. (1997) Regenera-tion systems from immature embryos of Bulgarian pea genotypes. Plant CellTissue and Organ Culture 48, 139–142.

Kozlowska, H., Borowska, J., Fornal, J., Schneider, C.H. and Schmandtke, H. (1989)Preparation of faba bean (Vicia faba L. minor) products. Part IV. Effect of hydro-thermal treatment of faba bean on the quality of flour. Acta Alimentaria PolonicaXV, 161–169.

Kozlowska, K., Zdunczyk, Z. and Borowska, J. (1992) The influence of the seed hulland hydrothermal treatment on the chemical composition and nutrient valuesof faba bean seeds in rats. In: AEP (ed.) Proceedings of 1st European Conference onGrain Legumes, Angers. AEP, Paris, p. 525.

Krause, D.O., Easter, R.A. and Mackie, R.L. (1994) Fermentation of stachyose andraffinose by hind-gut bacteria of the weaning pig. Letters in Applied Microbiology18, 439–352.

Kremlicka, G.J. and Hoffmann-Ostenhof, O. (1966) Biosynthesis of cyclitols. X.Reduction of 5-O-methyl-myo-inosose-(3) by the enzyme of Trifoliumincarnatum. Hoppe-Seyler’s Zeitschrift für Physiologische Chemie 344, 261–266.

Kuchuk, N.V. (1989) Isolation and cultivation of protoplasts from five legumespecies. Fiziologija Rastenij (Russian) 36, 821–824.

Kudla, E. and Tomasik, P. (1992) The modification of starch by high pressure.Part I: Air and oven-dried potato starch. Starch 44, 167–173.

Kuipers, A.G.J., Jacobsen, E. and Visser, R.G.F. (1994) Formation and depositionof amylose in the potato tuber starch granule are affected by the reduction ofgranule-bound starch synthase gene expression plant. Plant Cell 6, 43–52.

Kumar, V. and Sharma, D.R. (1989) Selection and characterization of anL-thiazolidine-4-carboxylic acid resistant callus culture of Vigna radiata (L.)Wilczek var. radiata. Plant Cell Reports 7, 648–651.

Kumar, V.D., Kirti, P.B., Sachan, J.K.S. and Chopra, V.I. (1994) Plant regenerationvia somatic embryogenesis in chickpea (Cicer arietinum L.). Plant Cell Reports 13,468–472.

Kunakh, V.A., Alkhimova, E.G., Voitjuk, L.I. and Alpatova, L.K. (1984) Variation inchromosome number in callus tissues and regenerants of pea. Cytology andGenetics (Kiev) 18, 20–26.

Kuo, T.M. (1992) Isolation and identification of galactinol from castor oilseed meal.Journal of the American Oil Chemists’ Society 69, 569–574.

Kuo, T.M., VanMiddlesworth, J.F. and Wolf, W.J. (1988) Content of raffinose oligo-saccharides and sucrose in various plant seeds. Journal of Agricultural and FoodChemistry 36, 32–36.

Kuriyama, S. and Mendel, L.B. (1917) The physiological behaviour of raffinose.Journal of Biological Chemistry 31, 125–147.

278 References




Kurtzman, R.H. and Halbrook, W.U. (1970) Polysaccharides from dry navy beans,Phaseolus vulgaris: its isolation and stimulation of Clostridium perfringens. AppliedMicrobiology 20, 715–719.

Kvasnibka, F. (1995) Antinutritional saccharides in peas. Vyz. Potraviny 50, 3–4(in Czech).

Kvasnibka, F., Ahmadová-Vavrousová, R., Mrskot, M., Velítek, J. and Kadlec, P.(1994) Characteristics of pea varieties according to galactooligosaccharidescontent. In: Fenwick, G.R., Hedley, C., Richards, R.L. and Khokhar, S. (eds)Agricultural Food Quality. An Interdisciplinary Approach. The Royal Society ofChemistry, Cambridge, pp. 153–156.

Kvasnibka, F., Ahmadová-Vavrousová, R., Frias, J., Price, K.R. and Kadlec, P. (1996)A rapid HPLC method for determination of raffinose family oligosaccharidesin pea seeds. Journal of Liquid Chromatography 19, 135–147.

Kweon, M.R., Bhirud, P.R. and Sosulski, F.W. (1996) An aqueous alcoholic-alkalineprocess of corn and pea starches. Starch/Stärke 48, 214–220.

Kysely, W. and Jacobsen, H.J. (1990) Somatic embryogenesis from pea embryos andshoot apices. Plant Cell Tissue and Organ Culture 20, 7–14.

Kysely, W., Myers, J.R., Lazzeri, P.A., Collins, G.B. and Jacobsen, H.J. (1987) Plantregeneration via somatic embryogenesis in pea (Pisum sativum L.). Plant CellReports 6, 305–308.

Lacassagne, L., Francesch, M., Carre, B. and Melcion, J.P. (1988) Utilization oftannin-containing and tannin-free faba beans (Vicia faba) by young chicks:effects of pelleting feeds on energy, protein and starch digestibility. Animal FeedSciences and Technology 20, 59–68.

Lacassagne, L., Melcion, J.P., de Monredon, F. and Carre, B. (1991) The nutritionalvalues of faba bean flours varying in their mean particle size in young chickens.Animal Feed Sciences and Technology 34, 11–19.

Lahuta, L.B., Górecki, R.J. and Rejowski, A. (1995) Accumulation of sugars inmaturing field bean (Vicia faba minor L.) and pea seeds (Pisum sativum L.) inrelation to seed viability. In: AEP (ed.) Improving Production and Utilisation ofGrain Legumes. Proceedings 2nd European Conference on Grain Legumes. AEP, Paris,p. 44.

Lahuta, L.B., Jagielska, T., Górecki, R.J., Jones, A. and Hedley, C. (1998) Solublecarbohydrates in desiccation tolerance of germinating pea seeds of differentisolines. In: AEP (ed.) Opportunities for High Quality, Healthy and Added-valueCrops to meet European Demands. Proceedings 3rd European Conference on GrainLegumes, Valladolid. AEP, Paris, pp. 40–41.

Lai, F.-M. and McKersie, B.D. (1994) Regulation of starch and storage proteinaccumulation in alfalfa (Medicago sativa L.) somatic embryos. Plant Science 100,211–219.

Landgren, C.R. (1976) The influence of culture conditions on mitotic activity ofprotoplats derived from Pisum root cortical explants. Protoplasma 87, 49–69.

Landgren, C.R. (1981) Gibberellic enhancement of the enzymatic release of Pisumroot cell protoplasts. Physiology Plantarum 52, 349–352.

Larkin, P.J. and Scowcroft, W.R. (1981) Somaclonal variation – a novel source ofvariability from cell cultures for plant improvement. Theoretical and AppliedGenetics 60, 197–214.

References 279




Larsson, S., Johansson, L.A. and Svenningsson, M. (1993) Soluble sugars and mem-brane lipids in winter wheats (Triticum aestivum L.) during cold acclimation.European Journal of Agronomy 1, 85–90.

Latunde-Dada, A.O. and Lucas, J.A. (1983) Somaclonal variation and reaction toVerticillium wilt in Medicago sativa L. plants regenerated from protoplast. PlantScience Letters 32, 205–211.

Latunde-Dada, A.O. and Lucas, J.A. (1988) Somaclonal variation and resistance toVerticillium wilt in lucerne, Medicago sativa L., plants regenerated from callus.Plant Science 58, 111–119.

Leach, H.W., McCowen, L.D. and Schoch, T.J. (1959) Structure of the starchgranule. I. Swelling and solubility patterns of various starches. Cereal Chemistry36, 534–544.

Le Bansky, B.P., McKnight, T.D. and Griffing, L.R. (1992) Purification andcharacterization a secreted purple phosphatase from soybean suspensioncultures. Plant Physiology 99, 391–395.

Lee, E.Y.C. and Whelan, W.J. (1971) Glycogen and starch debranching enzymes. In:Boyer, P.D. (ed.) The Enzymes, Vol. 5. Academic Press, New York, pp. 191–234.

Lee, S.C. and Prosky, L. (1994) Perspectives on new dietary fibre definition. CerealFoods World 39, 667.

Le Guen, M.-P., Peyronnet, C. and Gabon, G. (1997) Nitrogen ruminaldegradability of pea and lupin. Grain Legumes 17, 14–17.

Lehle, L. and Tanner, W. (1972) Synthesis of raffinose-type sugars. Methods inEnzymology 28B, 522–530.

Lehmann, J. (1998) In: Carbohydrates Structure and Biology, translated by Haines, A.H.Thieme, Stuttgart, p. 288.

Lehninger-Mertens, R. and Jacobsen, H.J. (1989a) Protoplast regeneration andorganogenesis from pea protoplasts. In Vitro Cellular and Developmental Biology –Plant 25, 571–574.

Lehminger-Mertens, R. and Jacobsen, H.J. (1989b). Plant regeneration from peaprotoplasts via somatic embryogenesis. Plant Cell Reports 8, 379–382.

Leopold, A.C. and Vertucci, C.W. (1986) Physical attributes of desiccated seeds.In: Leopold, A.C. (ed.) Membranes, Metabolism and Dry Organisms. CornellUniversity Press, Ithaca, New York, pp. 22–34.

Leopold, A.C., Sun, W.Q. and Bernal-Lugo, I. (1994) The glassy state in seeds:analysis and function. Seed Science Research 4, 267–274.

LePrince, O., Deltour, R., Thorpe, P.C., Atherton, N.M. and Hendry, G.A.F. (1990).The role of free radicals and radical processing systems in loss of desiccationtolerance in germinating maize (Zea mays L). New Phytologist 116, 573–580.

LePrince, O., Hendry, G.A.F. and McKersie, B.D. (1993) The mechanism ofdesiccation tolerance in developing seeds. Seed Science Research 3, 231–246.

Leprince, O. and Walters-Vertucci, C. (1995) A calometric study of the glass transi-tion behaviors in axes of bean seeds with relevance to storage stability. PlantPhysiology 109, 1471–1481.

Leske, K.L., Jevne, C.J. and Coon, C.N. (1993) Effect of oligosaccharide additionon nitrogen-corrected true metabolizable energy of soy protein concentrate.Poultry Science 72, 664–668.

Leske, K.L., Zhang, B. and Coon, C.N. (1995) The use of low alpha-galactosideprotein products as a protein source in chicken diets. Animal Feed Sciences andTechnology 54, 275–286.

280 References




Letzelter, N., Wilson, R., Jones, D.A. and Sinnaeve, G. (1995) Quantitative determi-nation of the composition of individual pea seeds by Fourier Transforminfrared photoacoustic spectroscopy. Journal of Food Science and Agriculture 67,239–245.

Lewandowicz, G. and Mczynski, M. (1990a) Chemiczna modyfikacja skrobi. Cz.I.Modyfikacja skrobi ziemniaczanej. Chemik 1, 9–14.

Lewandowicz, G. and Mczynski, M. (1990b) Chemiczna modyfikacja skrobi. Cz.II.Reaktywnosc skrobi róznych gatunków roslin. Chemik 3, 71–89.

Lewandowicz, G., Soral-Smietana, M. and Fornal, J. (1998) New RS preparations –structure and physico-chemical properties. Proceedings of the VIII Inter-national Starch Convention, Cracow (Poland), 16–19 June.

Li, B.W. and Schuhmann, P.J. (1980) Gas–liquid chromatography analysis of sugarsin ready-to-eat breakfast cereals. Journal of Food Sciences 45, 138–141.

Li, B.W., Schuhmann, P.J. and Wolf, W.J. (1985) Chromatographic determinationof sugars and starch in a diet composite reference material. Journal of Agricul-tural and Food Chemistry 33, 531–536.

Libbenga, K.R. and Torrea, J.G. (1973) Hormone-induced endoreduplication priorto mitosis in cultured pea root cortex cells. American Journal of Botany 60,293–299.

Lichner, F.T. and Spanswick, R.M. (1981) Electrogenic sucrose transport in devel-oping soybean cotyledons. Plant Physiology 67, 869–874.

Lim, H.T., Song, Y.N. and Yeoung, Y.R. (1994) Plant regeneration from immatureembryo and thin layer of pea (Pisum sativum L.) via callus induction, somaticembryogenesis and organogenesis. Korean Journal of Breeding 26, 33–40.

Lin, T.-P. and Huang, N.-H. (1994) The relationship between carbohydrate compo-sition of some tree seeds and their longevity. Journal of Experimental Botany 45,1289–1294.

Lin, W. (1985a) Energetics of sucrose transport into protoplasts from developingsoybean cotyledons. Plant Physiology 78, 41–45.

Lin, W. (1985b) Linear sucrose transport in protoplasts from developing soybeancotyledons. Plant Physiology 78, 649–651.

Lin, W., Schmitt, M.R., Hitz, W.D. and Giaquinta, R.T. (1984) Sugar transport intoprotoplasts isolated from developing soybean cotyledons. Plant Physiology 75,936–940.

Lin, W., Schmitt, M.R., Hitz, W.D. and Giaquinta, R.T. (1984) Sugar transportinto protoplasts isolated from developing soybean cotyledons. II. Protoplastisolation and general characteristics of sugar transport. Plant Physiology 75,936–940.

Lintas, C., Cappelloni, M. and Ruggeri, S. (1992) Processing and legume poly-saccharides. In: AEP (ed.) Proceedings of 1st European Conference on Grain Legumes.AEP, Paris, pp. 441–442.

List, G.R., Mounts, T.L., Orthoefer, F. and Neff, W.E. (1996) Potential margarineoils from genetically modified soybeans. Journal of the American Oil ChemistsSociety 73, 729–732.

Liu, J.J., Odegard, W. and de Lumen, B.O. (1995) Galactinol synthase from kidneybean cotyledon and zucchini leaf. Plant Physiology 109, 505–511.

Lizotte, P.A., Henson, C.A. and Duke, S.H. (1990) Purification and characterizationof pea epicotyl α-amylase. Plant Physiology 92, 615–621.

References 281




Lloyd, J.R., Bull, V.J., Hedley, C.L. and Ring, S.G. (1996a) Determination of theeffect of r and rb mutations on the structure of amylose and amylopectin in pea(Pisum sativum L.). Carbohydrate Polymers 29, 45–49.

Lloyd, J.R., Wang, T.L. and Hedley, C.L. (1996b) An analysis of seed developmentin Pisum sativum XIX. Effect of mutant alleles at the r and rb loci on starch grainsize and on the content and composition of starch in developing pea seeds.Journal of Experimental Botany 47, 171–180.

Locker, A. and Ilan, I. (1975) On the nature of the hormonal regulation of amylaseactivity in cotyledons of germinating peas. Plant Cell Physiology 16, 449–454.

Loewus, F.A. (1990) Cyclitols. In: Dey, P.M. and Harbourne, J.B. (eds) Methods inPlant Biochemistry, Vol. 2. Carbohydrates. Academic Press, New York, pp. 219–233.

Loewus, F. and Dickinson, D.B. (1982) Cyclitols. In: Loewus, F.A. and Tanner, W.(eds) Encyclopaedia of Plant Physiology: Plant Carbohydrates 1: Intracellular Carbohy-drates, Vol. 13A. Springer-Verlag, Berlin, pp. 193–206.

Loewus, F. and Loewus, M. (1980). Biochemistry of myo-inositol. In: Stumpf, P. andConn, E. (eds) Biochemistry of Plants, Vol. 3. Academic Press, New York,pp. 43–76.

Loewus, F.A. and Loewus, M.W. (1983) myo-Inositol: its biosynthesis and metabo-lism. Annual Review of Plant Physiology 34, 137–161.

Login, A., Piotrowicz-Cieslak, A. and Górecki, R.J. (1995) α-Galactosidase inmaturing and germinating yellow lupine seeds. In: Come, D., Duczmal, K.,Górecki, R., Grzesik, M. and Lewak, S. (eds) Third French–Polish Symposium‘Current problems of seed physiology’ 18–20 July 1995. ART, Olsztyn, Poland, p. 21.

Loiseau, J., Marche, C. and Deunff, Y. Le. (1995) Effect of auxins, cytokinins, carbo-hydrates and aminoacids on somatic embryogenesis induction from shootapices of pea. Plant Cell Tissue and Organ Culture 33, 267–275.

Longstaff, M. and McNab, J.M. (1987) Digestion of starch and fibre carbohydratesin peas by adult cockerels. British Poultry Science 28, 261–285.

Lorberth, R., Ritte, G., Willmitzer, L. and Kossmann, J. (1998) Inhibition of astarch-granule-bound protein leads to modified starch and repression of coldsweetening. Nature Biotechnology 16, 473–477.

Lorenz, K. (1979) The starch of the faba bean (Vicia faba). Comparison with wheatand corn starch. Starch 31, 181–184.

Lorz, H. and Brown, P.T.H. (1986) Variability in tissue culture derived plants –possible origins, advantages and drawbacks. In: Horn, W. (eds) Genetic Manipu-lation in Plant Breeding. Walter de Gruyter Publishers, Berlin, pp. 513–534.

Low, A.G. (1985) Role of dietary fibre in pigs diets. In: Haresign, H. and Cole, D.J.A.(eds) Recent Advances in Animal Nutrition. Butterworths, London, pp. 87–112.

Lowell, C.A. and Kuo, T.M. (1989) Oligosaccharide metabolism and accumulationin developing soybean seeds. Crop Science 29, 459–465.

Lozovaya, V.V., Zabotina, O.A. and Widholm, I.M. (1996) Synthesis and turnover ofcell wall polysaccharides and starch in photosynthetic soybean suspensioncultures. Plant Physiology 111, 921–929.

Lu, D.Y., Cooper-Bland, S., Pental, D., Cocking, E.C. and Davey, M.R. (1983) Isola-tion and sustained division of protoplasts from cotyledons of seedlings andimmature seeds of Glycine max L. Zeitschrift für Pflanzenphysiologie 111, 389–394.

Lu, H., Gai, J. and Ma, Y. (1993) Soybean protoplast culture under differenthormone conditions and plantlet regeneration. Acta Agronomica Sinica 19,328–333.

282 References




Lutova, L.A. and Zabelina, Ye.K. (1988) Callus and shoot in vitro formation in differ-ent forms of peas Pisum sativum L. Genetics (Moscow) 24, 1632–1640.

Lyons, A.J., Pridham, T.G. and Hesseltine, C.W. (1969) Survey of some Actino-mycetales for α-galactosidase activity. Applied Microbiology 18, 579–583.

MacKintosh, C., Lyon, G.D. and MacKintosh, R.W. (1994) Protein phosphataseinhibitors activate antifungal defence responses of soybean cotyledons and cellcultures. Plant Journal 5, 137–147.

Macleod, M.R., Hedley, C.L., Martin, C.R. and Wang, T.L. (1991) Genetic analysisof new wrinkled-seeded pea mutants at the r locus. Aspects of Applied Biology 27,263–265.

Madhusudhan, M. and Tharanathan, R.N. (1995) Legume and cereal starches –why differences in digestibility? Part 1: Isolation and composition of legume(Greengram and Bengalgram) starches. Starch 49, 165–171.

Madhusudhan, M. and Tharanathan, R.N. (1996) Structural studies of linear andbranched fractions of chick pea and finger millet starches. Carbohydrate Research284, 101–109.

Makkar, H.P.S., Becker, K., Abel, H. and Pawezik, E. (1997) Nutrients content,rumen protein degradability and nutritional factors in some colour- andwhite-flowering cultivars of Vicia faba beans. Journal of the Sciences Food andAgriculture 75, 511–520.

Malik, K.A. and Saxena, P.K. (1991) Regeneration in Phaseolus vulgaris L. Promotiverole of N(6)-benzylaminopurine in cultures from juvenile leaves. Planta 184,148–150.

Malik, K.A. and Saxena, P.K. (1992a) Somatic embryogenesis and shoot regenera-tion from intact seedlings of Phaseolus vulgaris acutifolius A., P. aureus (L.)Wilczek, P. coccineus L. and P. wrightii L. Plant Cell Reports 11, 163–168.

Malik, K.A. and Saxena, P.K. (1992b) Thidiazuron induced high frequencyshoot regeneration in intact seedlings of pea (Pisum sativum), chickpea (Ciceraratinum) and lentil (Lens culinaris). Australian Journal of Plant Physiology 19,731–740.

Malik, K.A. and Saxena, P.K. (1992c) Regeneration in Phaseolus vulgaris L.: Highfrequency induction of direct shoot formation in intact seedlings byN(6)-benzylaminopurine and thidiazuron. Planta 186, 384–389.

Mallee, F.M. (1995) Starch-based texturizing agent. US Patent 5470391.Mallee, F.M., Stone, J.A. and Finocchiaro, E.T. (1996) Starch-based texturizing

agent. US Patent 554751.Malmberg, R.L. (1979) Regeneration of whole plants from callus cultures of diverse

genetic lines of Pisum sativum L. Planta 146, 243–244.Maltese, S., Atta, S. and Cousin, R. (1995) Determination of pea seed composition

by near infrared transmission spectroscopy. In: AEP (ed.) Improving Productionand Utilisation of Grain Legumes. Proceedings of 2nd European Conference on GrainLegumes, Copenhagen. AEP, Paris, p. 372.

Mangat, B.S., Pelekis, M.K. and Cassells, A.C. (1990) Changes in the starch contentduring organogenesis in in vitro cultured Begonia rex stem explants. PhysiologyPlantarum 79, 267–274.

Manners, D.J. (1989) Review paper. Recent development in our understanding ofamylopectin structure. Carbohydrate Polymers 11, 87–112.

Marks, G.E. and Davies, D.R. (1979) The cytology of cotyledon cells and the induc-tion of giant polytene chromosomes in Pisum sativum L. Protoplasma 101, 73–80.

References 283




Marshall, J., Sidebottom, C., Debet, M., Martin, C., Smith, A. and Edwards, A.(1996) Identification of the major starch synthase in the soluble fraction ofpotato tubers. Plant Cell 8, 1121–1135.

Martin, C. and Smith, A.M. (1995) Starch biosynthesis. The Plant Cell 7, 971–985.Matthews, R.H. (1989) Legumes Chemistry, Technology and Human Nutrition. Marcel

Dekker, New York.Mathews, V.H., Rao, P.S. and Bhatia, C.R. (1986) Somaclonal variation in cotyledon-

ary plants of mung bean. Zeitschrift für Pflanzenzucht 96, 169–173Mauch, F. and Staehelin, L.A. (1989) Functional implications of the subcellular

localization of ethylene-induced chitinase and glucanase in bean leaves. PlantCell 1, 447–457.

Mauch, F., Mauch Mani, B. and Boller, T. (1988) Antifungal hydrolases in peatissue: II. Inhibition of fungal growth by combination of chitinase andglucanase. Plant Physiology 88, 936–942.

Mazur, A., Remesy, C., Gueux, E., Levrat, A.-M. and Deminged, Ch. (1990) Effect ofdiet rich in fermentable carbohydrates on plasma lipids levels and on lipo-protein catabolism in rats. Journal of Nutrition 120, 1037–1045.

Margareta, E., Nyman, G.L., Svanberg, M.S.J. and Asp, N.G. (1994) Molecularweight distribution and viscosity of water-soluble dietary fibre isolated fromgreen beans, brussels sprouts and green peas following different types ofprocessing. Journal of the Science of Food and Agriculture 66, 83–91.

McCabe, D.E., Swain, W.F., Martinell, B.J. and Christou, P. (1988) Stable trans-formation of soybean (Glycine max) by particle acceleration. Bio-Technology (NewYork) 6, 923–926.

McCann, M.C. and Roberts, K. (1994) Changes in cell wall architecture during cellelongation. Journal of Experimental Botany 45 (special issue), 1683–1691.

McCutchen, C., Duffaud, G., Leduc, P., Peterson, A., Tayal, A., Khan, S. and Kelly,R. (1996) Characterization of extremely thermostable enzymatic breakers(alactosidase and annanase) from the hyperthermophilic bacterium Thermo-toga neapolitana 5068 for hydrolysis of guar gum. Biotechnology Bioengineering 52,332–339.

McDougall, G.J., Morrison, I.M., Stewart, D., Weyers, J.D.B. and Hillman, J.R.(1993) Plant fibres: botany, chemistry and processing for industrial use. Journalof the Science of Food and Agriculture 62, 1–20.

McIntyre, A., Gibson, P.R. and Young, G.P. (1993) Butyrate production from dietaryfibre and protection against large bowel cancer in a rat model. Gut 34, 386–391.

McKee, H.R., Robertson, R.N. and Lee, J.B. (1955) Physiology of the pea fruits. I.The developing fruit. Australian Journal of Biological Sciences 8, 137–162.

McKently, A.H., Moore, G.A. and Garden, F.P. (1990) In vitro regeneration ofpeanut. Crop Science 30, 192–196.

McKentley, A.H., Moore, G.A., Doostdar, H. and Niedz, R.P. (1995) Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and thedevelopment of transgenic plants. Plant Cell Reports 14, 699–703.

Mercier, C. (1979) Les α-galactosides des graines de leguminouses. In: INRA (ed.)Les Materies Premiers et Alimentation des Volailess. Seances de Travail, Paris,pp. 79–90.

Meredith, F.I., Thomas, C.A., Snook, M.E., Himmelsbach, D.S. and van Halbeek, H.(1988) Soluble carbohydrates oligosaccharides and starch in lima bean seeds.Journal of Food Science 53, 768–771.

284 References




Messner, B. and Boll, M. (1993) Elicitor-mediated induction of enzymes of ligninbiosynthesis and formation of lignin-like material in a cell suspension cultureof spruce (Picea abie). Plant Cell Tissue and Organ Culture 34, 261–269.

Meuser, F., Pahne, N. and Moller, M. (1995) Extraction of high amylose starch fromwrinkle peas. Starch/Stärke 2, 56–61.

Mikhailov, O.F. and Bessonova, V.P. (1975) Some data on the cytogenetic analysis ofcallus obtained from regenerating cotyledons of pea. Problemy OnkologiiTeratologii Rastenij, Nauka, Leningrad, pp. 52–54.

Mikhailova-Krumova, A.B., Gavrilyuk, M.I., Azarkovich, R.G. and Butenko, R.G.(1991) Study of somaclonal variations in pea seed proteins. Plant Physiology(Moscow) 38, 521–529.

Miklas, P.N., Grafton, K.F., Secor, G.A. and McClean, P.E. (1992) Use of pathogenfiltrate to differentiate physiological resistance of dry bean to white molddisease. Crop Science 32, 310–312.

Minagawa, K., Ohkura, T., Goshima, G., Miwa, Y. and Tsuge, H. (1987) Hystereticeffect of step by step heating on the restriction of gelatinization of legumestarch. Starch 39, 241–246.

Minami, Y., Yazawa, K., Tamura, Z., Tanaka, T. and Yamamoto, T. (1983) Selectivityof utilization of galactosyl oligosaccharides by bifidobacteria. Chemical andPharmaceutical Bulletin 31, 1688–1691.

Mital, B.K. and Steinkraus, K.H. (1975) Utilization of oligosaccharides by lactic acidbacteria during fermentation of soya milk. Journal of Food Science 40, 114–118.

Mital, B.K. and Steinkraus, K.H. (1979) Fermentation of soya milk by lactic acidbacteria. A review. Journal of Food Protection 42, 895–899.

Mizuno, K., Kawasaki, T., Shimada, H., Satoh, H., Kobayashi, E., Okumura, S.,Arai, Y. and Baba, T. (1993) Alteration of the structural properties of starchcomponents by the lack of an isoform of starch branching enzyme in rice seeds.Journal of Biological Chemistry 268, 19084–19091.

Modgil, R. and Mehta, U. (1996) Effect of Callobruchus chinensis L. (ColeopteraBruchidae) on carbohydrate content of chickpea, green gram and pigeon pea.Nahrung 40, 41–43.

Mohamed, A.A. and Rayas-Duarte, P. (1995) Nonstarch polysaccharide analysis ofcotyledon and hull of Lupinus albus. Cereal Chemistry 72 (6), 648–651.

Mohamed, M.F., Coyne, D.P. and Read, P.E. (1993) Shoot organogenesis in callusinduced from pedicel explants of common bean (Phaseolus vulgaris L.). Journalof American Society for Horticultural Science 118, 158–162.

Mohamed, M.F., Read, P.E. and Coyne, D.P. (1992) Dark preconditioning, CPPUand thidiazuron promote shoot organogenesis on seedling node explants ofcommon and faba beans. Journal of American Society for Horticultural Science 117,669–672.

Mol, J., Van der Krol, A., Van Tunen, A., Van Blokland, R. and de Lange Stuitje, A.(1990) Regulation of plant gene expression by antisense RNA. FEBS 268,427–430.

Molnar-Perl, I., Pinter-Szakacs, M., Kovago, A. and Petoroczy, J. (1984) Gas–liquidchromatographic determination of the raffinose family of oligosaccharides andtheir metabolites present in soy beans. Journal of Chromatography 295, 433–443.

Molvig, L., Tabe, L.M., Eggum, B.O., Moore, A.E., Craig, S., Spencer, D. andHiggins, T.J.V. (1997) Enhanced methionine levels and increased nutritivevalue of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a

References 285




sunflower seed albumine gene. Proceedings of the National Academy of Sciences USA94, 8393–8398.

Monerri, C., Garcia Luis, A. and Guardiola, J.L. (1986) Sugar and starch changes inpea cotyledons during germination. Physiologia Plantarum 67, 49–54.

Mongeau, R. and Brassard, R. (1994) Comparison and assessment of the differencein total dietary fibre in cooked dry legumes as determined by five methods.Journal AOAC International 77, 1197–1202.

Mongeau, R. and Brassard, R. (1995) Importance of cooking temperature andpancreatic amylase in determination of dietary fibre in dried legumes. JournalAOAC International 78, 1444–1449.

Monma, M., Sugimoto, T., Monma, M., Kawamura, Y. and Saio, K. (1991) Starchbreakdown in developing soybean seeds (Glycine max cv. Enrei). AgricultureBiology Chemistry 55, 67–71.

Moore, P.J., Swords, K.M.M., Lynch, M.A. and Staehelin, L.A. (1991) Spatialreorganization of the assembly pathways of glycoproteins and complexpolysaccharides in the Golgi apparatus of plants. Journal of Cell Biology 112,589–602.

Morohashi, Y., Katoh, H., Kaneko, Y. and Matsushima, H. (1989) Control of α-amylase development in cotyledons during and following germination of mungbean seeds. Plant Physiology 91, 253–258.

Morris, P., Scragg, A.H., Smart, N.J. and Stafford, A. (1985) Secondary productformation by cell suspension cultures. In: Dixon, R.A. (ed.) Plant Cell Culture – aPractical Approach. IRL Press, Oxford, pp. 127–134.

Morris, V.J. and Miles, M.J. (1994) Birefringent techniques. In: Ross-Murphy, S.B.(ed.) Physical Techniques for the Study of Food Polymers. Blackie Academic &Professional, London, pp. 215–275.

Morrison, I.M. (1972) Improvements in the acetyl bromide technique to determinelignin and digestibility and its application to legumes. Journal of the Science ofFood and Agriculture 23, 1463–1469.

Morrison, W.R. and Karkalas, J. (1990) Starch. In: Dey, P.M. (ed.) Methods in PlantBiochemistry, Vol. 2. Academic Press, San Diego, pp. 323–352.

Moscoso, W., Rourne, M.C. and Hood, L.F. (1984) Relationships between thehard-to-cook phenomenon in red kidney beans and water absorption,puncture force, pectin, phytic acid, and minerals. Journal of Food Science 49,1577–1583.

Mroginski, L.A. and Kartha, K.K. (1981) Regeneration of pea (Pisum sativum L. cv.Century) plants by in vitro culture of immature leaflets. Plant Cell Reports 1,64–66.

Mueller-Roeber, B., Kossmann, J., Hannah, L.C., Willmitzer, L. and Sonnerwald, U.(1990) Only one of two different ADP-glucose pyrophosphorylase genes frompotato responds strongly to elevated levels of sucrose. Molecular General Genetics224, 136–146.

Mueller-Roeber, B., Sonnerwald, U. and Willmitzer, L. (1992) Inhibition of ADP-glucose pyrophosphorylase in transgenic potatos leads to sugar-storing tubersand influences tuber formation and expression of tuber storage protein genes.EMBO Journal 11, 1229–1238.

Muir, J.G. and O’Dea, K. (1992) Measurement of resistant starch: factors affectingthe amount of starch escaping digestion in vitro. American Journal of ClinicalNutrition 56, 123–127.

286 References




Muir, J.G. and O’Dea, K. (1993) Validation of an in vitro assay for predication theamount of starch that escapes digestion in the small intestine of humans.American Journal of Clinical Nutrition 57, 540–546.

Muir, J.G., Birkett, A., Brown, I., Jones, G. and O’Dea, K. (1995) Food processingand maize variety affects amounts of starch escaping digestion in the smallintestine. American Journal of Clinical Nutrition 61, 82–89.

Müller, H.L., Kirchgessner, M. and Roth, F.X. (1989) Energy utilisation ofintracaecally infused carbohydrates and casein in sows. In: Van der Honning, Y.and Close, W.H. (eds) Energy Metabolism of Farm Animals. Pudoc, Wageningen,pp. 123–126.

Muller-Rober, B. and Kossmann, J. (1994) Approaches to influence starch quantityand starch quality in transgenic plants. Plant Cell Environment 17, 601–613.

Munster, van I.P., Tangerman, A. and Nagengast, F.M. (1994) Effect of resistantstarch on colonic fermentation, bile acid metabolism, and mucosal pro-liferation. Digestive Diseases and Sciences 39, 834–842.

Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth andbioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497.

Murch, S.J. and Saxena, P.K. (1997) Modulation of mineral and free fatty acid pro-files during thidiazuron mediated somatic embryogenesis in peanut (Arachishypogeae L.). Journal of Plant Physiology 151, 358–361.

Murthy, B.N.S., Victor, J., Singh, R.P., Fletcher, R.A. and Saxena, P.K. (1996). Invitro regeneration of chickpea (Cicer arietinum L.): stimulation of directorganogenesis and somatic embryogenesis by thidiazuron. Plant GrowthRegulation 19, 233–240.

Mutaftschiev, S., Macaya, A., Prat, R., Devillers, P. and Golberg, R. (1993) Earlyeffects of plant cell wall fragments on plant cell growth. Plant Physiology andBiochemistry 31, 459–467.

Muthukumar, B., Mariamma, M., Veluthambi, K. and Gnanam, A. (1996) Genetictransformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp.)using Agrobacterium tumefaciens. Plant Cell Reports 15, 980–985.

Muzquiz, M., Rey, C., Cuadrado, C. and Fenwick, G.R. (1992) Effect of germination onthe oligosaccharide content of lupin species. Journal of Chromatography 607, 349–352.

Muzquiz, M., Burbano, C., Pedrosa, M., Folkman, W. and Gulewicz, K. (1999) Lupinas potential source of raffinose family oligosaccharides. Preparative method fortheir isolation and purification. Industrial Crops and Products 19, 183–188.

Naczk, M., Myhara, R.M. and Shahidi, F. (1992) Effects of processing on oligo-saccharides of oilseed and legume protein meals. Food Chemistry 45, 193–197.

Naczk, M., Amarowicz, R. and Shahidi, F. (1997) α-Galactosides of sucrose in food:composition, flatulence-causing effect, and removal. In: Shahidi F. (ed.)Antinutrients and Phytochemical in Food. ACS, Washington, DC, Series 662,pp. 127–151.

Naivikul, O. and D’Appolonia, B.L. (1978) Comparation of legumes and wheatflour carbohydrates. I. Sugar analysis. Cereal Chemistry 55, 913–918.

Natali, L. and Cavalini, A. (1987a) Regeneration of pea (Pisum sativum L.) plantletsby in vitro culture of immature embryos. Plant Breeding 99, 172–176.

Natali, L. and Cavalini, A. (1987b) Nuclear cytology of callus and plantlets regener-ated from pea (Pisum sativum L.) meristems. Protoplasma 141, 121–125.

Natali, L. and Cavalini, A. (1989) Grafting in vitro regenerated pea (Pisum sativumL.) shoots as a method for obtaining mature plants. Plant Breeding 102, 341–343.

References 287




Nauerby, B., Madsen, M., Christiansen, J. and Wyndaele, R. (1991) A rapid and effi-cient regeneration system for pea (Pisum sativum), suitable for transformation.Plant Cell Reports 9, 676–679.

Nawracal, J., Konieczny, G. and Kazmierczak, M. (1996) Regeneration of soybeanplants from embryonic axes. Soybean Genetics Newsletter 23, 117–119.

Nche, M.I.R., Nout, M.J.R. and Rombouts, F.M. (1994) Fast production byClostridium perfringens as a measure of the fermentability of carbohydrates andprocessed cereal- legume foods. Food Microbiology 11, 21–29.

Nelke, M., Nowak, J., Wright, J.M. and McLean, N.L. (1993) DNA fingerprinting ofred clover (Trifolium pratense L.) with Jeffrey’s probes: detection of somaclonalvariation and other applications. Plant Cell Reports 13, 72–78.

Nestares, T., Urbano, G., Lopez-Frias, M. and Barrionuevo, M.J. (1997) Nutritionalassessment of magnesium from raw and processed chickpea (Cicer arietinum L.)in growing rats. Journal of Agriculture Food Chemistry 45, 3138–3142.

Newell, C.A. and Luu, H.T. (1985) Protoplast culture and plant regeneration inGlycine canescence F. J. Herm. Plant Cell Tissue and Organ Culture 4, 145–149.

Nicolas, P., Gertsch, I. and Parisod, C. (1984) Isolation and structure determinationof an α-D-galactosyl-α-D-galactosyl-α-D-galactosyl-D-pinitol from the chickpea.Carbohydrate Research 131, 331–334.

Nielsen, S.V.A., Poulsen, G.B. and Larsen, M.E. (1991) Regeneration of shoots frompea hypocotyl explants. Physiologia Plantarum 82, 99–102.

Nisbet, G.S. and Webb, K.J. (1990) Transformation in legumes. In: Bajaj, Y.P.S.(ed.) Biotechnology in Agriculture and Forestry, Vol. 10. Legumes and Oilseed Crops 1.Springer-Verlag, Berlin, pp. 38–48.

Nnanna, I.A. and Phillips, R.D. (1988) Changes in oligosaccharide content, enzymeactivities and dry matter during controlled germination of cowpeas (Vignaunguiculata). Journal of Food Science 53 (6), 1782–1786.

Noah, L., Guillon, F., Bouchet, B., Buleon, A., Gallant, D.J., Colonna, P., Molis, C.,Faisant, N., Gelmiche, J.P. and Champ, M. (1995) Digestion of carbohydratecomponents of dry beans (Phaseolus vulgaris) in healthy humans. In: Proceedingsof the 2nd AEP Conference Improving Production and Utilisation of Grain Legumes,Copenhagen. AEP, Paris, pp. 276–277.

Noah, L., Guillon, F., Bouchet, B., Buleon, A., Molis, Ch., Gratas, M. and Champ, M.(1998) Digestion of carbohydrate from white beans (Phaseolus vulgaris L.)in healthy humans. Journal of Nutrition 128, 977–985.

Nowak, J. and Steinkraus, K.H. (1988) Effect of tempen fermentation of peas ontheir potential flatulence productivity as measured by gas production andgrowth of Clostridium perfringens. Nutrition Report International 38, 1163–1171.

Nwokolo, E. and Smartt, J. (eds) (1996) Food and Feed from Legumes and Oilseeds.Chapman & Hall, London.

Obendorf, R.L. (1997) Oligosaccharides and galactosyl cyclitols in seed desiccationtolerance. Seed Science Research 7, 63–74.

Obendorf, R.L., Horbowicz, M. and Taylor, D.P. (1993) Structure and chemicalcomposition of developing buckwheat seed. In: Janick, J. and Simon, J.E. (eds)New Crops. John Wiley & Sons, New York, pp. 244–251.

Obendorf, R.L., Moon, H., Hildebrand, D.F., Torisky, R. and Collins, G.B. (1996) Acomparison of pinitols in somatic and zygotic soybean embryos (Abstract).Molecular and Cellular Biology of the Soybean 6, 40.

288 References




Obendorf, R.L., Horbowicz, M., Dickerman, A.M., Brenac, P. and Smith, E. (1998)Soluble oligosaccharides and galactosyl cyclitols in maturing soybean seeds inplanta and in vitro. Crop Science 38, 78–84.

Ochatt, S., Pasquis, B., Pontecaille, C. and Rancillac, M. (1998) Rapid plant regener-ation from juvenile explants of protein legumes. In: AEP (ed.) Opportunities forHigh Quality, Healthy and Added-Value Crops to Meet European Demands. Proceedings3rd European Conference on Grain Legumes, Valladolid. AEP, Paris, p. 367.

Odunfa, S.A. (1983) Carbohydrate changes in fermenting locust bean (Parkiafilicoidea) during iru fermentation. Qualitas Plantarum Plant Foods and HumanNutrition 32, 3–10.

Oelck, M.M., Rao, P.S. and Ozias-Akins, P. (1983) Protoplast regeneration fromsome legume species. Experimentia (supplement) 45, 50–51.

Ogawa, K., Watanabe, T., Ikeda, Y. and Kondo, S. (1997) A new glycoside, 1D-2-O-α-D-galactopyranosyl-chiro-inositol from jojoba beans. Carbohydrate Research 302,219–221.

Ogura, H. (1982) Studies on the genetic instability of cultured tissues and theregenerated plants – effects of auxins and cytokinins on mitosis of Vicia fabacells. In: Fujiwara, A. (ed.) Plant Tissue Culture. Maruzen, Tokyo, pp. 433–434.

Ogura, H. (1990) Chromosome variation in plant tissue culture. In: Bajaj, Y.P.S.(ed.) Biotechnology in Agriculture and Forestry, Vol. 11. Somaclonal Variation in CropImprovement 1. Springer-Verlag, Berlin, pp. 49–84.

Okamura, J.K., Jofuku, K.D. and Goldberg, R.B. (1986) Soybean seed lectingene and flanking nonseed protein genes are developmentally regulated intransformed tobacco plants. Proceedings of the National Academy of Sciences USA89, 8240–8244.

Oku, T. (1994) Special physiological functions of newly development mono-and oligosaccharides. In: Goldberg, I. (ed.) Functional Foods – Designer Foods,Pharmafoods, Nutraceuticals. Chapman & Hall, New York, pp. 202–218.

Olive, M.R., Ellis, R.J. and Schuch, W.W. (1989) Isolation and nucleotide sequencesof cDNA clones encoding ADP-glucose pyrophosphorylase polypepetides fromwheat leaf and endosperm. Plant Molecular Biology 12, 525–538.

Olson, A.C., Evans J.J., Frederick, D.P. and Jansen, E.F. (1969) Plant suspensionculture media macromolecules. Pectic substances, protein and peroxidase.Plant Physiology 44, 1594–1600.

Ondøej, M., Rakousky, S. and Schutzner, K. (1998) Transgenic crops tolerant toherbicides. Czechoslovak Journal of Genetics and Plant Breeding 34, 31–42.

Orman, B.A. and Schumann, R.A. (1991) Comparison of near-infrared spectros-copy calibration methods for the prediction of protein, oil and starch in maizegrain. Journal of Agricultural and Food Chemistry 39, 883–886.

Oruna-Concha, M.J., Gonzalez-Castro, M.J., Lopez-Hernandez, J. and Simal-Lozano,J. (1996) Evolution of sugar, starch, pectin and insoluble fibre contents infrozen green beans and padron peppers. Deutsche Lebensmittel-Rundschau 92,278–281.

Ozcan, S., Barghchi, M., Firek, S. and Draper, J. (1992) High frequency adventitiousshoot regeneration from immature cotyledons of pea (Pisum sativum L.). PlantCell Reports 11, 44–47.

Ozias-Akins, P., Schnall, J.A., Anderson, W.F., Singsit, Ch., Clemente, T.E., Adang,M.J. and Weissinger, A.K. (1993) Regeneration of transgenic peanut plantsfrom stably transformed embryogenic callus. Plant Science 93, 185–194.

References 289




Padgette, S.R., Kolacz, K.H., Dellannay, X., Re, D.B., LaVallee, B.J., Tinius, C.N.,Rhodes, W.K., Otero, Y.I., Barry, G.F., Eichholtz, D.A., Peschke, V.M.,Nida, D.L., Taylor, N.B. and Kishore, G.M. (1995) Development, identificationand characterization of a glyphosate soybean line. Crop Science 35, 1451–1461.

Pahl, H. (1998) Potential and constraints in production and end-uses of grainlegumes in Europe – grain legumes and economics. In: AEP (ed.) Opportunitiesfor High Quality, Health and Added-value Crops to Meet European Demands.Proceedings of 3rd European Conference on Grain Legumes. AEP, Paris, pp. 3–5.

Pandey, R. and Ganapathy, P.S. (1984) Isolation of sodium chloride-tolerant callusline of Cicer arietinum L. cv. BG-203. Plant Cell Reports 3, 45–47.

Papes, D., Jelaska, S., Tomaseo, M. and Devide, Z. (1978) Triploidy in callus cultureof Vicia faba L. investigated by the Giemsa C-banding technique. Experientia 34,1016–1017.

Paredes-Lopez, O., Maza-Calvino, E.C., Gonzalez-Castaneda, J. and Montes-Rivera,R. (1988) Starch 40, 11–15.

Parrott, W.A., Hoffman, L.M., Hildebrand, D.F., Williams, E.G. and Collins, G.B.(1989) Recovery of primary transformants of soybean. Plant Cell Reports 7,615–617.

Parrott, W.A., Bailey, M.A., Durham, R.E. and Mathew, H.V. (1992) Tissue cultureand regeneration in Legumes. In: Moss, J.P. (ed.) Biotechnology and CropImprovement in Legumes. ICRISAT, Patancheru, India, pp. 115–148.

Parrott, W.A., Merkle, S.A. and Williams, E.G. (1993) Somatic embryogenesis:potential for use in propagation and gene transfer systems. In: Murray, D.R.(ed.) Advanced Methods in Plant Breeding and Biotechnology. CAB International,Wallingford, UK, pp. 58–200.

Parrott, W.A., All, J.N., Adang, M.J., Bailey, M.A., Boerma, H.R. and Stewart, C.N. Jr.(1994) Recovery and evaluation of soybean plants transgenic for a Bacillusthuringiensis var. Kurstaki insecticidal gene. In Vitro Cellular and DevelopmentalBiology – Plant 30, 144–149.

Paszkowski, J., Peterhans, A., Schlupmann, H., Basse, C., Lebel, E.G. and Masson, J.(1992) Protoplasts as tool for plant genome modifications. PhysiologiaPlantarum 85, 352–356.

Pate, J.S. (1984) The carbon and nitrogen nutrition of fruits and seed-case studiesof selected grain legumes. In: Murray, D.R. (ed.) Seed Physiology, Vol. 1. Develop-ment. Academic Press, London, pp. 41–82.

Pate, J.S., Sharkey, P.J. and Lewis, O.A.M. (1974) Phloem bleeding from legumefruits – a technique for study of fruit nutrition. Planta 120, 229–243.

Patel, A.R., Patel, M.R., Patel, N.R., Patel, K.C. and Patel, R.D. (1986) Graft copoly-mers of starch and polyacrylonitrile. Effect of source. Starch/Stärke 38, 235–237.

Patrick, J.W. and McDonald, R. (1980) Pathway of carbon transport withindeveloping ovules of Phaseolus vulgaris L. Australian Journal of Plant Physiology7, 671–684.

Patrick, J.W. and Offler, C.E. (1995) Post-sieve element transport of sucrose indeveloping seeds. Australian Journal of Plant Physiology 22, 681–702.

Patzelt, W.J. (1974) Polarized-light Microscopy. Principles, Instruments, Applications.Ernst Leitz, Wetzlar.

Paul, A.A. and Southgate, D.A.T. (1985) In: Widdowson, E.M. (ed.) The Compositionof Foods, 4th edn. Elsevier/North-Holland Biomedical Press, Amsterdam.

290 References




Pearson, D. (1976) Sugar and preserves. In: Pearson, D. (ed.) The Chemical Analysisof Food. Churchill Livingstone, Edinburgh, pp. 107–157.

Pedroso, M.C. and Pais, M.S. (1995) Factors controlling somatic embryogenesis.Plant Cell Tissue and Organ Culture 43, 147–154.

Penel, K. and Greppin, H. (1996) Pectin binding proteins: characterization of thebinding and comparison with heparin. Plant Physiology and Biochemistry 34,479–488.

Perez, S. and Imberty, A. (1996) Structural features of starch. Carbohydrates in Europe15, 17–21.

Periago, M.J., Ros, G. and Casas, J.L. (1997) Non-starch polysaccharides and in vitrostarch digestibility of raw and cooked chick peas. Journal of Food Science 62,93–96.

Petek, F., Villarroya, E. and Courtois, J.E. (1966) Isolation of two galactosides ofmyo-inositol from vetch seeds. Comptes Rendus de l’Academie des Sciences (Paris)Serie D: Sciences Naturelles 263, 195–197.

Petek, F., Villarroya, E. and Courtois, J.E., (1969) Purification and properties ofα-galactosidase in germinating Vicia sativa seeds. European Journal of Biochemistry8, 395–402.

Peterbauer, T. and Richter, A. (1998) Galactosylononitol and stachyose synthesis inseeds of adzuki bean. Purification and characterization of stachyose synthase.Plant Physiology 117, 165–172.

Peterbauer, T., Mucha, J., Mayer, U., Popp, M., Gloessl, J. and Richter, A. (1999)Stachyose synthesis in seeds of adzuki bean (Vigna angularis): molecularcloning and functional expression of stachyose synthase. The Plant Journal 20,509–518.

Peters, J.E., Padock, E.F., Tegenkamp, I. and Tegenkamp, T. (1977) Haploid calluscells from anthers of Phaseolus vulgaris. Phytomorphology 27, 79–85.

Petruzelli, L. and Taranto, G. (1989) Wheat ageing: the contribution of embryonicand non-embryonic lesions to loss seed viability. Plant Physiology 76, 289–294.

Peyronnet, C., Bastianelli, D. and Gatel, F. (1996) Factors of pulse value. GrainLegumes 11, 20–21.

Phansiri, S., Miyake, H., Maeda, E. and Taniguchi, T. (1993) Ultrastructural analysisof cell wall formation and cell division of soybean (Glycine max) protoplasts.Japanese Journal of Crop Science 62, 429–437.

Pharr, D.M., Hendrix, D.L., Robbins, N.S., Gross, K.C. and Sox, H.N. (1987)Isolation of galactinol from leaves of Cucumis sativus. Plant Science 50, 21–26.

Phillips, R.D. (1989) Composition and flatulence-producing potential of commonlyeaten Nigerian and American legumes. Food Chemistry 33, 271–281.

Pickardt, T., Saalbach, I., Waddell, D., Meixner, M., Muntz, K. and Schieder, O.(1995) Seed specific expression of the 2S albumin gene from Brazil nut(Bertholetia excelsa) in transgenic Vicia narbonensis. Molecular Breeding 1, 295–301.

Pigeaire, A., Abernethy, D., Smith, P.M.C., Simpson, K., Fletcher, N., Lu, C.Y.,Atkins, C.A. and Cornish, E. (1997) Routine transformation of a grain legumecrop (Lupinus angustifolius L.) via Agrobacterim tumefaciens-mediated genetransfer to shoot apices. Molecular Breeding 3, 341–349.

Piotrowicz-Cieslak, A.I., Górecki, R.J. and Frias, J. (1995) Changes in galactosidescontent during maturation of yellow lupin (Lupinus luteus L.) and lentil (Lensculinaris L.) seeds. In: AEP (ed.) Improving Production and Utilisation of GrainLegumes. Proceedings 2nd European Conference on Grain Legumes. AEP, Paris, p. 378.

References 291




Pizzoferrato, L., Capelloni, M., Carnovale, E., Clementi, A., Ceccarelli, G. andTolomeo, M. (1995) Evaluation of starch digestibility in mottled beans andlentils. In: AEP (ed.) Improving Production and Utilisation of Grain Legumes.Proceedings 2nd European Conference on Grain Legumes. AEP, Paris, p. 278.

Plant, A.R. (1984) Studies of the protein bodies of Lupinus angustifolius. DissertationAbstracts International, C- European Abstracts 45, 367.

Pokorny, J. and Dostalova, J. (1996) Legumes – their consumption and nutritionalvalue. Vyz. Potraviny 51, 133–135 (in Czech).

Poncet, C., Remond, D., Bernard, L. and Peyronnet, C. (1998) Nitrogen value ofprotein rich leguminous seed in ruminants: ruminal degradability of rawand extruded pea and lupin. In: AEP(ed.) Improving Production and Utilisationof Grain Legumes. Proceedings of 3rd European Conference on Grain Legumes. AEP,Paris, pp. 16–17.

Polanco, M.C. and Ruiz, M.L. (1997) Effect of benzylaminopurine on in vitro andin vivo in lentil (Lens culinaris Medik). Plant Cell Reports 17, 22–26.

Polisetty, R., Paul, J., Deveshwar, J.J., Khetarpal, S., Suresh, K. and Chandra, R.(1997) Multiple shoot induction by benzyladenine and complete plantregeneration from seed explants of chickpea (Cicer arietinum L.). Plant CellReports 16, 565–571.

Ponsamuel, J. Huhman, D.V., Cassidy, B.G. and Post-Beittenmiller, D. (1998) Invitro regeneration via caulogenesis and brassin-induced shoot conversion ofdormant buds from plumular explants of peanut (Arachis hypogeae L. cv.Okrum). Plant Cell Reports 17, 373–378.

Potrykus, I. and Spangenberg, G. (1995) Gene Transfer to Plants. Springer-Verlag,Berlin.

Power, J.B. and Chapman, J.V. (1985) Isolation, culture and genetic manipulationof plant protoplasts. In: Dixon, R.A. (ed.) Plant Cell Culture – a PracticalApproach. IRL Press, Oxford, pp. 37–66.

Preiss, J. and Levi, C. (1980) Starch biosynthesis and degradation. In: Pridham J.B.(ed.) The Biochemistry of Plants, Vol. 3. Academic Press, New York, pp. 371–423.

Price, K.R., Lewis, J., Wyatt, G.M. and Fenwick, G.R. (1988) Flatulence causes,relation to diet remedies. Nahrung 32, 609–623.

Pridham, J.B. and Dey, P.M. (1974) The nature and function of higherplant α-galactosidases. In: Pridham, J.B. (ed.) Plant Carbohydrate Biochemistry.Academic Press, New York, pp. 83–96.

Proceedings of the 1st European Conference on Grain Legumes (1992). AEP, Paris.Proceedings of the 3rd European Conference on Grain Legumes. Opportunities for High Qual-

ity, Healthy and Added-Value Crops to Meet European Demands (1998). AEP, Paris.Proceedings of the International Conference Euro Food Tox IV on Bioactive Substance in Food

of Plant Origin (1994). Oloztyn, Poland. FECS event 193.Prosky, L., Asp, N.G., Furda, I., De Vries, J., Schweizer, T.F. and Harald, B.F. (1985)

Determination of total dietary fibre in foods and food products: collaborativestudy. Journal of the AOAC 68, 677–679.

Puonti-Kaerlas, J. and Eriksson, T. (1988) Improved protoplast culture and regener-ation of shoots in pea (Pisum sativum L.). Plant Cell Reports 7, 242–245.

Puonti-Kaerlas, J., Eriksson, T. and Engstrom, P. (1990) Production of transgenicpea (Pisum sativum L.) plants by Agrobacterim tumefaciens-mediated gene trans-fer. Theoretical and Applied Genetics 80, 246–252.

292 References




Quemener, B. and Brillouet, J.M. (1983) Ciceritol, a pinitol digalactoside fromseeds of chickpea, lentil and white lupin. Phytochemistry 22, 1745–1751.

Quemener, B. and Thibault, J.F. (1990) Assessment of methanolysis for thedetermination of sugars in pectins. Carbohydrate Research 206, 277.

Quigley, M.E. and Englyst, H.N. (1992) Determination of neutral sugars andhexosamines by high performance liquid chromatography with pulsedamperometric detection. Analyst 117, 1715–1718.

Rackis, J.J. (1975) Oligosaccharides of food legumes: alpha-galactoside activityand flatus problems. In: Allen, J. and Heilge, J. (eds) Physiological Effect of FoodCarbohydrates. American Chemical Society, Washington, DC, pp. 207–222.

Raffi, J., Agnel, J.P., Dauberte, B., d’Urbal, M. and Saint-Lebe, L. (1981a) Gammaradiolysis of starches derived from different foodstuffs, Part I. Study on someinduced carbonyl derivatives. Starch 33, 188–194.

Raffi, J., Frejaville, C., Dauphin, J.F., Dauberte, B., d’Urbal, M. and Saint-Lebe, L.(1981b) Gamma radiolysis of starches derived from different foodstuffs, Part II.Study of induced acidities. Starch 33, 235–240.

Raffi, J., Agnel, J.P., Dauberte, B. and Saint-Lebe, L. (1981c) Gamma radiolysis ofstarches derived from different foodstuffs, Part III. Study of induced hydrogenperoxide. Starch 33, 269–271.

Raffi, J., Dauberte, B., d’Urbal, M., Pollin, C. and Saint-Lebe, L. (1981d) Gammaradiolysis of starches derived from different foodstuffs, Part IV. Study ofradiopolymerization. Starch 33, 301–306.

Rajasekaran, K. and Pellow, J.M. (1997) Somatic embryogenesis from cultured epi-cotyls and primary leaves of soybean (Glycine max L. Merr). In Vitro Cellular andDevelopmental Biology – Plant 33, 88–91.

Ralet, M.C., Saulnier, L. and Thibault, J.F. (1993) Raw and extruded fibre from peahulls. Part II: Structural study of the water-soluble polysaccharides. CarbohydratePolymers 20, 25–34.

Ramsay, G. and Middlefell-Williams, J.E. (1992) Regeneration from seedlingexplants in Vicia. In: AEP (ed.) Proceedings of 1st European Conference on GrainLegumes. AEP, Paris, pp. 105–106.

Rani, S., Jood, S. and Sehgal, S. (1996). Cultivar differences and effect of pigeon peaseeds boiling on trypsin inhibitor activity and in vitro digestibility of protein andstarch. Nahrung 40, 145–146.

Rao, P.U. and Belevady, B. (1978) Oligosaccharide in pulses: varietal differencesand effects of cooking and germination. Journal of Agricultural Food Chemistry 26,316–319.

Rassi, Z.E. and Mechref, Y. (1996) Recent advances of capillary electrophoresis ofcarbohydrates. Electrophoresis 17, 275–301.

Ravindran, G. and Palmer, J.K. (1984) Polysaccharides of winged beans (Pospho-carpus tetragonolobus L. DC), Variety TPT-2. Journal of Food Science 49, 70–71.

Ray Chaudhuri, A., Hait, N.C., DasGupta, S., Bhaduri, T.J., Deb, R. and Majumder,A.L. (1997) L-myo-Inositol 1-phosphate synthase from plant sources: character-istics of the chloroplastic and cytosolic enzymes. Plant Physiology 115, 727–736.

Reddy, C.G., Susheelamma and Taranathan, R.N. (1989) Viscosity pattern of nativeand fermented black gram flour and starch dispersions. Starch 41, 84–88.

Reddy, N.R., Balakrishna, C.V. and Salunkhe, D.K. (1978) Phytate phosphorus andminerals changes during germination and cooking of black grain (Phaseolusmungo) seeds. Journal of Food Science 43, 540–543.

References 293




Reddy, N.R., Pierson, M.D., Sathe, S.K. and Salunkhe, D.K. (1984) Chemical,nutritional and physiological aspects of dry bean carbohydrates. A Review ofFood Chemistry 13, 25–68.

Rees ap, T. and Morrell, S. (1990) Carbohydrate metabolism in developingpotatoes. American Potato Journal 67, 835–847.

Reeves, J.B. (1988) Near infrared spectroscopic analysis of lignin components insodium chlorite-treated and untreated forages and forage by-products. Journalof Dairy Science 71, 388–397.

Reichert, R.D. (1982) Air classification of peas (Pisum sativum) varying widely inprotein content. Journal of Food Science 47, 1263–1267.

Reichert, R.D. and Youngs, C.G. (1978) Nature of the residual protein associatedwith starch fractions from air-classified field peas. Cereal Chemistry 55, 469–480.

Reid, D.S., Carr, J.M., Sajjaanantakul, T. and Labavich, J.M. (1986) Effect of freez-ing and frozen storage on the characteristics of pectin extracted from cellwalls. ACS Symposium Series, American Chemical Society 310, 200–216.

Reid, J.S.G. (1985) Galactomannans. In: Dey, P.M. and Dixon, R.A. (eds) Bio-chemistry of Storage Carbohydrates in Green Plants. Academic Press, London,pp. 205–288.

Reisch, B., Duke, S.H. and Bingham, E.T. (1980) The genetic control of budformation from callus cultures of diploid alfalfa. Plant Science 20, 71–77.

Rentz, A. and Stitt, M. (1993) Substrate specificity and product inhibition ofdifferent forms of fructokinases and hexokinases in developing potato tubers.Planta 190, 166–175.

Revilla, M.A. and Tarango, J.F. (1986a) Scanning electron microscopy study onstarch granules in lentil cotyledons. Starch 38, 199–202.

Revilla, M.A. and Tarango, J.F., (1986b) Ultrastructure of starch grain breakdown incotyledone cells. Starch 38, 379–381.

Revilleza, M.J.R., Mendoza, E.M.T. and Raymundo, L.C. (1990) Oligosaccharides inseveral Philippine indigenous food legumes: determination, localization andremoval. Plant Foods for Human Nutrition 40, 83–93.

Roberts, K. (1996) Structures at the plant cell surface. Current Opinion in Cell Biology2, 920–928.

Robertson, J.B. and Horvath, P.J. (1993) Detergent analysis of foods. In: Spiller,G.A. (ed.) Handbook of Dietary Fibre in Human Nutrition. CRC Press, Boca Raton,Florida, pp. 49–52.

Rochat, C., Wuilleme, S., Boutin, J.-P. and Hedley, C.L. (1995a) ADPGPPase activityaffects storage product metabolism in rbrb pea seed coats. In: AEP (ed.) Proceed-ings of the 2nd European Conference on Grain Legumes, Copenhagen. ImprovingProduction and Utilisation of Grain Legumes. AEP, Paris, pp. 34–35.

Rochat, C., Wuilleme, S., Boutin, J.-P. and Hedley, C.L. (1995b) A mutation at the rbgene, lowering ADPGPPase activity, affects storage product metabolism of peaseed coats. Journal of Experimental Botany 46, 415–421.

Rodriguez, A., Martin, L., Alonso, J.R., Nicolas, G. and Villalobos, N. (1988)Scanning electron microscope study of starch granule degradation in chick-peacotyledons. Part I. Influence of embryonic axis on degradation process. Starch40, 211–214.

Roffs, C.H., Schon, H., Steffen, M. and Kindl, H. (1987) Cell suspension culture ofArachis hypogea L. Model system for specific enzyme induction in secondarymetabolism. Planta 172, 238–244.

294 References




Roper, W. (1979) Growth and cytology of callus and cell suspension cultures of Viciafaba. Zeitschrift für Pflanzen Physiology 93, 245–257.

Ross, J.K., English, C. and Perlmutter, C.A. (1985) Dietary fiber constituents ofselected fruits and vegetables. Journal of the American Dietetic Association 85,1111–1116.

Rossi, M., Germondari, I. and Casini, P. (1984) Comparison of chickpea cultivars.Chemical composition, nutritional evaluation and oligosaccharide content.Journal of Agricultural and Food Chemistry 2, 811–814.

Roth, E.J., Frazier, B.L., Apuya, N.R. and Lark, K.G. (1989) Genetic variation in aninbred plant: variation in tissue cultures of soybean [Glycine max (L.) Merrill].Genetics 121, 359.

Rubluo, A., Kartha, K.K., Mroginski, L.A. and Dyck, J. (1984) Plant regenerationfrom pea leaflets cultured in vitro and genetic stability of regenerants. Journal ofPlant Physiology 117, 119–130.

Rufty, T.W., Israel, D.W. and Wolk, R.J. (1984) Assimilation of 15NO3− taken up by

plants in the light and in the dark. Plant Physiology 76, 769–775.Rufty, T.W., Huber, S.C. and Volk, R.J. (1988) Alteration in leaf carbohydrate

metabolism in response to nitrogen stress. Plant Physiology 88, 725–730.Russell, D.R., Wallace, K., Bathe, J., Martinell, B. and McCabe, D. (1993) Stable

transformation of Phaseolus vulgaris via electric-discharge mediated particleacceleration. Plant Cell Reports 12, 165–169.

Russel, P.L., Berry, C.S. and Greenwell, P. (1989) Characterisation of resistantstarch from wheat and maize. Journal of Cereal Science 9, 1–15.

Ryan, C.A. and Farmer, E.E. (1991) Oligosaccharide signals in plants: a currentassessment. Annual Reviews in Plant Physiology and Plant Molecular Biology 42,651–674.

Rybczynski, J. and Podyma, E. (1993a) Preliminary studies of plant regeneration viasomatic embryogenesis induced on immature cotyledons of white lupin(Lupinus albus L.) Genetica Polonica 34, 249–257.

Rybczynski, J. and Podyma, E. (1993b) Micropropagation of some Lupinus speciesfrom seedling explants. Genetica Polonica 34, 237–247.

Saalbach, I., Pickardt, T., Machemehl, F., Saalbach, G., Schieder, O.N. and Muntz,K. (1994) A chimaeric gene encoding the methionine-rich 2S albumin of theBrazil nut (Bertholletia excelsa H.B.K.) is stably expressed and inherited intransgenic grain legumes. Molecular General Genetics 242, 226–236.

Saalbach, I., Pickardt, T., Waddell, D.R., Hillmer, S., Schieder, O.N. and Muntz, K.(1995a) The sulphur-rich Brazil nut 2S albumin is specifically formed intransgenic seeds of the grain legume Vicia narbonensis. Euphytica 85, 181–192.

Saalbach, I., Waddell, D., Pickardt, T., Schieder, O. and Muntz, K. (1995b) Stableexpression of the sulphur-rich 2S albumin gene in transgenic Vicia narbonensisincreases the methionine content of seeds. Journal of Plant Physiology 145,674–681.

Saalbach, L., Hofmeister, J. and Baumlein, H. (1997) Plant seeds as bioreactors:use of a heat-stable, bacterial amylase in transgenic seeds of legumes forliquefaction of seed starch. In: Proceedings 5th Symp. Renewable Resources. Schrift.Bundesminister. Ernahrung, Landwirschaft und Forsten. Reihe A, Angew.Wissenschaft., pp. 182–184.

Saeed, M. and Duke, S.H. (1990) Amylases in pea tissues with reduced chloroplastdensity and/or function. Plant Physiology 94, 1813–1819.

References 295




Sagara, A.P., Suhasini, K. and Krishnamurthy, K.V. (1993) Plant regeneration viasomatic embryogenesis in chickpea (Cicer arietinum L). Plant Cell Reports 12,652–655.

Saini, H.S. (1989) Legume seed oligosaccharides. In: Huisman, J., van der Poel,A.F.B. and Liener, I.E. (eds) Recent Advances of Research in Antinutritional Factorsin Legume Seeds. Pudoc, Wageningen, pp. 329–341.

Saini, H.S. and Knights, E.J. (1984) Chemical constitution of starch andoligosaccharide content of ‘desi’ and ‘kabuli’ chickpea (Cicer arietinum) seedtypes. Journal of Agricultural and Food Chemistry 32, 940–944.

Saini, H.S. and Gladstones, J.S. (1986) Variability in the total and componentgalactosyl sucrose oligosaccharides of Lupinus species. Australian Journal ofAgricultural Research 37, 157–166.

Sakai, K., Tachiki, T., Kumagai, H. and Tochikura, T. (1987) Hydrolysis of alpha-D-galactosyl oligosaccharides in soymilk by alpha-D-galactosidase of Bifido-bacterium breve 203. Agricultural Biological Chemistry 51, 315–322.

Sakurai, N. (1991) Cell wall functions in growth and development – a physical andchemical point of view. The Botanical Magazine, Tokyo 104, 235–251.

Salunkhe, D.K. and Kadam, S.S. (eds) (1989) CRC Handbook of World Food Legumes:Nutritional Chemistry, Processing Technology, and Utilization, Vol. 11. CRC Press,Boca Raton, Florida.

Salunkhe, D.K., Sathe, S.K. and Deshpande, S.S. (1989) French Bean. In: Salunkhe,D.K. and Kadam, S.S. (eds.) CRC Handbook of World Food Legumes: NutritionalChemistry, Processing Technology and Utilization, Vol. I. CRC Press, Boca Raton,Florida, pp. 23–63.

Sambuceti, M.E. and Zuleta, A. (1996) Resistant starch in dietary fiber values mea-sured by the AOAC method in different cereals. Cereal Chemistry 73, 759–761.

Sanago, M.H.M., Shattuck, V.I. and Strommer, J. (1996) Rapid plant regenerationof pea using thidiazuron. Plant Cell Tissue and Organ Culture 45, 165–168.

Sandberg, A.-S., Andersson, H., Carlsson, N.-G. and Sandström, B. (1993) Degrada-tion of soy bean oligosaccharides in the stomach and small intestine of humanileostomy subjects. In: Schlemmer, U. (ed.) Proceedings of the International Confer-ence on Bioavailability ‘93 – Nutritional, Chemical and Food Processing Implicationsof Nutrient Availability. Bundesforschungsanstalt für Ernährung, Karlsruhe,pp. 197–201.

Santangelo, E., Mosconi, P., Crino, P., Soressi, G.P. and Saccardo, F. (1997) In:INTAGRES and ICARDA. New Perspectives for an Ancient Species. Aleppo, Syria,pp. 73–83.

Saravitz, D.M., Pharr, D.M. and Carter, T.E. (1987) Galactinol synthase activity andsoluble sugars in developing seeds of four soybean genotypes. Plant Physiology83, 185–189.

Sarko, A. and Wu, H.-C.H. (1978) The crystal structures of A-, B- and C-polymorphsof amylose and starch. Starch/Stärke 30, 73–8.

Sasaki, K., Hicks, K.B. and Nagahashi, G. (1988) Separation of eight inositol isomersby liquid chromatography under pressure using a calcium-form, cation-exchange column. Carbohydrate Research 183, 1–9.

Sathe, S.K. and Salunkhe, D.K. (1981) Isolation and partial characterization of anarabinogalactan from the Great Nortin Bean (Phaseolus vulgaris L.). Journal ofFood Science 46, 1276–1277.

296 References




Sator, C. (1990) Lupins (Lupinus spp.). In: Bajaj Y.P.S. (ed.) Legumes and OilseedCrops I, Biotechnology in Agriculture and Forestry, Vol. 10. Springer-Verlag,Berlin-Heidelberg, pp. 288–311.

Saura-Calixto, F., Goni, I., Bravo, L. and Manas, E. (1993) Resistant starch in foods:modified method for dietary fibre residues. Journal of Food Science 58, 642–643.

Savage, G.P. (1988) The composition and nutritive value of lentil (Lens culinaris).Nutrition Abstract and Reviews (Series A) 5, 319–343.

Schaumann, A., Bruyant-Vannier, M.P., Goubet, F. and Morvan, C. (1993) Meta-bolism in suspension cultured cells of Flax (Linum usitatissinum L.). Plant CellPhysiology 34, 891–897.

Schlumbaum, A., Mauch, F., Vogeli, U. and Boller, T. (1986) Plant chitinases arepotent inhibitors of fungal growth. Nature 324, 365–367.

Schmitt, M.R., Hitz, W.D., Lin, W. and Giaquinta, R.T. (1984) Sugar transport intoprotoplasts isolated from developing soybean cotyledons. II. Sucrose transportkinetics, selectivity, and modelling studies. Plant Physiology 75, 941–946.

Schoch, T.J. and Maywald, E.C. (1968) Preparation and properties of variouslegume starches. Cereal Chemistry 45, 564–571.

Scholda, R., Billek, G. and Hoffmann-Ostenhof, O. (1964) Biosynthesis of cyclitols.VIII. Mechanism of conversion of myo-inositol to D-pinitol and D-chiro-inositol inTrifolium incarnatum. Monatshefte für Chemie 95, 1311–1317.

Schroeder, H., Schotz, A., Wardley-Richardson, T., Spencer, D. and Higgins, T.(1993) Transformation and regeneration of two cultivars of pea (Pisum sativumL.). Plant Physiology 101, 751–757.

Schroeder, H.E., Gollasch, S., Moore, A., Tabe, L.M., Craig, S., Hardie, D.C.,Chrispeels, M.J., Spencer, D. and Higgins, T.J.V. (1995) Bean (β-amylaseinhibitor confers resistance to the pea weevil (Bruchus pisorum) in transgenicpeas (Pisum sativum). Plant Physiology 107, 1233–1239.

Schrott, M. (1995) Selectable marker and reporter genes. In: Potrykus, I.and Spangenberg, G. (eds) Gene Transfer to Plants. Springer-Verlag, Berlin,pp. 325–336.

Schukla, T.P. (1996) Cereal grain and legume processing by extrusion. Cereal FoodsWorld 41, 35–36.

Schweizer, T.F., Horman, I. and Würsch, P. (1978) Low molecular weight carbohy-drates from leguminous seeds; a new disaccharide: galactopinitol. Journal of theScience of Food and Agriculture 29, 148–154.

Schweizer, T.F. and Horman, I. (1981) Purification and structure determination ofthree α-D-galactopyranosylcyclitols from soya beans. Carbohydrate Research 95,61–71.

Schweizer, T.F., Andersson, H., Langkilde, A.M., Reimann, S. and Torsdottir, I.(1990) Nutrients excreted in ileostomy effluents after consumption of mixeddiets with beans or potatoes. II. Starch, dietary fibre and sugars. EuropeanJournal of Clinical Nutrition 44, 567–575.

Scowcroft, W.R., Brettell, R.I.S., Ryan, S.A., Davies, P.A. and Pallotta, M.A. (1987)Somaclonal variation and genomic flux. In: Green, C.E. (ed.) Plant Tissue andCell Culture. Alan R. Liss, New York, pp. 275–286.

Selvendran, R.R. (1983) The chemistry of plant cell walls. In: Birch, G.G.and Parker, K.J. (eds) Dietary Fibre. Applied Science Publishers, London,pp. 95–147.

References 297




Selvendran, R.R. (1984) The plant cell wall as a source of dietary fibre: chemistryand structure. American Journal of Clinical Nutrition 39, 320–337.

Selvendran, R.R. and King, S.E. (1989) Structural features of the cell-wall polysac-charides of the parchment layers of the pods of mature runner beans. Carbohy-drate Research 195, 87–99.

Selvendran, R.R. and O’Neill, M.A. (1987) Isolation and analysis of cell walls fromplant material, In: Glick, D. (ed.) Methods of Biochemical Analysis. John Wiley &Sons, New York, pp. 25–154.

Selvendran, R.R. and Robertson, J.A. (1990) The chemistry of dietary fibre – anholistic view of the cell wall matrix. In: Southgate, D.A.T., Waldron, K.,Johnson, I.T. and Fenwick, G.R. (eds) Dietary Fibre: Chemical and BiologicalAspects. The Royal Society of Chemistry, Cambridge, pp. 27–43.

Selvendran, R.R., March, J.F. and Ring, S.G. (1979) Determination of aldoses anduronic acid content of vegetable fiber. Analytical Biochemistry 96, 282.

Selvendran, R.R., Stevens, B.J.H. and O’Neill, M.A. (1985) Developments in theisolation and analysis of cell walls from edible plants. In: Brett, C.T. andHillman, J.R. (eds) Biochemistry of Plant Cell Walls. Cambridge University Press,Cambridge, pp. 39–78.

Senaratna, T., McKersie, B.D. and Bowley, S.R. (1985) Antioxidant levels in germi-nating soybean axes in relation to free radical and dehydration tolerance. PlantPhysiology 78, 168–171.

Seve, P., Kerros, C., Lebreton, Y., Quemener, B., Gaborit, T. and Bouchez, P. (1989)Effect of the extraction of α-galactosides from toasted or raw soybean meal ondietary nitrogen and fat utilization in the young pig. In: Huisman, J., van derPoel, T.F.B. and Liner, I.E. (eds) Recent Advances of Research in AntinutritionalFactors in Legumes Seeds. Pudoc, Wageningen, pp. 276–280.

Shade, R.E., Schroeder, H.E., Pueyo, J.J., Tabe, L.M., Murdock, L.L., Higgins, T.J.V.and Chrispeels, M.J. (1994) Transgenic pea seeds expressing the α-amylaseinhibitor of the common bean are resistant to bruchid beetles. Bio-Technology(New York) 12, 793–796.

Shamina, Z.B. and Butenko, R.G. (1976) Optimization of nutrient medium fortissue culture of Vicia faba. Fiziologiya Rastenii (Plant Physiology) 23, 1264–1268.

Sharma, V.K. and Kothari, S.L. (1994) Genotypic differences of multipleshoot-forming capacity in soybean. Plant Tissue Culture 4, 17–24.

Shehata, A., El-shimi, N.M. and Mesallam, A.S. (1985) Pectic substances of fababeans and their relation to texture of cooked beans. Journal of Food Processingand Preservation 9, 65–74.

Shekib, L.A. (1994) In-vitro digestibility and microscopic appearance of germinatedlegume starches and their effect on dietary protein utilization. Food Chemistry50, 59–63.

Shekib, L.A.E., Zouil, M.M., Youssef, M.M. and Mohammed, M.S. (1985). Effect ofcooking on chemical composition of lentil, rice and their blend (kloshary).Food Chemistry 18, 163–168.

Shetty, K., Asano, Y. and Oosawa, K. (1992) Stimulation of in vitro shoot organo-genesis in Glycine max Merrill by alantoin and amides. Plant Science 81, 245–251.

Shewmaker, C.K., Boyer, C.D., Wiesenborn, D.P., Thompson, D.B., Boersig, M.R.,Oakes, J.V. and Stalker, D.M. (1994) Expression of E. coli glycogen synthase inthe tuber of transgenic potatoes (Solanum tuberosum) results in a highlybranched starch. Plant Physiology 104, 1159–1166.

298 References




Shoemaker, R.C. and Hammond, E.G. (1988) Fatty acid composition of soybean[Glycine max (L.) Merrill] somatic embryos. In Vitro Cellular and DevelopmentalBiology – Plant 24, 829–832.

Shoemaker, R.C., Amberger, L.A., Palmer, R.G., Oglesby, L. and Ranch, J.P. (1991)The effect of 2,4-dichlorophenoxyacetic acid concentration on somaticembryogenesis and heritable variation in soybean [Glycine max (L.) Merr.]. InVitro Cellular and Developmental Biology – Plant 27, 84–88.

Shukla, K.S. (1987) Quantitative determination of oligosaccharides in defattedsoybean products by high speed liquid chromatography. Fat Science Technology89, 75–79.

Sidduraju, P., Bravo, L. and Saura-Calixto, F. (1998) Chemical composition andcarbohydrate digestibility of common and underexploited legumes. In: AEP(ed.) Improving Production and Utilisation of Grain Legumes. Proceedings of 3rdEuropean Conference on Grain Legumes. AEP, Paris, pp. 380–381.

Sievert, D. and Pomeranz, Y. (1989) Enzyme-resistant starch. I. Characterisation andevaluation by enzymatic, thermoanalytical and microscopic methods. CerealChemistry 66, 342–347.

Sievert, D. and Pomeranz, Y. (1990) Enzyme-resistant starch. II. Differentialscanning calorimetry studies on heat-treated starches and enzyme-resistantstarch residues. Cereal Chemistry 67, 217–221.

Sievert, D., Czujachowska, Z. and Pomeranz, Y. (1991) Enzyme-resistant starch. III.X-ray diffraction of autoclaved amylomaize. VII. Starch and enzyme starchresidues. Cereal Chemistry 68, 86–91.

Siljeström, M. and Björck, I. (1990) Digestible and undigestible carbohydrates inautoclaved legumes, potato, and corn. Food Chemistry 38, 145–152.

Siljeström, M., Eliasson, A.C. and Björck, I. (1989) Characterisation of resistantstarch from autoclaved wheat starch. Starch/Stärke 41, 147–151.

Silvester, K.I., Englyst, H.N. and Cummings, J.H. (1995) Ileal recovery of starchfrom whole diets containing resistant starch measured in vitro and fermenta-tion of ileal effluent. American Journal of Clinical Nutrition 62, 403–411.

Simmonds, D.H. (1992) Plant cell wall removal: cause for microtubule instabilityand division abnormalities in protoplast cultures. Physiologia Plantarum 85,387–390.

Simonenko, Y.V., Gleba, Y.Y. and Kuchuk, N.V. (1999) Double transformation:producing transgenic phosphinotricin-resistant plants of commercial pealines. Russian Journal of Plant Physiology 46, 804–807.

Simoni, A., Mugnai, M., Storti, E., Bittini, P., Schipani, C., Simeti, C., Angelini, P.and Buiatti, M. (1995) Biochemical markers for early screening of tolerantgenotypes in the system Glycine max – Diaporthe phaseolorum var. Caulivora.Journal of Genetics and Breeding 49, 169–178.

Sims, I.M., Munro, S.L., Currie, G., Craik, D. and Bacie, A. (1996) Structural charac-terization of xyloglucan secreted by suspension-cultured cells of Nicotianaplumbaginifolia. Carbohydrate Research 293, 147–172.

Singh, K.B. (1997) Chickpea. Field Crops Research 53, 161–170.Sinnaeve, G., Jones, A., Merghem, R., Jay, M., Dardenne, P. and Biston, R. (1995)

Non destructive single seed analysis of peas by near infrared spectroscopy.In: AEP (ed.) Proceedings of 2nd European Conference on Grain Legumes: ImprovingProduction and Utilisation of Grain Legumes. AEP, Paris, France, pp. 370–371.

References 299




Sivak, M.N. and Preiss, J. (1995) Starch synthesis in seeds. In: Kigel, J. and Galli, G.(eds) Seed Development and Germination. Marcel Dekker, New York, pp. 139–168.

Skrabanja, V., Liljeberg, H.G.M., Hedley, C.L., Kreft, I. and Bjorck, I.M.E. (1999)The influence of genotype and processing on the in vitro rate of starch hydroly-sis and resistant starch formation in peas (Pisum sativum L.). Journal of Agricul-tural and Food Chemistry 47, 2033–2039.

Smietana, Z., Szpendowski, J. and Soral-Smietana, M. (1996) Modification of potatostarch by extrusion. Acta Academica Agricultura, Tech. Olstyn TechnologiaAlimentorum 29, 3–13.

Smiley, K., Hensley, D. and Gasdorf, H. (1976) Alpha-galactosidase production anduse in a hollow-fiber reactor. Applied Environmental Microbiology 31, 615–617.

Smith, A.M. (1988) Major differences in isoforms of starch-branching enzymebetween developing embryos of round- and wrinkled-seeded peas (Pisumsativum L.). Planta 175, 270–279.

Smith, A.M. and Denyer, K. (1992) Tansley Review No. 39. Starch synthesis indeveloping pea embryos. New Phytologist 122, 21–33.

Smith, A.M., Quinton-Tulloch, J. and Denyer, K. (1990) Characteristics of plastidsresponsible for starch synthesis in developing pea embryos. Planta 180,517–523.

Smith, M.K. and McComb, J.A. (1981) Effect of NaCl on the growth of whole plantsand their corresponding callus cultures. Australian Journal of Plant Physiology 8,267–275.

Smith, P.T., Kuo, T.M. and Crawford, C.G. (1991) Purification and characterizationof galactinol synthase from mature zucchini squash leaves. Plant Physiology 96,693–698.

Smythe, B.M. (1967) Sucrose crystal growth. Australian Journal of Chemistry 20,1097–1114.

Snedecor, G.W. and Cochran, W.G. (1973) Statistical Methods. Iowa State UniversityPress, Ames, Iowa.

Snoad, B. (1974) A preliminary assessment of ‘leafless peas’. Euphytica 23, 257–265.Somiari, R. and Balogh, E. (1995) Properties of an extracellular glycosidase of

Aspergillis niger suitable for removal of oligosaccharides from cowpea meal.Enzymatic and Microbial Technology 17, 311–316.

Song, H.S., Lim, S.M. and Widholm, J.M. (1994) Selection and regeneration ofsoybean resistant to the pathotoxic culture filtrates of Septoria glycines.Phytopathology 84, 948–951.

Sonnewald, U. (1992) Expression of E. coli pyrophosphatase in transgenic plantsalters photoassimilate partitioning. Plant Journal 2, 571–581.

Sonnewald, U., Hajiraezaei, M.R., Kossman, J., Heyer, A., Trethewey, R.N. andWillmitzer, L. (1997) Expression of a yeast invertase in the apoplast of potatotubers increases tuber size. Nature Biotechnology 15, 794–797.

Soral-Smietana, M. (1995) Faba bean monostarch phosphates. In: AEP (ed.) Proceed-ings of the 2nd AEP Conference. Improving Production and Utilisation of GrainLegumes. AEP. Paris, pp. 364–365.

Soral-Smietana, M. and Dziuba, Z. (1995) Changes in fraction structure offaba bean starch. In: AEP (ed.) Proceedings of the 2nd AEP conference. ImprovingProduction and Utilisation of Grain Legumes. Copenhagen. AEP, Paris, pp. 402–403.

Soral-Smietana, M., Fornal, J. and Wronkowska, M. (1998) Microstructure and func-tional properties of wheat and potato resistant starch preparates. Proceedings

300 References




of a Conference ‘Structure and Functionality of Food Products’. Polish Journalof Food Nutrition Science, 7, 131.

Sosulski, F., Waczkowski, W. and Hoover, R. (1989) Chemical and enzymaticmodifications of the pasting properties of legume starches. Starch/Stärke 41,135–140.

Sosulski, F.W., Elkowicz, L. and Reichert, R. (1982) Oligosaccharides in elevenlegumes and their air-classified protein and starch fractions. Journal of FoodSciences 47, 498–502.

Sosulski, F.W. and McCurdy, A.R. (1987) Functionality of flours, protein fractionsand isolates from field peas and faba bean. Journal of Food Science 52, 1010–1014.

Sosulski, R., Hoover, R., Tyler, R.T., Saskatoon Murray, E.D. and Winnipeg, B.D.A.(1985) Differential scanning calorimetry of air-classified starch and proteinfractions from eight legume species. Starch/Stärke 37, 257–262.

Sotomayor, C. (1997) Flours legumes obtained by technological procedures.Chemical and biological evaluation of starch. Doctoral Thesis, Madrid.

Souci, S.W., Fachman, W. and Krant, H. (1986) Food Composition and Nutrition Tables1986/1987. Wissenschaftliche Verlagsgellschaft GmbH, Stuttgart.

Southgate, D.A.T. (1991) Chapter 5: The measurement of unavailable carbo-hydrates: structural polysaccharides and dietary fibre. In: Determination of FoodCarbohydrates, 2nd edn. Elsevier Applied Science, London, p. 73.

Southgate, D.A.T. (1992) Starch and Dietary Fibre. Health Education Authority,London, p. 39.

Southgate, D.A.T. (1995a) The chemistry of dietary fibre. In: Belton, P.S. (ed.)Dietary Fibre Analysis. The Royal Society of Chemistry, Cambridge, pp. 14–28.

Southgate, D.A.T. (1995b) Chapter 3: Analytical strategies. In: Belton, P.S. (ed.)Dietary Fibre Analysis. The Royal Society of Chemistry, Cambridge, pp. 29–43.

Spaeth, S.C. and Sinclair, T.R. (1984) Soybean seed growth. I. Timing of growth ofindividual seeds. Agronomy Journal 76, 123–127.

Spiller, G.A. (1986) Handbook of Dietary Fibre in Human Nutrition. CRC Press, BocaRaton, Florida.

Stab, M.R. and Ebel, J. (1987) Effects of Ca2+ on phytoalexin induction by fungalelicitor in soybean cells. Archives of Biochemistry and Biophysics 257, 416–423.

Stahl, E. (1969) Thin Layer Chromatography. Springer-Verlag, Berlin.Stamp, J.A. (1987) Somatic embryogenesis in cassava: the anatomy and morphology

of the regeneration process. Annals of Botany 57, 451–459.Stark, D.M., Timmerman, K.P., Barry, G.I., Preiss, J. and Kishore, G.M. (1992) Regu-

lation of the amount of starch in tissues by ADP-glucose pyrophosphorylase.Science 258, 287–292.

Stark, D.M., Barry, G.F. and Kishore, G.M. (1996) Improvement of food qualitytraits through enhancement of starch biosynthesis. Annals of the New YorkAcademy of Science 792, 26–36.

Steadman, K.J., Pritchard, H.W. and Dey, P.M. (1996) Tissue-specific soluble sugarsin seeds as indicators of storage category. Annals of Botany 77, 667–674.

Steinhart, K.I., Jenkins, D.J.A., Mitchell, S., Cuff, D. and Prokipchuk, E.J. (1992)Effect of dietary fibre on total carbohydrate losses in ileostomy effluent.American Journal of Gastroenterology 87, 48–54.

Stejskal, J. and Griga, M. (1992) Somatic embryogenesis and plant regeneration inPisum sativum L. Biologia Plantarum 34, 15–22.

References 301




Stephens, P.A., Barwale-Sehr, U., Nickell, C.D. and Widholm, J.M. (1991) A cyto-plasmatically inherited, wrinkled-leaf mutant in soybean. Journal of Heredity 82,71–73.

Steup, M., Robeneck, H. and Melkonian, M. (1983) In-vitro degradation of starchgranules isolated from spinach chloroplasts. Planta 158, 428–436.

Stewart, C.N. Jr., Adang, M.J., All, J.N., Boerma, H.R., Cardineau, G., Tucker, D. andParrott, W.A. (1996) Genetic transformation, recovery, and characterization offertile soybean transgenic for a synthetic Bacillus thuringiensis crylAc gene. PlantPhysiology 112, 121–129.

Stikova, O., Sekavova, H., Mrhalkova, I. and Fronek, P. (1996) Food consumptionand prediction of future food demand (in Czech). Výzkumný Ústav ZemedelskéEkonomiky, Research Reviews 34, 1–40.

Stikova, O., Sekavova, H., Mrhalkova, I. and Baudisova, H. (1997) Food consump-tion and estimation of food demand (in Czech). Výzkumný Ústav ZemedelskéEkonomiky, Research Reviews 46, 1–60.

Stinard, P.S., Robertson, D.S. and Schnable, P.S. (1993) Genetic isolation, cloning,and analysis of a Mutator-dominant antimorph of the maize amylose extender1locus. Plant Cell 5, 1555–1566.

Stitt, M. (1996) Plasmodesmata play an essential role in sucrose export from leaves:a step towards an integration of metabolic biochemistry and cell biology. PlantCell 8, 565–571.

Stute, R. (1990a) Eigenschaften und Anwendungsmöglichkeiten von Erbsenstärken.Teil 1. Starch/Stärke 42, 178–184.

Stute, R. (1990b) Eigenschaften und Anwendungsmöglichkeiten von Erbsenstärken.Teil 2. Starch/Stärke 42, 207–212.

Sugimoto, H. and Buren, J.P. (1970) Removal of oligosaccharides from soy milk byan enzyme from Aspergillus saitoi. Journal of Food Science 35, 655–660.

Suhasini, K., Sagara, A.P. and Krishnamurthy, K.V. (1994) Direct somatic embryo-genesis from mature embryo axes in chickpea (Cicer arietinum L.). Plant Science103, 189–194.

Sun, W.Q. (1997) Glassy state and seed stability: the WLF kinetics of seed viabilityloss at T > Tg and the plasticization effect of water on storage stability. Annals ofBotany 79, 291–297.

Sun, W.Q. and Leopold, A.C. (1993) The glassy state and accelerated aging ofsoybeans. Physiologia Plantarum 89, 767–774.

Sun, W.Q. and Leopold, A.C. (1995) The Maillard reaction and oxidative stressduring aging of soybean seeds. Physiologia Plantarum 94, 94–104.

Sun, W.Q., Leopold, A.C., Crowe, L.M. and Crowe, J.H. (1996) Stability of dryliposomes in sugar glasses. Biophysical Journal 70, 1769–1776.

Sun, Z., Duke, S.H. and Henson, C.A. (1995) The role of pea chloroplastα-glucosidase in transitory starch degradation. Plant Physiology 108, 211–217.

Suzuki, H., Ozawa, Y. and Tanabe, O. (1966) Studies on the decomposition ofraffinose by α-galactosidase of Actinomycetes. Agricultural Biological Chemistry 30,1039–1046.

Švábová, L. and Griga, M. (1997) Utilization of some Fusarium filtrates in resistancebreeding programme of peas. Cereal Research Communications 25, 847–848.

Švábová, L. and Griga, M. (1998a) Fusarium spp. on peas – several in vitro techniquesused in resistance breeding. In: AEP (ed.) Opportunities for High Quality, Healthy

302 References




and Added-Value Crops to Meet European Demands. Proceedings 3rd EuropeanConference on Grain Legumes, Valladolid. AEP, Paris, pp. 235–236.

Švábová, L. and Griga, M. (1998b) In vitro cultures used for improvement of pearesistance to some fungal diseases. In: de Ron, M.A. (ed.) EUCARPIA Inter-national Symposium on Breeding of Protein and Oil Crops. BBG-CSIC, Pontevedra,Spain, pp. 81–82.

Švábová, L., Griga, M. and Ondrej, M. (1995) The application of in vitro cultures ofpeas for improvement of resistance to the filtrate of Fusarium semitectum. In:Abstracts of International Seminar Fusarium – Mycotoxins, Taxonomy and Pathogenic-ity. Martina Franca, Italy, pp. 121–122.

Švábová, L., Griga, M. and Ondrej, M. (1996) Effect of Fusarium semitectum culturefiltrate on the multiple shoot and callus cultures of pea. Biologia 51 (suppl. 4),27.

Švábová, L., Lebeda, A. and Griga, M. (1998) Comparison of in vitro testing methodsfor resistance screening of peas to Fusarium spp. Acta Phytopathology andEnthomology (Hungary) 33, 275–284.

Svanberg, S.J.M., Suortti, T. and Nyman, M.G.-L. (1997) Physicochemical changesin dietary fiber of green beans after repeated microwave treatments. Journal ofFood Science 62, 1006–1010.

Swain, R.R. and Dekker, E.E. (1966) Seed germination studies. II Pathways forstarch degradation in germinating pea seedlings. Biochemica Biophysica Acta 122,87–100.

Swinkels, J.J.M. (1985) Composition and properties of commercial native starches,chemical analysis of dietary fiber. Journal of the AOAC 77, 703–709.

Taha, R.S. and Francis, D. (1990) The relationships between polyploidy andorganogenetic potential in embryo- and root-derived tissue cultures of Viciafaba L. Plant Cell Tissue and Organ Culture 22, 229–236.

Takeda, Y. and Hizukuri, S. (1982) Location of phosphate groups in potatoamylopectin. Carbohydrate Research 102, 312–327.

Takeda, Y., Shirasaka, K. and Hizukuri, S. (1984) Examination of the purity andstructure of amylose by gel permeation chromatography. Carbohydrate Research132, 83–92.

Takeda, Y., Hizukuri, S. and Juliano, B.O. (1986) Purification and structure ofamylose from rice starch. Carbohydrate Research 148, 299–308.

Takeda, Y., Shitaozono, T. and Hizukuri, S. (1990) Structures of subfractions ofcorn amylose. Carbohydrate Research 199, 207–214.

Takeuchi, I., Tohbarn, M. and Sato, A. (1994) Polysaccharides in primary cell wallof rice cells in suspension culture. Phytochemistry 35, 361–363.

Tal, M. (1990) Somaclonal variation for salt resistance. In: Bajaj, Y.P.S. (ed.) Biotech-nology in Agriculture and Forestry, Vol. 11. Somaclonal Variation in Crop Improvement1. Springer-Verlag, Berlin, pp. 236–257.

Talbott, L.D. and Ray, P.M. (1992) Molecular size and separability features ofpea cell wall polysaccharides – implications for models of primary cell wallstructure. Plant Physiology 98, 357–368.

Talyer, R.T., Youngs, C.G. and Sosulski, F.W. (1981) Air classification of legumes.I. Separation efficiency, yield, and composition of the starch and proteinfractions. Cereal Chemistry 58, 144–148.

References 303




Tanner, W., Lehle, L. and Kandler, O. (1967) myo-Inositol, a cofactor in thebiosynthesis of verbascose. Biochemical and Biophysical Research Communications29, 166–171.

Taranathan, R.N. and Bhat, U.R. (1988) Scanning electron microscopy of chemi-cally and enzymatically treated black gram (Phaseolus mungo) and ragi (Elusinecoracana) starch granules. Starch 40, 378–382.

Täufel, K., Steinbach, K.J. and Vogel, E. (1960) Mono- und Oligosaccharide einigerLeguminosensamen sowie ihr Verhalten bei Lagerung und Keimung.Zeitschriften für Lebensmittel Untersuchungen Vorschungs 112, 31–40.

Tetu, T., Sangwan, R.S. and Sangwan-Norreel, B.S. (1990) Direct somatic embryo-genesis and organogenesis in cultured immature zygotic embryos of Pisumsativum L. Journal of Plant Physiology 137, 102–109.

Theander, O. (1995) Total dietary fiber determined as neutral sugar residues,uronic acid residues and klason lignin (The Uppsala method): collaborativestudy. Journal of Association of Official Analytical Chemists International 78,1030–1044.

Theander, O., Aman, P., Westerlund, E. and Graham, H. (1994) Enzymatic/chemical analysis of dietary fiber. Journal of Association of Official AnalyticalChemists International 77, 703–709.

Therman, E. and Murashige, T. (1984) Polytene chromosomes in cultured pearoots (Pisum, Fabaceae). Plant Systematics and Evolution 148, 25–34.

Thibault, J.F. (1979) Automatisation du dosage des substances pectiques par lamethode au metahydroxydiphenyl. Lebensmittel Wissenschaft und Technologie 12,247.

Thorne, J.H. (1979) Redistribution of assimilates from soybean pod walls duringseed development. Agronomy Journal 71, 812–816.

Thorne, M.J., Thompson, L.U. and Jenkins, D.J.A. (1983) Factors affecting starchdigestibility and the glycemic response special reference to legumes. AmericanJournal of Clinical Nutrition 38, 481–485.

Thynn, M., Wolff, A., Gorge, E. and Werner, D. (1989) Low concentrations ofphytoalexins correlate with resistance in regenerated plants from meristemcultures of Vicia faba L. Zeitschrift für Naturforsch 44c, 237–242.

Tipler, A. (1993) Gas chromatography instrumentation, operation, and experimen-tal considerations. In: Baugh, P.J. (ed.) Gas Chromatography: a Practical Approach.IRL Press, Oxford, pp. 15–70.

Tomomatsu, H. (1994) Health effects of oligosaccharides. Food Technology 48, 61–65.Torre, M., Rodriquez, A.R. and Saura-Calixto, F. (1991) Effect of dietary fibre and

phytic acid on mineral availability. Critical Reviews in Food Sciences and Nutrition1, 1–22.

Tovar, J. and Melito, C. (1996) Steam-cooking and dry heating produce resistantstarch in legumes. Journal of Agricultural Food Chemistry 44, 2642–2645.

Tovar, J., Björck, I.M. and Asp, N.G. (1990) Analytical and nutritional implicationsof limited enzymic availability of starch in cooked red kidney beans. Journal ofAgricultural Food Chemistry 38, 488–493.

Tovar, J., Björck, I.M. and Asp, N.G. (1992a) Incomplete digestion of legumestarches in rats: a study of precooked flours containing retrograded andphysically inaccessible starch fractions. Journal of Nutrition 122, 1500–1507.

304 References




Tovar, J., Granfeldt, Y. and Björck, I. (1992b) Effect of processing on blood glucoseand insulin responses to starch in legumes. Journal of Agricultural Food Chemistry40, 1846–1851.

Trethewey, R.N., Geigenberger, P., Riedel, K., Hajirezaei, M.R., Sonnewald, U.,Stitt, M., Riesmeier, J.W. and Willmitzer, L. (1998) Combined expression ofglucokinase and invertase in potato tubers leads to a dramatic reduction instarch accumulation and a stimulation of glycolysis. Plant Journal 15, 109–118.

Trick, H.N. and Finer, J.J. (1998) Sonication assisted Agrobacterium-mediatedtransformation of soybean [Glycine max (L.) Merrill] embryogenic suspensionculture tissue. Plant Cell Reports 17, 482–488.

Tricoli, D.M., Hein, M.B. and Carnes, M.G. (1986) Culture of soybean mesophyllprotoplasts in alginate beads. Plant Cell Reports 5, 334–337.

Trijatmiko, K.R. and Harjosudarmo, J. (1996) Plant regeneration of soybeanthrough somatic embryogenesis. Jurnal Bioteknologi Pertanian 1, 53–59.

Troszynska, A., Honke, J., Waszczuk, K. and Kozlowska, H. (1995) Oligosaccharidecontent in legume seeds and their changes during sterilization. In: AEP (ed.)Improving Production and Utilisation of Grain Legumes. Proceedings of the 2ndEuropean Conference on Grain Legumes. AEP, Paris, p. 288.

Trowell, H. (1972) Ischemic heart disease and dietary fibre. American Journal ofClinical Nutrition 25, 926–932.

Trowell, H. (1976) Definition of dietary fibre and hypotheses that it is a protectivefactor in certain diseases. American Journal of Clinical Nutrition 29, 417–427.

Trugo, L.C., Almeida, D.C.F. and Gross, R. (1988) Oligosaccharide contents in theseeds of cultivated lupins. Journal of the Science of Food and Agriculture 45, 21–24.

Trugo, L.C., Ramos, L.A., Trugo, N.M.F. and Souza, M.C.P. (1990) Oligosaccharidecomposition and trypsin inhibitor activity of P. vulgaris and the effect of germi-nation on the α-galactoside composition and fermentation in the humancolon. Food Chemistry 36, 53–61.

Tsukamoto, C., Kikuchi, A., Harada, K., Kitamura, K. and Okubo, K. (1993) Geneticand chemical polymorphisms of saponins in soybean seed. Phytochemistry 34,1351–1356.

Tuan, Y.-H. and Phillips, R.D. (1991) Effect of the hard-to-cook defect and process-ing on protein and starch digestibility of cowpeas. Cereal Chemistry 68, 413–418.

Turova, A.P. and Gladkych, A.S. (1964) Experimental and clinical pharmacology ofsaponins. Farmakologia and Toxikologia 27, 242–249.

Tyler, R.T. and Panchuk, B.D. (1982) Effect of seed moisture content on the airclassification of field peas and faba beans. Cereal Chemistry 59, 31–33.

Tyson, R.H. and ap Rees, T. (1988) Starch synthesis by isolated amyloplasts fromwheat endosperm. Planta 175, 33–38.

Uchiyama, T., Numata, M., Terada, S. and Hosino, T. (1993) Production andcomposition of extracellular polysaccharide from cell suspension cultures ofMentha. Plant Cell Tissue and Organ Culture 32, 153–159.

Ueno, Y., Hasegawa, A. and Tsachiya, T. (1973) Isolation of O-methyl-scyllo-inositolfrom mung bean seeds. Carbohydrate Research 29, 520–521.

Upadhyay, J.K. and Garcia, V.V. (1988) Effect of soaking and cooking on reductionof oligosaccharides of cowpea (Vigna unguiculata (L.) Walp.). Philippine Journalof Food Science and Technology 12, 21–28.

Urbano, G., Lopez-Jurado, M., Hernandez, J., Fernandez, M., Moreu, M.C., Frias, J.,Diaz-Pollan, C., Prodanov, M. and Vidal-Valverde, C. (1995) Nutritional

References 305




assessment of raw, heated, and germinated lentils. Journal of Agricultural FoodChemistry 43, 1871–1877.

Urooj, A. and Puttaray, S. (1994) Effect of processing on starch digestibility in somelegumes. An in vitro study. Die Nahrung 38 (1), 38–46.

Uzzan, A. (1988) Vegetable protein products from seeds: technology and uses in thefood industry. In: Hudson, B.J.F. (ed.) Developments in Food Proteins – 6. ElsevierApplied Science, London, p. 73.

Vahouny, G.V., Satchithanandam, S., Chen, I., Tepper, S.A., Kritchevsky, D.,Lightfoot, F.G. and Cassidy, M.M. (1988) Dietary fibre and intestinal adapta-tion: effects on lipids absorption and lymphatic transport in the rat. AmericanJournal of Clinical Nutrition 47, 201–206.

van der Poel, A.F.B., Aarts, H.L.M. and Stolp, W. (1989) Milling and air classifica-tion of two different types of peas – effect on the distribution of antinutritionfactors. Netherlands Journal of Agricultural Science 37, 273–278.

van der Poel, A.F.B., Gravendeel, S., van Kleef, D.J., Jansman, A.J.M. and Kemp, B.(1992) Tannin-containing faba bean (Vicia faba L.): effect of methods ofprocessing on ileal digestibility of protein and starch for growing pigs. AnimalFeed Sciences and Technology 36, 205–214.

Van Engelhardt, W., Roennau, K., Rechkemmer, K. and Sakata, T. (1989) Absorp-tion of short-chain fatty acids and their role in the hindgut of monogastricanimals. Animal Feed Science and Technology 23, 43–53.

Vanhoof, K. and De Schrijver, R. (1997) Consumption of enzyme resistant starchand cholesterol metabolism in normo- and hypercholesterolemic rats. NutritionResearch 17, 1331–1340.

Van Huystee, R.B. and Lobazzewski, J. (1982) An immunological study ofperoxidase released by cultured peanut cells. Plant Science 27, 59–67.

Van Huystee, R.B., Malko, M., Wan, K. and Guzen, M. (1994) Isolation ofperoxidase from bean and millet compared with that from peanut. CanadianJournal of Botany 72, 1432–1435.

Van Lonkhuijsen, H.J., Lichtendonk, W., Jetten, J. and Hammer, R.J. (1992)Non-starch polysaccharides of pulses composition and enzymatic breakdown.In: AEP (ed.) Proceedings of the 1st European Conference on Grain Legumes, Angers.AEP, Paris, pp. 405–406.

van Munster, I.P., Tangerman, A. and Nagengasr, F.M. (1994) Effect of resistantstarch on colonic fermentation, bile acid metabolism, and mucosal prolifera-tion. Digestive Diseases and Sciences 39, 834–842.

Van Soest, P.J. (1963) Use of detergents in the analysis of fibrous feeds. II. A rapidmethod for the determination of fiber and lignin. Journal of Association of OfficialAnalytical Chemists 46, 829–835.

Van Soest, P.J. (1978) Fibre analysis table. American Journal of Clinical Nutrition S31,284.

Van Soest, P.J. and Wine, R.H. (1967) Use of detergents in the analysis of fibrousfeeds. IV. Determination of plant cell wall constituents. Journal of Association ofOfficial Analytical Chemists 50, 50–55.

Van’t Hof, J. and McMillan, B. (1969) Cell population kinetics in callus tissues ofcultured pea root segments. American Journal of Botany 56, 42–51.

Vasanthan, T., Sosulski, F.W. and Hoover, R. (1995) The reactivity of nativeand autoclaved starches from different origins towards acetylation andcationisation starch. Starch/Stärke 47, 135–143.

306 References




Vasil, I.K. (1986) Relative stability of embryogenic cultures of Gramineae anduniformity of regenerated plants. In: Senal, J. (ed.) Somaclonal Variation andCrop Improvement. Martinus Nijhoff, Dordrecht, pp. 108–116.

Vazquez-Blanco, M.E., Lopez-Hernandez, J., Vazquez-Oderiz, M.L., Simal-Lozano, J.and Gonzalez-Castro, M.J. (1995) Determination of pectins by HPLC. AComparison of two methods applied to green beans (Phaseolus vulgaris L.).Journal of Chromatographic Science 33, 551–553.

Vecherko, L.P., Zinkevich, E.P. and Kogan, L.M. (1973) Structure of calendulosideF from Calendula officinalis roots. Chimija Prirodnych Soedinenij 561–562.

Veena, A., Urooj, A. and Puttaraj, S. (1995) Effect of processing on the compositionof dietary fiber and starch in some legumes. Die Nahrung 39, 132–138.

Veldman, A., Veen, W.A.G., Barug, D. and van Paridon, P.A., (1993) Effect ofα-galactosides and α-galactosidase in feed on ileal piglet digestive physiology.Journal of Animal Physiology and Animal Nutrition 69, 57–65.

Venkatachalam, P. and Jayabalan, N. (1997a) Effect of auxin and cytokinins onefficient plant regeneration and multiple-shoot formation from cotyledonsand cotyledonary node explants of groundnut (Arachis hypogeae L.) by in vitroculture technology. Applied Biochemistry and Biotechnology 67, 237–247.

Venkatachalam, P. and Jayabalan, N. (1997b) Selection of groundnut plants withenhanced resistance to late leaf spot through in vitro mutation technique. Inter-national Arachis Newsletter 17, 10.

Venkatachalam, P., Rao, K.S., Kishor, P.B.K. and Jayabalan, N. (1998) Regenerationof late leaf spot-resistant groundnut plants from Cercosporidium personatumculture filtrate-treated callus. Current Science (Bangalore) 74, 61–65.

Venketeswaran, S. (1963) Tissue culture studies on Vicia faba. II. Cytology. Caryologia16, 91–100.

Venketeswaran, S. and Spiess, E.B. (1963) Tissue culture studies on Vicia faba. III.Effect of growth factors on chromosome morphology. Cytologia 28, 201–212.

Venketeswaran, S. and Spiess, E.B. (1964) Tissue culture studies on Vicia faba. IV.Effect of growth factors on mitotic activity. Cytologia 29, 298–310.

Vernon, D.M., Tarczynski, M.C., Jensen, R.G. and Bohnert, H.J. (1993) Cyclitolproduction in transgenic tobacco. Plant Journal 4, 199–205.

Vertucci, C.W. (1990) Calorimetric studies of the state of water in seed tissues.Biophysical Journal 58, 1463–1471.

Vertucci, D.J. and Farrant, J.M. (1995) Acquisition and loss of desiccation tolerance.In: Kigel, J. and Galili, G. (eds.) Seed Development and Germination. MarcelDekker, New York, pp. 237–271.

Vicente, G. (1998) Antinutritional factors in pea. BSc Project, Agronomy Faculty,Madrid.

Vidal-Valverde, C. and Frias, J. (1991) Legume processing effects on dietary fibrecomponents. Journal Food Science 56, 1350–1352.

Vidal-Valverde, C. and Frias, J. (1992) Changes in carbohydrates during germina-tion of lentils. Zeitschrift für Lebensmittel Untersuchung und Forschung 194,461–464.

Vidal-Valverde, C., Frias, J. and Valverde, S. (1992a) Effect of processing on thesoluble carbohydrate content of lentils. Journal of Food Protection 55, 301–304.

Vidal-Valverde, C., Frias, J. and Esteban, R. (1992b) Dietary fibre in processedlentils. Journal Food Science 57, 1161–1163.

References 307




Vidal-Valverde, C., Frias, J. and Valverde, S. (1993a) Changes in the carbohydratecomposition of legumes after soaking and cooking. Journal of American DieteticAssociation 93, 547–550.

Vidal-Valverde, C., Frias, J., Prodanov, M., Tabera, J., Ruiz, R. and Bacon, J. (1993b)Effect of natural fermentation on carbohydrates, riboflavin and trypsininhibitor activity of lentils. Zeitschrift für Lebensmittel Untersuchung und Forschung197, 449–452.

Vidal-Valverde, C., Frias, J., Sotomayor, C., Diaz-Pollán, C., Fernandez, M. andUrbano, G. (1998) Nutrients and antinutritional factors in faba beans asaffected by processing. Zeitschrift für Lebensmittel Untersuchung und Forschung207, 140–145.

Vierling, E. (1991) The roles of heat shock proteins in plants. Annual Review of PlantPhysiology and Molecular Biology 42, 570–620.

Vijayakumari, K., Siddhuraju, P. and Jonardhanan, K. (1996) Effect of soaking,cooking and autoclaving on phytic acid and oligosaccharide contents of thetribal pulse, Mucuna monosperma DC. Ex. Wight. Food Chemistry 55, 173–177.

Villand, P., Aalen, R., Olsen, O.A., Luethi, E., Loennenberg, A. and Kleczkowski,L.A. (1992) PCR amplification and sequence of cDNA clones for the small andlarge subunit of ADPglucose pyrophosphorylase from barley tissues. PlantMolecular Biology 19, 381–389.

Villand, P., Olsen, O.A. and Kleczkowski, L.A. (1993) Molecular characterization ofmultiple cDNA clones for ADPglucose pyrophosphorylase from Arabidopsisthaliana. Plant Molecular Biology 23, 1279–1284.

Viola, R., Davies, H.V. and Chudeck, A.R. (1991) Pathways of starch and sucrosebiosynthesis in developing tubers of potato (Solanum tuberosum L.) and seeds offaba bean (Vicia faba L.): elucidation by carbon-13 NMR spectroscopy. Planta183, 202–208.

Visser, R.G.F. and Jacobsen, E. (1993) Towards modifying plants for altered starchcontent and composition. Trends in Biotechnology 11, 63–68.

Visser, R.G.F., Somhorst, I., Kuipers, G.J., Ruys, N.J., Feenstra, W.J. and Jacobsen, E.(1991) Inhibition of expression of the gene for granule-bound starch synthasein potato by antisense constructs. Molecular General Genetics 225, 289–296.

Vitaladasa, M.K. and Belavady, B. (1980) Unavailable carbohydrates of commonlyconsumed indian foods. Journal of Science Food Agriculture 31, 194–198.

von Arnold, S. and Eriksson, T. (1976) Factors influencing the growth and divisionof pea mesophyll protoplasts. Phisiologia Plantarum 36, 193–196.

von Arnold, S. and Eriksson, T. (1977) A revised medium for growth of peamesophyll protoplasts. Physiologia Plantarum 39, 257–260.

Vose, J.R. (1980) Production and functionality of starches and protein isolates fromlegume seeds (field peas and horsebeans). Cereal Chemistry 57, 406–410.

Vose, J.R., Basterrechechea, M.J., Gorin, P.A.J., Finlayson, A.J. and Youngs, C.G.(1976) Air classification of field peas and horsebean flours: chemical studies ofstarch and protein fractions. Cereal Chemistry 53, 928–936.

Wagner, J.R., Becker, R., Gumbmann, M.R. and Olson, A.C. (1976) Hydrogenproduction in the rat following ingestion of raffinose, stachyose andoligosaccharide-free bean residue. Journal of Nutrition 106, 466–470.

Wan, Y., Sorensen, E.L. and Liang, G.H. (1988) Genetic control of in vitro regenera-tion of alfalfa Medicago sativa L. Euphytica 39, 3–10.

308 References




Wang, G., Robertson, J., Parpia, B., Chen, J. and Campbell, T.C. (1991) Dietary fibrecomposition of selected foods in the People’s Republic China. Journal of FoodComposition and Analyses 4, 293–303.

Wang, H. and Holl, F.B. (1988) In vitro culture and the incidence of somaclonalvariation in regenerated plants of Trifolium pratense L. Plant Science 55, 159–167.

Wang, H., Cutler, A.J. and Fowke, L.C. (1989) High frequences of preprophasebands in soybean protoplast cultures. Journal of Cell Science 92, 575–580.

Wang, H.L., Patrick, J.W. and Offler, C.E. (1995) Cellular pathway of photosynthatetransfer in the developing wheat grain. III. A structural analysis and physiologi-cal studies of the pathway from the endosperm cavity to the starchy endosperm.Plant, Cell and Environment 18, 389–407.

Wang, T.L. and Hedley, C.L. (1991) Seed development in peas: knowing your three‘r’s’ (or four or five). Seed Science Research 1, 3–14.

Wang, T.L. and Hedley, C.L. (1993) Genetic and developmental analysis of theseed. In: Casey, R. and Davies, D.R. (eds) Peas, Genetics, Molecular Biology andBiotechnology. CAB International, Wallingford, pp. 83–120.

Wang, T.L., Cook, S.K., Francis, R.J., Ambrose, M.J. and Hedley, C.L. (1987a) Ananalysis of seed development in Pisum sativum. VI. Abscisic acid production.Journal of Experimental Botany 38, 1921–1932.

Wang, T.L., Smith, C.M., Cook, S.K., Ambrose, M.J. and Hedley, C.L. (1987b) Ananalysis of seed development in Pisum sativum, III. The relationship betweenthe r locus, the water content and the osmotic potential of seed tissues in vivoand in vitro. Annals of Botany 59, 73–80.

Wang, T.L., Hadavizideh, A., Harwood, A., Welham, T.J., Harwood, W.A., Faulks, R.and Hedley, C.L. (1990) An analysis of seed development in Pisum sativum L.XIII. The chemical induction of storage product mutants. Plant Breeding 105,311–320.

Wang, T.L., Bogracheva, T.Y. and Hedley, C.L. (1998) Starch: as simple as A, B, C?Journal of Experimental Biology 49, 481–502.

Wardlaw, I.F. (1990) The control of carbon partitioning in plants. Tansley reviewNo. 27. New Phytologist 116, 341–381.

Warkentin, T.D. and McHughen, A. (1993) Regeneration from lentil (Lensculinaris) cotyledonary nodes and potential of this explant for transformationby Agrobacterim tumifaciens. LENS-Newsletter (ICARDA), Lentil Experimental NewsService 20, 26–28.

Weber, H., Heim, U., Borisjuk, L. and Wobus, U. (1995a) Cell-type specific, coordi-nate expression of two ADP-glucose pyrophosphorylase genes in relation tostarch biosynthesis during seed development of Vicia faba L. Planta 195,352–361.

Weber, H., Borisjuk, L., Heim, U., Buchner, P. and Wobus, U. (1995b) Seed coat-associated invertases of faba bean control both unloading and storagefunctions, Cloning of cDNAs and cell type-specific expression. The Plant Cell 7,1835–1846.

Weber, H., Borisjuk, L. and Wobus, U. (1997) Sugar import and metabolism duringseed development. Trends in Plant Science 2, 169–174.

Weber, H., Heim, U., Golombek, S., Borisjuk, L. and Wobus, U. (1998) Metaboliccontrol of legume seed development: altering carbohydrate metabolism intransgenic Vicia narbonensis. In: AEP (ed.) Opportunities for High Quality, Healthy

References 309




and Added-Value Crops to Meet European Demands. Proceedings 3rd European Confer-ence on Grain Legumes, Valladolid. AEP, Paris, pp. 374–375.

Weightman, R.M., Renard, C.M.G.C. and Thibault, J.F. (1994) Structure andproperties of the polysaccharides from pea hulls. Part 1: Chemical extractionand fractionation of the polysaccharides. Carbohydrate Polymers 24, 139–148.

Wen, Q.B., Lorenz, K.J., Martin, D.J., Stewart, B.G. and Sampson, D.A. (1996)Carbohydrate digestibility and resistant starch of steamed bread. Starch/Stärke40, 180–185.

Wettlaufer, S.H. and Leopold, A.C. (1991) Relevance of Amadori and Maillardproducts to seed deterioration. Plant Physiology 97, 165–169.

Wheeler, P.G., Menzies, I.S. and Creamer, B. (1978) Effect of hypersmolar stimulicoeliac disease on the permeability of the human gastrointestinal tract. ClinicalScience and Molecular Medicine 54, 495–501.

Wiege, B., Bergthaller, W. and Lindhauer, M.G. (1995) Separation of starch,protein, fibre, and water soluble components from wrinkle pea flour in a pilotplant and processing of wrinkle pea starch by extrusion. Proceedings of 2ndEuropean Conference on Grain Legumes, Copenhagen. AEP, Paris, pp. 352–353.

Wiggins, H.S. (1984) Nutritional value of sugars and related compounds undigestedin the small intestine. Proceedings of the Nutrition Society 43, 69–75.

Wilkie, K.C.B. (1985) Chapter 1: New perspectives on non-cellulosic cell-wallpolysaccharides (hemicelluloses and pectic substances) of land plants. In:Brett, C.T. and Hillman, J.R. (eds) Biochemistry of Plant Cell Walls. CambridgeUniversity Press, Cambridge, London, pp. 1–37.

Williams, R.J. and Leopold, A.C. (1989) The glassy state in corn embryos. PlantPhysiology 89, 977–981.

Williamson, F.A., Fowke, L.C., Constable, F.C. and Gamborg, O.L. (1976) Labellingof concavaline-A sites on the plasma membrane of soybean protoplasts.Protoplasma 89, 305–308.

Williamson, J.D. and Quatrano, R.S. (1988) ABA-regulation of two classes ofembryo-specific sequences in mature wheat embryos. Plant Physiology 86,208–215.

Willison, J.H.M. (1985) Isolated protoplasts as laboratory tools in the study of cellwall deposition. In: Fowke, L.C. and Constabel, F. (eds) Plant Protoplasts. CRCPress, Boca Raton, Florida, pp. 77–89.

Willmot, D.B., Nickell, C.D., Widholm, J.M. and Gray, L.E. (1989) Evaluation of soy-bean resistance to Phialophora gregata culture filtrate in tissue culture. Theoreticaland Applied Genetics 77, 227–232.

Wink, M. (1984) Evidence for an extracellular lytic compartment of plant cellsuspension cultures: the cell culture medium. Naturwissenseshaften 17, 635–637.

Wink, M. (1994) The cell culture medium – a functional extracellular compartmentof suspension-cultured cell. Plant Cell Tissue and Organ Culture 38, 307–319.

Woitke, H.P., Kayser, J.P. and Hiller, L.M. (1970a) Fortschritte in der Erfotschungder Treiterpenesaponine. Pharmazie 25, 133–143.

Woitke, H.P., Kayser, J.P. and Hiller, L.M. (1970b) Fortschritte in der Erfotschungder Treiterpenesaponine. Pharmazie 25, 213–241.

Wolever, T.M.S. (1995) Dietary fibre and lipid metabolism in humans. In: Cherbut,C., Barry, J.L., Lairon, D. and Durand, M. (eds) Dietary Fibre: Mechanism of Actionin Human Physiology and Metabolism. John Libbey Eurotext, Paris, pp. 69–81.

310 References




Wolever, T.M.S. and Brand-Miller, J. (1995) Sugars and blood glucose control. Clini-cal Nutrition 62 (Suppl.), 212–221.

Wolever, T.M.S., Cohen, Z., Thompson, L.U., Thorne, M.J., Jenkins, M.J.A.,Prokipchuk, E.L. and Jenkins D.J.A. (1986) Ileal loss of available carbohydratein man: comparison of a breath hydrogen methods with direct measurementusing a human ileostomy model. American Journal of Gastroenterology 81,115–122.

Wrather, J.A. and Freytag, A.H. (1991) Selection of atrazine tolerant soybean calliand expression of that tolerance in regenerated plants. Plant Cell Reports 10,44–47.

Wright, D.J., Bumstead, M.R., Coxon, D.T., Ellis, H.S., DuPont, S. and Chan,H.W.-S. (1984) Air classification of pea flour – analytical studies. Journal ofScience of Food and Agriculture 35, 531–542.

Würsch, P., Del Vedovo, S. and Koellreutter, B. (1986) Cell-structure and starchnature as key determinations of the digestion rate of starch in legume. AmericanJournal of Clinical Nutrition 43, 25–29.

Xu, X.L., Li, X.H., Liu, W.H., Li, J.L. and Wang, Y. (1996) Study of transferring thesoybean mosaic virus coat protein (SMV-CP) gene into soyabeans using theRi-plasmid. Soybean Science 15, 279–288.

Xu, Z.H., Davey, M.R. and Cocking, E.C. (1982) Callus formation from root proto-plasts of Glycine max (soybean). Plant Science 24, 111–115.

Xu, Z. and Yang C. (1993) Influential factors of somatic embryogenesis of Vicia faba,Pisum sativum and Phaseolus vulgaris. Southwest China Journal of AgriculturalSciences 6, 42–47.

Yadav, S. and Khetarpaul, N. (1994) Indigenous legume fermentation: effect onsome antinutrients and in-vitro digestibility of starch and protein. Food Chemistry50, 403–406.

Yaklich, R.W. (1985) Effect of ageing on soluble oligosaccharide content in soybeanseeds. Crop Science 25, 701–704.

Yamaguchi, F., Ota, Y. and Hatanaka, C. (1996a) Extraction and purification ofpectic polysaccharides from soybean okara and enzymatic analysis of theirstructures. Carbohydrate Polymers 30, 265–273.

Yamaguchi, F., Kojima, H. and Muramelo, M. (1996b) Effects of hexameta-phosphate on soybean pectic polysaccharide extraction. Bioscience, Biotechnologyand Biochemistry 60, 2028–2031.

Yamaguchi, M., Kainuma, K. and French, D. (1979) Electron-microscopic observa-tions of maize starch. Journal of Ultrastructure Research 69, 249–261.

Yamane, Y. (1975) Chromosomal variation in calluses induced in Vicia faba andAllium cepa. Japanese Journal of Genetics 50, 353–355.

Yamasaki, Y. and Konno, H. (1989) α-Glucosidases of suspension-culturedsugar-beet cells. Phytochemistry 28, 2583–2585.

Yang, L.J., Barratt, D.H.P., Domoney, C., Hedley, C.L. and Wang, T.L. (1990) Ananalysis of seed development in Pisum sativum. X. Expression of storage proteingenes in cultured embryos. Journal of Experimental Botany 41, 283–288.

Yasui, T. (1980) Identification of a new galactosyl cyclitol from seeds of Vignaangularis Ohwi et Ohashi (Adzuki bean). Agricultural and Biological Chemistry 44,2253–2255.

References 311




Yasui, T., Tateishi, Y. and Ohashi, H. (1985) Distribution of low molecular weightcarbohydrates in the subgenus Ceratotropis of the genus Vigna (Leguminosae).Botanical Magazine Tokyo 98, 75–87.

Yasui, T., Endo, Y. and Ohashi, H. (1987) Infrageneric variation of the lowmolecular weight carbohydrate composition of the seeds of the genus Vicia(Leguminosae). Botanical Magazine Tokyo 100, 255–272.

Yazdi-Samadi, B., Rinne, R.W. and Seif, R.D. (1977) Components of developingsoybean seeds: oil, protein, sugars, starch, organic acids and amino acids.Agronomy Journal 69, 481–486.

Yellowless, D. (1980) Purification and characterization of limit dextrinase fromPisum sativum L. Carbohydrates Research 83, 109–118.

Yook, C., Sosulski, F. and Bhirud, P.R. (1994) Effects of cationization on functionalproperties of pea and corn starches. Starch 46, 393–399.

Young, D.H. and Kauss, H. (1983) Release of calcium from suspension-culturedGlycine max cells by chitosan, other polycations and polyamines in relation toeffects on membrane permiability. Plant Physiology 73, 698–702.

Young, D.H., Kohle, H. and Kauss, H. (1982) Effect of chitosan on membrane per-meability of suspension-cultured Glycine max and Phaseolus vulgaris cells. PlantPhysiology 70, 1449–1454.

Yue, S.X., Fu, J.H., Li, L.C., Yuan, H.L., Zhai, W.X., Chen, H., Su, W.Q. and Wang,Y.L. (1996) Atrazine-resistant gene introduced into Heilongjiang soyabeanvarieties and its expression and heredity. Scientia Agricultura Sinica 29, 78–83.

Yuste, P., Longstaff, M.A., McNab, J.M. and McCorquodale, C. (1991) The digest-ibility of semipurified starches from wheat, cassava, pea, bean and potato byadult cockerels and young chicks. Animal Feed Science and Technology 35,289–300.

Zagorska, N.A. (1995) Induced adrogenesis and somaclonal variation in economi-cally important species. DSc thesis, Bulgarian Academy of Sciences, Sofia.

Zagorska, N.A., Shamina, Z.B. and Butenko, R.G. (1974) The relationship ofmorphogenetic potency of tobacco tissue culture and its cytogenetic features.Biologia Plantarum 16, 262–274.

Zalewski, K. and Lahuta, L.B. (1998) The metabolism of ageing seeds. Changes inthe raffinose family oligosaccharides during storage of field bean (Vicia fabavar. minor Harz) seeds. Acta Societatis Botanicorum Poloniae 67, 193–196.

Zamora, F.A. and Fields, M.L. (1979) Nutritive quality of fermented cowpeas (Vignasinensis) and chickpeas (Cicer arietinum). Journal of Food Science 44, 234–236.

Zamski, E. (1995) Transport and accumulation of carbohydrates in developingseeds: The seed as a sink. In: Kigel, J. and Galli, G. (eds) Seed Development andGermination. Marcel Dekker, New York, pp. 25–44.

Zdunczyk, Z., Juskiewicz, J., Frejnagel, S., Wroblewska, M., Krefft, B. and Gulewicz,K. (1999) Influence of oligosaccharides from lupin seeds and fructooligo-saccharides on utilisation of protein by rats and absorption of nutrients in thesmall intestine. Proceedings of 9th International Lupin Conference, 20–24 June 1999.Klink Müritz, Germany.

Zdunczyk, Z., Juskiewicz, J., Frejnagel, S. and Gulewicz, K. (1998) Influence ofalkaloid and oligosaccharides from white lupin seeds on utilization of dietsby rats and absorption of nutrients in the small intestine. Journal of Animal andFeed Sciences 72, 143–154.

312 References




Zdunczyk, Z., Godycka, I. and Amarowicz, R. (1997) Chemical composition andcontent of antinutritional factors in Polish cultivars of peas. Plant Foods forHuman Nutrition 50, 37–45.

Zdunczyk, Z., Juskiewicz, J., Flis, M. and Frejnagel, S. (1996) The chemical composi-tion and nutritive value of low-alkaloid varieties of white lupin. 2. Oligosaccha-rides, phytates, fatty acids and biological value of protein. Journal of Animal andFeed Science 5, 73–82.

Zdunczyk, Z., Juskiewicz, J., Frejnagel, S., Flis, M. and Godycka, I. (1994) Chemicalcomposition of the cotyledons and seed coat and nutrition value of whole andhulled seed of yellow lupin. Journal of Animal and Feed Science 3, 141–148.

Zebrowska, T., Dlugolecka, Z., Pajak, J.J. and Korczynski, W. (1997) Rumendegradability of concentrate protein, amino acid and starch, and their digest-ibility in the small intestine of cows. Journal of Animal and Feed Sciences 6,451–470.

Zhang, G.F. and Staehelin, L.A. (1992) Functional compartmentalization of theGolgi apparatus of plant cells. An immunochemical analysis of high pressurefrozen and freeze substituted sycamore maple suspension cultured cells. PlantPhysiology 99, 1070–1083.

Zhang, Z.Y., Coyne, D.P. and Mitra, A. (1997) Factors affecting Agrobacteriummediated transformation of common bean. Journal of American Society forHorticultural Science 122, 300–305.

Zieg, R.G. and Outka, D.E. (1980) The isolation, culture and callus formation ofsoybean pod protoplasts. Plant Science 18, 105–114.

Ziegler, P. (1988) Partial purification and characterization of the majorendoamylase of mature pea leaves. Plant Physiology 86, 659–666.

Ziegler, P. (1995) Carbohydrate degradation during germination. In: Kigel J.and Galili, G. (eds) Seed Development and Germination. Marcel Dekker, New York,pp. 447–474.

Zimniak-Przybylska, Z. (1992) Characterization of catalytic properties of Pisum seedα-amylases. Genetica Polonica 33, 203–208.

Zobel, H.H. and Stephen, A.M. (1995) In: Stephen, A.M. (ed.) Food Polysaccharidesand their Applications. Marcel Dekker, New York, pp. 19–55.

Zrenner, R., Salanoubat, M., Willmitzer, L. and Sonnewald, U. (1995) Evidence ofthe crucial role of sucrose synthase for sink strength using transgenic potatoplants (Solanum tuberosum L.). Plant Journal 7, 97–107.

References 313




IndexIndexIndex

Index

α-amylase 31, 46–49, 139acid detergent fibre (ADF) 48–49, 57adzuki bean 19–20, 32α-D-galactosyl moiety 125α-galactosidase genes 199–201

thermostable α-galactosidase gene200

α-galactoside 17–19, 31, 63, 68, 71–73,80

consumption 69–70, 82–83animal diets 69–70food 82–83

content in animal diet 70content in legume dishes 83content in seeds 63–64, 67–68

beans 63chickpea 63–64faba bean 63–64, 68lentil 63–64, 68lupin 68pea 63, 68

losses during cooking 83nutritional properties 71–74

antinutritional effect 61elimination of negative effect

72–73gas production 78

ileal digestibility 72–73, 80intestinal transit 73monogastric nutrition 72–73nutrients absorption 73

see also unavailable carbohydratesα-galactoside biosynthesis gene(s)

199–201galactinol synthase (GS) gene

200stachyose synthase gene 200

α-D-glucose-6-phosphate 126α-glucosidase 139–140α-glucosidic bonds 140aglycon 28, 30agronomy 209–232

agronomic characters 228–231chemical characters 232technological characters 231

ajugose 17aldoses 16, 53amorphous 94, 96, 103–104, 106β-amylase 139amyloglucosidase 46amylopectin 23–24, 130amylose 23, 47–48, 130arabinose 26–27Arachis hypogaea 30

315

A3867: AMA: Hedley: First Proof:14-Nov-00 Index



available carbohydrates 79, 84–85classification 79nutritional properties 84–85

glycaemic index 84see also mono- and disaccharides,

starch

bean 8adzuki 19–20, 32broad 26, 122brown 26butter 30common 3, 123dry 20faba 3, 8, 17, 122, 130field 30garden 20green 29haricot 29jack 3kidney 21, 29–30lima 29, 123mung 2, 20, 29–30, 32, 123navy 29pinto 29runner 29

D-bornesitol 19breeding 209–232

contradiction of goals 210goals 209–211, 235pedigree 211physical selection 222selection methods 220–222

Brabender viscograph 100, 103broad bean 26, 123brown bean 26butter bean 30

Cajanus cajan 2, 20calculation and statistical analysis 37,

40, 43Canavalia ensiformis 3capillary zone electrophoresis (CZE) 42

advantages 43disadvantages 43recommended method 43

carbohydrates 5, 12, 22, 26, 28, 30–31,34, 37–43, 45, 49, 53–54, 56,120

accumulation 122biosynthesis 125–127chemistry 15extraction procedure 34GC determination 35physiological role 131–132,

134–138unloading of 118

cell wall 25–28, 47, 49–50, 52, 54preparation 50

cell wall components 127, 202biosynthesis 159–160, 202–203oligosaccharides 203–204

cellulose 25, 50, 52, 55content in seeds 65

chemical analysis 31chickpea 2, 8, 19–21, 30, 32, 122D-chiro-inositol 18–19, 23, 32, 133Cicer arietinum 2, 8, 20–21, 30, 32,

214ciceritol 20–22, 32, 126, 236

content in seeds 63–64chickpea 63–64lentil 63–64

common bean 3consumption of grain legumes 7–11cooking of legume starch 112–113

extrusion 113high pressure 112low pressure 112

cowpea 3, 19–20, 122, 123crude fibre 48crystallinity 94, 96–97, 100–101

biaxial crystalline polymers 94cyclitols 31–32, 127, 135–137, 143

occurrence of 32

debranching enzymes 140delignification 54, 57desiccation 131

injury 143stress 124, 132tolerance 131, 141

developmental stages 120

316 Index




dietary fibre (DF) 25, 46, 48differential scanning calorimetry DSC

98digalactosyl myo-inositol 133disaccharides 16, 45, 71, 124

see also sucrosedishes 62, 83, 86–87

amount of legumes 87consumer acceptance 87flatulence 62, 86intake of α-galactosides 83preventive effect 62

dry bean 20

embryocomposition 119role 119

endospermdegradation 119development, role 119

enzymic method 45–46, 49exoamylase 139extraction 31

recommended procedure 34solvents 31, 34temperature 31

faba bean 3, 8, 17, 20, 29, 122, 130fagopyritol B series 23fagopyritol B1 19–20, 23, 32fagopyritol B2 20, 23fagopyritol B3 23fibre 11, 25, 46, 48, 58, 65–67, 83,

85–86, 234, 237composition 69consumption in food 83content in seeds 65–67fibre fractions 65genetic variation 216physiological effect 85see also non-starch polysaccharides,

unavailable carbohydratesfield bean 30field pea 34food application of legume starches

109–110

free radicals 136fructose 16–17, 26–27, 31, 44

content in seeds 63–64faba bean 63–64lentil 63–64

see also monosaccharides 71fucose 26functional properties 98–101

gelatinization 98melting 98pasting 98

fungal disease(s) 228–230

galactinol 19–21, 32, 125–126synthase 125

galactinol series 21galactociceritol 20galacto-cyclitols 20, 31di-galacto-inositol 20galactomannans 26, 135galacto-ononitol 20, 32galactopinitol A 19–22, 32galactopinitol A series 22galactopinitol B 22galactopinitol B series 22galactopinitols 19galactose 19, 26–27, 31galactoside moieties 140galactosyl cyclitols 127galactosyl ononitol 126

galactosyl ononitol series 21galactosyl pinitol 126D-galacturonic acid 26–27, 56galacturonosyl residues 28garden bean 20gas chromatography GC 35, 57–58

advantages 37, 43disadvantages 37, 43recommended procedure 35

gelatinization 23–24germplasm banks 213

Pisum 213glucomannans 25, 54glucose 16–17, 24–27, 31, 44–45Glycine max 2, 20, 26, 30, 32, 214glycosidic bond/linkage 16, 23–24,

26–27, 57

Index 317

A3867: AMA: Hedley: First Proof:30-Oct-00 Index



granular structure of legume starches93–97

amylose 93–94, 97, 102–103,106–108, 110–111, 116

amylopectin 94, 97, 103, 107, 111double helix 94–95, 97, 103

A-type polymorph 95–97B-type polymorph 95–97C-type polymorph 95–97

green bean 29

haricot bean 29, 30hemicellulose 16, 25, 28, 50, 52, 54–55

content in seeds 65extraction 54

hexoses 16, 50high performance anion chromatography

(HPAC) 39Dionex system 39

high performance liquid chromatography(HPLC) 38, 57–58

advantages 41, 43affinity chromatography 40disadvantages 41, 43ion moderate partition 39normal phase 39, 56recommended method 40reverse phase 39

inositol 18invertase 16in vitro cultures 146–148

cell suspension culture 201–208isolation of native starch 89

dry processing 89–91wet processing 89–90

jack bean 3

ketoses 16, 53kidney bean 21, 29–30

lactose 16, 31, 43LEA proteins 132

legume seeds 17–20, 28–29Leguminosae 19, 36Lens culinaris 3, 8, 30, 32, 214lentil 3, 8, 17, 19–21, 29–30, 32, 122leucaenitol 19lignin 25, 28, 50, 54–55

content in seeds 65determination 57

lima bean 29, 121, 123lucerne 20, 21, 32, 135lupin 2, 8, 17, 19–21, 26Lupinus albus 2, 20, 26Lupinus angustifolius 2Lupinus luteus 2, 20, 45Lupinus mutabilis 2Lupinus spp. 8

Maillard’s reactions 136maltase 16maltose 16, 31mannans 25mannose 26–27, 54Medicago sativa 20, 32melezitose 31methyl cyclitols 32mid-infrared spectroscopy 225mimositol 32modified starch 101–109

biotechnological 108–109fermentation 109, 114–115germination 108, 114hydrolysis 108

chemical 104–107acetylation 104cationization 107cross-linking 106hydroxipropylation 106phosphorylation 105

physical 102–104annealing 103extrusion 103gamma irradiation 103steaming 102

monosaccharides 16, 31, 34, 57, 71free monosaccharides 124fructose 63–64, 120, 124, 136, 143galactose 124, 136glucose 124, 136, 139–140, 143

318 Index




muco-inositol 18mung bean 2, 20, 29–30, 32, 123myo-inositol 18, 20–21, 31–32, 36, 125,

128D-myo-inositol-1-phosphate 126

Nastar R 116Nastar R Instant 116native starch 102, 104, 106, 110, 116navy bean 29near-infrared (NI) spectroscopy 224neutral detergent fibre NDF 48non-food use 98non-starch polysaccharides 69, 76–78

composition 69, 77content in seeds 69–70, 76nutritional properties 76–78

digestibility of energy 78eliminate of negative effect

76energetic effect 76gas production 78ileal digestibility 76–77intestinal transit 77nutrient absorption 77–78

see also fibre, unavailablecarbohydrates

oligomers 126oligosaccharides 16, 25, 45, 71, 124

accumulation 121degradation 140α-galactosides 63, 68, 71–73, 80rathinose 120, 123, 125, 132, 135,

137, 140, 143RFO 125–126, 132–134, 136, 140stachyose 120, 123, 125, 132, 135,

137, 140, 143verbascase 120, 123, 132, 137, 140

ononitol 126D-ononitol 19–21, 32optical rotation 44–45

pea 3, 8, 20, 26, 29–30, 122, 123breeding programmes 210, 211hulls 29

mutant 17production 209

peanut 30, 123pectic acid 27pectic substances 26–27, 55

occurrence of 29pectin 16, 25–26, 28, 50, 52, 55pectinic acid 27pentoses 16, 50Phaseolus aureus 30Phaseolus coccineus 30Phaseolus lunatus 30Phaseolus vulgaris 3, 6, 20, 26, 30, 214phenyl α-D-glucoside 31photoassimilates, supply of 119pigeon pea 2, 19–20, 122D-pinitol 19–20, 22, 31–32, 126pinto bean 29Pisum sativum 3, 8, 20, 22, 26, 30,

214plant genetic transformation, methods

181–195Agrobacterium-mediated

transformation 183electroporation 184microinjection 184particle bombardment 183–184

plant regeneration 148–156organogenesis 151–153pea regeneration 149somatic embryogenesis 150–151

polymerization, degree of 103, 108polysaccharides 15–16, 22, 27, 45

cellulosic 25non-cellulosic 25

protopectin 27protoplasts culture 156–158

raffinose 16–19, 31–32, 36, 40–41,63–65, 68, 71

antinutritional effect 71content in seeds 63–64, 68

beans 63chickpea 63–64faba bean 63, 65, 68lentil 63–64, 68lupin 68pea 63, 68

Index 319




raffinose continuedsynthase 125see also α-galactoside 63, 68, 71–73,

80raffinose family of oligosaccharides

(RFO) 11, 16–18, 31, 33,40–42, 125, 233

rapid visco-analyser (RVA) 100reducing sugar method 44resistant starch (RS) 46, 80–83,

110–111, 235Berry method 47consumption in food 83content in legume products 81–82definition 80determination 46method of determination 81nutritional properties 80retrograded starch (RS III)

110–111see also fibre, unavailable

carbohydratesrhamnopyranose 28rhamnose 26round pea 36rug3 17, 234runner bean 29

saccharides 16, 42sample clean-up 38sample preparation 31sapogenin 28sapogenol 58saponins 28, 58

determination 58extraction from seeds 58genetic variation 219

scyllo-inositol 18, 32seed

coat 34, 131colour 210components 117desiccation tolerance 131–132,

135–136, 143development 119–122, 225–227full maturity 121, 130germination 142orthodox storage 137

quality 211recalcitrant 138stage of development 120size 210storage 137structure 117viability 137vigour 137yield 210zygotic 131

seedsdesiccation tolerance 131mature 140

sequoyitol 19, 32, 128soaking 112soluble carbohydrates 11, 15–16, 31,

235accumulation 122biosynthesis 125–127extraction from seeds 31physiological role 131–132,

134–138soluble sugars 63–64, 67–68

content in seeds 63–64, 67–68beans 63chickpea 63–64faba bean 63–64, 68lentil 63–64, 68lupin 68pea 63, 68

genetic variation 216, 219see also oligosaccharides,

α-galactosidesomaclonal variation 162–181

biochemical changes 173cytological instability 165, 170,

172–173product quality changes 181stress resistance 170–172, 180yield characters 177–179

somatic embryos 135soybean 2, 19–20, 26, 29–30, 32, 120,

122, 123spectrophometric method 45stachyose 16–19, 31, 33, 36, 40–41,

63–65, 68content in seeds 63–64, 68

beans 63chickpea 63–64

320 Index




faba bean 63, 65, 68lentil 63–64, 68lupin 68pea 63, 68

synthase 125see also oligosaccharides,

α-galactosidestandards 31starch 11, 16, 22, 63–65, 74–76, 80–83,

234, 237accumulation 120, 128, 130animal nutrition 74–76biochemistry 130calcium chloride method 46consumption in food 82–83content in seeds 63–65, 68, 121

beans 63chickpea 63–64faba bean 63–65, 68lentil 63–64, 68lupin 68pea 63–64, 68

degradation 138–139determination 45feed processing 75–76genetic variation 216–219glucose hydrolysis method 46granules 23, 90, 92–96, 98,

101–104, 106, 108, 111,115, 130

size of 90nutritional classification 80

rapidly digestible 80resistant starch 80–81, 235slowly digestible 80

nutritional properties 74–76,80–81

energetic value 76ileal digestibility 74–76, 81

rapidly digestible (RDS) 47retrograded 47role 128slowly digestible (SDS) 47see also available carbohydrates

starch biosynthesis genes 195–199ADP-glucose pyrophosphorylase

gene(s) 195genes influencing starch 198–199

invertase gene 198–199

pyrophosphatase gene 199starch phosphorylation gene

198sucrose synthase gene

198–199starch synthase gene(s) 196–197starch branching enzyme gene(s)

197–198starch ethers 104stress proteins 132substitution 104, 106–107sucrose 11, 16–19, 31, 33, 36, 41,

63–64, 67–68, 118content in seeds 63–64, 67–68

beans 63chickpea 63faba bean 63, 68lentil 63, 68lupin 68pea 63, 68

see also disaccharidesugar 16, 44, 56–57sweet lupin 2Swelite R 116swelling properties of 92, 100–103,

105–106, 109–111

temperature stress 136test kits

glucose 44medical/food 44Megazyme 47

testa 34role 117–118

thin layer chromatography (TLC) 42,58

advantages 42recommended method 42

transgenic grain legume plants, fieldtrials 192–195

transgenic traits 184–195herbicide tolerance 184–195insect resistance 189–190nutritional quality 191–192virus resistance 190–191

tri-galactopinitol 20, 22tri-galactopinitol A 22, 135tri-galactopinitol B 22

Index 321




trimethylsilylation TMS 35–36, 59triterpene glycosides 30

unavailable carbohydrates 79–80,85–86

classification 79–80nutritional properties 85–86

cholesterol metabolism 85glycaemic index 84

see also α-galactoside, fibreuronic acid 25–26, 50

verbascose 16–18, 31, 36, 40, 63–64, 68content in seeds 63–64, 68

beans 63chickpea 63–64faba bean 63, 68lentil 63–64, 68lupin 68pea 63, 68

see also α-galactosideVicia faba 3, 8, 20, 26, 30, 45, 119, 120,

214

Vigna angularis 20, 32Vigna radiata 2, 20, 32Vigna unguiculata 3, 20viscosity 97–98, 100–104, 106, 109–110,

116

water-insoluble cell wall components78

content in seeds 78nutritional properties 78

digestibility of energy 78gas production 78

see also fibre, unavailablecarbohydrates

white lupin 2

xylans 25–26xyloglucans 25–26xylose 25–27

yellow lupin 2, 122, 132–134

322 Index




carbohydrates in grain legume seeds improving nutritional quality and

Documents