in vitro regeneration and protein changes associated with

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LSU Historical Dissertations and Theses Graduate School

1987

In Vitro Regeneration and Protein ChangesAssociated With Two Cultivars of Agrostis PalustrisHuds. Cultured Under High Sodium-ChlorideConditions.John Casper HovanesianLouisiana State University and Agricultural & Mechanical College

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Recommended CitationHovanesian, John Casper, "In Vitro Regeneration and Protein Changes Associated With Two Cultivars of Agrostis Palustris Huds.Cultured Under High Sodium-Chloride Conditions." (1987). LSU Historical Dissertations and Theses. 4361.https://digitalcommons.lsu.edu/gradschool_disstheses/4361

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International

IN VITRO R E GE NE RA TI ON AND P R O TEIN CHANGES A S S O C I A T E D WITH TWO CULT I VARS OF A q r o s t is p a 1 u s t r is H u d s .

CULTURED UNDER HIGH SO D I U M CHLORIDE CONDITIONS

A D 1 ssertation

Submitted to the Graduate Faculty of the Louisiana State U n i v ersity and

Agricultural and Mechanical College in partial fulfilIment of the

req u i r e m e n t s for the degree of Doctor of Philosophy

1 n

the Department of Hor t i c u l t u r e

by

John Casper H o v a n e s i a nBA, Univer s i t y of Connecticut, 1972BA, New E n g l a n d University, 1979

MS, University of S o u t h e r n Mississippi, 198 1May, 1987

ACKN O W L E D G M E N T S

I wish to thank the members of my committee, Drs. A l

bert C. Purvis, chairman, W i lliam J. Blackmon, James F. F o n

tenot, Ear 1 P. Barrios Jr., Donald W. Newsom, David H.

Picha, and Kenneth C. Torres for g u i d a n c e throughout the

various stages of this research. My a p p r e ciation is e x

tended to Ms. Rhonda Parche~ S o r b e t w h ose efficiency and

warmth has made the laboratory we spent so much time in a

pleasure, to Mr. Selim Cetiner with whom I participated in

many valuable academic and extra-curricular activities, and

to Mr. Paul C. St. Amand and Dr. Paul W. Wilson, who se wi 1 1 -

ingness to share their computer knowledge saved so many

hours. I would like to e x p ress my g r atitude to the D e p a r t

ment of H o r t i cu lt u re at Louisiana State University for

being a place which e n c o u r a g es close professional and p e r

sonal relationships. A special note of thanks to my very

good friend, James R. Ault, for thinking the way I do on

many i s s u e s .

My most heartfelt a p p r e c i a ti on goes to Dr. William M.

Randle, whose high professional and moral standards will

i i

always serve as g u i d elines in my career and in my personal

life. Without the many hours we spent preparing for the

next day's work, the q u a l i t y of life in Baton Rouge would

not have been the same.

There is always a person in one's life who provides the

warmth and lave that makes one feel special. It is to Ms.

Nancy Gordon Barker that I owe this debt. She has had a

profound and flattering influence in reshaping many of the

ways I now think and behave. Finally* to my mother,

Luciene* and to my father, A r s h a g ( who have always been

there with love and as s i s t a n c e throughout my life.

1 1 1

Table of Contents

Page

A c k n o w l e d g e m e n t s .................................................... i i

List of T a b l e s ....................................................... v

List of F i g u r e s ......................................................vi

Abs t r ac t v 1 i 1

I n t r o d u c t i o n ........................................................... 1

Chapter'

L i t e r a tu re R e v i e w ........................................ A

L i t e r a t u r e C i t e d ........................................ 17

I In Vitro Se l e c t i o n for Sodium ChlorideResistance* Regeneration, and Solution Culture E v a l u a t i o n of Two Cultivars of flqro s t 1 s palustr is H u d s ............................... 33

Abs tr ac t ..............................................33I n t r oduc t i o n ........................................ 35M aterials and M e t h o d s ............................ 36Resu I t s ............................................... A3D i s c u s s i o n ...........................................A 7L iterature C i t e d ................................... 51

II Syn t h e s i s of Salt Shock Proteins in CallusCultures of A q r o s 1 1 s p a l u s tris Huds. As a R e s p o n s e to So d i u m Chloride S t r e s s ................ 77

Abstrac t ..............................................77I n t r o d u c t i o n ........................................ 79M a t erials and M e t h o d s ............................ BOResu I t s ...............................................BAD i sc uss i o n ........................................... B5Literature C i t e d ................................... B6

Append l ............................................................... ^3

V i t a ................................................................... 10^

1 v

LIST DF TABLES

PageTab 1 e

Chapter 1 . In vitro selection -for sodium chloride resistance* regeneration, and solution c u lture e v a l u a t i o n s of two cul t i v a r s of A q r o s t 1 s palustr is Huds.

1 E f fects of 51A mM NaCl and generation upont r ansfer rable oalli of A_;_ palustr isPen n e a g l e ( ' P E ’ ) and Seaside ( 'S S ’ ) .......... .55

2 Effects of 51A mM NaCl on the p ro du c t i o n ofshoots and roots from callus cultures ofA . pal us t rls Pen n e a g l e < 'P E ' ) and Seaside( ' S S ’ ) on callus induc tion medlurn d u r i nydark i n c u b a t i o n ..........................................56

3 The effects of 51A mM NaCl on the percentageof cultures of A_ p a l u s t r i s Penneaq 1 e ( * PE ’ ) and Seaside ( ' S S ’) which had visiblyi nc r e a s e d ................................................... 57

A The effects of 51A mM NaCl an the perrentageof cultures of A_ palustr is P e n neagle <'P E ’ > and S e a side ( ' S S ’ ) with varying degrees o f nec r os i s ................................................ 5B

5 The effect of 51A mM NaCl on the productionof shoots and roots from callus c u l t u r e s of A . palustris P e nn ea gl e ( ' P E ’ > and Seaside( ' SS ’ ) on r eg en e r a t i o n m e d i u m ........................ 59

6 The effects of 51A mM NaCl on 1y o p h i 1 i zedweight and c o n c e n t r a t i o n of selected and nonselected callus cultures of A_. pa 1 u s t n s P en n e a g l e ( ' P E ’ ) and Seaside < 'S S ’ )................60

V

LIST OF FIGURES

F iqure

1

a

3

6

5

Page

Chapter 1. In vitro s e 1ec tlo n for sadlum c hloride resistance, regeneration, and s olution culture e v a l u a t i o n s of two cultivars of ftqros t i s p alustris Huds.

Visual rating chart for determining percent necrosis of pal us t r is' P e n n e a g l e ’ and ' S e a s i d e ’ calli cultured at 0 mM and 516 mM NaCl. From left to right: 0*/. ( + >, 1-35 (++), E 6 - 50 V. (+ + +),and greater than 51V. (+ + ++ )........................... 63

Respanse of A p al u s t r i s 'P e n n e a g 1e ’ and ’S e a s i d e ’ plants germin a t e d from caryopses ( ED > . pi an ts r egener a ted from calli cultured on O mM NaCl supplemented medium ( <^> ), and plants regenerated from c a l 1i selected from 516 mM NaCl supplemented medium c X ) , grown h y d r o p o n i c a l l y in 0 mM NaCl in H o a g l a n d ’s #3 nutrient m e d i u m ........................ 66

R esponse of A . p a l u s t r i s , ’P e n n e a q l e ’ and ’S e a s i d e ’ plants germinated from caryopses, cultured h yd ro po n i c a l l y in H o a g l a n d ’s HE nutrient med i u m at 0 mM NaCl ( ED > and at 516 mM NaCl ( )......................................... 66

R esponse of A . p a 1 us t r l s ■ ' P e n n e a q l e ’ and ' S e a s i d e ’ plants regenerated from calli cultured on O mM NaCl supplemented medium, grown h y d r o p o m c a l ly in H o a g l a n d ’s #3 nutrient medium at O mM ( ED 1 and at 516 mM ( \^> >N a C l ................. 6B

Response of A_;_ p a lust r i s , ' P e n n e a g l e ’ and ‘S e a s i d e ’ plants regen e r a t e d from calli selected from 516 mM NaCl supplemented medium, grown h y d r o p o n i c a l l y l n _ H o a g 1 a n d ’s #E nutrient me d i u m at 0 mM NaCl ( ED and and 516 mM NaCl ( )................................... VC)

Callus cultures from A . palustris ’P e n n e a g l e ’(t o p ) and 'Seas i d e ’ (bottom) cultured on a medium supplemented with 516 mM NaCl. Lighter areas are portions of cultures wher e certain cells are actively p r o l i f e r a t i n g .................... 73

v 1

P a g eF 1 qur e

7 R es p o n s e of A_^ pal us t r i s . ' Penneaq 1 e ' (topleft), and ' S e a s i d e ' (top right) plants re g enerated from N a C l - adapted c a l 1i , cultured h y dr op o n i c a l l y in H o a g l a n d ’s #2 nutrient medium at O mM NaCl- On the bottom are ft. palustr is plants regenerated from N a C l — adapted calli in 257 mM N a C l ............ 7ft

0 ftqrost t s palustr is, ' P e n n e a g l e ’ and' S e a s i d e ’ pla n t l e t s regenerated on medium supplemented with 51ft mM N a C l - Note succulence, dark coloration, and stunted morphology of many plants as a typical symptom of severe salt s t r e s s ................................... 7<b

Chapter 2. S y n thesis of salt shock proteins in callus cultures of ftqrostis p a l u s tris Huds. as a response to sodium chloride stress

1 One dimensional SDS p o 1y a c r y 1 amide gel slab depicting p r otein banding patterns of control and NaCl-adapted calli of ft_ p alustris ' P e n n e a g l e ’ and ' S e a s i d e ’ over threeg e n e r a t i o n s ...................................................93

v l l

Abstract: In vitro r e g e n e r a t i o n and p r o tein changes

associated with two cultivars of

Agrost i s palustr is Huds. cultured

under high sodium chloride conditions

Calli from ' P e n n e a g l e ’ and ' S e a s i d e ’ creeping bentgrass

(Aqr os t 1 s pa 1 us t r i s Huds. ) were selected for enhanced

ability to p r o l i f e ra te on media supplemented with 51A mM

NaCl. Based on dry matter produc t i o n and vigor, growth of

plants r e g enerated from NaC l - a d a p t e d calli of both cultivars

in saline hydroponic nutrient medium, was superior to that

of pl a n t s regenerated from non-adapted c a l 1i . At P57 mM

NaCl, plants from N a C l - s en si ti ve calli died or were unable

to p r o duce measur a b l e dry matter whereas plants from N a C 1 -

adapted calli grew well. Growth of plants from NaCl-adapted

calli was inhibited at 3A0 mM NaCl. At 51A mM NaCl, r e s i s

tant plants no longer produced measur a b l e dry matter, a l

though they remained alive.

The extra c t a b l e soluble protein content of NaC 1 -

adapted calli was less than that of n o n -adapted cultures.

E 1ectrophoretic banding patterns of soluble proteins from

N a C l - a da pt e d calli were compared with those from non-adapted

calli using one dimensional p o 1y a c r y 1 amlde gel e l e c t r o

phoresis. One particular band at 31.5fcd was unique to the

v i i i

N a C l - a da p t ed calli. The intensity of some bands increased!

while others decreased with increasing levels of N a C l -

adaptation. One g e ne ra ti on (30 days of dark incubation)

after being transferred to a medium without NaCl, p o l y p e p

tide banding patterns of NaCl-adapted calli were similar to

that of n o n-adapted calli.

INTRODUCTION

It has been predi c ted that there will soon be more

demand for food nearly e v e r y w h e r e in the world (20, 72, 73,

101). A c c ordlng to Tal (101), h o w e v e r , food production is

actually declining. The scarcity of arable land, the limits

of mod e r n technology, the high cost of energy, and decreases

in the funding of agricultural research have all contributed

to this downward trend ( EO » 72, 73, 101>. The opening of

marginally arable land to alleviate some anticipated food

shortages has, therefore, encouraged plant breeders to

produce cul t i v a r s which can tolerate environmental extremes

(20, 72, 73, 10 1).

Sodium chloride inhibition of plant growth is one of the

oldest and most common agricultural problems in the world

(16, 26). Nearly every irrigated region on earth, to some

extent, is affected by high salt a c c u m ulation (26, 29, 90,

9 9, 72, 73). Each year, many ac res of valuable farml and 1 n

the United States are taken out of production or will suffer

severe crop damage b e c ause of high soil salinity or sodicity

( 79 , B2 ) . Sod ium toxicity has bee o me a p r o blem on 50*/. o f

the irrigated land in the western United States, actually

r e stricting crop p r o d u c ti on on 25*/. of this land (20, 72, 73,

75, 85). Moreover, b e cause of excess salinity, on

1

2

millions of hectares of arable land throughout the world, it

will soon become difficult to produce food crops e c o n o m i

cally (01, 82). Many attempts have been made to alleviate

soil and crop losses from high salinity; however, little has

been accomplished. In mast cases, the d e s a 1 i n 1 zat 1 on of

irrigation water or the r e clamation of saline or sodic soils

is impractical and cost p r o h i b i t i v e (EB, 1O 1). The opening

of a g r i c u 1tura 1 1y marginal lands to p r o d u c t i o n is an equally

ex p e n s i v e and resource intensive program which often leads

to u n a c c e p t a b l e losses of valuable native habitat. One e x

ample of this is the d e s t r u c t i o n of much of the Costa Rican

tropical rainforest habitat.

Many researchers believe that tissue culture tech

niques can be used along with conventional breeding methods

for developing salt tolerant crops ( E 0 t 85, 100).

D e m o n s t r a b l e NaCl tolerance has been reported in tissue c u l

tured cell lines of many cultured plant species (IE, El, 83,

2*+, 2b, 3b, bE, 71, 7*+, 85, BO, 100, 106). In addition to

salinity resistance, other stress tolerant cell cultures

have been obtained (1*4, IB, 20, 21, 25, 39, 99, 102). In

order for an agricultural breeding program to be successful,

however, plants must be produced which have commercial or

aesthetic applications. Breeding programs which utilize

cell c u l ture techniques must strive for these goals.

Although r e ge n e r a t i o n from N a C 1-resistant cell lines of

many plant species has been achieved <20, 5A , 68* 72, 73,

75 * 76 * B A , B A , B 5 , 86, 1O O , 103), permanent heritable NaCl

resistance in plants regenerated from tissue cultured cell

lines has not been reported (72,73,75). Nevertheless, r e

searchers are optimistic that with continued efforts, they

will be able to produce cultivars which transmit increased

salt tolerance to their offspring (20, A3, 70, 72, 73, BA,

B 5 , 100). In the following review of the literature, the

effects of salinity stress on plants and some contributions

of tissue and cell culture to recent studies of the

genetics and physio l o g y of plant adaptation to environmental

REVIEW OF THE LITERATURE

□ne of the unifying concepts of environmental stress is

cel lular dehyd ration ( 93 > . Many of the effects of salinity

stress are manifested similarly to the effects caused by

drought stress <97). Pl a n t s suffering from cellular d e h y d r a

tion are wilted, stunted, and in extreme cases, necrotic

(97). Few plants are obligate halophytes, or can tolerate

extremely high salt c o n c e n t r a t i o n s <10). According to

Bernstein (10), even salt resistant plants lining in e n

vironments with a soil conductivity greater than 9 mmho/cm ‘

in the soil solution extract, will be affected by high

salinity.

The major ions cont r i b u t i n g to soil salinity are Cl •

SO.,'' , H C O V , N a ‘ . Ca;‘' , and Mg1" <10, 109). Of all the

ions r e sponsible for salinity, Na' and Cl cause the most

severe crop damage (16, 36, 93, 75). Sodium chloride

toxicity alone affects over one-third of the irrigated land

w or ld - w i d e (16, 69, 73, 73).

High soil salinity results in soil solutions with low

water potentials. Few plants can maintain a negative o s

motic gradient against an external medium with a low water

potential (31, 39, 83). Many plants are able to avoid

9

5

injury or death by e q u i l i b r a t i n g their internal water

p o t e n t i a ls to that of their environmen t . Many salt s e n s i

tive p la n t s are able to m a i n t a i n favorable water activity

for turgor pressure and cell e l o n g a t i o n by taking u p « or

synt h e s i z i n g o s m otically active solutes from the external

medium ( 10, 31 , 3*+ , 37, 97, 99, 95, 96, 97) . Sodium and

p o ta ss iu m are the main c a tions r e sponsible for o s m o r e g u l a

tion, but at high concentrations, they can strongly inhibit

en z ym e and other metabolic activities <32, 33, 3 B , 79, 96,

97). When radical osmotic adjustments are required, plant

metabolic processes are subject to stress.

The effects of salt on plant growth, development, and

p r o d u c ti vi ty have been e x tensively studied (10, 31, 32, 37,

9B, 99, 69, 81, B 2 , 89, 93, 101). Damages due to excess

soil and water salinity still remain a serious concern to

agriculturists. Excess salinity profoundly affects the

biochemistry, anatomy, and morphology of plants. Sufficient

information to cor r e l a t e s a l t — induced alterations to

specific metabolic events, however, is present 1y u n a v a i l

able (01). Accor d i ng to St rogonov < 98) and more recently,

W a l s e 1 <109), tnese c h a nges i n c 1u d e :

a. increased succulence;

b . changes in number and size of stomata;

c. thickening of the cuticle;

6

d . ex tens i ve deve 1opmen t of I y 1a s e s ;

p - ear lier occur re nee of ] i g m f icat ion!

f . inhibition of differentiation;

g. c h a nges in diameter and number of xylem vessels; and

h. stunting at various levels of organization.

In addition to structural alterations, plants affected

by salt stress undergo numerous biochemical changes. A c

cording to Pol j a k o f f-Ma y b er (91 , 92) and Jennings < *9 ) ,

these changes are:

a. increased respiration;

b. decreased germination;

c. reduced CO,-, a s s i m i l a t i o n (due to stomatal closure);

d. decreased p r o tein content;

e. reduced water flow through the plant;

f . 1owered water activity;

g. reduced t r a n s 1o c a t l o n of photosynthate!

h. decreased nutrient availability; and

l. a l t e r a t i o n s in memb r ann per m e a b i 1 i t y .

Damages caused by excess NaCl may be osmotic, toxic, or

nutritional (10). Osmotic stress causes severe internal

plant water deficits. These conditions lead to reduced

water activity, a l t e r a t i o n of m a c r o m o 1e c u 1ar structures,

7

c on c e n t r a t i o n and p r e c i p i t a t i o n of ion;,, and reduced rates

of photosynthesis* cell division and cell expansion (97,

98). Specific ion toxicity occurs when the concentratlon of

nsiTiotical ly active solutes accumulated to effect o s m o r e g u l a

tion become too great <1C). Bernstein <10) describes the

major nutritional effects of salinity stress as those a s

sociated with cation nutrition. With increasing salinity,

c o m p e t i t i v e ion ab sor p tion favors the uptake of Na ’ (10).

In many cases* salt-induced nutritional stresses can be

overcome by additions of C a '' * ( 1O ) .

Plants differ widely in their mechanisms to tolerate

excess salinity (2* 26* 27, 3 1, 39 , 95, 99, 96, 97). Plants

are separated into two catego r i e s based on their abilities

to resist salt stress: h al op h y t e s and g l ycophytes (31, 27).

H a l o p h yt es are plants which increase in dry weight content

in the presence of high conce n t r a t i o n s of electrolytes (at

least 300 m M ) in their environment, while showing no

obligate requirement for high salinity (lO, 31).

Ha l o p h yt es which accumu l a t e salts to effect osmotic a d

justment are called e u ha l o p h y t e s (10, 31). Halophytes which

accumulate organic solutes such as praline, betaine* and

choline for osmotic adjustment are known as qlycahalophvtes

(10, 31). Glyco p h y t e s are plants which cannot survive in

salt sol u t i o n s greater than 0.5*/,. Some, however, do have a

a

limited ca p a b i l i t y for a s m o r e g u l a t i o n (37, 95, 92, 97).

One of the most d e le te ri ou s effects of sodium chloride

stress on crop plants is stunted growth (10). Salt s e n s i

tive plants remain stunted in spite of osmotic adjustment or

ion c a m p a rtmenta 1i z a t i o n . P o 1 jakoff-Mayber (02) and J e n

nings (99) suggest several possible reasons for this: ( 1 )

energy acquired from photo s y n t h e s i s is diverted to active

ion uptake and compar tmenta 1 i z a t io n , syn t h e s i s of organic

salutes, and m a i n t e n a n c e of osmor e g u l a t o r y mechanisms; (2)

the high energy costs of repairing damage to enzymes and

tissues resulting from d e h y d r a t i o n and salt ions; (3)

stomatal closure resulting from temporary loss of turgor

which restricts CO;' uptake; and ( 9 ) dec r e a s e s in anabolic

nitrogen m e ta bo l i s m resulting from all of the above.

According to several authors (17, 31, 3B, 9 9, 50, 82)

NaCl stress alters p r otein synthesis. Interference with

protein synthesis may occur at several levels since: (1)

membrane p e rm ea b i l i t i e s are altered by NaCl, preventing u p

take of organic nitrogen and other n i trogenous compounds

(90, 99, 82); (2) an excess of NaCl lnh ibits at least some

enzyme activity, diss o c i a t e s r ibosomes (11), and leads to

reduct i o n s in p o l y r i b o s o m e complexes (33, 99, 52); (3) NaCl

may cause permanent changes in the genome of plants, a l t e r

ing the synthesis of RNA and the translation of proteins

9

(11, 52, 67, 101); and (A) plant hormones are affected by

salinity which could lead to protein alterations (11* BE).

Tissue and cell c u lture techniques have become useful

to plant breeders for the isolation of p o t entially lmpo r t a n t

p h e n o t y p e s (EO, 70, 100). Another a pp l i c a t i o n of tissue

c ul tu re is in physiological studies for investigating the

tissue and cellular basis of stress tolerance. This a p

proach is not without limitations. According to Tal (100),

using cell culture techniques in studies of this nature has

several unique problems associated with them. One of the

problems is that variants in culture may be expressed only

at the cellular or tissue levels of organization, and not in

whole plants. Likewise, variability may be an epigenetic

change resulting from the selective agent in the absence of

which there is no phenotypic expression. Another problem

assoclat ed with the appli c a t i o n of in v i tro t ec h niqu e s is

the progr e s s i v e decrease in r e g e n e r a b l 1 lty exhibited by

cells and tissues in c u l ture for extended periods of time.

A fourth i nh er ent p r o b l e m associated with virtually all

tissue c u lture research is the absolute necessity for

screening at the whole plant level of crganilation.

The high incidence of variation in plant tissue and

cell c u l t u r e s is a useful trait. Model systems designed for

the investigation of genotypic and phenotypic stability

io

under normal and stressed conditions take advantage of this

p h e n o m e n o n (61, 70). fhe two major sources of heritable

cellular variation are, mutation and epigenetic change

(70). V a riability from s p o ntaneous or induced mutation is

rare and the me c h a n i s m s c o ntrolling epigenesis are still not

fully understood.

The primary source for obtaining phenotypic diversity in

culture, according to Meins (70), is selection. In recent

years, tissue culture scientists have exploited this p r i n

ciple to screen vast numbers of plant cells for desirable

traits <61, 66, 100). Differen t i a t i n g b e t ween physiological

adaptations, epigenetic changes, and directed genomic a l

terations is a fundamental problem inherent in this type of

research ( 19, £?0, 61 , 70, 100) . A method for distinguishing

the e f fects of these sources of variability on phenotype

would be of great practical advantage to plant breeders.

It has been well e s t ablished that the combination of

gene action, epigenetics, and environmental interactions

d e t e r m in es phenotype (IS, 100, 101). How these factors r e

late to salt tolerance still is uncertain. It is likely

that plant responses to salinity stress are regulated at

both the cellular and molecular levels <11, l'?, k3, 100 ,

1 0 1 , 1 0 5 ) .

The c h anges which occur in plants from excess salinity,

11

such as an increased synthesis of o r g a n i c solutes, are often

considered physiological a d a p t a t i o n s that may have survival

advantages under these condit i o n s <B1 ) . These changes have

alterna t i v e l y been d e scribed as pe r t u r b a t i o n s of normal m e

tabolic activi t i e s of living o r g a n i s m s and represent damage

imposed by saline c o n d i t i o n s (BE, 101). Whichever of these

e x p l a n at io ns most accurately a c c o u n t s for the changes in

plants in response to excess salt is not known.

A unique class of polypeptides, o f t e n referred to as

"shock proteins" <9, 51) has a t t r a c t e d a great deal of a t

tention recently for their puta t i v e r o l e in environmental

stress tolerance. These proteins a r e synthesized in many

plants and animals in response to environmental changes. It

is unc e r t a i n when and where the p r o t e i n s are synthesized,

how long these proteins remain in the absence of selective

pressure, and what the nature of their genetic regulation

l s .

Numerous articles report substantial, guantifiable

changes in the p r o te in complement of s o m e plants and animals

in response to various applied physiological stresses (4, b,

11, IP, 15, PE, 51, 53, 107,). Several of these studies

cite p r otein changes directly a t t r i b u t a b l e to osmotic stress

(A, E9 , 30, 91). These supposedly s t r e s s-induced proteins

are synthesized in callus tissue and at the who l e plant

12

level of o r g a n i z a t i o n in response to c e r tain changes in the

environment. Heat, salt, a n a e r o b i o s i s » drought, and cold

are among the stresses eliciting the induction of novel

p rotei ns from numerous plants and animals. It is not known

whether shock proteins actually enhance the ability of an

o rganism to tolerate radical changes in their environment,

however, is not known.

A common theory explaining stress tolerance is not

available. The ability of plants to withstand stress may be

the result of gene action, epigenetic interactions and

physiological adaptation, or spontaneous or directed m u t a

tion. Enhanced ability to withstand stress may be

ephemeral, or transferable, but it is certainly species

spec 1 fic .

One of the most co m m o n methods for the analysis of

proteins in solution is polyac r y l a m i d e gel e l ectrophoresis

(PAGE). Electrophoretic techniques presently are being used

in num erous laboratories for the analysis of the proteins in

cells and tissues of cultured plants. The a p p lication of

these techniques e n ables researchers to determine a great

deal of q u a l i t a t i v e and q u a n t i t a t i v e information concerning

the nature of proteins in solution. One-dimensional sodium

d o d e c y 1 sulfate gel e l e c t r o p h o r es is (SDS PAGE) is the most

widely used technique for p r o tein analysis (^1, 95). The

1 3

type, number, relative abundance, and molecular weights of

p ro t e i n s in a complex m i x t u r e can be identified us 1nq 5DS

PAGE. W he n sodium dodecyl sulfate (an ionic detergent) is

reacted with proteins before electrophoresis, every 1.9

grams of SDS will denature and bind approximately 1 gram of

pr otein (91, 92>. These S DS - p r o t e i n complexes are generally

soluble and ( in an electric field) migrate towards the anode

through an a c r y 1 amide —b 1 s a c r y 1 amide gel matrix. The rate of

m i gr at i o n is generally inversely proportional to the

logarithm of these sds- p r o t e i n c o m p l e x e s ’ molecular weiqhts

(91, 92 ) .

Two factors det e r m i n e the mobility of proteins in an

el e c t r o phoretic system. First, the strength of the electric

field has a direct effect on the speed of p r o t e i n migration.

The second factor is the frictional compon e n t s in the qel

and the shape of the individual proteins. The higher the

viscosity of the gel, the more resistance a protein will e n

counter. The larger and more globular a p r otein is, the

greater the resistance it will encounter <91, 92). The

positions of known molecular weight markers within a gel can

be used to e s ti ma te the molecular weights of unknown

proteins .

Although numerous SDS electrophoretic protocols are

available, the method of Laemmli (60) is the most widely

1 9

used. Laemmli developed a two part gel system which

resolves p r o tein bands better than previous systems which

utilize a one part gel only. The acrylamide c o n centration

of the top or stacking gel is less dense (2-5'/.) than the

acryla m i d e content of the separating or running (l o w e r ) gel

(5-207,J. This differential c o n ce n t r a t i o n gradient favors

increased p r otein band resolution b e c a u s e proteins will

"stack" together at the interface of the two q e 1s

(l s o t a c h o p h o r e s i s ) before they migrate through the running

gel. The c o n c e n t r a t i o n of acrylamide and N ,N ’—m e t h y 1ene

b i s a c r y 1 amide (bis) can be adjusted to facilitate optimal

protein m i g r a t i o n d e pending on the molecular weights of the

proteins being assayed. Some recent studies using SDS PAGE

have linked substantial changes in the banding patterns of

extracted proteins from cells of some species of N i c o t i a na

tabacum with increasing levels of N a C 1 adapta t i o n (29* 30*

91* 107). There has been no evidence linking changes in the

p o l y p e p t i d e complement of cells having N a C 1-reslstance with

a survival a d v antage under high salt conditions, though the

a s s ociation is oft e n made (29, 91).

E f forts towards increasing N a C 1 resistance in crops

should be continued. It has been d e m o n strated with tissue

culture that stable v a riation can be obtained. Cells and

tissues in c u lture inherently exhibit extremely high rates

15

of spontaneous, persistent, and her i t a b l e variation, which

ai e cften transferable in sexual crosses (70) . There is

reason to believe that r e searchers utilizing tissue culture

techniques and conventional breeding methods will be able to

develop superior cultivars.

N 1c o t i a n a tabacum spp. (a dicotyledon) has t r a d i

tionally been used for investigations into the tissue and

cellular mechan i s m s plants have for toleratinq environmental

stress <18, 1 A , 30, 51, 73, 100). Many of the w o r l d ’s most

important horticultural crops, however, are m o n o c o t y 1e d o n s .

Moreover, cereals (also monocotyledons) are among the

w o r l d ’s most important food crops. Creeping bentgrass <A._

pal us t r is Huds.) is a mon o c o t y l e d o n o u s plant. particularly

well suited for use in model systems designed for the study

of i.n v itro stress physiology. Its hormonal, nutritional,

and environmental r e q u i rements for tissue and cel 1 culture

have been established (3, 55, 56, 57, 58). This genus c o n

tains some relatively halatolerant cultivars, but it is

generally considered a glycophytic group of plants with

limited g e netically control led ion e x clusion mechanisms <1,

0, Callus p ro l i f e r a t i o n and plant let regeneration have

been reported from at least four cul t i v a r s of this plant

species (*f6, 57, 58).

A g r o s tis palustris cultivars are highly regarded as

horticultural crops. They are extremely tine turfgrasses

with many des i r a b l e traits and are extensively used as golf*

tennis and bowling greens (0). Creeping bentgrass grows

from vigorous stolons and is one of the most hardy cool

season turfgrasses (0). When mowed closelyi A . p a 1 u s t r i_s

for ms one of the flnest quality sods known < 0 ) . A cultivar

with strong persistent salinity tolerance would be a valu

able commodity.

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IN VITRO SEL E C T I O N FOR S O D I U M CHLORIDE RESISTANCE,

REGENERATION, AND S O L U T I O N CULTURE E V A L U A T I O N S OF

TWO CULTIVARS OF Ag r o s tis p a 1 u s t. ris Huds .

Ab s t r a c t :

Calli from two cul t i v a r s of creepinq he n t g r a s s (Aq r os t 1 s

plustjri^s Huds.) were selected for enhanced ability to

p ro li te ra te on media supplemented with 51A mM N a C l . The t o

tal s o luble p r o t e i n c o n c e n t r a t i on of calli s e 1ec ted from

control media (0 mM NaCl) was greater than soluble protein

c on ce nt ra ti on of calli selected from NaCl s t r e s s - 1 n d u c 1 nq

media. Non- s e l e c t e d calli from N a C 1-stressed and control

media contained less soluble protein than selected calli

exposed to the same treatments. Percent dry weights of

lyophilired callus tissue selected from the control medium

were lower than percent dry weights of 1y o p h i 1 lzed callus

tissue selected from 51A mM N aC l- su p p l e m e n t e d media.

Growth of plants r e g enerated from N a C l - a da pt ed calli in

saline liquid nutrient c u lture <as dry weight and vigor) was

superior to that of plants regenerated from non-adapted

calli. Plants regen e r a t e d from salt s en si t i v e calli died or

were un a b l e to p r oduce m ea su r a b l e dry matter in liquid c u l

ture containing 257mM NaC 1 , whereas pl a n t s regenerated front

N a C l - a d a pt ed calli grew well in 257 mM NaCl* Growth of

33

3^

plants regenerated from salt-adapted calli was inhibited at

NaCl c o n c e n t r a t i o n s greater than 3^0 m M . At 51^ mM NaCl,

plants regenerated from salt resistant calli could no longer

produce measur a b l e dry matter, although they remained alive.

Abb r e v i a t l ons : 2,^-D: 2 , '♦-di ch lorophenoxyacet ic-ac id , B6: N-

(p h e n y 1- m e t h y 1 )- 1- H - p u r i n e - 6 - a m i n e , MS: Mur a s h i g e and SIooq

(5) ,P M S F : p h e n y 1 methy 1s u 1f o n y 1f 1u o r i d e , EDTA (disodium

salt): e t h y 1enediamene t e t r a a c e t i c acid, D T T : d i t h i o t h r e i t o l ,

HEPE5: N - p - h y d r o k y e t h y 1piper a 2 l n e - N '- E - e t h a n e s u 1fonic acid,

TR1S: t r i s t h y d r o x y m e t h y l )a m l n o m e t h a n e , Tween 20: P o 1yoxy-

e t h y l e n e sorbitan monolaurate.

35

INTRODUCTION

Many r e searchers b e l i e v e that tissue c u l ture techniques

can be used along with conventional breeding methods for

d e v e loping salt tolerant crops (2, 13. 15). Several species

of plants have already b ee n regenerated from sodium chloride

resistant cell lines (2. £ , 7 , 9, 11, 10, 12. 13). In vitro

r e g e n er at io n of salt resistant plants from liquid suspension

(9, 19) and callus cul tures (9, 10, 17) has been achieved;

however, permanent NaCl tolerance has not been accomplished

from tissue cultured cell lines. R e s e a r c h e r s are optimistic

that continued e f forts will produce cul t i v a r s transmit

he r i t a b l e salt resist a n c e (2, 11, 13, 15).

Cereals are among the most important food crops in the

world, and like most other monocots, do not tolerate hiqh

c o n c e nt ra ti on s of NaCl in the soil solution. One objective

of this study was to investigate the p os s i b i l i t y of s e l e c t

ing callus lines of the monocots, creeping bentgrass

( Aqrost i_s p a l u s tr i s Hud s. > which can d e m onstrate an enhanced

ability to proli f e r a t e on media supplemented with NaCl and

to s u b s e quently regen e r a t e plantlets from these calli.

Another obj e c t i v e of this research was to evaluate rooted

plants regenerated from NaCl - a d a p t e d calli in liquid

36

nutrient culture for any enhanced ability to tolerate NaCl

stress at the whole plant l e v e l .

M A T E R I A L S AND METHODS

C allus es t a b l i s h m e n t

Callus cultures of 'Penneagle' and ' S e a s i d e ’ creeping

b e n t q r a s s were e s t a b l i s h e d on modified MS medium (5),

sup p l e m e n t e d with 1 mg/1 BA, 5 m g /1 P,6-D, 30 g/1 sucrose,

and 10 g/1 agar. The pH of the media was adjusted to 5.7

with 2 N NaOH prior to a u toclaving (1 k g /c m ; , 121° C for 15

minutes), checked and readjusted after autoclaving if n e c e s

sary. Car y o p s e s were s u r face sterilized by immersing them

in 95*/. ethyl alcohol for 5 minutes followed by immersion in

1.05*/, sodium h yp oc h l o r i t e ( 20'/. commercial bleach solution),

sup p l e m e n t e d with 0.01 7. Tween 20 (surfactant) for 20

minutes. Following s t e r 1 1 i z a t i o n , car y o p s e s were rinsed 3

times in sterile, double distilled water. Two to 5

ca r y o p s e s were e s t ablished in 25 x 150 mm c u lture tubes on

25 ml of medium and incubated in the dark for 30 days. A 1 1

c ultures were cleaned of debris, such as hulls from

car y o p s e s and any eti o l a t e d germinating shoots after 30 days

and transferred to fresh a medium. The remaining callus

material was incubated for an additional 30-days in the

dark.

37

Sel e c t i o n for Salt T o l erance

Stock calli from caryopses, weighing a p proximately 50-75

mg ,were subcultured onto fresh media supplemented with

either 0 or 51^ mM (30 g /1 ) NaCl. Ac tively prol lfpratinq

calli (and portions o+ calli) from both treatments were

selected, rated, and transferred following a sinqle 60 day

dark incubation period. Calli were selected for N a C 1 -

re s i s t an ce based on visual o b s e r v a t i o n s (Figure 1). Those

calli which had not increased in mass beyond their initial

transfer size were given a ( + ) rating. Those calli which

had visibly increased in mass, but less than double their

initial transfer size were given a (++) rating; and those

calli which had doubled their initial transfer size or

greater were g i ven a (+++) rating. Calli which had no

necrotic areas were g i ven a < + > rating; those with 1-25 V.

necrosis a (++) rating; those with 26-50*/. necrosis, a <+++);

and those with greater than 51*/., a (+ + + +) ratinq. Calli

with shoots were given a <+) rating; and those without

shoots a ( — ) rating.. The same rating system was applied to

calli developing roots. All ratings given as (+ or -) were

transformed into p er c e n t a g e s of the original number of c u l

tures in each treatment at the beginning of the transfer

period for subsequent data analysis.

30

E x t r ac ti on and e s t i m a ti o n of soluble proteins

Calli were selected at random from each treatment at

the end of the each transfer period, frozen in liquid

nitrogen, l y o p h i 1 lzed overnight, and stored at -00° C until

needed for p r o tein extraction. One gram of each l y o p h i 1 ized

sample Mas ground with a Thomas motorized tissue homoqeni;er

at full speed in 10 volumes ot chilled a c etone (- ? 0 ° C ) in an

ice bath, suction filtered, and placed in a desiccator

(under vacuum) until all of the acetone had evaporated.

S o luble proteins were ext r a c t e d with a Thomas motorized

tissue homogenizer in an icebath for 30 sec from 100 mg

samples of the acetone powder in 2 ml of lysis buffer c o n

taining 50 mM HEP E S (pH 7.5 at A ° C), 65 mM DTT, 17 mM 2 —

m e r c a p t o e t h a n o 1, 2 mM EDTA, and 1 mM P M S F . The homogenized

samples were transferred to 30 ml Corex c en tr i f u g e tubes and

c en t rif uqed a t 5 0 » GOO x G for 20 min at P ° C. Supernatants

were then car e f u l l y pipetted into screw top microcentr ifuqe

tubes for storage.

One hundred ul a li q u o t s were removed from each of the

samples for p r otein quantification. The remaining soluble

p r o tein extract was frozen in liquid nitroqen and stored at

-00° C far subsequent analyses. Total soluble protein in

each sample was d et er m i n e d by the B i o —Rad protein assay

method. Bovine gamma g l o b ul in was used to generate a

39

standard c a l i b r a t i o n curve for p r otein quantification. A b

sorbance at 595 nm was read usi ng a Bausch and Lomb

Spectranic 21 spectrophotometer.

L y o p h i 1 ized weight d e t e r m in at io n

Each of the stock callus c u l t u r e s generated from caryopses

was divided into ap p r o x i m a t e l y equal segments weighinq b e

tween 75-100 mg and labeled for identiflcation ( i .e. 1 and

1 ’). One segment from each pair was exposed to N a C 1 and

the other segment was used as a control . Since each of the

callus pairs origin a t e d from a common callus culture. they

were referred to as sister c a l l i . For example, cultures 1

and 1 ’ wouId be sister calli; 2 and 2 ’ would be sister

c a 1 1 l . At the be g i n n i n g of each weeL , calli from NaCl

treatments selected for transfer and its co r r e s p o n d l n q c o n

trol were chosen at random, placed in two petri dishes and

mixed thoroughly. Sodium chloride treated calli were placed

in one petri dish and control calli were placed in another.

Two gram samples from each treatment were collected, frozen

in liquid nitroqen, and lyophilized. L y o p h i 1 i zed weiqhts

were compared at the end of three transfer periods.

Plantlet r e g e n e r a t io n and establishment

At the end of three passages, s a l t - adapted and non-

AO

adapted calli (controls) were transferred to r e g e n eration

media. The r e g e n eration medium had the same components as

callus 1 n d u c t 1 on med i u m , ex cep t for the omission of an auxin

source. Calli selected from N a C 1- s u p p 1emented media were

r e generated with NaCl in the growth medium. Controls were

regenerated on reg e n e r a t i o n medium without NaCl. Plantlets

that were regenerated from salt-adapted calli before the end

of the third genera t i o n were set aside and kept on fresh

N a C l -s up pl em en te d media. Embryogenic cultures from all

three g en e r a t i o n s were placed on r e g e n e r a t i o n media without

NaCl for shoot elonga t i o n at the end of the third transfer

period. Plants were acclimated when the majority of shoots

had elongated beyond 20 mm and roots had elongated beyond 10

mm. R e g enerated pla n t l e t s were acclimated off by removinq

culture tube caps and exposing the succulent plant tissue to

the atmosphere. A cc l i m a t i o n took 3 days. Acclimated plants

were estab l i s h e d in 3 inch plastic pots filled with s t e r i l

ized v e r m i c u l i te. Potted plants were placed inside of p l a s

tic ch ambers with ap p r o x i m a t e l y 90*/. relative humidity for

five days, before being moved into a growth chamber. Two

grams of Mi 1o r g a n i t e , a slow release fertilizer, was used as

the source of nutrition. Plantlets were watered with d i s

tilled water every 3 days. This schedule was followed for 1

m o n t h .

U !

L iquid nutrient evaluation

E stab l i s h e d plants were evaluated in hydroponic c u l

ture using H o a g l a n d ’s #2 nutrient solution as modified by

P eterson (13), supplemented with 0 or 51^ mM NaCl. Controls

were acclimated to saline nutrient c u lture in a stepwise

fashion, beginning with 85 NaCl mM the first two weeks, 170

mM NaCl the second and third weeks, 257 mM NaCl the fifth

and sixth weeks. As all control plants were dead at the end

of the sixth week, the liquid nutrient solution was not a d

justed to 51L mM NaCl for the seventh and eighth wee)s for

the controls. Plants regenerated from salt-adapted calli

were acclimated at different rates. The results from

p r e l i m i n a r y experiments indicated that plants regenerated

from salt-adapted calli could tolerate radical chanqes in

the osmotic potential of their medium. The initial

hydroponic medium they were initially exposed to contained

257 mM NaCl. During weeks three and f o u r,plants regenerated

from s a l t - adapted calli were placed in 3^0 mM NaCl, fallowed

by 51A mM NaCl for the fifth through eighth weeks. Two inch

(ID) polyvinyl chloride (P V C ) pipes with holes bored in the

tops were used to support the plants while their roots were

bathed in nutrient solution (Figure 1). Sump pumps were

used to d is tr i b u t e nutrient solutions through the PVC pipes.

The nutrient solutions were aerated

ge

by gravity as the liquids fell back into the reservoirs.

Visual o b s e r v at io ns of the plants were made daily- Growth

was determi ned by dry wel gh t . Each clump of plants was

clipped to a height of 10 cm, and the clippings dried

weiqhed, and recorded. This procedure was repeated for 0

weeks, unless the plants had died. All of the evaluations

were conducted in growth chambers with 16 hours light at 06°

C temperature, and 0 hours of dark at 18° C temperature.

Liqht intensity inside of the growth chambers was 5000

m ic r o E i n s t e l n s /c m ■V s e c . No humidifiers were used. Relative

humidity was a p proximately 0 0 ’/..

RESULTS

E f fects of Salt

Sodium c hl o r i d e had a highly significant effect on the

percent of callus cultures transferred (Table 1). At the

end of the first generation, 95.3'/. of the non — stressed

’P e n n e a q l e ’ c ul t u r e s and 90.3% of the ' S e a s i d e ’ cultures

were selected for transfer. Of the cultures transferred,

92.77, of the nan-stressed 1 Penneagle ’ cultures and 967. of

the ’S e a s i d e ’ cultures were selected for transfer a second

time. At the end of the second transfer period, 90.77, of

the ' P e n n e a g l e ’ cultures and 86.77. of the ' S e a s i d e ’ cultures

43

were selected and transferred. After the first g e n e r a t i o n

on NaC 1 -supp 1 emented medium, 00.7V. of the 'Penneagle' calli

could be transferred and 66V, of the 'Seaside' cultures were

selected for t r a n s f e r . At the end of the second transfer

period, only 7 V. of the ' P e n n e a g l e ’ calli and A . 1V. of the

' S e a s i d e ’ cultures were suitable for transfer to fresh

m e d i um for a third generation. A total of B1V. of the callus

cultures an control media were transferred during this e x

periment over three g e n e r a t i on s and used in the regeneration

studies, while only 0.02V. of the callus c u l t u r e s exposed to

514 mM NaCl remained after the third selection/transfer and

were s uitable for the r e g e n e r a ti on studies.

There was a highly significant difference between the

number of cultures producing shoots and roots on control

m edium and the number of cultures producing shoots and roots

on a N a C 1- s u p p 1emented m e d iu m (Table 2). Sodium chloride

depressed the p r o d uc t i o n of itinerant shoots (in both

cultlvars) on callus m a i n t e n a n c e medium (Table 2). Neither

cultivar produced shoots on 514 mM N a C 1- s u p p 1emented

medium, but both cu l t i v a r s produced roots on the 514 mM

NaCl media. There was a highly significant inhibition of

root pr o d u c t i o n by calli of both cultivars on the NaCl-

med ium (Table 2).

Salt also had a highly significant effect on the

AA

visible increase in mass (size) that both cu l t i v a r s gained.

Both c u ltivars produced s i gnificantly less mass increase on

media supp l e m e n t e d with SlAmM NaCl than an control media

over three g e n e r a t i o n s (Table 3).

Growth media s u p p l emented with 5 1 A mM NaCl had a highly

significant effect on n e c r o s i s over 3 generations. There

was c o n s i d e r a b le variability among cultivars and the p e r

centage of necrosis. ' P e n n e a g l e 7 had the greatest number of

cultures with zero necrosis (Table A) over 3 transfer

periods and ' S e a s i d e ’ had the greatest number of cultures

with 1-25V, necrosis (Table A). Alternatively* ' P e n n e a g l e 7

p roduc ed the greatest number of c u l t u r e s with 86-50*'.

necrosis (Table A). At the level of 51 percent or greater

necrosis (Table A) there was no significant difference b e

tween cultivar response.

S odium chloride strongly reduced the number of callus

cultures remaining from both cultivars at the end of three

transfer periods. Less than IV. of the initial cultures e x

posed to 51A mM NaCl were selected for r e g e n eration

Sodium chloride had a highly significant effect on the

number of calli from both cul t i v a r s r e g e n erating shoots over

three g e ne ra ti on s (Table 5). There was a significant inter

action b e t ween g e n e ra t i o n time and the number of cultures

pro d u c i n g shoots. More calli from both cul t i v a r s produced

A5

shoots during the first transfer period on r e g e neration

media than during the third generation (Table 5). There was

also a significant d i f f e r e n c e among cultivar and treatment

n N a C 1 or no NaCl ) . Sodlum chl o r i d e - a d a p t e d 'P e n n e a q 1 e ’ c u l

tivars produced far more cultures with shoots on 51A mM

N a C 1—s u p p 1emented r eg e n e r a t i o n media than 'Seaside". while

nonadapted ' S e a s i d e ’ calli produced a greater number of

em b ryoqenic cultures on control media.

So d i u m chloride also had a significant effect on the

number of cultures pro d u c i n g roots. Roots were produced by

both cul t i v a r s in a s i g n i f l c a n t 1y greater number of cultures

on N aC l - s t r e s s inducing media than on control media (Table

5). There was also a highly significant cultivar by salt or

nonsalt interaction (C X S), Seaside' calli produced a s i q -

nifica n t l y greater number of cultures with roots than

'Penneagle* however, 'Penneagle' produced more cultures with

roots on control media (Table 5).

A total of 1 <b2 NaC l - a d a p t e d ’P e n n e a g l e ’ cultures u l

timately produced rooted plants on 51A mM supplemented

media. Only A3 N a C l - a da pt ed ' S e a s i d e ’ calli produced rooted

plants on salt stressed media.

The mean l y a p h i 1i zed weights of the control treatments

were lower than NaCl treatments (Table 6). The protein c o n

c e n t r a ti on in control treatm e n t s was only slightly higher

A 6

per 100 mg of lyophilized tissue compared to protein c o n

c e n t r a t i o n s of lyophilized NaCl treatments. Samples from

selected N a C 1-stressed and non-stressed calli of both cul-

tivars had protein c o n c e n tr at io ns that were significantly

higher than samples from nonselected salt-stressed and non-

s t r essed calli.

Normally regenerated plants of 'Penneaqle' and ' S e a s i d e 1

produced dr y weight more slowly than plants from normally

germinated caryopses or plants regenerated from salt-adapted

calli (Figure E ) . The normally r egener a t ed plants obtained

the same level of g r o w t h ( however, by the end of B weeks.

Plants either from normally germinated caryopses or

regenerated from n o n -adapted calli adjusted enough at 85 mM

N a C 1 to show an appre c l a b l e growth in terms of dry matter

(Figures 3 and A) . At 173 m M , plants from plants from both

of these groups had either died or were showing absolutely

no growth (Figures 3 and A). Plants r e g enerated from both

cultivars of NaCl - a d a p t e d bentgrass calli grew and added dry

matter at E57 mM NaCl (Figure 5), but were strongly in

hibited at NaCl c on ce nt r a t i o n s higher than this. At 51A mM

NaCl they were still alive but unable to p r oduce measurable

dry mat ter .

A7

DISCUSS!ON

The results of this study indicate that it is possibile

to isolate N a C 1 - to 1erant callus cultures of two cultivars of

Aqrostj^ P#J_y?tr_is with enhanced ability to tolerate hiqh

salinity conditions. Sodium chloride at a concentration of

5 1A mM closely approximates the salinity of seawater. This

is an extremely high c o n ce nt ra ti on of saiti and one which

would unlikely be encountered. Figure 6 illustrates calli

of ' P e n n e a g l e ’ (top) and ' S e a s i d e ’ (bottom) which have been

exposed to 5 1^ mM NaCl. There are c e rtain cells or qroups

of cells which appear very healthy (translucent and friable)

and actively p r o liferating at this concentration. One might

expect that plants regenerated from these NaCl-adapted calli

could have some selective advantage to withstand higher c o n

c en t r a t i o n s of NaCl in the soil solution than plants

regenerated from non-adapted calli, or from plants normally

qermin a t e d from caryopses. To some degree, the results of

this study indicate this. Plants r e generated from salt-

adapted calli maintained superior growth compared with n o n

adapted plants at NaCl c on ce nt r a t l o n s up to B57 m M . They

remained alive but did not grow at c o n e e n t r a t ions up to 51k

mM NaCl (Figure 7).

The selection of calli or the r e g e n e r a t i o n of plants

from N a C 1-resistant calli at NaCl concentrations as high as

<40

51 <4 mM has not been reported. Previously, Kochba et al . (5)

obtained C itrus s i nensi s callus resistant up to 170 mM;

Nabors (10, 11) obtained NaCl-tolerant N i c o t i a na tabacum

s p p . at m a x imum c o n c e n t r a t i o n s of 150 mM. Croughan (3),

S a l q a d o - G a r c i g l i a et al. (17), and Tyagi O l ) all selected

for N a C 1-resistance in calli of alfalfa, sweet potato, and

Jimson at 170 m M . It was possible to regene r a t e plants from

s alt-a dapted calli at c o n c e n t r a t i o n s of 51 <4 mM NaCl. Nabors

(11) was not able to r e g e n e r a t e plants from N a C 1 -reslstant

calli unless they were placed on a non-stressed medium

Tyagi et al. (21) r egener a ted NaCl - a d a p t e d D a tur a i nno i a

calli at 170 m M . Mathur et al. (6) obtained regeneration of

K i c k_x i a ramo s i s s i ma at 120 mM NaCl c o n c e n t r a t i o n s from in-

ternodal sections, not from NaCl-adapted calli.

□ne of the more promising aspects of this work is that

calli be c a m e embr yogenic whlie exposed to the s e 1ec 1 1 ve

agent for which they were screened. Acc o r d i n g to Chandler

and Thorpe (2), this is an extremely des i r a b l e trait in

breeding programs for salinity resistance. It is likely,

from their appearance, that plantlets r e g enerated from salt

adapted calli on 51 <4 mM NaC 1 -supp 1 emented r e g e n eration media

were under salt stress conditions. They exhibited the same

symptoms as whole plants grown under salt under salt stress

c ondit i o n s in the field (figure B>. The regenerated

99

pla nt le ts were dark green or dark greenish*blue, succulent,

and stunted (1). Plants regenerated from selected calli

did not show any superior growth in control liquid culture

(Figure 2 > . However, they were superior to plants qrown

from normally germin a t e d car y o p s e s (Figure 3), and plants

re g enerated from non-adapted calli (Figure 9) in saline

hydrop o n i c medium. They appeared have an enhanced ability

for o s m or eg ul at io n under N aC l - s t r e s s conditions.

Pl a n t s regenerated from salt adapted calli of both c u l

tivars, performed similarly in liquid culture, alt. houqh,

' S e a s i d e ’ is consid e r e d to be the more halotolerant of the

two cultivars. It is p os s i b l e that superior 'Penneaqle'

q e no ty pe s were selected and regenerated as a result of the

s c r e e m n q procedure.

It was not possible to grow plants f r o m normally g e r

minated caryopses, nor normally regenerated plants in 519 mfi

NaCl liquid culture. However, plants regenerated from sodium

chloride selected calli were capable of w i t h standing this

shock and recovered. ' P e n n e a g l e 1 calli transferred over 3

generations, therefore may have superior genetic salt r e s i s

tance mechanisms. This sel e c t i o n procedure is a method for

singling out these individuals.

Data from the mean weights from 2.0 g samples of

lyophilized tissue indicates that Na C l - s t r e s s e d tissue is

50

higher than tissue of control calli in dry matter. The

lyophilized weights from selected and non-selected tissue

was not a p p reciably different from their corresponding

selected sister calli. The p r o t e i n c o n c e n t r a t i o n from n o n

selected calii from both treatments were significantly less

than from select ed calli. An e s t i m a t i o n of total soluble

proteins* therefore) may facilitate rapid screening for

callus tissue with enhanced ability to tolerate osmotic

Whether any enhanced ability to tolerate high NaCl is

lost in the absence of the selected pressure or is t r a ns

ferred to progeny is not presently known. Further studies

will have to be performed to det e r m i n e this.

LITERATURE CITED

1. Catarino, F.M. and A.J. Trevawas. 1970* Metabolic

changes in nucleic acids associated with the development

of succulence. Phytochemistry. 9: 1807-1B09.

2. Chand ler , 5. F . and T. Thorpe. 1986 . Var i a t i o n from plant

tissue cultures: B i o t e chnalog ical a pp li ca ti on to improv

ing salinity tolerance. Biotech. Adv. 9: 117-135.

3. Crnuqham, T.P.i 5.J. Stavarek, and D.W. Rains. 19 6 Q .

S e le ct io n of NaCl tolerant line of cultured alfalfa

cells. Crop S c l . IB: 959-963.

9. Epstein* E. 1972. Mineral nutrition of plants: p r i n

ciples and perspectives. John Wiley and S o n s » N. Y.

5. Kochba, J.> G. Ben-Hayim, P. Spiegal-Roy, S. Saad , and

H. Neumann. 1982. Sel e c t i o n of stable salt-tolerant c a l

lus lines and embryos in Clt r u s s lnens i s and C. auran-

tium. Z. Pflanrenphysiol . 106: 1 1 1 - 1 1B .

6. Mathur, A.K., P . 5. Ganapathy, and B .M . J o h r l , 19 B 0 .

Isolation of sodium chlori d e - t o l e r a n t pla n t l e t s of Kick-

x l a ramosjssi^na under in vitro conditions. Z. P f l a n z e n -

physiol . 99 i 207-299.

7. Murashiqe, T. and F. S k o o g . 1962. A revised medium for

rapid qrowth and b io as s a y s with tobacco tissue

cultures. Physiol. Plant. 15: 973-997.

51

8. Nabors, M.W. 1983. Increasing the salt and drought

tolerance of crop plants. p. 165-189. In: D. Randall

< e d C u r r e n t topics of plant bioc hemistry and p h y s i o l

ogy, Vo 1 -P, Univ. Missouri Press, St. Louis, MO.

9. Nabors, M.W. and T.A. Dykes. 1909. Tissue culture of

cereal cultivars with increased salt, drought, and acid

tolerance. p. 181-139. In: B i o technology in internat

ional agricultural research. proce e d i n g s of the inter-

centera seminar on international agricultural research

(LARCS ) and biotechnology. International Research In

stitute Manila, Philippines, pp. 121-139.

10. Nabors, M.W., A. Daniels, L. Nadolny, and C. Brown.

1975. Sodium chloride tolerant lines of tobacco cells.

Plant Sci. Let. 9: 155-159.11. Nabors, M . W . , S.E. Gibbs,

C.5. Bernstein, and M.E. M e i s . 1980. N a C 1 - t o 1erant

tobacco plants from cultured cells. Z . Pflanzenphysiol.

97: 13-17.

12. Nabors, M.W., C.S. Kroskey, and D.M. McHugh. 19B2. Green

spots are predic t o r s of high callus growth rates and

shoot formation in normal and salt stressed tissue c u l

tures of oat (A v e na sativa L .t . Z. P f l a n z e n p h y s i o l . 105:

391-399.

13 .

19 .

15 .

16 .

17.

1 B .

19 .

53

Norlyn, J.D. 1900. Breeding salt tolerant plants.

Bi o s a l i n e Res. S3: S93-309.

Rains, D.W., T.P Croughan, and S . J . Stavarek , 1979.

Sel e c t i o n of salt tolerant plants using tissue culture,

p. 257-S79. In: The genetic engineering of o s m o r e g

u l ation impact on plant p r o d u ctivity for food, c h e m i

cals, and energy. P l e nu m Press, N. Y.

Rains, D.W. 1902, D e v e loping salt tolerance. Calif. Aqr .

31-33.

Rang a n , T.S. and I .K . Vasil. 1983. Sodium chloride

to lerant emb r y o g e m c cell li nes of j V H 1

amer ic an u m (L. ) K. Schum. Annals Bat. 52: 59-69.

S a 1g a d o - G a r c l g 1 i a , R., F. L o p e z - G u t ie r r e z , and N.Ochoa-

Alejo. 19 B 5 . N a C 1 -resistant variant cells isolated from

sweet potato cell suspensions. Plant Cell Tissue Organ

Culture. 5: 3-12.

Tal, M. 1903. S e l e c t i on for stress tolerance. p. 961-

988. In: D.A. Evans, W.R. Sharp, P.V. Ammirato, Y.

Yamada, ( e d s . ) , Handbook of plant cell culture

Vol. 1. M a c m i l l a n P ub li s h i n g Co., N. Y.

Tal, M. 1905. G e n e t i c s of salt tolerance in higher

plants: Theoretical and practical considerations. Plant

and S o i 1. 89: 199-226.

5 9

20. T e m p l e t o n - S o m e r s , K.M., W.R.S. Sharp, and R.M. P f s t e r .

1901. Se l e c t i o n of c o l d - resistant cell lines of carrots.

Z. Pflanzenphysiol. 103: 139-198.

21. Tyagi, A . k'. , A. Rashid, and S.C. Maheshwar i . 19B1.

Sodium chloride resistant cell line from haploid Datura

i n no k a Mill. A resistant trait carried from cell to

plantlet and vice versa. Protoplasma. 105: 327-332.

Taale :. Effects of 5l«t Nall and generation upon transferable call s of calustr:s P e n - p e ; ! e i ' P t ’ .' a n d S E a o i a p ‘ E S 1 .

Ferrentaae if call: tr ir,sferred froa earh qeneratur

Ni ia:t Bolt

tiener at; o -

:uliua 1OS ; , ; p

S3.3-

PB.’T'B.'l re.

“ 0 . 7 . 3 . B

B'.7 3.3.'

o c. 0 7.5 .

M'. 11.r

*1 . . .J,£' it .3.5.1

♦ dear ar.d standard er-cr ■' ftr percentage cultures of k palus.tr :s ’Perioaole' anc 'Be-side'selected and transferred ( o r 3 oeneraticns. Per: pr>t sqes represent data free 3 rep 1:: a 11 :.n-;.

Table 3. Ef fee 15 of IN a!' S t l i or the production o' sheets an: roots '"rest ca.l: n.ito-es :.f ft. p a 1 ur t r 1 s fenne.-tble ■ arid Geaside ■ 'as' ■ on callusi id-..: 11 on a p d i u a Our: rc a r k i n c u D i m n .

Perze'.taSf c u i t i ' e s p r r ea c jn q sheets anc r c c t s £••• c a l l u s mauc t i : r. i s c . e . i

Nv_5dj_! S a i t

_ _ _ _ _ C - e r e r a t if 1 _ _ _ _ _ _ _ _ _ _ _ _ G er e a 11 c r _ _ _

L'.iti.a 1 6 3 1 3 3

•ft ■

Snot- ”s 1.3 116c: ts 33.','1 ; ■ 39.0 L;

9 ,j3t . O . 11/

'j11.3 ■.

911 . 6 3t . r !

1 -ft' 1 1 3h.l :H 1

* Kesr ard s t a i d i ' d e r ' i r i for pe r c e n t c u l t o r e s of p a i u s t - 1 s ' Fen ' ea : ; - : - ' a r : ’ S e a s i d e ‘m: th s t o a t s and r o o t s c a l c u l a t e d at t he bee t ' t i n : of each t r a n s i t

p c r : rd f t ' 3 c e n e - a t i t -s. Fe c e n t a l s r e p r e s e - t da t a ' r o t 3 r e p l i c a t i o n s .

57

Table 3. The effects of 514 mM NaCl on the percentage of cultures of A_;_ pa lustr is Penneagle ('PE') and Seaside ('5S’) which had visibly increased in size.

No Sa1t 5a 11

C v ./Gen.

( + )Callus Growth( ++ ) ( + + + >

Callus Growth ( + ) ( ++ ) (+ + + >

‘PE '

1

2

3

4.0(2) 13.5(2) SI.7(2)

1.8(2) 11.0(2) 87.2(3)

4,2(2) 17.0(4) 7B.8(3)

45.3(15) 41.3(h) 3.3(1)

54.7(4) 42.7(4) 2.7(1)

36.0(7) 62.3(6) 4.B(3)

'SS’

1

2

3

0 6.3(1) 93.7/1 ) 40.7(7)

0 5.7(2) 93.2(2) 44.7(5)

0.07 12.3(1) 87.7(1) 34.0(7)

53.3(7) 3.3(1)

56.0(3) 1.0(1)

62.3(6) 3.7(1)

* Mean and standard error ( ) for percent cultures of A . pa lust rls‘P E ’ and 'S S ’ which had not visibly increased in size beyond their initial transfer size (+>; those cultures which had visibly increased beyond their initial transfer sizei but not doubling (++>; and those cultures which doubled in beyond their initial transfer size (+++>. Percentages represent data from 3 generations and 3 replications/ generation.

5B

Table 9. The effects of 519 mM MaCJ on the percentage of A . palustr1s Penneagle <'P E ’> and Seaside ('SS’) callus cultures with varing degrees of necros 1s .

No Salt Salt

Cv , /Gen.

0*/. Nec r os i s 1-25 26-50 >51

’PE ’

1 88.7(E) 6.3(2) 11.0(1) 0.1

B 95.0(1) 5.0(1) 0.1

3 99.0(3) 5.3(3) 0.7

0

O

’/. Necrosis0 1-25 Bb-50 >51

7.3(E) 19.3(E) 69.3(9) 9.0(5)

11.0(1) 31.3(E) 50.7(1) 7.0(E)

8.7(3) 3E.6(9) 9? . 3(11) 13.0(5)

'SS’

1 96.0(S) 3.0(E) 1.0(1)

E 88.0(E) 10.7(3) 1.9(1)

3 83.3(3) 16.0(3) 0.7(1)

0 9.7(2) 69.7(6) E0.7(0) 3.3(1)

0 5.7(1) 63.7(6) 27.3(6) 6.0(E)

0 9.7(E) 35.3(9) E9.0(3) 30.3(E)

* Mean and standard error for percent cultures of pa 1ustris ’P E ’and ' SS ’ with 0'/., 1-E5’/., 26-50’/.* and >51*/. necrosis over 3 genrations. Percentages represent data from 3 replications.

T a t 1 e 5 , ' t i e e v f t o f 51** ftt* ha C i o n t h e d r o c i . : t i o n o f s h . t t s a n d r o d s f r o n r a l . u s c : : l t u r e s of t n l i : £ t ' i 5 ' V m e a d l p • ' P E ' a n d S e a s i d e ' . ‘ S o t o r r e d e n t r a t i o n * e d i j « .

P e rte-:tai;e of c o ! t u res p r o d u c i n g snoots and re n t s or r e c e r e ra t i o n e e d u a

Ho S a i t S a i l

6 enp* at ion bene- a t : or

l o_: t; v a ’ !_______ 3___ 3 ________ 1________ c ____ 3 _

' f£'

a l ' oc t s s » . " 3- a l . u 1! ' j 5 - j ■ -+ ■ Hn. t i 1- Si 3 e . " ' t : S ? . 3 1 r ■

k o : t s 7 1 . y i r i <* 3 . v 5 3 “ . 7 i i ■ *< 0 . ■ 4 1 4 9 . 1 ' ! ■' 4 4 . ' J 1 1 .

sf.oots :s '■-■,*''0 ‘7“,'7C;1 IS.jj li.t 5 4.!1.**.1Pools Ea.a hO.' • Se.j'ii HE.3’3 "E.vS)

t Hear and standard error it for percent cultures :f palustr 1 s ' F E ’ and 'SS' fro* ttierMtip.a:ec on regeneration aedie wricf; p'odurec snorts and roots c*f 1- oer er a t;o * s. fei:ertfdes represent dat; f:c 3 rephceticrs.

0‘.

Table t . The effects cf 5S •t' Nail or 1 vophiii;ed neiqnt and p-ctem, concert'at i:r. c* selected ;nc no os elected callus cultures of Fenneeq-e i'cE'.' ar.p Seaside 1'55 ■.

Sel ec ted heree!ec ted

P r Ci t e i n

1 [-,1 Trea*»e'' t s.■■ op" - Wt. CO ":C .

uc .1Lytc-,. Wt. Icn: ,

u: ■ u:

’ff

0. le? 3.’

■j.252 l.e

Cc.nt: [■ i

5N»* ' J . CS4 J . Z

1 . 2

l.C

* Mean anc standard error i 1 cf ivt-ptiil i:ea we:oM and protein ccrtent_at it n selected and nonselected h. paiusris 'sister calli" for 3 generations. oscair.ec froa ;.0 arari satples fresf weight tissue.Soluble protens eitracted fro* ;0u aq cf lyophi ii:ed tissue.Standard error for Lvoph: 1:red weig tt p'ctem corccertration r 0 for ail testej.

61

Fig. 1. Visual rating chart for determining percent necrosis of ft.pa 1ustris 'Penneagle’ or ’Seaside’ calli cultured at 0 mM and 51^ mM NaCl. From left to right: 0’/. ( + ), 1-35*/. (++>,36-50*/. (+ + +>, and greater than 51'/. (+ + ++).

63

Fig. 2. Dry weight of A_;_ palustr i & ’Penneagle’ and 'Seaside' plants germinated from caryopses ( Q ), plants regenerated from calli cultured on 0 mM NaCl supplemented medium ( ) « and plants regenerated from calli selected from 5ig mM NaCl supplemented medium * X grown hydroponica 1 1 y in 0 mM Haagland’s #2 nutrient medium. Bars represent standard error.

Dfi

Y W

EIG

HT

CAIN

(g

) S

RY WEICHT (g)

64

' PENNEAGLE' O m M

3 -

20 64 1 0W E E K S t N C U L T U R E

O N C C O m M 4 N R F O m M * S E L E C T IO N S O m M

' S E A S I D E ' O m M€

5

4

3

2

1

02 100 6 84

O N C C O m MW E E K S IN C U L T U R E

4 N R P O m M * S E L E C T I O N S O m M

4.5

Fig. 3. Response of A_. paiustr is. 'Penneagle' and 'Seaside'plants germinated from caryopses cultured hydroponica11y in Hoagland’s #3 nutrient medium with 0 mM NaCl < <^>) or with 357 mM NaCl ( Q ). Salinization procedeed i n a stepwise gradient. At week 3 plants were exposed to 05 mM NaCl; at week A plants were exposed to 173 mM; at week 6 plants were exposed to 257 mM NaCl; and at week 0* if any plants were alive, they were exposed to 51 A mM NaC1.

CRY

W

EIG

HT

CAIN

(g

) D

Pr

WE!

a}!T

C

AJf

* (9

)

66

NORMALLY GERMINATED CARYOPSES' P E N N E A G L E '

S

4

3

Z

1

00 2 4 e 6 t o

W E E K S I N C U L T U R E o N C C O m M * N C C ft* m M

NORMALLY GERMI NAT ED CARYOPSES

\7 3

2 . S -

0.6

e 1 o60 z 4

W E E K S I N C U L T U R E D N C C O m M ♦ N C C 5 t A m M

67

Fig. k . Response of A^ pa 1 us t r i s, 'Penneagle' and 'Seaside' regenerated from calli cultured on 0 mM NaCl supplemented medium, grown hydroponica 11y in Hoagland's #2 nutrient medium with 0 mM NaCl ( Q ) or at 51 * mM NaCl ( > - Salinization procedeed in a stepwise gradient.At week 2 plants were exposed to 85 mM NaCl; at week A plants were exposed to 173 mM; at week 6 plants were exposed to 257 mM NaCl; and at week 8, if any plants were alive, they were exposed to 5lA mM NaCl.

DRY

If

EIG

HT

CASH

tg

) D

RY

WE

1C SI

T CA

SH

(g)

68

NORMALLY R E G E N E R A T E D PLANTS' P E N N E A C L E '

€

S

3

2

T

00 2 4 £ 8 1Q

W E E K S C U L T U R E D S R P O m M * M R P S f 4 m M

NORMALLY R E G E N E R A T E D PLANTS€

5

4

3

f

O0 2 4 6 a to

W E E K S i r t C U L T U R E D b l R F O m M ♦ b f R P 5 M mi/

69

Fig. 5. Response of A_ pa 1ustr is. 'Penneagle' and 'Seaside' plants regenerated from calli selected from 51A mM supplemented medium, grown hydroponica 11y in Hoagland's #2 nutrient medium with 0 mM NaCl ( Q ) or at 51^ mM NaCl ( . Salinization proceeded in a stepwisegradient. At week 8 plants were exposed to 85 mM NaC1; at week A plants were exposed to 173 mM; at week 6 plants were exposed to 857 mM NaCl; and at week 8, if any plants were alive, the> were exposed to 51^ mM NaCl.

DRY

W

EIG

HT

CAIN

(g

) D

RY

WEI

GH

T CA

IH

(g)

70

S E L E C T I O N S O m M V S . 5 1 3 m MPENNEAGLE'

6

85 257

S

.j y

J

2

1

02Q 4 G a t o

W E E K S I N C U L T U R E o S E L E C T I O N S O m M ♦ S E L E C T I O N S 5/4 mJ/

SEL ECT IONS O mM V S . 5 1 3 m M€

2 5 7

S

3

Z

1

00 Z 4 6 6 t o

W E E K S I N C U L T U R E D S E L E C T I O N S O m M * S E L E C T I O N S S t A m M

71

Fig. i. Callus cultures from ft. palustris 'Penneagle'(top) and 'Seaside’ (bottom) cultured an a medium supplemented with 51^ mM NaCl. Lighter areas are portions of cultures where cells are actively proliferating. Such areas were selected and transferred.

73

Fig. 7. Response of pa 1ustris. 'Penneagle' (top left), and’Seaside’ (top right) plants regenerated from NaCl- adapted talli cultured hydroponica 11y in Hoagland’s #3 nutrient medium at 0 mM NaCl. On the bottom are A . palustris plants regenerated from NaCl-adapted calli in 057 mM NaC1.

75

Fig- 9. ftqrostis palustr 1 s. 'Seaside’ plantlets regenerated on medium supplemented with 51^ mM NaCl. Note succulence, dark color, and stunted morphology of plants as a typical symptom of salt stress.

It

/i*

SY N T H E SI S OF SALT SHOCK PROTEINS IN CALLUS CULTURES OF

flg r o s t i s p a 1 u s t r is H u d s . IN RESPONSE

TO NACL STRESS

Ab s t r ac t :

Callus cultures of Aq r o s tis p a l u s tr is Huds. grown on a

me d i u m sup p l e m e n t e d with 51L mM NaCl produced several unique

pr otei n bands on one dimensional SDS p o l y a c r y l a m i d e qel

slabs compared to calli grown on medium without NaCl. A p

parent increases in the density of some already pxistinq

pr o tein bands and reduct i o n s in the density of others were

also observed. Induced synthesis of novel proteins was o b

served after the second week on N a C 1- s u p p 1emented media.

After sub c u l t u r i n q N a C 1-adapted calli onto media without

NaCl for one transfer period <30 days), the production of

stress induced proteins ceased, or could not be detected on

one dimensional gel slabs.

77

7B

A b b r e v i a t i o n s :SDS PAGE: sodium dodecyl sulfate p o l y a c r y l

amide gel electrophoresis, MB: Mur a s h i g e and Skoog (13),

PMRF : p h e n y 1 m e t h y 1s u 1f o n y 1 fluoride, EDTA: ethylene-

d l a m l n e t e t r a a c e t ic acid d i s o d i u m salt, DTT: d i th lothrei tol i

HEPEB: N - 2 - h y d r a x y e t h y 1piper a 2 i n e - N ’- 2 - e t h a n e s u 1fonic acid,

TRI5: t r i s (h y d r a x y m e t h y 1)a m l n o m e t h a n e , Tween 20: polyoxy-

ethyle le sorbitan monolaurate.

7 9

INTRODUCTION

A stress-inducedi unique class of p r o t e i n s ( often

referred to as ‘’shock proteins"* are presently being r e

searched for a putative role in the tolerance of plants to

environmental stresses <1* 9, 9, 10). Although no strong

co i'relation b e t ween the production of stress-induced

proteins and any increased survival advantage under c o n d i

tions of environmental extremes has been established yet,

the asso c i a t i o n is often made (10).

Much of the current research with "shock proteins" is

in three areas. R e s e a r c h e r s in these areas are seeking to

det e r m i n e the location in the cell and the time when stress

p roteins are synthesized! how long s ho ck - i n d u c e d proteins

remain in the absence of a selective pressure (stress); and

c h a r a c t e r i z i n g the nature of the genetic regulation of the

proteins.

Significant a l t erations in the p r o t e i n c om p o s i t i o n of

many plants and animals in response to changes in their e n

vironments have been reported (9, 5, t, 7, 9* 10, 11, It,

IB). Sever al studies have reported c hanges in p o 1ypep tides

that are directly a t t r ibutable to osmotic stress (7, 8, IB).

Shock or stress prot eins have been observed at the whale

plant level of o r g a n i z a t i o n (11) and in cultured tissues and

80

cells (7, 18).

Nico t i ana to b a c u m , a dicotyledon, has traditionally been

used in model systems for i n vitro stress physioloav r e

sear c h ( 1 A , 15, 19). E r i c k s o n and Alfinito ( 7 ) , and Sinqh

et. al (18) used tobacco suspen s i o n cultures to investigate

p rot e i n s associated with NaCl stress. Many of the w o r l d ’s

most valuable horticultural and agronomic crops, however,

are monocotyledons, and little work has been done to

e l u c i d a t e some of the p r o t e i n changes occ u r r i n g in monocots

as a result of salt stress.

The purpose of this study was to utilize a

m o n o c o t y l e d o n o us flqrostls palustris, to investigate the

synthesis of salt shock proteins contained in the total

soluble protein fraction of NaCl - a d a p t e d and nan-adapted

calli. Two member s of A . p a l u s tr is, 'P e n n e a q 1e ’ , a s a 1 t -

sensitive cultivar and ' S e a s i d e ’, a relatively salt-

tolerant cultivar were compared.

Ma t e r i a l s and Methods

Establi s h m e n t of be n t g r a s s callus

Callus cultures of ' P e n n e a g l e ’ and ' S e a s i d e ’ creeping

bentgrass (Aqro s t i s p alu s t ris H u d s . ) wer e established on

modified MS medium (13), supplemented with 1 m g /1 BA, 5 m g /1

2,A-D, 30 g/1 sucrose, and 10 g /1 agar. Prior to autoclav-

a 1

i nq (1 kq/cm1', 121° C for 15 min) , the pH was adjusted to

5.7 with 2N N a O H . After autoclavinq pH was checked and

readju s t e d with NaOH if necessary. Car y o p s e s were surface

st e r i l i z e d by immersion in 95 Vi ethyl alcohol for 5 minutes

followed by immersion in 1.05*/. sodium h y p o c h l o r i t e ( 20'/. c om-

mercial bleach), s u p p l emented with 0 . 0 1 V. Tween 20

(surfactant) for 20 min. Following sterilization, caryopsps

wer e rinsed 3 times in sterile, d i stilled w a t e r . T wo to 5

c a r y o p s e s were e s t a b l i s h e d in 25 * 150 mm culture tubes and

incubated in the dark at 20° C for 30 days. After 30 days,

cultures were transferred to fresh medium, then incubated in

the dark for an additional 30-days.

Induction of sodium c h l o r i d e stress proteins

At the end of the second 30-day dark incubation period,

callus cultures gen e r a t e d from caryopses were divided into

a p p r o u m d t e l y equal segments weighing b e t w e e n 75-100 mq and

labeled. Since the 2 a p p r o x i m a t e 1y equal segments of

tissue o r i g i n a t e d from a common culture, each pair was

referred to as sister calli. One sister callus was s u b c u l

tured onto fresh media supplemented with no NaCl and the

other sister callus onto 5 1 g mM <30 g /1) N a C 1. On a daily

basis far the first week, then on a weekly basis for an 12

additional weeks, calli selected for transfer because of

82their ability to p r o l i f e r at e on NaCl* and their c o r r e s p o n d

ing sister cultures from the control treatment were c o l

lected, Calli collected from N a C 1- s u p p 1emented medium and

from control medium were placed into 2 different petri

dishes (according to treatment) and mixed thoroughly. Two

gram samples from each treatment were removed. frozen in

liquid nitrogen, and lyophilized.

Sample p r e p a r a t i o n and e x t r a c t i o n of soluble proteins

One gram of each lyophilized sample was ground with a

Thomas motorized tissue homogenizer at full speed in 10

volumes of chilled acetone (- B O ° C ) in an ice bath, suction

filtered, and placed in a desiccator (under vacuum) until

all of the acetone had evaporated. Soluble proteins were

extracted with a Thomas motorized tissue homogenizer in ~n

icebath for 30 sec from 100 mg samples of the acetone powder

in 2 ml of lysis buffer c o n t aining 50 mM HEPES (pH 7.5 at q 0

C ) , mM DTT, 17 mM 2 - m e r c a p t o e t h a n o 1, 2 mM EDTA, and 1 mM

P M S F . The h o mogenized samples were transferred to 30 ml

Corex ce n t r i f u g e tubes and centr i f u g e d at 50,000 x G for 20

min at U 0 C. One hundred ul aliquots were removed from each

of the samples. The remaining supernatants were then c a r e

fully pipetted into individual screw top m i c r o c e n t r i f u g e

tubes, frozen in liquid nitrogen, and stored at -80° C for

03subsequent analysis.

Pr otein e s t i m a t i o n

The 100 ul aliquots p r e v iously removed from each of the

samples were analyzed for protein concentration. Total

soluble p r otein in each sample was determined by the Bio-Rad

pr o tein assay method. Bovine gamma g l o b u l i n was used to

qenerate a standard c a li br at io n curve for protein q u a n

tification. Absorb a n c e at 595 nm was read using a Bausch

and Lomb Spectronic PI spectrophotometer.

E l e c t r o phoretic co n d i t i o n s

Samples from the experimental calli containing 25 uq of

d is s o l v e d p r o tein in total volumes of 20 ul of buffer were

loaded into the we 11s of a 13’/. p o l y a c r y la mi de qel slab.

E l e c t r o p h o r e s i s was con d u c t e d according to the method

described by Laemmli < 1 2 > , however, a di s c o n t i n u o u s TRIS-

q l y cine buffer was used. Samples were e 1e c t rophoresed for

IP hours at 20 mAmps constant current in a refriqerated

chamber at 5° C. Molecular weight markers provided in W-SDS-

17 and M W - 5 D S - 2 0 Q p r otein molecular marker kits of the Sigma

Chemical Company were used. A total of 12 standards ranging

from 2 0 0,000 to 2 , 510 d a ltons were e 1ec t r o p h o r e s e d alonq

with the samples. Gels were then stained in 0.1257.

0 L

Coo m a s s i e RS50 for 0 hours* destained in 50*/. methyl alcohol

and 1OV. glacial acetic acid for 1 hr and then finally

cleared in 5 V. methyl alcohol and 77. glacial acetic acid

overnight. The gels were rinsed for one hour in distilled

water and then placed in a 0.17, solution of silver nitrate

for 30 min. Silver was developed in 3V. sodium carbonate and

0.05 7. f or ma l d e h y d e solution.

RESULTS

P r otein patterns of s a l t - adapted and control calli

Lanes 1 and 2 of the gel contain soluble proteins of

' P e n n e a g l e ’ and ' S e a s i d e ’ control calli* respectively

(Figure 1). Lanes 3 and ^ c o n tain the p ro t e i n s of salt-

adapted callus lines from ' P e n n e a g l e ’ . Lanes 5 and 6 c o n

tain the proteins of s a l t- a d a p t e d ' S e a s i d e ’. The first a p

parent d if fe r e n c e in p r otein banding occurred at a p

p ro x i m a t e l y 85.7-kd. There is a reduced p r otein band at this

location on the gel in both ' P e n n e a g l e ’ and ' S e a s i d e ’ salt-

adapted callus lines c om p a r e d with the corresp o n d i n q control

callus lines. There are several other p r otein differences

among cul t i v a r s and treatments* but the most strikinq d i f

ference was found in the protein bands welqhing a p

pr oximately 31.5-kd. It is at this molecular weiqht that

unique p ro t e i n s are synth e s i z e d in salt-adapted calli from

85

bath c u l tivars compared with their c o rresponding controls.

At ap p r o x i m a t e l y 19.1— kd and 18.2 kd» there appeared to be

cultivar p r otein differences. ' P e n n e a g l e ’ produced enhanced

levels of proteins at these molecular w e i q h t s . Since e q u i v

alent amounts of total p r otein were loaded into each lane,

it is assumed that diffe r e n c e s in the densities of the d i f

ferent bands represent differences in proteins. At a p

p ro x i m a t e l y 1B .9 - k d , calli from controls of both cultivars

produced enhanced levels of proteins compared to proteins

from their salt-adapted lines. ’S e a s i d e ’ produced increased

amounts of 18.2-kd proteins compared with both ’P e n n e a g l e ’

control and s a 1t -stressed lines and ' S e a s i d e ’ control

calli. There appeared to be additional d i fferences in the

lower molecular weight banding range.

Thirty days after being placed on control media, N a C 1 -

adapted calli from both cultivars failed to produce d e t e c

table p ol yp ep t id e banding at the 31.5-kd site on gels.

However, when placed back on N a C 1 -s u p p 1emented media their

growth was only slightly inhibited.

DISCUSS I ON

The results of this research corroborate some of the

findings of studies using tobacco < N i c o t i ana t ab ac u m s p p . >

liquid cell suspensions. E ri c k s o n and Alfinito (7), working

with N aC l - r e s i s t a n t tobacco cell suspensions, found that

there was enhanced p o l y p e p t i d e banding in the molecular

weight range of 32-kd and 20-kd. Singh et a l . <18) found in

creased levels of 21-kd, 19.5-kd, and 10-kd proteins in

N a C 1-adapted cell lines of cultured tobacco. Increased

levels of 19.1-kd and 18.2-kd proteins were detected in

' P e n n e a g l e ’ and 1B .5 — kd in ' S e a s i d e ’. However, whereas both

Singh et al. and Erickson a nd Alfinito ( 7 ) found a protein

unique to salt-adapted lines at 56-kdi the novel protein was

consistently found at 31.5-kd in both cul t i v a r s of creepinq

bentgrass. Another apparent differ e n c e was the decrease in

the 18.9-kd protein in NaCl -adapted Aqr os t is cultivars,

whereas Singh et al. (IB) reported an increase in protein of

that molecular weight range. Erickson and Alfinito (7)

found that when the N a C 1-adapted cell lines were transferred

to control medium, they lost their ability to grow in salt-

contai n i n g media and behaved like normal cells. There was a

complete loss in the p r o du c t i o n of both S O — kd and 3S-kd

p r o t e i n s after IS days (approximately 1 transfer period).

It took 2 to 3 passages for the 26-kd p r otein to become u n

detectable. Likewise, it took 30 days (one transfer period)

for the 31.5-kd salt-adapted bentgrass callus protein to b e

come undetectable. The presence of the unique 31.5-kd

pr otein in be n t g r a s s calli was detected after 2 weeks on

8 7

N a C l - s u pp le me nt ed media, and became more apparent with in

creasing levels of adaptation. It is, therefore, suqqested

that for b e n tgrass calli the 3 1 . 5 — k d protein is a salt

s t r e s s - 1 nduced protein. It may also be p os s i b l e that the

enhanced levels of 16.5-kd proteins play some role in the

a da pt a t i o n of 'Seaside' to N a C 1- s t r e s s .

LITERA T U R E CITED

Altschuler, M. and J.P. Mascarenhas. 1982. Heat shack

Mo 1 . Biol. 1 : 103-115.

Barnett, T., M. Altschuler, C.N. McDaniel, and J.P. M a s

carenhas. 1980. Heat shock induced p r o t e i n s in plant

cells. Deve. Gen. 1: 331- 39 0.

Baszczynski, C.L. and B.B. Walden. 1981. Regulation of

gene e x p r es si o n in c o r n (Zea mays L.) by heat shock.

Can. J. Biochem. 60: 560-579,

Bewley, J.D. and M.J. Oliver. 1983. Responses to a

c h anging environment at the molecular l e v e l : Does des-

s i c a tlo n modulate p r o t e i n synthesis at the t r a n s c r i p

tional or t r a n s 1 a t i o n a 1 level in a tolerant p l a n t 7 p.

195-169. In: D. Ran d a 11 (e d . > . Cur rent topics of plant

b i o c h e m i s t r y and physiology, Vol. 2. Univ. Missouri

Press. St. Louis, M D .

Brown, I.R. and S.J. Rush. 1989. Induction of a stress

p r otein in intact m a m m a l i a n organs after the intravenous

a d m i n i s t r a t i o n of sodium arsenite. Biochem. Biophys.

Res. 120: 150-155.

Cooper, P. and T.H.D. Ho. 1983. Heat shock proteins in

maize. Plant Physiol. 7 1: 215-222.

of heat shock in plants. Plant

09

7. Erickson* M.C. and S.H. Alfinito. 1989. Proteins

produced during salt stress in tobacco cell culture.

Plant P h y s i o l . 79: 506-509.

8. Fleck* 3., A. Durr, C. F n t s c h , T. Vernet , and L.

Hirth. 19 0 P . □ s m o t l c -shock ’s t r e s s - p r o t e in s ’ in

proto p l a s t s of M ic o t lana s y 1v e s t r i s . Plant 9cl . Let. 86:

159-165.

9. Hahn, G.M. and G.C. Li . 1982. Thermo to 1erance and heat

shock proteins in m a mm al ia n cells. Radiat. R e s . 92: 952-

957 .

10. K a n a b u s , 3 . » C.S. Pikaard, and J.H. Cherry. 1989. Hea t

shock proteins in tobacco cell s u s p e n s i o n during growth

cycle. Plant Physiol. 75: 639-699.

11. Key, J.L., C.V. Lin, and Y.M, Chen. 1981. Heat shock

p ro t e i n s of higher plants. Proc. Nat. Acad. S c l . U.S.A.

70: 3526-3531.

12. Laemmli, U.K.. 1970. Cleavage of structural proteins

during the assembly of the head b a c t e r i o p h a g e T-9. N a

ture. 227: 6B0-6B5.

13. Murashige, T. and F. Skoog. 1962. A revised medium for

rapid growth and b i o a s s a y s with tobacco tissue cultures.

Physiol. Plant. 15: 973-997.

1*+.

15.

1 6 .

17 .

18.

1 9 .

9 0

Nabors, li. W . 1983. Increasing the salt and drought

tolerance of crop plants. p. 165-189. In: D. Randall

Cur rent topics of plant bioc hemlstry and physiology,

Vol. E, Univ. Missouri Press. S t . Louis, MO.

Nabors, M.W. and T.A. Dykes. 1989. Tissue culture of

cereal c u lt iv a rs with increased salt, drought, and acid-

tolerance. p . 1E 1 - 139. In: Biotec h n o l o g y in i nt e r n a t l o n a 1

agricultural research. proce e d i n g s of the intei— center

seminar on international agricultural research (L A R C S )

and biotechnology. International Research Institute

Ph i 1 1 i p i n e s .

P o 1 j a k o f f - M a y b e r , A. 1900. Biochemical and p h y s io 1oqica 1

res p o n s e s of higher plants to salinity stress. Biosaline

Res. E 3: E95-E69.

Sachs, M., M. Freeling, and R. Qkimoto. 1980. The

anaerobic proteins of maize. Cell E 0 : 761-767.

Singh, N.K., A.K. Handa, P.M. Hasagawa, and R.A. Bres-

s a n . 1985. P r o t e i n s associated with adapta t i o n of c u l

tured tobacco cells to N a C 1. Plant Physiol. 79: 1E6-137.

Tal, M. 1983. S e l e c t i o n for stress tolerance. p.

961-988. In: D.A. Evans, LI .R . Sharp, P.V. Ammirato, Y.

Yamada, (eds.), Handbook of plant cell culture. Vol. 1.

M a c m i 1lan Pub 1 i sh i ng C o . , N . Y .

91

Fig. 1. One dimensional 5D5 Polyacrylamide gel slab. In a total volume of 20 ul, including sample buffert each lane contains 25 ug of soluble protein.Proteins from calli of ft . pa 1ust r is . 'Penneagle' and 'Seaside’ cultured at 0 mM NaCl are in lanes 1 and 2, respectively. Proteins from 'Penneagle’ calli selected over three generations on medium supplemented with 51^ mM NaCl are in lanes 3 and ^ . Proteins from calli selected over three generations on medium supplemented with 51^ mh NaCl are in lanes 5 and 6. Molecular weiqht standards are at the far left.

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APPENDIX

9 3

A N O V A TA B LE 1. P e r c e n t p a l u s t r 1s 'Penneagle' and 'Seaside' c a l l u sc u l t u r e s s e l e c t e d and t r a n s f e r r e d ove r t h r e e g e n e r a t i o n s .

SOURCE OFVARIATION DF 55 MS F-5TAT

Cu 1tivar (C ) 1 156. 3 156.3 1 .3S a 1t/Non (S ) 1 0556.3 0556.3 71.7**Generatn (G) 0 950. A A76.0 A .00 X S 1 000.0 000.0 1 .8C X G 0 535.0 067.6 P.PS X G 0 3A6.P 173. 1 1 .5C X S X G 0 1000.1 5A1 .0 A. 5*Error PA P866 .0 1 19 .ATotal 35 1 . Ae + OA

* S i g n i f i c a n t at .05** S i g n i f i c a n t at .01

95

ANOVA TABLE E?. Percent A . pa 1ust r i s 'Penneagle' and 'Seaside' cacultures with shoots. Resu1ts fcom three generat

SOURCE OFVAR 1A T ION___ DF S S MS F-STAT

Cultivar (C) 1 3.7 3.7 8.1**Sa 1t/Nan (S ) 1 31.9 31.9 70.8**Generatn (G) 2 6.5 3.3 7.3*C X 5 1 3.7 3.7 B. 1 **C X G 2 E. 1 1 . 1 2.3S X G a 6.5 3.3 7.3*C X S X G a a. i 1 .0 2.3Error 10.0 0.5Total 35 67.3

* Significant at .05* * Significant at .OtG

A N O V A T AB LE 3. P e r c e n t A^_ pa 1u s t r 1s ' P e n n e a g l e ’ a nd ' S e a s i d e ’ c al luc u l t u r e s w i th r oo ts . R e s u I t s f r o m t hr e e g e n e r a t 1o n s .

SOURCE OFVARIATION DF S5 MS F-STAT

C u 1t ivar (C > 1 831 -A 031 .9 27 . 0**Sa1t/Non (5) 1 3863.3 2862.3 93 . 1 **Generatn (G) 2 91 .6 95.B 1.5C X S 1 1100.1 1100.1 35.8**C X G 2 780.3 360. 1 11 .7**S X G 2 280.0 109.0 3.6C X S X G 2 179.2 87. 1 2.8Error 29 73B.0 30.8Total 35 6735.6


97

ANOVA TABLE 9. Growth of A_ pa 1 ustr is 'Penneagle' and 'Seaside' callus cultures over three generations which had not increased in mass beyond original transfer size.

SOURCE OF VARIATION DF SS MS F-STA1

Cu1tlvar 1 92. B 92. B 0.9Sa1t/Non 1 1.6e + 09 1.6e+09 157.3##Gener a t n 2 337. 1 1SS.5 1 .7A X B 1 0.9 0.9 0.0A X C 3 90.9 20.2 0.2B X C 3 902 . 6 201 .3 2.1A X B X C 3 102.0 51.0 0.5Error 3h 2925. B 101.1Total 35 1 .9e+09

* S i g n i f i c a n t at .05## S i g n i f i c a n t at .01

98

ANOVA TABLE 5. Growth of A^ pa 1ustris 'Penneagle' and 'Seaside' callus cultures over three generations which increased in mass» but not double transfer size.

50URCE OFVAR 1 AT ION DF SS MS F-STA'

Caltlvar (C ) 1 33. 1 33. 1 0.6Salt/Non (S) 1 1 . 5e+0A I.5e+0A 873.8Generat n < G ) 8 688.5 311.8 5.5C X S 1 585.8 585.8 9.AC X G 8 86.0 13.0 0.2S X G 8 1 10.6 55.3 1 .0C X S X G 8 AA . A 82.8 0. AError 2A 1338.8 55.8Total 35 1 . 8e + 0A

» S i g n i f i c a n t at .05#* S i g n i f i c a n t at .01

99

ANOVA TABLE 6. Growth of A^ pa 1 ustris 'Penneaq1e ' and 'Seaside’ calluscultures over three generations which had doubled in mass from transfer size.

SOURCE OFVARIATION DF ss MS F-STAT

Cultjvar (C) 1 1 3 3 . 6 1 2 3 . 6 1 9 . 6 * *Sa11/Non (S ) 1 6.9e + 09 6. 9e + 09 7 6 2 5 . 0 * *Generatn (G) a 39.6 19.0 a . 3C X S i 305. 1 a e s . 1 33.0**C X G a 9.6 a . 9 0 . 3S X G a 153.9 80. u 9.1**C X S X G a 16.0 8.0 1 .0Error PR aoa.7 0.9Total 35 6.5e + 09


100

ANOVA TABLE 7. Percent A_ pal ustr i s 'Penneagle' and 'Seaside calluscultures over three generations with 0 percent necrosis.

SOURCE OFVARIATION DF SS MS F-STAT

C u 11 1 var (C ) I 66.7 66.7 *r-in

Sa 11 /'Non ( 5 ) 1 6 . 2e + 04 6 .2e + 0A 52A3.1**Generatn (G > 2 ino 20.2 1 . C X S 1 1 A .6 1A .7 1 .3C X G 2 29A . 1 1A7.0 12.5**S X G 2 3.2 1 .6 0. 1C X S X G 2 212.7 1 06. A 9. 0**Error a u 202.7 U .BTotal 35 6.3e+OA

* Significant at .05*# Significant at .01

101

ANOVA TABLE B. Percent A_;_ pa 1 ustr i s 'Penneagle' and 'Seaside' callus cultures over three generations with 1-25 percent necros 1s .

SOURCE OFVARIAT ION DF SS MS F-STAT

C u 1tivar (C ) 1 2176.2 2176.2 55.6**S a 1t/Non (S ) 1 1 . 0e + 09 1.0e + 09 257.1*«Generatn (G) 2 192.5 96.2 2.5C X S 1 1 139 .6 1139.6 20. 0# *C X G 2 362.5 101 .2 9 .6*S X G 2 969.2 239 .6 *oin

C X S X G 2 1222.7 611.9 15.6**Error 29 939. A 39. 1To ta 1 35 1.7e*09

* S i g n i f i c a n t at .05** S i g n i f i c a n t at .U1

102

ANOVA TABLE 9. Percent A_ pa 1 us t r 1 s 'Penneagle' and 'Seaside' callus cultures over three generations with 26-50 percent necrosis.

SOURCE OF VARIAT ION DF SS MS F-STAT

C u 1t lvar 1 197B.B 1970.B 37.6* *Salt/Nan 1 1 .2e+09 1.2e+09 228.5*#Genera t n 2 236. 1 110.1 2.2A X B 1 1285.2 12B5.2 29 .9**A X C 2 675.0 337 .9 6. 2*B X C 2 16.9 B . 2 0.2A X B X C 2 155. 1 77.5 i 1“1 M

Error 29 1269.1 52.7Total 35 1 . 0P + O9


103

ANOVA TABLE 10. Percent A pa 1ustr 1 s ’Penneagle’ andcultures over three generations with percent necrosis.

SOURCE OFVARIA T ION DF SS MS

Cultivar < C ) 1 32.3 32.3Salt/Non (S ) 1 1 1B2.2 1 102.2Generat n < G > 2 461 .3 230 .6C X S 1 31 .9 31.9C X G 2 216.9 100.5S X G 2 460 . 2 230. 1C X S X G 2 217.9 109.0Error 24 344 . 0 14.3Total 35 2946.7

'Seaside ’ call us greater than 50

F-STAT

£.302.5**16.1**2 . 2 7.6*

16.1**


109

A N O V A TABLE 11. Percent A^ palustris 'Penneagle' and 'Seaside'regenerated calli over three generations producing shoots.

SOURCE OFVAR IAT ION DF S S M5 F-STAT

Cultivar (C ) 1 113.0 113.0 1 .35 a 1t/Non (5) 1 1 . 9e+09 1 . 9e+09 153.3*#Generatn (G) 2 093.5 921 .0 9.6#C X S 1 9312.1 9312.1 97.5#*X X G 2 909. 1 969 .5 5. 1*5 X G 2 O ru **4Oru 0.2C X S X G B 220.9 1 10.2 1.2Error 29 2100.0 90.0Total 35 2.3e+09

* S i g n i f i c a n t at .05## S i g n i f i c a n t at .01

105

ANOVA TABLE IE. Percent A_ pa 1 ustr is 'Penneagle' and 'Seaside'regenerated calli over three generations producing roots.

SOURCE OF VARIATION DF SS MS F-STAT

C u 11 i var 1 3i>A .2 36A. 2 A . 0Sa 11/Non 1 5A0.6 5A0.6 5.9*Genera tn a 60A .6 302.3 3.3A X B i IA12.5 1A 12 .5 15.5**A X C 2 5A7.2 273.6 3.0B X C 2 37A1.3 1070.7 20.5**A X B X C 2 A A . a 22. 1 0.2Error PA El 87.2 91.1Tota 1 35 9AA 1 .7


VI TA

John Casper Hovanesian, the son of Luc iene and Ar-

shag H o v a n e s 1 a n , was born in New Britain, Co nnec ticut on

November 8, 1998. He was reard in Connecticut and M a s

sachusetts where he attended public schools for his

elementary and secondary education. In 1978, He

received a B.A. degree in History from the University of

Connecticut. In 1979, he received a B.A. degree in

M a r i n e Science from New England University (Saint F r a n

cis College). He was graduated the top ranking student

in the Department of Marine Science. His M.S. degree in

Biology was earned from the University of Southern M i s

sissippi in 1981. I n i 9 B 2 , he entered Louisiana State

Univer s i t y and anticipates receiving his Ph.D. degree in

H o r t i c u l t u r e with minor diciplines in Botany and Plant

P h y s i o l o g y in May, 1987.

1 06

DOCTORAL EXAMINATION ANI) DISSERTATION REPORT

C a n d i d a t e : John Casper Hovanesian

M a j o r Eield: Horticulture

T i t l e Of D i s s e r t a t i o n . IN VITRO REGENERATION AND PROTEIN CHANGES ASSOCIATED WITH TWO CULTIVARS OF Agrostis palustris Huds. CULTURED UNDER HIGH SODIUM CHLORIDE CONDITIONS

A pproved .

Major Professor and Chairman I k n m f r D . i t y s

I XT'Dean of the Graduate Ret tool

E X A M I N I N G C O M M I T T E E :

>-David H. Picha

( j , < T J ~ ^ - James F. Fontenot

0 <%/* Wk'-CV'—Blackmbn

t* t ; t. L L L- i' — -

Lowell E. Urbatschy L

Fogg

D a t e of E x a m i n a t i o n :

March 23, 1987

in vitro regeneration and protein changes associated with

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