TRANSFORMATION OF ‘GALIA’ MELON TO IMPROVE FRUIT QUALITY

By

HECTOR GORDON NUÑEZ-PALENIUS

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2005

Copyright 2005

by

Hector Gordon Nuñez-Palenius

This document is dedicated to the seven reasons in my life, who make me wake up early all mornings, work hard in order to achieve my objectives, dream on new horizons and

goals, feel the beaty of the wind, rain and sunset, but mostly because they make me believe in God: my Dads Jose Nuñez Vargas and Salvador Federico Nuñez Palenius, my Moms Janette Ann Palenius Alberi and Consuelo Nuñez Solís, my wife Nélida Contreras

Sánchez, my son Hector Manuel Nuñez Contreras and my daughter Consuelo Janette Nuñez Contreras.

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ACKNOWLEDGMENTS

This dissertation could not have been completed without the support and help of

many people who are gratefully acknowledged here.

My greatest debt is to Dr. Daniel James Cantliffe, who has been a dedicated advisor

and mentor, but mostly an excellent friend. He provided constant and efficient guidance

to my academic work and research projects. This dissertation goal would not be possible

without his insightful, invariable and constructive criticism. I thank Dr. Daniel J.

Cantliffe for his exceptional course on Advanced Vegetable Production Techniques

(HOS-5565) and the economic support for living expenses during my graduate education

in UF.

I extend my appreciation to my supervisory committee, Dr. Donald J. Huber, Dr.

Harry J. Klee, and Dr. Donald Hopkins, for their academic guidance. I am very thankful

to Dr. Donald J. Huber for his excellent advice on postharvest guidance, and also for his

excellent lectures in Postharvest Physiology (HOS-6331). I am very grateful to Dr. Harry

J. Klee for his constant supervision and help on plant molecular issues, as well as for his

outstanding teaching in Molecular Biology of Plant Hormones (HOS-5306). I express my

gratitude to Dr. Donald Hopkins for his welcoming assistance.

I would like to express my deepest appreciation to Dr. Richard Lee and Dr. Charles

Niblett for being part of my committee, although they had to leave before this dissertation

was completed. I would like to extend an special thank to Dr. Charles Niblett for all his

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help when I arrived at UF, and for being an excellent mentor, but mostly for his

exceptional friendship.

I extend my appreciation to Dr. Mark Settles for allowing me to use his lab to

accomplish some of the plant molecular issues, as well as to Diego Fajardo for his

technical advice and untied camaraderie.

I would like to make a special acknowledgement to my friends in the Seed

Physiology Lab for their help and support during these years: Reggie Salazar, Dr. Do

Kim, Dr. Javier Castellanos, Nicole Shaw, Dr. Ivanka Kozareva, Ashwin Paranjpe,

Yousef Al-Dlaigan, Juan Carlos Rodriguez, Elio Jovicich, Jennifer Bonina, JeanMarie

Mitchell, Teddy McAvoy, Jimmy Webb, Ji-Young and Cecil Shine III.

I would like to thank my friends in the Plant Molecular Lab: Dr. Mark G. Taylor,

Dr. Joseph A. Ciardi, Dawn Bies, Stephanie Maruhni, Michele Auldridge, Michele

Zeigler, Anna Block, Patricia Moussatche, and especially Dr. Denise M. Tieman and

Bryan Kevany for their outstanding technical advice and friendship to accomplish this

dissertation.

I would like to show appreciation to my friends in the Postharvest Lab: Dr. Steve

A. Sargent, James Lee, Muharem Ergun, Yasart Karakurt, Kim Cordasco, Adrian Berry,

Brandon Hurr and Daniel Stanley.

I would like to express my deepest appreciation to the National Council for Science

and Technology (CONACYT-México) and EDUCAFIN (Guanajuato-México) which

have supported my scholastic and living expenses during my graduate education in UF.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS ................................................................................................. iv

LIST OF TABLES............................................................................................................. ix

LIST OF FIGURES ........................................................................................................... xi

ABSTRACT..................................................................................................................... xiv

CHAPTER

1 INTRODUCTION ........................................................................................................1

2 LITERATURE REVIEW .............................................................................................4

2.1. Importance of Melon .............................................................................................4 2.1.1 Introduction ..................................................................................................4 2.1.2 Botany and Origin of Cucumis melo ............................................................5 2.1.3 Classification and Cultivars..........................................................................7

2.1.3.1 Naudin’s categories for Cucumis melo L. are listed below:...............9 2.1.3.2 Guis’s categories: ...............................................................................9

2.1.4 Climateric and Non-climateric Fruits .........................................................11 2.2 Postharvest Physiology of Melon .........................................................................11

2.2.1 Physiological Changes During Ripening....................................................11 2.2.2 Ethylene Production ...................................................................................14 2.2.3 Biochemical Changes During Ripening .....................................................15

2.2.3.1 Introduction ......................................................................................15 2.2.3.2 Carbohydrate Metabolism ................................................................16 2.2.3.3 Organic Acids...................................................................................19 2.2.3.4 Volatiles ...........................................................................................20 2.2.3.5 Cell Wall Degradation......................................................................23 2.2.3.6 Pigments ...........................................................................................26

2.2.4 Ethylene and Molecular Changes During Ripening...................................28 2.2.4.1 Introduction ......................................................................................28 2.2.4.2 Biosynthesis, Perception and Effects of Ethylene............................28

2.3 Melon Biotechnology ...........................................................................................36 2.3.1 Genetic Improvement .................................................................................36

2.3.1.1 Traditional Breeding ........................................................................36 2.3.1.2 Improvement Through Genetic Engineering....................................37

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2.3.1.3 Melon Biotechnology.......................................................................42 2.3.2 In vitro Regeneration..................................................................................43

2.3.2.1 Genetic Control ................................................................................44 2.3.2.2 Polyploidization and Somaclonal Variation.....................................46 2.3.2.3 Vitrification ......................................................................................51

2.3.3 Regeneration by Organogenesis .................................................................53 2.3.3.1 Medium Composition.......................................................................54 2.3.3.2 Environmental Factors .....................................................................60

2.3.4 Regeneration by Somatic Embryogenesis ..................................................61 2.3.4.1 Medium Composition.......................................................................65 2.3.4.2 Environmental Factors .....................................................................66

2.3.5 Haploid Plants and Embryo Culture...........................................................67 2.3.6 Genetic Transformation..............................................................................70

2.3.6.1 Improvement of Disease Resistance ................................................76 2.3.6.2 Improvement of Tolerance to Physical Factors................................78 2.3.6.3 Improvement of Postharvest Characteristics....................................78

2.4 ‘Galia’ melon ........................................................................................................82 2.4.1 Introduction ................................................................................................82 2.4.2 Botany and Origin ......................................................................................83 2.4.3 Postharvest Physiology...............................................................................86 2.4.4 Genetic Improvement by Conventional Methods and Biotechnology .......89

3 EFFECT OF EXPLANT SOURCE ON REGENERATION AND TRANSFORMATION EFFICIENCY IN ‘GALIA’ MELON (Cucumis melo L.) MALE AND FEMALE PARENTAL LINES ............................................................95

3.1 Introduction...........................................................................................................95 3.2 Materials and Methods .........................................................................................97

3.2.1 Plant Material .............................................................................................97 3.2.2 Agrobacterium Inoculation and Plant Transformation...............................97 3.2.3 Histochemical Staining for GUS Activity and GFP Detection ..................99 3.2.4 PCR Assay................................................................................................100 3.2.5 Experimental Design and Statistical Analysis..........................................100

3.3 Results and Discussion .......................................................................................101 3.3.1 Effect of Explant Origin on Regeneration Efficiencies............................101 3.3.2 Plant Transformation ................................................................................102 3.3.3 Summary...................................................................................................110

4 TRANSFORMATION OF A MUSKMELON ‘GALIA’ HYBRID PARENTAL LINE (Cucumis melo L. var. reticulatus Ser.) WITH AN ANTISENSE ACC OXIDASE GENE .....................................................................................................111

4.1 Introduction.........................................................................................................111 4.2 Materials and Methods .......................................................................................113

4.2.1 Plant Material ...........................................................................................113 4.2.2 Plant Regeneration....................................................................................113 4.2.3 Agrobacterium Inoculation and Plant Transformation.............................114

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4.2.4 Flow Cytometry Analysis.........................................................................115 4.2.5 Detection of Transgenes ...........................................................................116 4.2.6 Southern Blot Analysis.............................................................................117 4.2.7 Segregation Analysis of Transgenes in Primary Transformants ..............118

4.3 Results and Discussion .......................................................................................118 4.3.1 Transformation Efficiency........................................................................118 4.3.2 Ploidy Level of Primary Regenerants.......................................................120 4.3.3 Southen Blot Analysis ..............................................................................123 4.3.4 Transgene Inheritance in the T1 Progenies of Primary Transformants ....123 4.4.1 Conclusion................................................................................................124 4.5.1 Summary...................................................................................................127

5 EMBRYO-RESCUE CULTURE IN ‘GALIA’ MALE PARENTAL LINE MELON (Cucumis melo L. var. reticulatus Ser.)....................................................129

5.1 Introduction.........................................................................................................129 5.2 Materials and Methods .......................................................................................130

5.2.1 Plant Material ...........................................................................................130 5.2.2 Embryo Culture ........................................................................................131 5.2.3 Experimental Design and Statistical Analysis..........................................134

5.3 Results and Discussion .......................................................................................134 5.3.1 Embryo Development...............................................................................134 5.3.2 Regeneration Efficiency ...........................................................................136

5.4 Summary.............................................................................................................146

6 FRUIT RIPENING CHARACTERISTICS IN A TRANSGENIC ‘GALIA’ MALE PARENTAL LINE MUSKMELON (Cucumis melo L. var. reticulatus Ser.)...........................................................................................................................148

6.1 Introduction.........................................................................................................148 6.2 Materials and Methods .......................................................................................149

6.2.1 Plant Material ...........................................................................................149 6.2.2 Determination of Fruit Size ......................................................................151 6.2.3 Determination of Physical and Biochemical Characteristics ...................151 6.2.4 Experimental Design and Statistical Analysis..........................................154

6.3 Results.................................................................................................................155 6.3.1 ACC Oxidase Activity In Vivo ................................................................155 6.3.2 Ethylene Production .................................................................................155 6.3.3 Firmness, Rind Color and TSS.................................................................157 6.3.4 Mesocarpic Titratable Acidity, pH and Ripening Index ..........................159 6.3.5 Determination of Fruit Size and Seed Number ........................................161

6.4 Discussion...........................................................................................................164 6.5 Summary.............................................................................................................175

LIST OF REFERENCES.................................................................................................177

BIOGRAPHICAL SKETCH ...........................................................................................215

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LIST OF TABLES

Table page 2.1 Production of various cucurbits on different countries in 2004 (1,000 metric

tons). ...........................................................................................................................5

2.2 Similarities between conventional and biotechnological methods for melon plant improvement.............................................................................................................38

2.3 Melon regeneration (shoots, roots and/or complete plants) through direct organogenesis. ..........................................................................................................55

2.4 Melon regeneration through indirect organogenesis. ...............................................58

2.5 Melon regeneration through somatic embryogenesis...............................................63

2.6 Genes transferred to melon by plant genetic transformation. ..................................73

2.7 ‘Galia’ Type Muskmelon Imports to U.S.................................................................84

2.8 ‘Galia’ Type Muskmelon Unit Value Imports to U.S. .............................................85

3.1 Regeneration efficiency in ‘Galia’ muskmelon parental lines depending on explant source.........................................................................................................103

4.1 Transformation efficiency of ‘Galia’ male parental line with CMACO-1 antisense construct..................................................................................................120

5.1 Components of E-20A nutrient medium. ...............................................................133

5.2 Regeneration efficiency of ‘Galia’ male parental line immature embryos that underwent development to form normal plants......................................................141

5.3 Regeneration efficiency of ‘Galia’ male parental embryos cultured on two different media. ......................................................................................................145

5.4 Regeneration efficiency of ‘Galia’ male parental embryos depending on harvesting dates. .....................................................................................................145

5.5 Regeneration efficiency of ‘Galia’ male parental embryos depending on inoculation system (IS)...........................................................................................145

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6.1 Postharvest fruit characteristics of ‘Galia’ male parental line. ..............................163

6.2 Postharvest fruit characteristics of ‘Galia’ male parental line. ..............................165

6.3 Postharvest fruit characteristics of ‘Galia’ male parental line. ..............................166

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LIST OF FIGURES

Figure page 2.1 Global land area of cultivated transgenic crops.. .....................................................40

2.2 Yearly increase in global land area of transgenic crops...........................................41

2.3 Top Ten Countries growing GMOs in the World in 2004. From James, C. (2004). ......................................................................................................................42

3-1 Map of the T-DNA in pMON17204 and pCAMBIA 2202-sGFPS65T...................99

3.2 Organogenetic response of ‘Galia’ muskmelon parental lines depending on explant source.........................................................................................................101

3.3 Time schedule for de novo shoot regeneration in hypocotyl, cotyledon and true-leaf explants of ‘Galia’ muskmelon parental lines.................................................104

3.4 Stable expression of GFP and GUS genes in T0 shoots and roots of ‘Galia’ muskmelon parental lines.......................................................................................106

3.5 Transformation efficiency on ‘Galia’ muskmelon parental lines. GUS reporter gene was used to transform different explant sources............................................107

3.6 Transformation efficiency on ‘Galia’ muskmelon parental lines. GFP reporter gene was used to transform different explant sources............................................107

3.7 PCR assay for transgenic GUS ‘Galia’ melon male and female line plants. .........108

3.8 Time schedule for cotyledon and hypocotyl transformation systems in ‘Galia’ melon parental lines ...............................................................................................109

4.1 T-DNA region of binary vector pCmACO1-AS. ...................................................116

4.2 PCR assay for putative transgenic ‘Galia’ muskmelon male line plants. ..............119

4.3 Stable expression of β-D-Glucuronidase (GUS) gene in T0 shoots (A and B) and roots (C and D) of ‘Galia’ muskmelon male parental line. ....................................121

4.4 Transgenic (left) and non-transgenic (right) in vitro ‘Galia’ male line explants. ..122

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4.5 Flow cytometry analysis of propidium iodide-stained nuclei from wild type leaf tissue of ‘Galia’ male parental line. .......................................................................125

4.6 Flow cytometry analysis of propidium iodide-stained nuclei from transgenic leaf tissue of ‘Galia’ male parental line. .......................................................................126

4.7 Southern blot hybridization assay of male muskmelon transgenic plants. ............127

5.1 ‘Galia’ male parental line fruit at 4 DAP stage (a and b). (c) in vitro embryo development from 4 DAP stage. ............................................................................136

5.2 ‘Galia’ male parental line fruit at 10 DAP stage (a)...............................................137

5.3 ‘Galia’ male parental line fruit at 17 DAP stage (a)...............................................138

5.4 ‘Galia’ male parental line fruit (a and b) and embryo (c) at 24 DAP stage. ‘Galia’ male parental line fruit (d and e) and embryo (f) at 30 DAP stage............139

5.5 Normal ‘Galia’ male parental line seedlings obtained from embryo rescue, having well-developed cotyledonary (red arrows) and true leaves (blue arrows). 140

5.6 Percentage of ‘Galia’ male parental line immature embryos that developed to form normal plants. ................................................................................................143

5.7 Percentage of ‘Galia’ male parental line immature embryos that developed to form normal plants. ................................................................................................144

6.1 Wild type and TGM-AS ‘Galia’ male parental line fruit at 50 DAP. ....................152

6.2 Flesh size and seed cavity size determination in ‘Galia’ male parental line fruit.. .......................................................................................................................154

6.3 ACC oxidase activity in vivo in WT (∗), TGM-AS-1 ()), TGM-AS-2 (→), azygous-AS (=) and T-GUS (■) fruits....................................................................156

6.4 Ethylene production from WT (∗), TGM-AS-1 ()), TGM-AS-2 (→), azygous-AS (=) and T-GUS (■) fruits. .................................................................................157

6.5 Regression analysis between ACC oxidase activity and ethylene production in ‘Galia’ male parental fruits. ...................................................................................158

6.6 Ethylene production from WT (∗) and TGM-AS ()) fruits compared to firmness of WT (■) and TGM-AS (□) intact fruits...............................................................160

6.7 Ethylene production from WT (∗) and TGM-AS ()) fruits compared to rind color of WT (■) and TGM-AS (□) intact fruits......................................................161

6.8 Rind color in WT (A) and TGM-AS (B) ‘Galia’ male muskmelon fruit. ..............162

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6.9 Ethylene production from WT (∗) and TGM-AS ()) fruits compared to TSS content of WT (■) and TGM-AS (□) intact fruits. .................................................162

xiv

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

TRANSFORMATION OF ‘GALIA’ MELON TO IMPROVE FRUIT QUALITY

By

Hector Gordon Nuñez-Palenius

December 2005 Chair: Daniel J. Cantliffe Major Department: Horticultural Science

Stable genetic transformation in ‘Galia’ muskmelon has not been achieved to date.

The objectives of this study were to obtain an efficient and reliable in vitro regeneration

system for ‘Galia’ muskmelon (Cucumis melo L. var. reticulatus Ser.) parental lines, and

to attain a dependable transformation protocol as depicted through reporter genes such as

β-glucuronidase (GUS) and green fluorescent protein (GFP). Once this was accomplished

the ACC oxidase gene (CMACO-1) in antisense orientation was inserted into both

‘Galia’ parental lines using an Agrobacterium-mediated transformation system. By

reducing ACC oxidase, it was hoped that ripening would be delayed thus allowing a

longer time-frame to ship fruit in a firm condition. Three protocols using different melon

explants, i.e. cotyledon, hypocotyl and true-leaf, and several plant hormonal balances

were used on ‘Galia’ male and female parental lines with the aim to induce de novo shoot

in vitro regeneration. The best explant to induce in vitro regeneration was cotyledon

using 1 mg.L-1 benzyladenine (BA) and 0.001 mg.L-1 α-Naphthaleneacetic acid (NAA) as

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plant hormones. Efficient transformation rates as high as 13% were achieved when

hypocotyl and the GFP reporter gene were used, however, more than 27 weeks were

needed to attain seedlings. Using cotyledon as explants and either the GUS or GFP gene,

transformation rates of 8-10% were observed, and transgenic seedlings were obtained in

fewer than 11 weeks. The ACC oxidase gene from melon (CMACO-1) in antisense

orientation was used to transform ‘Galia’ male and female parental lines. Two (TGM-

AS-1 and TGM-AS-2) independent ‘Galia’ male transgenic diploid plants were obtained

using the ACC oxidase gene. A postharvest evaluation was carried out on ACC oxidase

transgenic and wild type fruits. Antisense fruits had reduced ACC oxidase enzyme

activity, as well as lower ethylene production compared to wild type fruits. Moreover,

fruit softening was delayed in CMACO-1. The results of this research indicated that the

insertion of an ACC oxidase gene in antisense orientation in the ‘Galia’ male parental

inbred reduced ethylene synthesis in transgenic (TGM-AS) T0 fruits. As a result of low

ethylene production by TGM-AS fruits, several parameters such as yellowing of the rind,

ripening index, and fruit softening were delayed by as much as 10 days. Other non-

ethylene dependent traits, such as fruit size, seed development, and mesocarpic total

soluble solids, titratable acidity and pH were not affected by the transgene presence.

TGM-AS-1 and TGM-AS-2 T1 seeds, obtained from these T0 evaluated fruits, will be

used to obtain and select future improved lines with extended shelf-life.

1

CHAPTER 1 INTRODUCTION

Melon (Cucumis melo L.) belongs to the Cucurbitaceae family, and is an important

worldwide commodity (Food and Agriculture Organization [FAO], 2005). Within

Cucumis melo species several important horticultural groups can be identified:

cantalupensis, inodorus, flexuosus, conomon, dudaim, and momordica (Robinson and

Decker-Walters 1999). Melons belonging to these horticultural groups vary in their field

performance and postharvest characteristics.

Developed from a breeding program initiated in the mid-1960s ‘Galia’ muskmelon

was the first Israeli melon hybrid. This commodity was released in 1973, and has

exceptional characteristics such as fruit quality with 13-15% total soluble solids (TSS),

bold flavor and a distinct musky aroma. Galia’s high-quality characteristics increased

local market popularity within a short time. In fewer than 10 years, ‘Galia’ melons were

distributed almost all over Western Europe, and became a unique and special market

category of muskmelon (Karchi, 2000). The main disadvantage of ‘Galia’ is its short

storage life, since it is harvested near peak maturity for optimum flavor. Storage is

limited to two or three weeks, even when it is maintained in low temperature (6-8oC).

Different strategies have been used in order to delay fruit softening in ‘Galia’ including

the use of controlled atmospheres plus adding an ethylene absorbent (Aharoni et al.,

1993a), reducing the storage time during transportation by using airfreight systems,

instead of seafreight shipping (Bigalke and Huyskens-Keil, 2000), and applying

inhibitors of ethylene action, such as 1-methylcyclopropene (Ergun et al. 2005).

2

Using traditional breeding methods and long-shelf life melon cultivars, breeders

have been able to develop a strategy to insert gene(s) which have increased ‘Galia’

muskmelon shelf-life. However, this approach has resulted in a loss of favorable fruit

quality characteristics (Karchi, personal communication). Using a plant biotechnology

approach by means of plant transformation is a feasible alternative to introduce novel or

native genes, which likely will increase ‘Galia’ muskmelon shelf life, without affecting

its unique characteristics. This approach was used to delay fruit ripening in Cantaloupe

melon (cv. Védrantais), by inserting the ACC oxidase gene in antisense orientation with

promising results (Ayub et al. 1996; Guis et al. 1997b; Silva et al. 2004). However, in

order to attain a consistent plant transformation protocol, it is imperative to have a

reliable and efficient in vitro plant regeneration system. Nevertheless, ‘Galia’ muskmelon

has not been easily in vitro cultivated and full regenerated plants are especially difficult

or are impossible to obtain (Leshem, 1989; Leshem et al. 1994a; 1994b; Gaba et al. 1994;

Gaba et al. 1996; Edriss et al. 1996; Kintzios and Taravira, 1997; Galperin et al. 2003a).

Moreover, Gaba et al. (1999) reported ‘Galia’ muskmelon to be recalcitrant to

transformation by Agrobacterium tumefaciens.Therefore, the research on plant in vitro

parameters to induce de novo shoot regeneration in ‘Galia’ muskmelon will provide

valuable information to attain a consistent and practical in vitro protocol, in order to

obtain fully regenerated ‘Galia’ muskmelon plants. Moreover, the research to find

favorable conditions for successful Agrobacterium-mediated transformation will enable

us to insert any gene of interest into this recalcitrant muskmelon cultivar, specifically

with the final aim to increase its shelf-life.

3

The objective of this research was to obtain an efficient and reliable in vitro

protocol to induce de novo plant regeneration in ‘Galia’ muskmelon parental lines, as

well as to attain a consistent and practical transformation methodology aimed to insert the

ACC oxidase gene in antisense orientation, and delay the fruit ripening process. In order

to achieve the objective of this research, several experimental approaches were taken.

These were to 1) examine the effect of several plant hormones and their balances, as well

as different melon explant sources on de novo shoot and root regeneration in ‘Galia’ male

and female parental lines; 2) establish the favorable conditions for Agrobacterium

tumefaciens-mediated transformation using two reporter genes, i.e., GUS and GFP in

‘Galia’ male and female parental lines; 3) transform ‘Galia’ male parental line with the

ACC oxidase gene in antisense orientation (CMACO-1); and 4) evaluate transgenic

CMACO-1 ‘Galia’ male fruit since a postharvest approach.

4

CHAPTER 2 LITERATURE REVIEW

2.1. Importance of Melon

2.1.1 Introduction

The Cucurbitaceae family consists of mostly frost sensitive, principally tendril-

bearing vine plants which are found in sub-tropical and tropical regions around the world

(Robinson and Decker-Walters, 1999). Nevertheless, some species within this family are

well adapted to low temperatures and xerophytic conditions (Wien, 1997). Plants

belonging to the Cucurbitaceae family are commonly well known as Cucurbits, and,

according to their geographic origin they can be classified as new world and old world

species. Two well-defined subfamilies, eight tribes, about 120 genera and more than 800

species are found in this family (Jeffrey, 1990). They are largely cultivated as vegetables

and several parts of the plants are utilized for foodstuff. Fruits are the most commonly

eaten part of the plant, but seeds, flowers, tendrils, very young shoots and roots are also

used for food. In addition, Cucurbits are exploited as medicines, such as Cucurbita

andreana, which has chemical compounds (cucurbitacins) with anticancer and

antiinflamatory activities (Jayaprakasam et al. 2003). In addition, in China, fruits and

roots of Cucumis melo are taken as emetic, leaves and seeds for hematoma, and stems to

reduce hypertension. Cucurbits are utilized for unusual purposes as well, such as to store

food (Gourd) or to be used as a sponge (Loofah) (Robinson and Decker-Walters, 1999).

The most important cultivated cucurbits –based on total production and harvestable

area– around the globe are watermelon (Citrullus lanatus Thunb.), cucumber (Cucumis

5

sativus L.), melon (Cantaloupe and other melons) (Cucumis melo L.), pumpkin

(Cucurbita spp.), and squash (Cucurbita spp.) (Table 2.1) (FAO, 2005).

Table 2.1 Production of various cucurbits on different countries in 2004. Region Watermelon Cucumber Melon Pumpkin & squash Asia 81,157 33,038 19,537 12,122 Europe 3,955 3,923 3,160 2,177 N&C America 3,135 2,059 2,440 1,942 Africa 3,800 1,074 1,601 1,782 South America 1,315 77 565 722 World 93,481 40,190 27,371 19,016

(1,000 metric tons). (FAO, 2005).

Among the major cucurbit vegetables, Cucumis melo has one of the highest

polymorphic fruit types and botanical varieties. This is as a consequence of genetic

diversity in this species (Mliki et al. 2001). Therefore, some melon fruits can have

excellent aroma, variety of flesh colors, deeper flavor and more juice compared to other

cucurbits (Goldman, 2002).

2.1.2 Botany and Origin of Cucumis melo

Most melons are trailing indeterminate length vines and up to 15 m long;

nevertheless some modern cultivars with shortened internodes, bushy appearance and

concentrated-yield have been bred (Paris et al. 1982, Paris et al. 1985, McCollum et al.

1987, Paris et al. 1988). All melons are frost-sensitive, but many differ in their ability to

survive cold and hot environments (Wien, 1997).

The main stem is almost round in shape. Stems may have some pubescence or not,

but when present it is not so pronounced as in other cucurbits (Zitter et al. 1996). Leaves

are simple, either three- or five-lobed, and borne singly at the nodes and commonly they

may have a great variation in size, color, and shape (Kirkbride, 1993). Tendrils are borne

in leaf axis and are simple (unbranched).

6

Sex expression in Cucumis melo is controlled by genetic factors, as well as by

environment (Wien, 1997; Robinson and Decker-Walters, 1999). According to Wien

(1997), at least four environmental factors, such as light energy, photoperiod, water

supply, and temperature, have a strong influence on sex expression. Normally,

physiological conditions which favor the increase of carbohydrates within the plant, such

as low temperature, low nitrogen availability, short photoperiod and high moisture

accessibility, promote female sex expression (Robinson and Decker-Walters, 1999).

These environmental factors affect plant hormonal balance, which in turn determines sex

expression. In general, gibberellins promote male flower development, whereas auxins

and ethylene induce female flower production (Karchi, 1970). Melon plants bear perfect

or imperfect flowers in several combinations: perfect (hermaphroditic) flowers are

capable of self-pollination, and imperfect flowers are either pistillate (female) or

staminate (male) (Zitter et al. 1996). Most melon cultivars are andromonoecious

(hermaphroditic and staminate flowers present at the same plant), although monoecious

(pistillate and staminate flowers) forms are found as well (Seshadri and More, 2002).

Melon fruits are generally classified as an indehiscent ‘pepo’, with three ovary

sections or locules. According to Robinson and Decker-Walters (1999), a ‘pepo’ is a

fleshy fruit with a leathery, non-septate rind derived from an inferior ovary. The edible

flesh is derived from the placentae or mesocarpic tissue (Seymour and McGlasson, 1993).

Among the different parts of a melon plant, fruit has the highest diversity in size, form,

external ornamentation, and internal and external color (Kirkbride, 1993). For instance,

Périn et al. (2002b) reported that fruits as short as 4 cm long (Cucumis melo L. var.

agrestis) and as long as 200 cm (Cucumis melo L. var. flexuosus), attaining weights of

7

between 50 g and more than 15 kg (a 300-fold variation size), are known (Naudin, 1859).

Variation is also expressed in flesh color (orange, orange light or pink, green, white or

even mixture of these colors), rind color (green, yellow, white, orange, red, gray or blend

of these colors), rind texture (smooth, warty, striped, netted, rough or combination of

these textures), form (round, flatten or elongated), and size (from 4 up to 200 cm)

(Kirkbride, 1993; Goldman, 2002). Some melon fruits (depending of the cultivar) when

ripe have an abscission layer at the attachment zone between the fruit and stem, whereas

others remain attached to the stem even after they are ripe (Kirkbride, 1993).

Africa and Asia have been suggested as possible sites of origin (Robinson and

Decker-Walters, 1999). Nevertheless, Kerje and Grum (2000) reported, based on genetic

studies, crossing attempts with other Cucumis species, and world distribution of melon

varieties, that the origin of melon appears to be Africa. The domestication process of

melon started in Egypt over 3,000 years ago (Pangalo, 1929). From this area, melon

species dispersed throughout the Middle East and Asia, where a secondary domestication

and diversification development may have ocurred (Kerje and Grum, 2000).

2.1.3 Classification and Cultivars

According to Jeffrey (1990), melon classification may be listed as follows:

Class: Dycotyledoneae

Subclass: Dilleniidae

Superorder: Violanae

Order: Cucurbitales

Family: Cucurbitaceae

Tribe: Melothrieae

Subtribe: Cucumerinae

8

Genus: Cucumis

Subgenus: Melo

Species: Cucumis melo

The high polymorphism of fruits in cultivated melons has lent botanists to propose

different infraspecific classifications. An excellent, updated and complete study on

Cucumis genus was undertaken by Dr. Joseph H. Kirkbride, Jr. (1993) from USDA. His

book titled “Biosystematic Monograph of the Genus Cucumis (Cucurbitaceae)” is a

cornerstone in melon classification.

In 1859 the french botanist Charles Naudin, using mostly living plants grown in the

gardens of the Natural History Museum of Paris, proposed the first useful system of

infraspecific categorization for Cucumis melo L. Naudin (1859) subdivided this species

into ten groups, which were later revised by Munger and Robinson (1991), proposing

trinomial names. However, several classifications have been reported (Alefeld, 1866;

Cogniaux and Harms, 1924; Whitaker and Davis, 1962; Pangalo, 1958; Filov, 1960;

Grebenščikov, 1986; Kirkbride, 1993; Pyzhenkov and Malinina, 1994; Robinson and

Decker-Walters, 1999). Recently, Pitrat et al. (2000) proposed a complete synthesis of

infraspecific classification of melon. They identified the synonymous epithets used in

several publications in order to propose their classification. These authors recognized 16

groups and denominated them as varietas or variety.

On the other hand, Smith and Welch (1964) and Robinson and Decker-Walters

(1999) considered that Naudin's categories are horticultural groups and not botanical

varieties based in phylogeny.

9

2.1.3.1 Naudin’s categories for Cucumis melo L. are listed below:

1. Cantalupensis group. Cantalupe and muskmelon. Medium size fruits with netted, warty or scaly surface, flesh usually orange but sometimes green, flavor aromatic or musky. Fruits with abscission layer at maturity. Usually andromonoecious plants.

2. Inodorus group. winter melons: honeydew, canary, casaba and crenshaw. Fruits usually larger, later in maturity and longer keeping than those of the Cantalupensis group. Rind surface smooth or wrinkled, but not netted, flesh typically white or green and lacking amusky odor. Fruits do not detach from the peduncle when mature. Typically andromonoecious plants.

3. Flexuosus group. Snake melon or Armenian cucumber. Fruits are very long, slender and often ribbed. They are used when inmmature as an alternative to cucumber. Monoecious plants.

4. Conomon group. Makura uri and Tsuke uri (pickling melons). Small fruits with smooth, tender skin, white flesh, early maturity and usually with little sweetness or odor. They are used as a pickling, but also eaten fresh or cooked. Andromonoecious plants.

5. Dudaim group. Pomegranate melon, chito melon, Queen Anne’s pocket melon and mango melon. Small, round to oval fruits with white flesh and thin rind.

6. Momordica group. phoot and snap melon. Small fruits with oval to cylindrical shape. Flesh is white or pale orange, low in sugar content. Smooth surface. Most of the cultivars are monoecious.

Alternatively, Guis et al. (1998) reported a new categorization of horticultural

important melons. These authors based their classification on a previous biosystematic

monograph of the genus Cucumis (Cucurbitaceae) reported by Kirkbride in 1993, who

used morphological, cytological and macro-distributional data to systematize that genus.

2.1.3.2 Guis’s categories:

7. C. melo var. cantaloupensis Naud. Medium size fruits, rounded in shape, smooth surface or warty, and often have prominent ribs and sutures, if there is netting, is sparse. Orange flesh, aromatic flavor and high in sugars.

8. C. melo var. reticulatus Ser. Medium size fruits, and netted surface. If ribs are present, they are not-well marked, flesh color from green, white to red orange. Most are sweet and have a musky odor.

9. C. melo var. saccharinus Naud. Medium size fruits, round or oblong shape, smooth with grey tone sometimes with green spots, very sweet flesh.

10

10. C. melo var. inodorus Naud. Smooth or netted surface, flesh commonly white or green, lacking the tipical musky flavor. These fruits are usually later in maturity and longer keeping than cantaloupensis or reticulatus.

11. C. melo var. flexuosus Naud. Long and slender fruit, green rind and finely wrinkled or ribbed. Green flesh and usually eaten as an alternative to cucumber. Low level of sugars.

12. C. melo var. conomon Mak. Small fruits, smooth surface, crisp white flesh. These melons ripe very rapidly, develop high sugar content but little aroma.

13. C. melo var. dudaim Naud. Small fruits, yellow rind with red streak, white to pink flesh.

On the other hand, Stepansky et al. (1999) proposed an intraspecific classification

of melons based on phenotypic and molecular variation. They studied a collection of 54

accessions representing diverse melon genotypes (cantaloupensis, inodorus, conomom,

chito, dudaim, momordica, flexuosus, agrestis and some non-defined varieties) from

more than 20 countries, building with their data a “botanical-morphological” dendogram.

Likewise, DNA polymorphism among the accessions was assessed using inter-SSR-PCR

and RAPD techniques. They concluded that the molecular phylogeny agreed, broadly,

with the classification of melon into two subspecies, and it did not contradict the division

into “horticultural varieties.”

Recently, Liu et al. (2004) concluded after an extensive evaluation of 72 melon

accessions belonging to six melon varieties -cantaloupensis, reticulatus, inodorus,

acidulus and saccharinus- that accessions which were previously classified in the same

variety by traditional taxonomy were also located closely to each other using Principal

Component Analysis (PCA) approach in 35 different morphological and physiological

plant characters.

In general, both Naudin’s and Guis’ categorizations have more common features

than contrasting ones; therefore both are well accepted among scientists.

11

2.1.4 Climateric and Non-climateric Fruits

Fruit in general can be classified as either climacteric or non-climacteric on the

basis of their respiration pattern and autocatalytic ethylene production peak during

ripening (Tucker, 1993, Hadfield et al. 1995). Climateric fruits, such as tomato, peach,

avocado, apple and pear, have a respiratory burst and a pronounced autocatalytic ethylene

production while the ripening process is proceeding. Non-climateric fruits, such as bell

pepper, watermelon, strawberry, grape, and citrus, do not show evidence of an increased

ethylene evolution or respiratory rise coincident with ripening (Seymour and McGlasson,

1993). This fruit categorization might not be completely strict for all species. Within a

species there could be both climateric and non-climateric fruits. As a general rule, melon

fruits have been considered as a climateric type; usually reticulatus and cantaloupensis

melon varieties belong to this group. However, non-climateric melon fruits are available

as well, most of them fitting in inodorus variety (Seymour and McGlasson, 1993; Zheng

and Wolf, 2000; Périn et al. 2002a).

Therefore, it is not easy to define a set of criteria that may be used to predict the

ripening-related respiratory and ethylene evolution performance of specific fruit and then

extrapolate that behavior for another fruit related-cultivar or species.

2.2 Postharvest Physiology of Melon

2.2.1 Physiological Changes During Ripening

In order to achieve a typical melon fruit growth pattern, pollination, satisfactory

double fertilization and a normal development of the ovules have to take place (Wien,

1997). Fruit growth patterns among melon cultivars can be similar or quite diverse. In

1971 Pratt reported that both the ‘Honey Dew’ and ‘cantaloupe’ types reached half of

their total fruit growth almost at the same time (15 and 20 days after anthesis); however,

12

the ‘Honey Dew’ melon attained four times as much as size fruit than ‘cantaloupe’.

Likewise, McCollum et al. (1987) described a comparable fruit growth in two melon

genotypes (NY and D26) for the first 14 days after anthesis, but from 21 days after

anthesis to full slip, NY had greater fresh weight than did D26 fruits. McCollum et al.

(1988) measured fruit growth in ‘Galia’ and ‘Noy Yizre’el’ muskmelon cultivars, and

reported that both fruits had sigmoidal growth curves. However, differences between the

cultivars were apparent; i.e. ‘Galia’ fruits were larger than ‘Noy Yizre’el’ fruits at each

stage of development and continued to grow until the time of abscission.

Due to genetic diversity, melon fruits have a wide variation in ripening behavior.

Fruits belonging to the reticulatus and cantaloupensis varieties have a quick climacteric

at, or close to, the time of fruit maturity and abscission, although the abscission process is

absent in some muskmelon (reticulatus) varieties (Sakata and Sugiyama, 2002). On the

other hand, inodorus and saccharinus type fruits may have the climacteric process

extended up to several days or it may be absent (Miccolis and Saltveit, 1991; Aggelis, et

al. 1997a; Liu et al. 2004).

The moment of fruit maturation as well as the beginning of fruit ripening depends

upon the melon variety (Liu et al. 2004). In reticulatus and cantaloupensis varieties the

abscission characteristic is one of the most practical standards to estimate the harvest

maturity (Pratt et al. 1977; Larrigaudiere et al. 1995). Other indexes of muskmelon

harvest maturity include fruit color and appearance of the netted pattern. A muskmelon

fruit color chart has been prepared for ‘Galia’, which categorized six different levels of

maturity: 1, very dark green; 2, green; 3, light yellow with some green areas; 4, light

yellow; 5, yellow; and 6, dark yellow to orange peel (Fallik et al. 2001). On the other

13

hand, in those melon varieties, such as inodorus, flexuosus and saccharinus, where an

abscission layer is not formed, other characteristics are used to assess harvest maturity.

For example according to Portela and Cantwell (1998), at commercial melon production

level a variety of subtle changes in external color (green to white), peel texture (hairy to

smooth), aroma at the blossom end (none to detectable), and fruit density (low to high)

are used in order to assess the harvest maturity point.

Fruit ripening is a genetically determined event that involves a series of changes in

color, texture, and flavor (Hashinaga et al. 1984, Keren-Keiserman et al. 2004). Flavor is

a multifaceted human perception, which involves taste and aroma (Shewfelt, 1993).

According to Tucker (1993), fruit flavor depends on the complex interaction of sugars,

organic acids, phenolics and a wide variety of volatile compounds. In general, the quality

of melon fruit is mostly associated to both elevated sugar level and excellent flavor in

mesocarpic tissue (McCollum et al. 1988; Shewfelt 1993; Wyllie et al. 1995). In netted

melons, final fruit quality is also influenced by shading of the melon plant (Nishizawa et

al. 2000). It was reported by Pratt et al. (1977) that in the State of California, for ‘Honey

Dew’ melon a minimum of 10% soluble solids is legally required for market, but high

quality melons can even reach soluble solids content as high as 17% (Pratt et al. 1977;

Bianco and Pratt, 1977). Regarding the melon fruit volatile compounds, a comprehensive

study on the key aroma compounds in melon as well as their development and cultivar

dependence has been described previously (Wyllie et al. 1995).

Fruit storage shelf life is dissimilar among melon varieties. Cantaloupensis and

reticulatus fruits have a shorter shelf life compared to fruits belonging to inodorus and

saccharinus varieties (Liu et al. 2004). Muskmelon and netted melons have a storage life

14

of approximately 10-14 days at cool temperatures (6-9oC) and proper humidity (90-95%)

conditions (Gull, 1988). Muskmelon and netted melons can be kept at storage conditions

of 2-5o C and a relative humidity of the air of 90-95%. Netted melon fruits are more

prone to lose moisture. This may be a result of the presence of fissured epidermal tissue

(netted), which is an elaborated system of lenticels, therefore allowing a more rapidly

water loss as a result of evaporation (Webster and Craig, 1976). ‘Honey Dew’ type

melons should be stored at 7 to 10o C and 85-90% relative humidity up to three or four

weeks, but lower temperatures of 6o C can cause chilling injury (Gull, 1988; Suslow et al.

2001, Lester et al. 2001).

2.2.2 Ethylene Production

Ethylene is a plant gas hormone, which is involved in the melon fruit ripening

process (Giovannoni, 2001). A burst of ethylene production coincides with ripening in

climateric melon fruits (Pratt et al. 1977). Kendall and Ng (1988) measured ethylene

from two netted (reticulatus variety) and three non-netted (Casaba type-inodorus variety)

muskmelon cultigens and their hybrids immediately after harvest and found that the

netted muskmelon fruits synthesized considerable quantities of ethylene at or close

harvest. Conversely, the non-netted fruits did not produce ethylene until as late as 20 days

after harvest. Hybrids were generally intermediate to the parental lines in rate and time of

ethylene production. These results suggested that ethylene production in Cucumis melo

fruit is regulated by both genetic and developmental factors.

Because netted fruit melons produce ethylene during ripening, they do not require

exogenous ethylene application after harvest (Pratt, 1971). However, inodorus fruit types

may require exogenous ethylene application after harvest, in order to obtain a more

uniform and rapid ripening, as well as better development of color, wax, and aroma (Gull,

15

1988, Suslow et al. 2001). Likewise, inodorus fruit types must be harvested when they

have already acceptable soluble solids content, because melon fruit generally do not

increase their sugar content after harvest (Bianco and Pratt, 1977).

In general, orange- or green-fleshed and netted rind fruit melons produce higher

amounts of ethylene than green- or white-fleshed and smooth rind fruits (Zheng and

Wolff, 2000; Liu et al. 2004). However, exceptions to this generalization may be found in

netted melons. Shiomi et al. (1999) measured the ethylene biosynthetic capacity in two

netted cultivars, ‘Earl’s Favourite’ and ‘Andes’, and found that ehylene production in

‘Earl’s Favourite’ fruit remained low even at their commercial harvest maturity stage,

whereas ‘Andes’ fruit exhibited a typical climacteric pattern with a high ethylene

production. They concluded that the ‘Earl’s Favourite’ fruit used in that experiment

behaved like a non-climacteric fruit.

2.2.3 Biochemical Changes During Ripening

2.2.3.1 Introduction

Major biochemical changes take place in fruit during maturation and ripening

(Jiang and Fu, 2000; Giovannoni, 2001, Lelievre et al. 2000, Pech et al. 2002). The melon

fruit ripening process requires a high metabolic activity, i.e. synthesis and/or degradation

of new structural, soluble and enzymatic proteins, novel mRNAs, changes in plant

hormones levels, and DNA transcription, as well as accumulation of original pigments,

organic acids and sugars, and the release of volatile compounds (Bianco and Pratt 1977;

Miccolis and Saltveit 1995; Larrigaudiere et al. 1995; Dunlap et al. 1996; Guillén et al.

1998; Aggelis et al. 1997a; Sato-Nara et al. 1999; Flores et al. 2001a; 2001b; Villanueva

et al. 2004). All these anabolic and catabolic events need both energy and a carbon-

nitrogen-framework for building blocks, which are supplied via respiration. The two most

16

important respiratory substrates found in melon fruit are sugars and organic acids

(Seymour and McGlasson, 1993). Likewise, ethylene is the major plant hormone

involved in the melon fruit ripening process (Bianco and Pratt, 1977; Leliévre et al. 1997;

Sato-Nara, 1999).

2.2.3.2 Carbohydrate Metabolism

Sweetness is the most important edible quality attribute of ripe melon fruits

(Yamaguchi, et al. 1977; Lester and Shellie, 1992; Artes et al. 1993). Sucrose, glucose

and fructose are the major sugars found in the mesocarp of ripe melon fruits. High levels

of sucrose attribute fruit sweetness in melon (McCollum et al. 1988; Hubbard et al. 1990;

Burger et al. 2003; Villanueva et al. 2004).

Muskmelon fruit do not store starch as some other fruits do (i.e. apple, and banana),

therefore the fruit requires a constant supply of translocated photoassimilate from the leaf

canopy for sugar use and accumulation during development and ripening (Pratt, 1971;

Hubbard et al. 1989; Hubbard et al. 1990). Consequently, any factor which has an effect

on photoassimilate translocation during fruit development will reduce sucrose content

(Hubbard et al. 1990). For example, presence of viral infections, such as cucumber

mosaic virus, in melon plants causes an alteration in carbon metabolism in source leaves,

and in resource partitioning among the various plant organs because there is an increased

in respiration, and a decrease in net photosynthetic rate in infected leaves (Shalitin and

Wolf, 2000; Shalitin et al. 2002).

In Cucumis melo, sucrose is not the only translocated photoassimilate carbohydrate,

since galactosyl-sucrose oligosaccharides raffinose and stachyose can be found in the

phloem (Mitchell et al. 1992; Gao and Schaffer, 1999; Gao et al. 1999; Volk, et al. 2003).

17

Mitchell et al (1992) reported maximal amounts of sucrose (60 mM), stachyose (50 mM),

and raffinose (10 mM) in sap phloem measurements.

Sugar continues to accumulate during fruit development (Pratt, 1971; Pratt et al.

1977). Beginning at early fruit enlargement and reaching its maximum at full maturity

(McCollum et al. 1988; Seymour and McGlasson, 1993; Burger et al. 2002; Burger et al.

2003). The trait for sugar accumulation is controlled by a single recessive gene, called

suc (Burger et al. 2002). Therefore, sucrose accumulation is controlled through several

hormones and enzymes, as well as compartmentation processes (McCollum et al. 1988;

Hubbard et al. 1989; Ofosu-Anim and Yamaki, 1994; Lee et al. 1997; Ofosu-Anim et al.

1998; Gao et al. 1999; Gao and Schaffer, 1999; Feusi et al. 1999; Carmi et al. 2003; Volk

et al. 2003). Likewise, sugar accumulation is affected quantitatively by environmental

and physiological factors as well, such as salinity, nutrient availability, shading, cellular

size in the fruit, and available foliar area (Hubbard et al. 1990; del Amor et al. 1999;

Nishizawa et al. 2000; Nishizawa et al. 2002; Kano, 2002; Kano, 2004).

Netted, muskmelon, and Honeydew fruits have similar, but not identical, patterns

of sugar accumulation (Seymour and McGlasson, 1993). For instance, Bianco and Pratt

(1977) reported that both ‘Honey Dew’ and ‘PMR-45’ fruits have a parallel pattern for

sugar accumulation including total sugars, sucrose, glucose and fructose. Likewise,

McCollum et al. (1988) reported that ‘Galia’ and ‘Noy yizre’el’ fruits accumulated

glucose and fructose, in nearly equal amounts, during the first 24 days after anthesis.

Sucrose accumulation built up 24 days after anthesis and it was the predominant sugar at

the ripe stage. Similar results were obtained one year later by Hubbard et al. (1989), who

measured the concentrations of sucrose, raffinose saccharides, glucose, fructose, and

18

starch in one orange-fleshed netted melon and three green-fleshed muskmelons, two of

them categorized as sweet melons and one as a non-sweet type (‘Birds Nest’).

In an extensive study, Stepansky et al. (1999) found considerable variation in sugar

content and composition in mature flesh of melon fruits from 56 different genotypes

belonging to cantaloupensis, inodorus, conomom, chito, dudaim, momordica, flexuosus,

agrestis, and some non-defined varieties. Among the 14 genotypes classified as

cantaloupensis, total sugars ranged between 40-100 mg/gfw, and sucrose was 50-70% of

the total sugar, although a few accessions had lower levels. Within the inodorus group,

both low and high sucrose-accumulating genotypes were observed. Some genotypes

reached only ∼30mg/gfw total sugar, mostly glucose and fructose, whereas others had a

high sucrose accumulation (∼50 mg/gfw). Among the six conomon genotypes analyzed,

there were fruits with almost no sucrose (line 85-893) accumulation as well as genotypes

with intermediate and high sucrose levels. In the chito and dudaim varieties five

genotypes were evaluated, four out of five genotypes accumulated less than 10 mg/gfw

sucrose, but interestingly, the last one (PI 164320) had an unusual sugar pattern profile as

it accumulated high levels of total sugar, due mostly to elevated glucose and fructose

levels. Most members of the agrestis group accumulated extremely low levels of sugars,

however, two accessions (PI 164493 and PI 436532) had high total sugars (41 and 58

mg/gfw respectively). The momordica and flexuosus genotypes did not accumulate

significant amounts of sucrose or hexose. These authors also indicated that in the sweeter

melon varieties, sucrose was generally the most significant component that contributed to

variation in total sugars.

19

The physiological and biochemical aspects of sucrose accumulation in melon fruit

has been investigated extensively (McCollum et al. 1988; Hubbard et al. 1989; Hubbard

et al. 1990; Gao et al. 1999; Gao and Schaffer, 1999; Feusi et al. 1999; Carmi et al. 2003;

Volk et al. 2003; Burger et al. 2003; Villanueva et al. 2004). As previously mentionated,

melon plants translocate sucrose, stachyose and raffinose as main soluble sugars, which

are used as the carbon supply for sucrose synthesis in the fruit. Two enzymes, acid

invertase (EC 3.2.1.26) and sucrose phosphate synthase (SPS) (EC 2.4.1.14) have been

implicated as the determinants of sucrose accumulation in melon fruit (Hubbard et al.

1989; Stepansky et al. 1999). Both enzymes are inversely related in melon sink tissues,

such as fruits (Hubbard et al. 1989; Hubbard et al. 1990; Gao et al. 1999). During sucrose

accumulation, acid invertase activity decreases, as a result less sucrose degradation. At

the same time, SPS activity begins to increase significantly (Hubbard et al. 1989;

Hubbard et al. 1990; Gao et al. 1999). In addition, SPS activity is higher in sweet melon

fruit compared with non-sweet genotype fruits, suggesting its function in sucrose

accumulation (Hubbard et al. 1990).

Stepansky et al. (1999) stated “the final content of sucrose in the fruit mesocarp of

sweet melon is a function of two factors: the rate of sucrose accumulation, coupled with

the duration of the accumulation period until abscission or harvest.”

2.2.3.3 Organic Acids

Organic acids are compounds regularly found at low amounts in sweet ripe melon

fruit types, such as inodorus, cantaloupensis and reticulatus varieties (Yamaguchi et al.

1977; Seymour and McGlasson, 1993). On the other hand, non-sweet ripe melon fruits,

(flexuosus variety) are able to accumulate higher amounts of organic acids (Stepansky et

al. 1999; Pitrat et al. 1999). For instance, Burger et al. (2003) reported that the high-

20

organic acid fruit content characteristic is conferred by a single dominant gene, called So,

which is found only in melon varieties that do not accumulate high levels of sugars and

which are used for non-dessert purposes. In the recessive condition (so), melon fruits

have a low-organic acid attribute. Furthermore, these authors stated that the evolution of

horticultural sweet melon varieties required the sequential selection of three recessive

mutations: first a recessive mutation that allowed for non-bitter fruit (bif), then a

recessive mutation for low-acid fruit (so), followed by a recessive mutation for high

sucrose fruit (suc) (Burger et al. 2003). Despite the fact that low-organic acid level is a

genetically regulated feature, several envionmental factors, such as salinity can affect

quantitatively, the organic acid level in melon fruit (del Amor et al. 1999).

Citric and malic acids are the most important organic acids found in the flesh of

different melon varieties (Leach et al. 1989; Flores et al. 2001b; Burger et al. 2003). In

1989, Leach et al. studied the organic acid fractions from 12 melon cultivars, and

reported that citric acid was the major component in all melon cultivars that they

analized. Similarly, Flores et al. (2001b) found that the major organic acids found in wild

type and transgenic cantaloupe melon fruit were citric and malic acids. Artes et al. (1993)

found that titratable acidity in four melon varieties varied from 0.14% in ‘Tendral’ up to

0.50% in ‘Galia’ melon fruits.

2.2.3.4 Volatiles

The aroma or fragance of melon fruits are essential quality factors, for consumer

quality (Yamaguchi, 1977), and they are strongly linked to the ripening process (Wang et

al. 1996; Beaulieu and Grim, 2001). Unlike sugar accumulation in the ripe fruit, the

aroma fruit caused by volatile production continues after harvest (Wyllie et al. 1995). The

volatile profile, as well the identification of the main ‘melon odor’ substances in melon

21

fruits have been the subject of a considerable amount of research (Kemp et al. 1972;

Yabumoto et al. 1977; Yabumoto et al. 1978; Buttery et al. 1982; Horvart and Senter,

1987; Leach et al. 1989; Wyllie and Leach, 1990; Homatidou et al. 1992; Wang et al.

1996; Ueda et al. 1997; Bauchot et al. 1998; Yahyaoui et al. 2002; Aubert and Bourger,

2004).

Early studies reported that the volatile ester pattern of ripe muskmelon (reticulatus

varieties) and Honey Dew (inodorus varieties) type fruit were extremely similar, except

for ethyl butyrate, which was more abundant in muskmelon (Kemp et al. 1972;

Yabumoto et al. 1977; Yabumoto et al. 1978). The volatile profile of melon fruit was

made of around 35-50 volatile compounds (Kemp et al. 1972; Yabumoto et al. 1977;

Yabumoto et al. 1978; Buttery et al. 1982). With the advance and improvement of

extraction methods (Beaulieu and Grimm, 2001), such as Solid Phase Microextraction

(SPME), as well as the analytical and detection techniques (Aubert and Bourger, 2004),

such as sniffing port analysis, have shown that the volatile compound content responsible

for ‘melon aroma’ is diverse and cultivar dependent. Indeed, Aubert and Bourger (2004)

were able to differentiate statistically long-shelf life cultivars from wild and mid-shelf life

melon cultivars, based merely upon volatile compound profiles. Moreover, Beaulieu and

Grimm (2001) affirmed that roughly 240 volatile compounds have been reported from

muskmelon fruit.

In the particular case of ‘Arava’ melon, which is a ‘Galia’-type melon, various

volatile acetates were identified in the ripening fruit, including nine aliphatic, four

aromatic, and one compound containing a sulfur moiety (Shalit et al. 2000). Benzyl

acetate was the most abundant volatile compound in the headspace of this cultivar,

22

however, hexyl acetate and 2-methyl butyl acetate were also found in considerable

amounts.

The aroma and taste of most melon fruits are influenced considerably by ester

compounds, as well as to a certain extent by sulphur compounds (Yabumoto et al. 1977;

Wyllie and Leach, 1990; Homatidou et al. 1992). Even though, Kemp et al. (1972)

suggested that four unsaturated esters found in muskmelon fruit did not contribute

significantly to the ‘melon aroma’. Yabumoto et al. (1977) using three different

extraction methods for melon fruit volatiles stated that it was probable that the large

quantities of volatile esters also play a critical role in the integrated flavor of melons, and

that they are necessary for the strong and characteristic fruity aroma. According to

Yabumoto et al. (1978), the volatile ester profile of ripe reticulatus variety (‘PMR-45’

and ‘Top Mark’) and inodorus variety (‘Honey Dew’ and ‘Crenshaw’) fruit were similar,

and they fit into two groups, depending on the pattern exhibited by the production of that

compound. One group had a continuously accelerating rate of production (ethyl esters)

and another increased rapidly and then plateaued (acetate esters).

The major compounds responsible for ‘Honey Dew’ melon aroma are ethyl 2-

methylbutyrate, ethyl butyrate, ethyl hexanoate, hexyl acetate, 3-methylbutyl acetate,

benzyl acetate, (Z)-6-nonenyl acetate, and possibly (E)-6-nonenol and (Z,Z)-3,6-

nonadienol (Buttery et al. 1982). Horvat and Senter (1987) identified eight novel volatile

compounds from ‘Saticoy’ melon (reticulatus). In ‘Galia’-type melon cultivars C8 and

5080, six important aroma volatiles during ripening were found: ethyl acetate, isobutyl

acetate, butyl acetate, 2-methylbutyl acetate, hexyl acetate, and 3-hexenyl acetate (Fallik

et al. 2001).

23

The biochemical connection between the development of aroma volatiles and free

amino acid content in melon ripening fruit is well established (Yabumoto et al. 1977;

Wyllie and Leach, 1992; Wyllie et al. 1995). Wang et al. (1996) proposed that several

amino acids, such as valine, isoleucine, methionine, and alanine may be the precursors of

the mayority of the esters found in melon fruit, providing the branched alkyl chain

moiety, which is present in a significant proportion of volatile ester compounds.

The aroma volatile profile in each type of melon is a genetically controlled

characteristic (Ueda et al. 1997; Yahyaoui et al. 2002). The presence or absense of seeds

in the fruit cavity can modify the final aroma volatile profile (Li et al. 2002). Likewise,

the developmental fruit stage may have a strong influence on aroma characteristics

(Beaulieu and Grimm, 2001).

In summary, similar to other fruits the melon fragance is made of complex mixtures

of volatiles compounds. Both their production and profile in melon fruits is a genetically

controlled attribute, which is associated with the ripening process (Wang et al. 1996), and

which is regulated by ethylene (Bauchot et al. 1998; 1999). Therefore, the volatile

compound profile is a cultivar-dependent characteristic (Aubert and Bourger, 2004).

2.2.3.5 Cell Wall Degradation

The plant cell wall is a dynamic structure, which determines cell shape and

contributes to the functional specialization of cell types (Carpita and McCann, 2000).

The plant cell wall is a highly organized structure composed of several polysaccharides,

proteins, and aromatic compounds. Likewise, the new primary cell wall comes from the

cell plate during cell division, and after differentiation many cells are able to develop

within the primary wall, a secondary cell wall (Carpita and McCann, 2000). According to

Bennett (2002), a simplest form of the structural model of the plant cell wall can be

24

pictured as a core structure of cellulose microfibrils embedded in two coextensive

networks of pectin and hemicellulose.

Fruit softening observed during ripening is associated with textural changes that are

believed to result from modification and disassembly of the primary cell wall (Fischer

and Bennett, 1991). Fruit softening and the underlying cell wall structural changes are

complex. Softening or loss of firmness of the edible mesocarp of melon fruit starts in the

middle (around 30-45 days after anthesis, depending on cultivar) of the development

cycle, along with other typical changes connected with the ripening process (Lester and

Dunlap, 1985). Some general events during melon fruit softening are (Lester and Dunlap,

1985; Lester and Bruton, 1986; McCollum et al. 1989; Ranwala et al. 1992; Fils-Lycaon

and Buret, 1991; Simandjuntak et al. 1996; Rose et al. 1998; Hadfiled et al. 1998; Rojas

et al. 2001; Bennett, 2002):

1. There is no a significant change in total pectins (measured as total polyuronides) as a percentage of cell wall material, rather a substancial change in the relative solubility and depolimerazation of pectin levels is observed as fruit softening proceed, as well as a decrease in pectin molecular size. Quoting McCollum et al. (1989) “Quantitative changes in pectin content are apparently less important than are qualitative changes in the softening process.”

2. It seems likely that polygalacturonase (PG) enzyme(s) might not be involved in that solubilization process during the early ripening stages, however some PG-dependent developments may contribute to overall pectin disassembly at later stages.

3. Other enzymes could be involved in that early pectin solubilization process, for instance β-galactosidases and / or β-galactanases.

4. Hemicellulose polymers undergo important modifications, such as changes in the degree of solubility and modifications from large molecular size to smaller size, and loss of specific sugars.

5. Most of the non-cellulosic neutral sugars decrease significantly in the mesocarp of ripening fruits, regularly Galactose, Mannose and Arabinose, whereas other neutral sugars such as Xylose might or not increase during fruit softening.

25

6. Other enzymatic activities, such as pectin methylesterase, which have been associated with pectin metabolism in other fruits (Harriman et al. 1991; Tieman et al. 1992), and/or other proteins, such as expansins, which have been proposed to disrupt hydrogen bonds within the plant cell wall polymer matrix (Rose et al. 1997; Civello et al. 1999), could also be involved in melon fruit softening. More evidence has appeared regarding the expansins’ role in early fruit softening. Brummell et al. (1999) obtained two types of transgenic tomato plants, some were suppressed and anothers were overexpressed in the LeExp1 protein. Tomato fruit in which Exp1 protein accumulation was inhibited by 3% were firmer than control fruit throughout the ripening process. Conversely, fruit overexpressing high amounts of LeExp1 protein were much softer than control fruit, even in mature green stage before the ripening event had commenced.

Rose et al. (1998) and Bennett (2002) proposed a complete model of the temporal

sequence of cell wall changes, pectinase activity, and PG-mRNA expression in ripening

‘Charentais’ melon fruit at defined developmental stages, unfortunately they did not

include the role of expansins in cell wall degradation in their model.

Among the different plant hormones which are involved in fruit development,

ethylene has the main role during melon fruit softening (Rose et al. 1998). Internal

ethylene concentrations inside ‘Charentais’ melon fruit cavity increases, concomitant

with a loss of flesh firmness during ripening. Furthermore, Guis et al. (1999) using

antisense ACC-oxidase transgenic melon plants to reduce ethylene production, reported

that plants did not have substancial changes in pectin molecular mass observed in the

wild type fruit. Moreover, exogenous ethylene application to those transgenic fruits

resumed both accelerated fruit softening and a downshift in the size of cell wall

polymers. Additionally, transgenic melon plants (antisense ACC oxidase) were also used

to study the role of ethylene in regulating cell wall-degrading enzyme activities (Botondi

et al. 2000). In transgenic ‘Charentais’ melon, cell-wall degradation process is regulated

by both ethylene-dependent and ethylene-independent mechanisms. In support of a fruit

softening-ethylene involvement, it was established that ripening-regulated expansin

26

gene(s) in tomato were influenced directly by ethylene, and the expression of that gene

parallels the pattern of xyloglucan disassembly, and early fruit softening (Rose et al.

1997).

The exact hormonal, molecular and enzymatic mechanisms by which all these

processes take place in melon ripening fruit, and finally develop to the fruit softening

event are not well understood. Maybe by using updated molecular and genetic

techniques, such as cDNA microarrays (Fonseca et al. 2004), in order to monitor the gene

expression during fruit development and ripening, there will be more evidence to

understand and manipulate the melon fruit softening process. What's more, as Bennett

(2002) proposed: ‘future research should focus on using genetic strategies to assess the

potential for synergistic interactions by suppression of both hemicelluloses and pectin

disassembly in ripening fruit.’

2.2.3.6 Pigments

Flesh color of melons is another important quality attributes for a consumer appeal

(Yamaguchi, 1977). In general, four basic and distinctive flesh colors can be observed in

melon fruits: orange, light-orange or pink, green, and white (Watanabe et al. 1991;

Goldman, 2002).

According to Seymour and McGlasson (1993) the principal pigments in orange-

fleshed melons are: β-carotene (84.7%), δ-carotene (6.8%), α-carotene (1.2%),

phytofluene (2.4%), phytoene (1.5%), lutein (1.0%), violaxanthin (0.9%) and traces of

other carotenoids. Likewise, Watanabe et al. (1991) evaluated nine different melon

cultivars belonging to the four basic and distinctive flesh colors. They found that the

orange-fleshed colored melon cultivars 'Iroquois', 'Blenheim Orange', 'Birdie Red',

27

'Quincy' and 'Tiffany' contained about 9.2 to 18.0-µg/g β-carotene as the major pigment,

as well as a small amount of phytofluene, α-carotene, ζ-carotene and xanthophylls. They

also measured pigments in light-orange-fleshed ‘Hale’s Best’ melon, which contained

about 4.0-µg/g β-carotene, and phytofluene; α-carotene, ζ-carotene and xanthophylls

were also present but in small amounts. Finally, in the green-fleshed melon 'Earl's

Favourite' and 'Fukunoka', and white-fleshed colored melon 'Barharman', their main

components were β-carotene and xanthophylls.

Chlorophyll and carotenoid changes in developing fruit muskmelon were studied

earlier by Reid et al. (1970). They evaluated three melon cultivars: ‘Crenshaw’, ‘Persian’,

and ‘PMR 45’. In all the fruits, cholorophyll content decreased to an intermediate level

five weeks after anthesis, and they suggested that chlorophyll loss was probably due to

dilution through growth, because there were no more chlorophyll synthesis, but an

enlargement of the fruit occurred. In ‘PMR 45’ and ‘Chenshaw’ fruits, however, there

was a successive rapid decrease, which was concurrent with the ripening process.

Carotenoids content increase steadily three weeks after anthesis to high levels at full

maturity. The development of orange pigmentation was a gradual event, starting at the

placentae and progressing outward through the mesocarp, until the flesh was uniformly

orange at full maturity.

Forbus et al. (1992) used Delayed Light Emission (DLE), a nondestructive method,

to study physical and chemical properties related to fruit maturity in Canary melons.

They found that cholorophyll and yellow pigments decreased with fruit development,

having a high correlation with maturity index (IM). Flügel and Gross (1982) studied

pigment and plastid changes during ripening of the green-fleshed ‘Galia’ muskmelon

28

fruit. They observed that the carotenoid profile in the exocarp and mesocarp did not

change during development. Also, relatively low levels of chlorophyll and carotenoids

were found in the flesh. Yellowing of the exocarp was due to increased chlorophyll

degradation during ripening, as well as a partial decrease in total carotenoids took place.

In conclusion, it seems that pigment profile accumulation and degradation in melon

fruit is a cultivar-dependent characteristic, which is expressed during fruit maturity.

2.2.4 Ethylene and Molecular Changes During Ripening

2.2.4.1 Introduction

There are ethylene-dependent and ethylene-independent biochemical and

physiological pathways throughout melon fruit ripening (Pech et al. 1999; Hadfield et al.

2000; Srivastava, 2002; Silva et al. 2004), which both coexist at the same time in the

climacteric fruit. Likewise, besides ethylene several plant hormones, such as indole-3-

acetic acid (IAA) and abscisic acid (ABA), are involved in melon fruit ripening

(Larrigaudiere et al. 1995; Dunlap et al. 1996; Guillén et al. 1998; Martínez-Madrid et al.

1999).

2.2.4.2 Biosynthesis, Perception and Effects of Ethylene

Ethylene biosynthesis goes from methionine, through S-adenosylmethionine

(SAM), then to 1-aminocyclopropane-1-carboxylic acid (ACC), and finally to ethylene

(Yang and Baur, 1969; Adams and Yang, 1979; Yang, 1980; Yang, 1982; Yang and

Hoffman, 1984). Two regulatory enzymes in this pathway are ACC synthase (ACS) (EC

4.4.1.14) and ACC oxidase (ACO) (EC 1.14.17.4). The latter enzyme was formely

known as ethylene-forming enzyme (EFE) by Adams and Yang (1979) because the

reaction mechanism was not known at that time. ACC synthase is generally considered as

the rate-limiting step in ethylene biosynthesis (Yang and Hoffman, 1984).

29

Both ACS and ACO melon enzymes are coded by a multigene family (Miki et al.

1995; Yamamoto et al. 1995; Lasserre et al. 1996; Lasserre et al. 1997), therefore several

isoenzymes are recognized in melon tissues. For instance, Miki et al. (1995) and Ishiki et

al. (2000) isolated three cDNAs for ACC synthase from wounded mesocarp tissue of

melon fruits. Lasserre et al. (1996) reported the isolation and categorization of three

genomic clones, identified by screening a melon genomic DNA library with the cDNA

pMEL1, corresponding to three putative members of the ACC oxidase gene family in

cantaloupensis melon. These authors in addition determined the entire sequence of these

genes and found that they were all transcriptionally active. One genomic clone, named

CM-ACO1, presented a coding region with four exons and interrumted by three introns.

The other two genes, CM-ACO2 and CM-ACO3, were only interrupted by two introns, at

same positions as CM-ACO1. The degree of DNA homology in the coding regions of

CM-ACO3 relative to CM-ACO1 was 75%. In contrast, the degree of DNA homology of

CM-ACO2 relative to both CM-ACO1 and CM-ACO3 were 59% in their coding region.

ACS and ACO melon multigenes are differentially activated and expressed by

several environmental and developmental factors (Yamamoto et al. 1995; Lasserre et al.

1996; Shiomi et al. 1999; Zheng et al. 2002). Yamamoto et al. (1995) using tissue

printing and immunoblot analysis with antibodies specific for ACO, were able to identify

in which part of the fruit the ethylene synthesis started at the early stages of ripening.

They reported that the rate of ethylene production (accumulation of ACO protein) in

melon fruits increased initially in the placental tissue, then in mesocarp tissue and finally

at the rind. They also concluded that levels of ACO mRNA and protein were low in

unripe fruit stage, but became detectable in placental tissue at the pre-climacteric period,

30

and their levels increased in the mesocarp at the climacteric stage. All these results

suggested that the central region of melon fruit (placental tissue and seeds) plays a major

role in the production of ethylene during the early stage of fruit ripening.

A RT-PCR assay was used by Lasserre et al. (1996) to detect the differential

expression of ACO melon genes (CM-ACO1, CM-ACO2, and CM-ACO3). They found

that these three genes were differentially expressed during development, ethylene

treatment and wounding. CM-ACO1 was induced during fruit ripening, and also in

response to wounding and ethylene treatment in leaves. CM-ACO2 was detectable at low

levels in etiolated hypocotyls, whereas CM-ACO3 was expressed in flowers and it was

not induced by any treatment tested.

Lasserre et al. (1997) found that the regulation of the CM-ACO1 gene was

connected preferentially to stress responses, while the CM-ACO3 gene seemed to be

associated with developmental routes. Moreover, Bouquin et al. (1997) using the 5’-

untranslated region of the CM-ACO1 gene fused to the β-glucuronidase (GUS) reporter

gene were capable of measuring the transcriptional activation in leaves of the CM-ACO1

gene after wounding and ethylene stimulation. Their results suggested that induction of

CM-ACO1 gene expression occurs via two direct and independent signal transduction

pathways in response to both stimuli. Zheng et al. (2002) studied some genetic aspects of

ethylene production and its relationship to the RFLPs of the ACC oxidase and ACC

synthase genes in two melon cultivars. One cultivar had high ethylene production during

fruit ripening (‘TAM Uvalde’) and another had low levels of ethylene production (‘TAM

Yellow Canary’). Their results of single-copy-reconstruction assays suggested that the

31

CMACO-1 gene was present as a single copy, whereas the CMACS-1 gene was a

component of a multigene family in both melon cultivars.

It has been suggested that differences in ethylene production among melon fruits

might be the result of transcriptional changes in ACS and ACO genes (Shiomi et al.

1999). These authors measured the ACS-1, ACO-1 and ACO-2 mRNA expression pattern

in exocarp, mesocarp and placental tissues of ‘Earl’s Favorit’ (recognized as non-

climacteric) and ‘Andes’ (known as climacteric) fruit cultivars at different stages of

maturity, finding that mRNA CMACS-1 transcripts accumulated only in the mesocarp

and placentae of ‘Andes’ fruit at 50 DAP (commercial harvest maturity stage). This

accumulation was coincident with increases in ACS activity, ACC content and maximum

ethylene production. In contrast, CMACO-1 mRNA accumulated in elevated levels in the

mesocarp and placentae of both cultivars at 50 DAP, but in ‘Andes’ cultivar those

transcripts were more abundant than in ‘Earl’s Favorit’ fruit. In the exocarp, the

CMACO-1 mRNA level was low for both cultivars. CMACO-2 mRNA was constitutively

expressed in placentae and mesocarp at low levels, and non-detectable in the exocarp.

These results suggested that the difference in ethylene-forming ability between these two

cultivars may result from the expression of CMACS-1 mRNA and CMACO-1 mRNA

during the fruit ripening process.

Ethylene perception is mediated by specific receptors, which have been cloned and

completely described for several plants, such as Arabidopsis (Fluhr and Mattoo, 1996;

Johnson and Ecker, 1998), tomato (Tieman et al. 1999; Tieman et al. 2000), tobacco

(Terajima et al. 2001), and carnation (Reid and Wu, 1992; Shibuya et al. 2002). In

Cucumis melo, ethylene-receptor-like homolog genes have been reported as well (Sato-

32

Nara et al. 1999, Takahashi et al. 2002; Nukui et al. 2004; Cui et al. 2004). Sato-Nara et

al. (1999) isolated and characterized two cDNAs, which were described as putative

ethylene receptors, from muskmelon using the Arabidopsis ethylene receptor genes ETR1

and ERS1 sequences. These authors measured the expression pattern of these cDNAs

during fruit enlargement and ripening by means of Northen blot assay, finding that both

clones were expressed in a stage- and tissue-specific manner. They named their cDNAs

as Cm-ETR1 (Accession No. AF054806) and Cm-ERS1 (Accession No. AF037368).

Three years later (Takahashi et al. 2002), polyclonal antibodies against melon receptor,

Cm-ERS1, were prepared in order to determine the temporal and spatial expression

pattern of Cm-ERS1 protein during melon fruit development. They reported that Cm-

ERS1 protein formed a disulphide-linked homodimer and it was present in microsomal

membranes but not in soluble fractions. In addition, their results revealed that a post-

transcriptional regulation of Cm-ERS1 expression affects stage- and tissue-specific

accumulation of this protein. That transition pattern was not cultivar-dependent because it

was observed in two different melon cultivars, i.e. ‘Fuyu A’ and ‘Natsu 4’.

The cloning and characterization of two melon ethylene receptor genes has allowed

their use as molecular genetic tools in heterologous systems with promising results

(Nukui et al. 2004; Cui et al. 2004). The overexpression of a missense mutated melon

ethylene receptor gene, Cm-ETR1/H69A, in a heterologous plant, Nemesia strumosa,

confered reduced ethylene sensitivity (Cui et al. 2004), making transgenic plants that had

a significantly extended flower longevity compared with the wild type counterpart. On

the other hand, because ethylene inhibits the establishment of symbiosis between rhizobia

and legumes, a point mutated Cm-ERS1/H70A gene was used to transform Lotus

33

japonicus plants in order to examine how and when endogenous ethylene inhibits that

rhizobial infection and nodulation (Nukui et al. 2004). Endogenous ethylene in L.

japonicus roots inhibits rhizobial infection at the early stages (primary nodulation), and

suggested that ethylene perception also assists negative feedback regulation of secondary

nodule initiation.

Ethylene has a profound influence on climacteric fruit ripening, and in some cases

it induces biochemical and physiological changes in non-climacteric fruit (Leliévre et al.

1997; Giovannoni, 2001; Périn et al. 2002a). In research with 63 different cultivars from

eight market types of melon, belonging to Cantaloupensis and Inodorus varieties, Zheng

and Wolff (2000) were able to demonstrate a significant correlation between RFLP

polymorphisms and ethylene production in the fruit. These RFLPs were associated with

flesh color, rind texture and postharvest decay characteristics in the melon genotypes

examined. Low ethylene production and green- and white-flesh color were associated

with the presence of a putative RFLP-MEL1 allele Ao (15kb), whereas high ethylene

production and orange-flesh color were associated with allele Bo (8.5 kb) in the

homozygous condition. Some melon cultivars, such as ‘Honeybrew’ and ‘HD Green

flesh’, did not accumulate any detectable ethylene. Likewise, Périn et al. (2002a) reported

that in the non-abscission melon fruit PI 161375, exogenous ethylene failed to stimulate

abscission, loss of firmness, ethylene production and expression of ethylene-inducible

genes. These authors obtained a recombinant population of Charentais X PI 161375

inbred lines segregating for fruit abscission and ethylene production. Genetic analysis

showed that both characters are controlled by two independent loci. They concluded that

34

the non-climacteric phenotype in fruit tissues was attributable to ethylene insensitivity

conferred by the recesive allelic forms from PI 161375.

As it was previously pointed out, ethylene-dependent and ethylene-independent

biochemical and physiological pathways take place and coexist during melon fruit

ripening. Hadfield et al. (2000) made a differential screening of a ripe melon fruit cDNA

library and identified 16 unique cDNAs corresponding to mRNAs whose accumulation

was estimulated by ripening. Expression of fifteen, out of sixteen cDNAs, was ripening

regulated, and twelve of them were fruit specific. Three patterns of gene expression were

observed when the expression of cDNA clones was examined in transgenic ACC oxidase

melon fruit. One group of cDNAs corresponded to mRNAs whose abundance was

reduced in transgenic fruit but still inducible by ethylene treatment. The second group of

mRNAs was not significantly altered in the transgenic fruit and it was not affected by

ethylene treatment, indicating that these genes are regulated by ethylene-independent

factors. The third group of cDNAs had an unexpected pattern of expression, low levels of

mRNA in transgenic fruit and even remaining low after ethylene treatment. Obviously,

the regulation of this third group of genes appears to be ethylene-independent.

Pech et al. (1999) described some ethylene-dependent events in cantaloupe

Charentais melon fruits and divided them into two main groups. One group was

considered as general metabolism, such as yellowing of the rind, fruit softening, volatile

production, presence of climacteric respiration, abscission of the fruit, and susceptibility

to chilling injury. The other group included enzyme activities, such as galactanase, α-

arabinosidase, β-galactosidase, endo-polygalacturonase, ACC synthase (negative

feedback) and ACC N-malonyltransferase. Likewise, these authors categorized ethylene-

35

independent characteristics into the same two groups, one group included in general

metabolism, such as coloration of the flesh, accumulation of sugars and organic acids,

accumulation of ACC, and loss of acidity. Within enzyme activities non-induced by

ethylene were pectin methyl esterase, exo-polygalacturonase and ACC synthase

(induction at onset of ripening). This classification of ethylene-dependent and ethylene-

independent events provides valuable basic information, which might be used to design

biochemical and/or molecular strategies with the aim to control melon fruit ripening.

Ethylene regulates fruit ripening by coordinating the expression of several genes

(Leliévre et al. 1997; Aggelis et al. 1997a; Aggelis et al. 1997b; Yang and Oetiker, 1998;

Jiang and Fu, 2000; Périn et al. 2002a). The most common genes which are frequently

regulated by ethylene during fruit ripening embrace some members of the ACS and ACO

multigene family, phytoene synthase, endo-polygalacturonase, galactanase, one

homologue of S-adenosyl-L-homocysteine hydrolase (SAHH) and even a mRNA, which

is ripening-specific, named MEL2 of unidentified function (Karvouni et al. 1995; Aggelis

et al. 1997a; Aggelis et al. 1997b; Hadfield et al. 1998; Pech et al. 1999; Guis et al. 1999;

Périn et al. 2002a).

New insight knowledge will be available on ethylene role(s) in melon fruit

development and ripening, when the molecular techniques which have produced

information from model systems, such as Arabidopsis and tomato, might some day be

applied to different melon genotypes. Moreover, that information could also be retrieved

from wild and landraces melons, in order to find ripening genes to be transferred to

commercial varieties.

36

2.3 Melon Biotechnology

2.3.1 Genetic Improvement

2.3.1.1 Traditional Breeding

For several thousands of years, human beigns have altered the genetic background

of plants, which have been used as crops (Fehr et al. 1987; Snustad et al. 1997). This

genetic alteration was achieved by first selecting one type of plant or seed in preference

to another, instead of randomly taking what nature provided (Fehr et al. 1987; Snustad et

al. 1997). Subsequently, human selection for specific traits such as faster plant growth,

larger and more nutritious seeds or sweeter fruits dramatically changed the domesticated

plant species compared to their wild type counterparts (Fehr et al. 1987). The improved

plant characteristics selected by those early agriculturalists were transmited genetically to

the succeeding plant generations, and later on, plant breeding methods as an art and

science discipline was born (Suslow et al. 2002).

Cucumis melo plants were not the exception to that human selection and plant

breeding effort. Traditional breeding methods in melon have led to a considerable varietal

improvement. Strong sexual incompatibility barriers at the interspecific and intergeneric

levels have restricted the use of that genetic potential to develop new and enhanced

melon cultivars (Niemirowicz-Szcztt and Kubicki 1979; Robinson and Decker-Walters,

1999). Melon plant improvement by traditional hybridization is slow and limited to a

restricted gene pool (Pitrat et al. 1999). It is possible to produce viable intraspecific

melon hybrids between wild type melons and commercial melon varieties, with the aim

to transfer some particular melon genetic traits, such as disease resistance to fungi,

bacteria, virus and insects, or tolerance to environmental factors, such as salinity,

flooding, drought, and high or low temperature, to commercial melon varieties (Dane,

37

1991). Commercial melon varieties, with sweet, non-bitter and low-acidic fruits, carry

three genes (suc/suc, so/so, bif/bif), which control high-quality-fruit traits, in recessive

form (Burger et al. 2003). Therefore, any intraspecific crosses, using traditional breeding

methods, between melon land races (Seshadri and More, 2002) and commercial melon

cultivars will produce hybrid fruit with low quality characteristics, because of the effect

of dominant genes controlling low-sweetness, high-acid, and high-bitterness level in the

melon land race fruit.

In conclusion, it is highly desirable to have other genetic breeding tools, besides

traditional hand crossing, in order to obtain improved melon cultivars. The same results

obtained from conventional breeding methods can be developed using biotechnology

strategies (Table 2.2). It is well documented that using genetic engineering strategies it is

feasible to overcome most of the genetic barriers among plants, which are unsurpassable

by traditional breeding techniques (Vasil, 1990; 1996; 1998; 2003).

2.3.1.2 Improvement Through Genetic Engineering

According to the ‘FAO statement on biotechnology’, The Convention on Biological

Diversity (CBD) defines biotechnology as: “any technological application that uses

biological systems, living organisms, or derivatives thereof, to make or modify products

or processes for specific use” (FAO, 2004). This scientific discipline in its broad sense

covers many of the tools and techniques that are commonplace in agriculture and food

production, whereas in its narrow or molecular sense it considers only the new DNA

techniques, molecular biology and reproductive technological applications, including

differente technologies such as gene manipulation, gene transfer, DNA typing and

cloning of genes, plants and animals (FAO, 2004).

38

Table 2.2 Similarities between conventional and biotechnological methods for melon plant improvement.

Steps in Breeding Conventional Methods Biotechnological Methods

1. Collection and evaluation of genetic resources

Genetic analysis of valuable traits

Cloning of valuable gene

2. Generation of variation Intraspecific crosses Genetic transformation, embryo culture, somatic cell fusion, polyploidy, anther culture, and somaclonal variation

3. Selection of desirable variants

Growth and evaluation PCR, selectable markers, DNA markers, etc.

4. Production of fixed lines

Self-pollination Self-pollination and haploid production by gynogenesis

5. Seed production Growth in controlled conditions

DNA markers to test seed purity and growth in controlled conditions

Modified from Ezura (1999).

During the 1970s, molecular biology and genetic engineering research laid the

foundation for the development of transgenic plants in 1983 using the Ti plasmid from

the soil bacterium Agrobacterium tumefaciens (Herrera-Estrella et al. 1983). This

bacterium transfers a specific fragment of the Ti plasmid (T-DNA) which can be

engineered to contain a selectable marker and /or genes of interest, into the plant nuclear

genome under in vitro conditions. Once inserted, nontransformed plants can be killed in

culture by the toxic substance the marker gene codes resistance to. Within the plant

biotechnology discipline, plant tissue culture methods has had an essential role, allowing

the development of transgenic plants with a number of desirable agronomic, pest

resistance and food traits. It is commonly accepted that the term “plant tissue culture”

refers to in vitro cultivation on nutrient media of any plant part, a single cell, tissue or an

39

organ under a sterile environment, leading to a whole de novo regenerated plant (Nunez-

Palenius et al. 2005a).

After the milestone plant tranformation achievement by Herrera-Estrella et al.

(1983), a number of technological difficulties were surpassed, allowing the cloning and

insertion of different genes and engineering of transgenic plants with: a) resistance to

plant viruses, fungi and insects, tolerance to herbicides, salinity, drought, heavy metals

and low/high temperatures, b) improved nutritional quality (proteins, oils, vitamins, and

minerals among others), shelf life of fruits and vegetables, flavor and fragance, c) novel

production of vaccines, pharmaceuticals, and therapeutic and prophylactic proteins, d)

reduced production of allergens, and e) phytoremediation activity (James and Krattiger,

1996; Vasil, 2002; 2003; Nuñez-Palenius et al. 2005). The first transgenic commercial

plant variety in the USA was released in May 1994, when Calgene marketed its Flavr-

Savr™ delayed ripening tomato (James and Krattiger, 1996). At the present time, more

than 50 transgenic crops have been approved for commercial planting, and at least 100

more are under field trials and/or regulatory review (Vasil, 2003).

The influence of plant genetic engineering on commercial crop production is

evident by the global increase of cultivated land with transgenic crops, also known as

Genetically Modified Organisms (GMOs). All this increase has happened in a relatively

short time span, i.e. less than a decade. According to James (2003), the global land area

of transgenic crops continued to grow for the seventh consecutive year in 2003, 15%, 9

million hectares, compared with 12% in 2002. In 2003, 25% of the aggregate area of four

main crops, i.e. soybean, maize, cotton and canola, totaling over one quarter billion

hectares was GMOs. The market value of transgenic crops is expected to increase from

40

$450 million in 1995 to over $7 billion by 2005 (James and Krattiger, 1996; James,

2003), for instance, the total market for transgenic seed now exceeds $3 billion (Vasil,

2003).

Throughout an eight-year period, from 1996-2003, the land area of transgenic crops

in the world increased 40 times, from 1.7 million hectares in 1996 up to 67.7 million

hectares in 2003 (Figure 2.1). Plants containing herbicide (Roundup) tolerance was the

dominant trait used, followed by insect resistance conferred by Bacillus thuringiensis

(Bt) toxin.

Global Land Area of Transgenic Crops

0102030405060708090

1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

M h

ecta

res

TotalIndustrial CountriesDeveloping Countries

Figure 2.1 Global land area of cultivated transgenic crops. From James and Krattiger,

(1996), James (1997; 1998; 1999; 2000; 2001; 2002; 2003; 2004).

The highest yearly increase of global land area cultivated with transgenic crops was

observed during the period 1997-1999 (Figure 2.2). Afterward, in 2000, a substancial

decline in the rate of yearly increase took place. Nevertheless, it seems from most recent

data (2001 to 2003) that global land area cultivated with GMOs will be increased and it

might be able to attain again similar levels as previous years (James, 2001; 2002; 2003).

41

13.3

96.1

8.44.3

12.1

1.79.3

16.8

0

10

20

30

40

50

60

70

80

90

1996 1997 1998 1999 2000 2001 2002 2003 2004

M h

ecta

res

IncreaseoverPreviousYearPreviousYear

Figure 2.2 Yearly increase in global land area of transgenic crops. Numbers above bars

indicate the increased amount in Mhectares for that year. From James and Krattiger, (1996), James (1997; 1998; 1999; 2000; 2001; 2002; 2003; 2004).

USA cultivated more land area (42.8 million hectares) of transgenic crops than any

other nation (Figure 2.3). In the same year a growth rate of 10% in planted area of

transgenic crops was reflected a strong increase in both Bt toxin maize and herbicide

tolerant maize, and continued growth in herbicide tolerant soybean (James, 2003). These

transgenic plants, for the period of 2003, were grown by 7 million farms in 18 different

countries, two million more of farms and two more countries than in 2002. Globally in

2003, the most common and commercialized transgenic crops were: soybean (41.4

million hectares), maize (15.5 million hectares), cotton (7.2 million hectares), and canola

(3.5 million hectares). Similarly in 2003, herbicide tolerance installed in soybean, maize,

canola and cotton occupied 49.7 million hectares (73%) of the global 67.7 million

hectares.

42

Figure 2.3 Top Ten Countries growing GMOs in the World in 2004. From James, C.

(2004).

Thus, world land area planted with GMOs is increasing substancially every year.

According to FAO (2004), global population reached 6 billion on October 12, 1999, and

in view of the current human population growth rates (1.5%), it is expected that human

beings in this world will be approximately 11 billion for the year 2050 (Swaminathan,

1995). Doubling or tripling of the world food and fiber production by 2050 cannot be

achieved using existing crop technology (James and Krattiger, 1996). Therefore, more

research with plant molecular tools and transgenic crops must be accomplished in order

to reach the needs to maintain such high human populations.

2.3.1.3 Melon Biotechnology

Since the first report of successful transformation of melon (Cucumis melo) (Fang

and Grumet, 1990), several valuable plant features, such as virus resistance (Fang and

2 ARGENTINA 16.2 Million Has. Soybean, Maize,

Cotton

3 CANADA 5.4 Million Has. Canola, Maize,

Soybean

1 USA 47.6 Million Has. Soybean, Maize,

Cotton, Canola

10 AUSTRALIA 0.2 Million Has.

Soybean

7 INDIA 0.5 Million Has.

Cotton

5 CHINA 3.7 Million Has.

Cotton

9 URUGUAY 0.3 Million Has. Soybean, Maize

8 SOUTH AFRICA

0.5 Million Has. Maize, Soybean,

Cotton

6 PARAGUAY 1.2 Million Has. Soybean, Maize

4 BRAZIL 5.0 Million Has.

Soybean

43

Grumet 1993; Yoshioka et al. 1993; Gonsalves et al. 1994; Huttner et al. 2001), tolerance

to salinity (Bordas et al. 1997) and fruit quality improvement (Ayub et al. 1996; Ezura et

al. 1997; Shellie, 2001; Silva et al. 2004; Nuñez-Palenius et al. 2003; 2004) have been

incorporated to wild type germplasm. The first US trial of field-tested virus resistance in

a commercial transgenic melon crop was done in 1993 and 1994 (Clough and Hamm,

1995). Although, a transgenic melon application was first registered in 1998 by the

USDA-APHIS service (2004), there is still no commercial transgenic melon cultivars

approved, despite of more than 140 transgenic melon field trials in the United States

during the period 1987-2001 (Grumet and Gifford, 1998; Grumet, 2002; Gaba et al. 2004;

Grumet, 2005. Personal communication). The lack of a commercial GMO melon is due to

unstable expression or partial loss of the transgene of interest (due somaclonal variation

and/or chromosomal rearrengements), and because the transgenic melon plant did not

have any commercial advantage compared to the wild type counter part.

2.3.2 In vitro Regeneration

In order to achieve a successful commercial application from biotechnology in

melon a competent de novo regeneration system from in vitro cultures is requiered (Guis

et al. 1998). In the last 25 years, more than 40 in vitro melon regeneration protocols have

been described, some using either organogenesis, somatic embryogenesis or both

regeneration pathways (Table 3.2, 3.3 and 3.4). Melon plant regeneration has been

reported from adventitious buds, somatic embryos, shoot primordia, protoplasts, and

axillary buds. Several biological and physical factors influence in vitro regeneration

efficiency rate, and all have to be considered in order to develop a reproducible and

reliable melon regeneration protocol.

44

2.3.2.1 Genetic Control

Because genetic variability in melon is highly diverse (Monforte et al. 2003),

genotype is the most important factor, determining regeneration potential. Melon

varieties (reticulatus, cantaloupensis, inodorus, flexuosus, etc.) and commercial cultivars

have differences on their regeneration ability under the same in vitro protocol and

environmental conditions (Orts et al. 1987; Oridate et al. 1992; Gray et al. 1993;

Ficcadenti and Rotino, 1995; Molina and Nuez, 1995a; Kintzios and Taravira, 1997;

Galperin et al. 2003a; 2003b). Likewise, organogenesis and somatic embryogenesis

responses in melon cultures are also genotype-dependent. For instance, Oridate et al.

(1992) and Gray et al. (1993) reported that reticulatus varieties were more prone to

produce in vitro somatic embryos than inodorus varieties.

Oridate et al. (1992) found significant differences in somatic embryogenic response

from melon seeds among 18 commercial cultivars. They made reciprocal crosses between

those cultivars, in order to obtain the F1 seeds and evaluate their embryogeny response.

Some lines produced a large number of somatic embryos whereas others did not produce

any somatic embryos or the response was very low. Moreover, these authors were able to

transfer, by sexual crosses, the embryogenic regeneration response from superior to

inferior responding cultivars, demonstrating that the capacity to de novo regenerate

through somatic embryogenesis by diferent melon cultivars was under genetic control.

These researchers were unable to determine the specific mode of inheritance of the

somatic embryogenic capacity due to variation in the range of somatic embryogenesis

from F2 seeds.

Gray et al. (1993) developed an improved protocol for high-frequency somatic

embryogenesis from melon seeds. Using the cultivar ‘Male Sterile A 147’, the authors

45

tested several factors, such as, changes in plant hormone levels and combinations, type of

culture media, and incubation time of explants in those media. This protocol was tested

on 51 commercial melon cultivars, where all underwent somatic embryogenesis, but

exhibited from 5% to 100% explant response and 0.1 to 20.2 embryos per explant,

indicating again a genetic factor in melon embryogenesis response.

Melon regeneration through organogenesis is also affected by melon genotype

(Orts et al. 1987; Ficcadenti and Rotino, 1995; Molina and Nuez, 1995a; Kintzios and

Taravira, 1997; Galperin et al. 2003a; 2003b). Orts et al. (1987) found significant

differences in the morphogenetic response of a diverse group of melon cultivars. The

percentage of calli with developed shoots ranged from 0 to 44.3 among cultivars.

Variability in morphogenetic responses was found between seed lots of the same cultivar.

Comparable results were obtained by Ficcadenti and Rotino (1995), who evaluated the

morphogenetic response of 11 melon cultivars belonging to the reticulatus and inodorus

genotypes. These authors found that melon morphogenetic response was affected by

genetic background, i.e. C. melo var. inodorus genotypes exhibited high narrow shoot

regeneration rates whereas wide differences were noted among the reticulatus types. The

number of shoots per explant ranged from 6.0 to 17.3 for reticulatus varieties and from

12.2 to 14.2 for inodorus genotypes.

A complete statistical approach was used by Molina and Nuez (1995a) to detect

genotypic variability of the in vitro organogenetic response (shoot regeneration) among

individual melon seeds. Their results clearly evidenced the presence of highly significant

differences for organogenetic response, among plants from a specific seed population.

These authors used data from stochastic simulation to study the accuracy of different

46

analysis to detect the presence of genotypic heterogeneity within a population. These

analyses, together with their experimental results, allowed the separation of seed

genotypes differing up to 5% in their regeneration ability. Afterward, Molina and Nuez

(1996) reported the inheritance of organogenesis response in melon cv. ‘Charentais’, by

studying the distribution of the shoot regeneration frequency in F1 and F2 generations

from parents representing extreme values for that in vitro organogenesis response. Their

results suggested a genetic model with two genes, partial dominance, independent

segregation and similar effects for both genes. Recently, Galperin et al. (2003b) claimed

that the high competence for adventitious regeneration in the BU-21/3 melon genotype

was controlled by a single dominant locus, without cytoplasmic interactions.

On the other hand, Kintzios and Taravira (1997) evaluated 14 commercial melon

cultivars for plant regeneration capability. Only six cultivars responded positively to a

shoot induction treatment. Similarly, Galperin et al. (2003a) screened 30 different

commercial melon cultivars for shoot de novo regeneration. In 24 out of 30 melon

genotypes, no detectable normal shoot growth was observed. Five of those which were

able to regenerate, exhibited very low regeneration efficiency. Only the genotype BU-21,

an inbred line, had profuse regeneration of multiple shoots.

In summary, melon in vitro response is under genetic control, however, other

factors should be taken into account for melon regeneration as well. Among them, plant

hormones have a paramount importance upon the melon in vitro response.

2.3.2.2 Polyploidization and Somaclonal Variation

Diploid melon plants have 24 chromosomes (haploid stage n=x=12), and a genome

size of 0.94 pg (454 Mbp/1C) (Arumuganathan and Earle, 1991). A natural and

spontaneous increase in the ploidy level has been observed in field-growing melon plants

47

(Nugent and Ray, 1992), nevertheless, this ploidy increase can also been induced in

muskmelon plants using chemical compounds, such as colchicine (Batra, 1952; Kubicki,

1962). In fact, polyploidy as a method of plant breeding received an increase amount of

attention in the late 1930s, when it was discovered that polyploidy could be induced with

colchicine treatment and the earlier demonstration that heat treatment in early

embryogeny stages could also induce a chromosome doubling (Batra, 1952).

Numerous tetraploid and triploid muskmelon plants have been obtained since the

1930s (Batra, 1952; Kubicki, 1962; Nugent and Ray, 1992; Fassuliotis and Nelson, 1992;

Ezura, et al. 1992a; Adelberg, 1993; Nugent, 1994a; Nugent, 1994b; Adelberg et al.

1995; Adelberg et al. 1999). According to Ezura et al. (1992b), Fassuliotis and Nelson

(1992) and Nugent (1994b), tetraploid melon plants are characterized by having large

male and hermaphrodite flowers, protrudent stigmas, low fertility, thickened and leathery

leaves, rounded cotyledons, highly hairy leaves and stems, short internodes, flat fruits,

large fruit blossom-end scar, increased number of vein tracts on the fruit, and round

seeds. Nonetheless, Shifriss (1941) had previously been reported that tetraploid melon

plants were highly fertile and no later in maturity than the ordinary diploids. Moreover,

Batra (1952) reported that the quality of melon tetraploids was superior to diploids in

certain varieties and that tetraploids were sufficiently fertile to be propagated readily

from seeds.

Regarding the fruit quality of tetraploid melon plants, Batra (1952) and Nugent

(1994a, 1994b) reported that tetraploid fruits were superior in sugar level, firmness, and

had better color than diploid fruits. However, tetraploid melon plants were less

productive, because they had smaller and flatter fruits than diploids, most cultivars had

48

low fertility, and the fruits had an increased tendency to suffer of easy cracking, therefore

reducing considerably their marketable properties.

As it was mentionated before, triploid melon plants have also been produced (Ezura

et al. 1993; Adelberg et al. 1995; Adelberg et al. 1999). Despite triploids plants grew

more vigorously than diploids, and their fruits were not as flat as tetraploids. These

triploids melon plants did not have any marketable advantage over diploids ones, because

still the percentage of cracking in those triploid fruits was greater than diploid fruits, and

their sugar content was lower. In addition, triploid plants required adjacent diploid

pollinators as a result that they did not set fruit when self-pollinated (Ezura et al. 1993;

Adelberg et al. 1995).

When modern biotechnology, specifically plant tissue culture, was applied to

Cucumis melo in order to obtain reliable regeneration protocols, somaclonal variation was

a common observable fact, therefore tetraploid, octaploid, mixoploid, and aneuploid

melon plants were easily recovered from in vitro cultures (Bouabdallah and Branchard,

1986; Fassuliotis and Nelson, 1992; Ezura et al. 1992a; 1992b; Debeaujon and

Branchard, 1992; Kathal et al. 1992; Ezura and Oosawa, 1994a; 1994b; Ezura et al.

1994). According to Ezura et al. (1995), somaclonal variation could be used to obtain

variants lines with low-temperature germinability in melon. Changes in fatty acid patterns

have been found in melon callus tissue (Halder and Gadgil, 1984), as well changes in a

repetitive DNA sequences during callus culture have been detected (Grisvard et al. 1990).

However, somaclonal variation has to be avoided in research, where genetic

transformation is involved because genomic stability in transgenic plants has to be

maintained in order to express the inserted transgene. In addition, regeneration of melon

49

plants has never been obtained from long-term calli tissue cultures of Cucumis melo

(Grisvard et al. 1990).

The production of tetraploid regenerated melon plants has been observed from

somatic embryogenesis (Ezura et al. 1992a; 1992b), organogenesis (Bouabdallah and

Branchard, 1986; Fassuliotis and Nelson, 1992; Ezura et al. 1992a), and protoplast

regeneration (Debeaujon and Branchard, 1992). Nevertheless, each morphogenetic

pathway has a diffferent effect on the frequency of recovered tetraploid plants, i.e.

somatic embryogenesis (31%), adventitious shoots (30%), shoot primordia (4%), and

axillary buds (0%) (Ezura et al. 1992a). Therefore, when a callus stage is involved in the

regeneration process the likelihood to augment the ploidy level in the regenerated plant is

increased as well. In addition, explant origin affects the frequency of tetraploid plants

from melon tissue cultures (Adelberg et al. 1994). Immature cotyledons produced more

tetraploid regenerants than mature cotyledons, while explants from apical meristems

produced fewer or no tetraploid plants (Adelberg et al. 1994).

Ezura and Oosawa (1994a; 1994b) reported that the capacity of diploid melon cells

to generate in vitro shoots was greater than tetraploid cells. The ability of tetraploid cells

to differentiate into somatic embryos was greater than diploid cells. These same authors

reported that the ability of somatic embryos to develop into plantlets decreased in the

following order: diploid>tetraploid>octaploid. Ezura and Oosawa (1994a) and Kathal et

al. (1992) reported the longer melon cells are kept under in vitro conditions, the greater

the possibility to increase the ploidy levels in those cells. The frequency of chromosomal

variation leading to aneuploid (hyperploid and hypoploid) plants at diploid, tetraploid and

octaploid levels also increases.

50

Melon plant ploidy levels can be determined by cytological methods, such as

counting the chromosome number using root squash tips (Ezura et al. 1992a; 1992b;

Kathal et al. 1992; Ezura et al. 1993; Ezura et al. 1994; Adelberg et al. 1995) or young

tendrils (Yadav and Grumet, 1994). These methods are very laborious and time

consuming, and are not completely reliable because melon chromosomes are smaller in

comparison with other plants, which complicated the easy chromosome observation.

Unconventional and indirect techniques have been developed in order to determine the

ploidy level of regenerated melon plants (Fassuliotis and Nelson, 1992; Adelberg et al.

1994; Adelberg et al. 1995). Among them, pollen grain shape and stomate length, as well

as the chloroplast number in guard cells from stomata have been commonly used. Diploid

plants have pollen grains with typical triangular-appearing shape and are tripolar,

whereas tetraploid plants produce many square tetraporous, round-monoporous or oval

biporous pollen grains (Fassuliotis and Nelson, 1992; Adelberg et al. 1994; Adelberg et

al. 1995). Likewise, Fassuliotis and Nelson (1992) reported that in diploid plants the

average stomate length was 22.1 ± 2.15 µm and the average number of chloroplast inside

the guard cell was 9.4 ± 1.5, while in tetraploid plants the average stomate length was

29.1 ± 2.15 µm and the average number of chloroplast inside the guard cell was twice the

diploid plant. All these data were measured in the epidermal layer from the third or fourth

expanded leaf from the apex.

Flow cytometry is the only absolute, reliable and precise technique to determine the

exact level of ploidy, and even it is useful to detect any chromosomal change or

polysomaty state in melon tissues (Brown, 1984; Delaat et al. 1987; Dolezel et al. 1998;

51

Gilissen et al. 1993; Dolezel et al. 2004). Neverthelesss, the economic constraint to

purchase and operate this equipment restricts its wide usage.

In order to avoid somaclonal variation and regenerate mostly diploid plants from

melon in vitro cultures, several strategies have been proposed. Among them, the

induction of shoot primordium aggregates from shoot-tips then cultivating them in liquid

medium shaken at low speed (Ezura et al. 1997b) was reported. Using this protocol, the

frequency of tetraploids and mixoploids regenerated plants was less than 8% after 4 years

of culture. The cryopreservation of shoot primordia cultures at low temperatures (liquid

nitrogen) using a slow prefreezing procedure has given excellent results as well (Niwata

et al. 1991; Ogawa et al. 1997). A reliable system for transformation of a cantaloupe

Charentais type melon leading to a majority of diploid regenerants was developed (Guis

et al. 2000). Unfortunatly, this regeneration system did not generate completely

developed transgenic shoots for other commercial melon cultivars (Nuñez-Palenius et al.

2002; Gaba, 2002, personal communication), again, a genetic factor is involved at some

stage in melon culture in vitro response.

In summary, it is particularly important to avoid in vitro-conditions which produce

polyploid melon plants or other induced somaclonal variation, in order to maintain

commercial value of the new genotype.

2.3.2.3 Vitrification

Woody and herbaceous explants are proned to suffer anatomical, morphological

and physiological abnormalities when they are cultivated in vitro. Several terms have

been used to describe those abnormalities, such as vitrification, translucency,

hyperhydration, succulency and glassiness (Paques and Boxus, 1987; Ziv, 1991).

52

Vitrification term is the most often used term to describe physical changes in leaves and

roots of cultured explants (Paques et al. 1987).

Melon in vitro cultures are very sensitive to undergo spontaneous vitrification, even

if explants are cultured on non-inductive media or conditions (Leshem et al. 1988a;

1988b). Different factors have been proposed to induce and maintain an explant in a

vitrificated state, such as, high relative humidity inside the in vitro container, high water

potential of the media, low agar level, deficiency in Ca2+ level, high NH4 concentration,

presence of ethylene within the flask, and high a level of cytokinins (mostly BA)

(Leshem et al. 1988a; 1988b; Paques and Boxus, 1987; Paques et al. 1987; Ziv, 1991).

Leshem et al. (1988a) studied the development of vitrification in melon shoot tips

cultured in solid and liquid media. These authors found that on solid medium the

vitrification process gradually increased with time, whereas on liquid medium it was an

‘all-or nothing’ effect. Cytokinins had the major effect on vitrification induction on

melon buds as well (Leshem et al. 1988b). Paques et al. (1987) and Kathal et al. (1994)

reported that vitrification process was an inducible and reversable physiological event. If

the tissues are frequently subcultured, vitrification may be avoided, however this may

induces somaclonal variation.

The following modifications in the culture media have been suggested to avoid

vitrification: increasing the agar concentration, diminishing chloride ions, reducing

potassium, increasing calcium, adding cobalt, modifying the plant hormone balance by

reducing the amount or type of cytokinins, suppressing the use of casein hydrolysate and

adenine sulphate, and adding pectin, phoroglucinol, or phloridzine (Paques and Boxus,

1987; Paques et al. 1987; Ziv, 1991). In vitro environmental conditions can also be

53

altered including: several treatments have been proposed: a cold treatment to the plants

before in vitro culture, reducing culture room temperature, increasing the daily dark

period, increasing the container-environment gas exchange, and reducing the relative

humidity within the flask (Paques and Boxus, 1987; Paques et al. 1987; Ziv, 1991).

2.3.3 Regeneration by Organogenesis

In addition to genotype, explant source or type has a main role on melon in vitro

regeneration (Adelberg et al. 1994; Ficcadenti and Rotino, 1995; Molina and Nuez,

1995b; Curuk et al. 2002b). Shoots, roots, and complete melon plants have been de novo

regenerated through organogenesis using diverse explants, among them: cotyledons (from

immature and quiescent seeds, and/or seedlings), hypocotyls, roots, leaves, protoplasts,

and shoot meristems have been reported (Table 3.2. and Table 3.3). This in vitro

organogenetic pathway may produce plants by direct regeneration (non-callus growth

between explant culture and de novo shoot induction) (Table 3.2) or indirect regeneration

(involving callus growth before de novo shoot induction) (Table 3.3).

Cotyledons and true leaves have commonly a higher regeneration frequency (above

80%) of de novo shoots using direct organogenesis compared to other melon explants

(Moreno et al. 1985a; 1985b; Kathal et al. 1986; Orts et al. 1987; Tabei et al. 1991;

Ficcadenti and Rotino, 1995; Yadav et al. 1996; Nuñez-Palenius et al. 2002, Galperin et

al. 2003a; 2003b). Likewise, Molina and Nuez (1995b) studied the variation in

regeneration ability among and within several populations of leaf, cotyledon and

hypocotyl melon explants. These authors reported that in vitro clonal selection to

improve the regeneration frequency from leaf explants also raises the organogenetic

response in other explant types. These results suggest the presence of a partial common

genetic system controlling the regeneration frequency of all type of explants.

54

In addition to explant type, other non-genotype in vitro culture components may

have some influence upon the efficiency of melon regeneration through organogenesis,

among them, environmental factors and media composition.

2.3.3.1 Medium Composition

It is generally accepted that medium composition has a greater effect upon

organogenic regeneration than environmental factors. This is due to the fact that plant

growth regulators have an enormous effect on in vitro cell development. In general, a

cytokinin/auxin ratio greater than 1 is used in order to induce de novo bud formation,

however, auxins are not always a prerequisite to achieve that goal (Table 2.3 and Table

2.4), and cytokinins alone are able to induce bud formation. Among cytokinins, 6-

benzylaminopurine (N6-benzyladenine, BA) is the most frequently used in high levels (1

mg/l or higher) to induce bud formation. BA concentration is lowered (0.5 mg/l or lower)

to allow shoot elongation. Elongated shoots are then transferred to a plant growth

regulator-free medium or with low-auxin level (NAA or IAA) to induce the rooting

process. If indirect regeneration is used, two- or even a three-step method has to be

utilized. First, an induction callus growth is stimulated on the explant by applying strong

cytokinins (TDZ) and auxins (2,4-D) to the medium culture. Second, those calli, which

have green nodules are transferred to a low-level plant hormone-medium to induce shoot

differentiation, and third, differentiated shoots are cultured in a low-cytokinin medium to

induce shoot elongation (Table 2.3). In general, indirect regeneration is longer in time

than direct regeneration to recover a whole regenerated plant. This is due to the several

subcultures are required for shoot elongation and two or three months are needed for

rooting using the indirect method (Moreno et al. 1985a; 1985b; Kathal et al. 1986; 1994).

55

Table 2.3 Melon regeneration (shoots, roots and/or complete plants) through direct organogenesis. Explant Source Cultivar Induction Medium

(Plant Growth Regulators) Reference

Cotyledon and primary leaf ‘Bananna’, ‘Dixie Jumbo’, Planters Jumbo’, ‘Morgan’, ‘Cavaillon Red Flesh’, and ‘Saticoy Hybrid’

BR-1 medium: 0.1mg/l NOA, 20 mg/l 2iP, and 0.1 mg/l CCC HC medium: 0.05 mg/l NOA, 10 mg/l 2iP, and 0.1 mg/l CCC

1

Hypocotyl from 11 to 13-day-old seedlings

‘Amarillo Oro’ 4.5 mg/l IAA 2

Leaf (0.3-0.5 cm) from 14-day-old seedlings

‘Pusa Sharbati’ 0.22 mg/l BA and 0.2 mg/l 2iP 3

Cotyledons ‘Halest Best’, ‘Iroquois’, and ‘Perlita’

NAA and BA 4

Cotyledons from mature seeds, and cotyledons and leaves from 5 to 7-day-old seedlings

‘Accent’, ‘Galia’, ‘4215’, ‘Preco’, and ‘Viva’

1 mg/l BA 5

Cotyledons from 4-day-old seedlings ‘Superstart’, ‘Hearts of Gold’, ‘Hale’s Best Jumbo’ and ‘Goldstart’

0.88 mg/l IAA and 1.13 mg/l BA 6

Cotyledons from 4-day-old seedlings

Not reported 0.2 mg/l BA 7

Cotyledons from 9 to 10-day-old seedlings

‘Topmark’ 1 mg/l 8

56

Table 2.3 Continued Explant Source Cultivar Induction Medium

(Plant Growth Regulators) Reference

Cotyledons from mature seed, cotyledons and hypocotyl from 10-day-old seedlings, and leaf segment and petioles from 3-weeks-old seedlings

‘Earl’s Favorite Harukei No.3’ 0.01 mg/l 2,4-D or 1 mg/l IAA and 0.1 mg/l BA 9

Cotyledons from 8-day-old seedlings ‘Charentais’ and ‘Gulfstream’ 1.12 mg/l BA for ‘Charentais’ and 1.12 mg/l BA and 1.75 mg/l IAA for ‘Gulfstream’

10

Cotyledons from 2-day-old seedlings ‘Sunday Aki’ 1 mg/l BA, 50-200 µM salycilic acid and 10 mM proline

11

Cotyledons from 7-day-old seedlings

Five Inbred lines from Teziers 0.1 mg/l NAA and 0.5 mg/l BA 12

Cotyledons from immature seeds ‘Miniloup’, ‘L-14’, and ‘B-Line’

2.25 mg/l BA 13

Cotyledons from mature seeds

‘Prince’and ‘Andes’ 1 mg/l BA 14

Cotyledons from 4-day-old seedlings

11 genotypes 0.63 mg/l BA and 0.26 mg/l ABA 15

3-4 cm diam expanded leaves ‘Hale’s Best Jumbo’ and ‘Ananas El Dokki’

0.87 mg/l IAA, 1.13 mg/l BA and 0.026 ABA 16

Cotyledons from 7-day-old seedlings

‘Pusa Madhuras’ 0.22 mg/l BA 17

57

Table 2.3 Continued Explant Source Cultivar Induction Medium

(Plant Growth Regulators) Reference

Cotyledons from 2-week-old seedlings 14 cultivars Embryogenesis: 1.98 mg/l 2,4-D and 4.99 mg/l Organogenesis: 0.01 mg/l 2,4-D and 0.059 mg/l BA

18

Leaves from 10 day-old seedlings ‘Védrantais’ 0.22 mg/l BA and 0.33 mg/l 2iP 19

Cotyledons from 2-day-old seedlings

‘Galia’ male and female parental lines

0.001 mg/l NAA and 1 mg/l BA 20

Cotyledons from mature seeds ‘Yellow Queen’, ‘Yellow King’, and ‘Hybrid AF-222’

1 mg/l BA 21

Proximal zone of the Hypocotyl from 4-day-old seedlings

‘Revigal’ 1 mg/l BA 22

Cotyledons from 4 to 5-day-old-seedlings

Some Turkish cultivars: ‘Hasanbey’ I, ‘Yuva’, ‘Kirkagac 637’, ‘Topatan’, ‘Kuscular’ and ‘Ananas’

Medium and plant growth regulators from reference 6 23

Cotyledons from mature seeds Thirty melon genotypes 1 mg/l BA 24 1. Blackmon et al. (1981a; 1982), 2. Moreno et al. (1985b), 3. Kathal et al. (1988), 4. Mackay et al. (1988), 5. Dirks and Van Buggenum, (1989), 6. Niedz et al. (1989), 7. Leshem et al. (1989), 8. Chee, (1991), 9. Tabei et al. (1991), 10. Fassuliotis and Nelson, (1992), 11. Shetty et al. (1992), 12. Roustan et al. (1992), 13. Adelberg et al. (1994), 14. Ezura and Oosawa, (1994a), 15. Ficcadenti and Rotino, (1995), 16. Yadav et al. (1996), 17. Singh et al. (1996), 18. Kintzios and Taravira, (1997), 19. Guis et al. (2000), 20. Nuñez-Palenius et al. (2002), 21. Stipp et al. (2001), 22. Curuk et al. (2002a), 23. Curuk et al. (2002b), 24. Galperin et al. (2003a).

58

Table 2.4 Melon regeneration through indirect organogenesis. Explant Source Cultivar Callus Induction Medium (CIM) and Shoot

Induction Medium (SIM) (Plant Growth Regulators)

Reference

Cotyledon-callus culture from 11 to 13-day-old seedlings

‘Amarillo Oro’ CIM: 1.5 mg/l IAA and 6.0 mg/l KIN SIM: 0.01mg/l NAA and 0.1 mg/l BA

1

Hypocotyl-callus culture from 7-day-old seedlings

‘Pusa Sharbati’ CIM: 1.0 mg/l IAA and 0.5 mg/l KIN SIM: 0.5 mg/l BA and 0.5 mg/l 2ip

2

Cotyledon-callus from 7 to 9-day-old seedlings

‘Charentais T’, ‘Doublon’, ‘CM 17 187’, and ‘Piboule’

CIM: 2 mg/l IAA and 2 mg/l KIN SIM: no plant growth regulators

3

Cotyledon-callus from 11 to 13-day-old seedlings

15 cultivars belonging to cantaloupensis, inodorus and reticulatus varieties

CIM: 6.0 mg/l KIN and 1.5 mg/l IAA SIM: Same as CIM

4

Cotyledon-protoplasts from 2-week-old seedlings

‘Hong-Xin-cui’ Protoplast Culture Medium and CIM: 0.5 mg/l 2,4-D, 0.5 mg/l Zeatin and 0.5 mg/l BA SIM: 0.3 mg/l 2,4-D, 1.0 mg/l Zeatin and 0.5 mg/l BA

5

Leaf segment (1.0 X 0.5 cm) from 8 to 10-day-old seedlings and petiole segment (0.4-0.8 cm) from 3 to 4-week-old seedlings

‘Cantaloupe PMR’ CIM: 5.0 mg/l NAA and 2.5 mg/l BA SIM: no plant growth regulators

6

Cotyledon-protoplasts from 2-week-old seedlings

‘Charentais’ Protoplast Culture Medium and CIM: 0.05 mg/l 2,4-D and 0.5 mg/l BA. SIM: 2 mg/l BA

7

59

Table 2.4 Continued Explant Source Cultivar Callus Induction Medium (CIM) and Shoot

Induction Medium (SIM) (Plant Growth Regulators)

Reference

Cotyledon-protoplasts from 12-day-old seedlings and fully expanded leaves-protoplasts from 3-week-old seedlings

‘Charentais T’ and F1 hybrid cv. ‘Preco’

Protoplast Culture Medium and CIM: 0.75 mg/l BA SIM: 1.0 mg/l 2,4-D and 0.1 mg/l BA

8

Root-callus culture from 21-day-old seedlings

‘Pusa Sharbati’ CIM: 0.61 mg/l 2iP and 0.68 mg/l BA SIM: 0.22 mg/l BA

9

Cotyledon and Hypocotyl from 11-13 day-old-seedlings

‘Charentais’ CIM: 2.5 mg/l NAA and 1 mg/l BA SIM: 0.01 mg/l NAA and 6 mg/l Kin

10

1. Moreno et al. (1985b), 2. Kathal et al. (1986), 3. Bouabdallah and Branchard, (1986), 4. Orts et al. (1987), 5. Li et al. (1990), 6. Punja et al. (1990), 7. Tabei et al. (1992), 8. Debeaujon and Branchard, (1992), 9. Kathal et al. (1994), 10. Molina and Nuez, (1995b).

60

Other plant hormones, such as gibberellins, and ABA (Kathal et al. 1986; Niedz et

al. 1989; Ficcadenti and Rotino, 1995), and/or plant additives, such as proline, proline

analogues, ornithine, salicylic acid, aspirin, and fish protein hydrolysates (Shetty et al.

1992; Milazzo et al. 1998; et al. 1999), Calcium antagonists (Leshem and Lurie, 1995),

silver nitrate (Niedz et al. 1989; Roustan et al. 1992; Yadav et al. 1996), have been used

to increase in vitro shoot regeneration frequency, nevertheless, the obtained results using

both types of components (plant hormones and plant additives) are either melon

genotype-dependent or are not consistent among reported results.

2.3.3.2 Environmental Factors

Environmental factors, such as light, temperature, nature of media gelling agent,

and relative humidity within flask, influence the efficiency of regeneration method

(Niedz et al. 1989; Ficcadenti and Rotino, 1995; Yadav et al. 1996; Kintzios and

Taravira, 1997; Curuk et al. 2003). For instance, Niedz et al. (1989) studied the effect of

temperature (22, 25 and 29oC) and light (0, 5, 10, 30, 60 and 3,000 µmol.m-2s-1) on the

percentage of bud initiation in cotyledonary explants of ‘Hale’s Best Jumbo’ melon. The

greatest bud initiation was obtained when explants were cultured at 29oC under a range of

light intensity of 5 to 30 µmol.m-2s-1. Conversely, lower temperatures (22 and 25oC),

darkness, and higher light intensities (60 and 3,000 µmol.m.2s-1) reduced bud initiation.

Similarly, Kintzios and Taravira (1997) tested two levels of light intensity, 50 and 250

µmol.m.2s-1 on shoot and root induction in 14 different melon cultivars. As expected,

lower light intensities induced a greater root induction in many melon cultivars. Also,

higher PPFD (250 µmol.m.2s-1) values adversely affected shoot induction from

cotyledonary explants. Interestingly, Curuk et al. (2003) recently described that

61

regeneration from hypocotyls (proximal part to the cotyledons) of Cucumis species does

not requiere light.

High relative humidity within the flask culture might induce ethylene

accumulation, which affects shoot regeneration in melon cotyledons (Roustan et al.

1992). These authors added several levels of silver nitrate (60-120 µM AgNO3) into

culture media to inhibit ethylene action. They were able to obtain a two-fold increase in

shoot regeneration by using silver. Furthermore, a transgenic antisense ACC oxidase line,

which had little ethylene production, displayed a 3.5-fold increase in regeneration

frequency compared to wild type line (Amor et al. 1998).

As it was pointed out previously, the nature of medium gelling agent also has an

important role in melon regeneration (Ficcadenti and Rotino, 1995; Yadav et al. 1996).

Ficcadenti and Rotino, (1995) described that using agar, instead of ‘gelrite’, they were

able to attain a better cotyledon organogenetic response. Likewise, Yadav et al. (1996)

preferred ‘phytagel’, as a substitute of agar for leaf organogenesis.

2.3.4 Regeneration by Somatic Embryogenesis

In addition to organogenesis, somatic embryogenesis is an alternative de novo

morphological pathway that it can be induced in explants to form and recover whole

dicots plants (Liu and Cantliffe, 1983). Somatic embryogenesis was described in melon

explants before organogenesis and used to recover complete melon plants (Table 2.5)

(Blackmond et al. 1981b). In general, cotyledonary tissue has been the most efficient

explant for the induction of melon somatic embryogenesis (Table 2.5).

An embryogenic response in melon is affected by the nature of explant and

genotype. Gray et al. (1993) reported significant differences in the frequency of

62

embryogenic muskmelon explants when they compared two commercial sources of the

same melon cultivar. High regeneration frequency, up to 100%, may be attained from

cotyledon explants with an average number of 20.2 embryos per explant (Gray et al.

1993). However, not all melon cultivars achieve that such a high regeneration frequency,

some as low as 5% with 0.1 embryos per explant (Gray et al. 1993). The conversion rate

of somatic embryos to plantlets might be a limiting step in some melon genotypes, for

example, Trulson and Shahin (1986) were able to recover only 5 melon plants from

hundreds of somatic embryos, Branchard and Chateau (1988) reported a 12% conversion

rate, and Homma et al. (1991) reported a conversion rate from 7% up to 61%, depending

on explant type.

Tabei et al. (1991) concluded that cotyledons were the best explant to induce

somatic embryogenesis in the melon cultivar ‘Earl’s Favorite Harukei No.3’ using high

concentrations of 2,4-D. Homma et al. (1991) tested the effects of explant shape on the

production of melon somatic embryos, finding that the most reproducible result was

obtained with explants consisted of radicle, hypocotyl and a proximal part of cotyledon.

Oridate et al. (1992) found significant differences in somatic embryogenesis capability

from 18 different melon cultivars from four genotypes. These authors concluded that

genetic differences in somatic embryogenic formation capacity existed among cultivars

rather than among genotypes.

Debeaujon and Branchard (1993) published a complete and extensive review on

somatic embryogenesis in Cucurbitaceae, including Cucumis melo, where they

concluded that even though somatic embryogenesis and plant recovery have been

obtained from numerous plant sources including protoplasts,

63

Table 2.5 Melon regeneration through somatic embryogenesis. Explant Source Cultivar Induction Medium (IM) and Development

Medium (DM) (Plant Growth Regulators)

Reference

Cotyledons from 5-day-old seedlings

‘Hale’s Best No.36’ and ‘Rocky Ford’

IM: 1.0 mg/l 2,4-D and 0.5 mg/l BA DM: 0.5 mg/l 2,4-D and 0.25 mg/l BA

1

Expanded cotyledons ‘Charentais T’ IM: 4.52 mg/l 2,4-D and 0.44 mg/l BA DM: no hormones

2

Cotyledons from mature seeds and cotyledons and hypocotyls from 10-day-old seedlings, leaves and petioles from 3-week-old plantlets

‘Earl’s Favorite Harukei No. 3’

IM: 2.0 mg/l 2,4,-D or 25 mg/l IAA DM: no hormones

3

Cotyledons and hypocotyls from mature seeds

‘Green Pearl’ and ‘Earl’s Favourite’

IM: 4 mg/l 2,4-D, 2 mg/l NAA, and 0.1 mg/l BA DM: no hormones

4

Hypocotyls from mature seeds ‘Earl’s Favourite’ IM: 1 mg/l 2,4-D, 1 to 4 mg/l NAA, and 0.1 mg/l BA DM: no hormones

5

Cotyledons from 1-day-old seedlings

‘Earl’s Favourite Haru 1’ IM: 1 mg/l 2,4-D, 1 mg/l NAA, and 0.1 mg/l BA DM: no hormones

6

Protoplasts from 12-day-old cotyledons

‘Charentais T’ and F1 hybrid ‘Preco’

IM: 1 mg/l 2,4-D and 0.1 mg/l BA DM: no hormones

7

64

Table 2.5 Continued

Explant Source Cultivar Induction Medium (IM) and Development Medium (DM)

(Plant Growth Regulators)

Reference

Cotyledons and hypocotyls from mature seeds

18 cultivars belonging to reticulatus, inodorus, makuwa and intermediated type between reticulatus and cantaloupensis varieties

IM: 3.0 mg/l 2,4-D and 0.1 mg/l BA DM: no hormones

8

Cotyledons from mature seeds 52 cultivars IM: 5 mg/l 2,4-D and 0.1 mg/l TDZ DM: no hormones

9

Mature seeds ‘Earl’s Favourite’ IM: 4 µg/l 2,4-D and 0.1 µg/l BA DM: no hormones

10

Hypocotyls from imbibed seeds ‘Prince’ and ‘Sunday Aki’ IM: 1 mg/l 2,4-D, 2 mg/l NAA and 0.1 mg/l BA DM: no hormones

11

Cotyledons from mature seeds ‘Védrantais’ IM: 2.2 mg/l 2,4-D and 0.11 mg/l BA DM: no hormones

12

Cotyledons from mature seeds ‘Yellow Queen’ and ‘Yellow King’

IM: 5.0 mg/l 2,4-D and 0.075 mg/l TDZ DM: no hormones

13

Mature seeds ‘Védrantais’ and ‘Earl’s Favoutire Fuyu A’

IM: 2.0 mg/l 2,4-D and 0.1 mg/l BA DM: no hormones

14

1. Trulson and Shahin, (1986), 2. Branchard and Chateau, (1988), 3. Tabei et al. (1991), 4. Homma et al. (1991), 5. Shimonishi et al. (1991), 6. Kageyama et al. (1991), 7. Debeaujon and Branchard, (1992), 8. Oridate et al. (1992), 9. Gray et al. (1993), 10. Hosoi et al. (1994) 11. Ezura and Oosawa (1994a), 12. Guis et al. (1997a), 13. Stipp et al. (2001), 14. Akasa-Kennedy et al. (2004).

65

the best results were observed with explants coming from seedling parts, especially

cotyledons and hypocotyls. These authors also reported that the genetic constitution of

donor plants seems to play a key role in the success of somatic embryogenesis.

2.3.4.1 Medium Composition

Media composition (mostly plant growth regulators) has a great effect on melon

somatic embryogenesis. The embryogenic pathway involves a two-stage methodology

process: first, explants are cultured onto an ‘induction’ medium, to which auxins have

been added, and second, ‘induced’ explants are transferred to development media, where

full and complete normal embryo development takes place in the absence of the induction

hormone. Auxins are prerequisite for induction of somatic embryogenesis (Tabei et al.

1991; Oridate et al. 1992; Debeaujon and Branchard, 1993; Gray et al. 1993; Guis et al.

1997a; Nakagawa et al. 2001).

In general, the most common and efficient auxin to induce somatic embryogenesis

in melon explants is 2,4-D (Oridate and Oosawa, 1986; Debeaujon and Branchard, 1993).

However, other auxins can be used, such as IAA and NAA, although NAA at high

concentrations can induce abnormal embryo growth (Tabei et al. 1991). Likewise, Tabei

et al. (1991) reported that IAA was the most efficient auxin to induce somatic

embryogenesis in ‘Earl’s Favorite Harukei No.3’ melon. Auxins can be used in

combination with cytokinins, such as BA and TDZ, and/or other hormones, such as ABA

(Trulson and Shahin, 1986; Tabei et al. 1991; Homma et al. 1991; Debeaujon and

Branchard, 1992; Gray et al. 1993; Guis et al. 1997a; Nakagawa et al. 2001). Hormones

are removed to mature the embryos (Table 3.4), nevertheless gibberellins can be added to

the culture medium (Tabei et al. 1991). ABA (10 mg.L-1) was supplemented into the

66

culture medium in order to control the desiccation process and to increase the survival

rate before cryopreservation (Shimonishi et al. 1991).

The type and concentration of carbohydrate in the media plays a role in somatic

embryogenesis in melon (Oridate and Yasawa, 1990; Debeaujon and Branchard, 1992;

Gray et al. 1993, Guis et al. 1997a). Oridate and Yasawa, (1990) reported that a complex

combination of different sugars, such as sucrose, glucose, fructose, and galactose, led to

the highest rate of somatic embryogenesis. Similarly, Gray et al. (1993) concluded that

the sucrose concentration in embryo induction and development media had a profound

effect on somatic embryogenesis, i.e. 3 % sucrose produced a greater explant response

than lower or higher levels of the carbohydrate. These authors found that sucrose

concentration also exerted an effect on the relative percentage of somatic embryo stages

recovered and on abnormal embryo development and precocious germination. Guis et al.

(1997a) tested the effects of several levels of sucrose, glucose and maltose, on inducing

melon somatic embryogenesis. Glucose enhanced the embryogenic response by almost

two-fold, whereas maltose at any level totally somatic embryogenesis. Nakagawa et al.

(2001) reported that the addition of mannitol to the initial media increased the frequency

of somatic embryogenesis in ‘Prince’ melon.

2.3.4.2 Environmental Factors

Several physical factors can affect melon embryogenesis, among them, presence

and quality of light, and physical state of media culture are the most important. In order

to induce somatic embryogenesis, melon explants are cultured in light (Trulson and

Shahin 1986; Branchard and Chateau 1988; Tabei et al. 1991; Homma et al. 1991;

Shimonishi et al. 1991; Ezura et al. 1992a; Debeaujon and Branchard, 1992; Oridate et al.

1992, Debeaujon and Branchard, 1993; Hosoi et al. 1994; and Nakagawa et al. 2001).

67

Somatic embryo formation has been augmented by pretreatment in a dark period, usually

one or two weeks before placing in light (Gray et al. 1993, Guis et al. 1997a). Culturing

on solid medium (Branchard and Chateau, 1988) is better than using liquid medium.

Different gelling agents have been used for this purpose, obtaining improved developed

embryos by using ‘gelrite’ and/or ‘phytagel’ as a substitute for agar (Branchard and

Chateau, 1988). Nevertheless, recently Akasaka-Kennedy et al. (2004) reported that

somatic embryos from ‘Vedrantais’ and ‘Earl’s Favourite Fuyu A’ melon cultivars

underwent development without vitrification, if agar was used instead of ‘gelrite’. Thus

different melon cultivars give diverse responses under similar in vitro conditions.

Kageyama et al. (1991) and Moreno et al. (1985a) reported that the vitrification state of

regenerated plants is increased if liquid cultures are used during the initial steps of

somatic embryogenesis. Kageyama et al. (1991) reported that consecutive washing of

somatic embryos with hormone-free MS medium with 0.5% activated charcoal increased

the number of somatic embryos two-fold.

2.3.5 Haploid Plants and Embryo Culture

Hybrid cultivars represent the F1 progeny of crosses that may involve inbred lines,

clones, or populations (Fehr et al. 1987). The most common type of hybrid cultivar is

produced by crossing two or more inbred lines, which have to be homozygous for certain

important traits (Fehr et al. 1987). The production of inbred lines in Cucumis melo

requires several generations, taking more than seven years of inbreeding in order to

obtain homozygosity (Yashiro et al. 2002). Through using a plant biotechnology

approach, such as production of haploid melon plants, is possible to reduce the amount of

time to obtain melon inbred lines. Diploids can be induced by chromosome doubling

agents, such as colchicine or oryzalin (Yetisir and Sari, 2003; Lotfi et al. 2003). This

68

approach has been used to obtain plants tolerant to diseases, such as virus or powdery

mildew (Kuzuya et al. 2000; 2002; 2003; Lotfi et al. 2003).

According to Guis et al. (1998), using androgenesis and gynogenesis have not been

successful to produce haploid melon plants. Using either gamma or soft X-ray irradiated

pollen will induce in situ gynogenetic haploid parthenogenesis in melon (Sauton and

Dumax de Vaulx 1987; Cuny et al. 1992; 1993; Yanmaz et al. 1999; Kuzuya et al. 2000).

Sauton and Dumax de Vaulx (1987) developed an in vitro technique (commonly named

embryo rescue or embryo culture) to recover muskmelon haploid plants. These authors

obtained haploid plants after pollination of hermaphrodite flowers with irradiated (Co60 γ-

rays) pollen and in vitro culture of ovules or immature embryos. They also developed a

new culture medium to allow further development of these embryos into plants, resulting

with an average number of 2.5 haploid embryos per 100 seeds. Sauton and Dumax de

Vaulx’s embryo culture technique has been applied not only to induce and rescue haploid

melon plants, but also to culture diploid embryos as well in numerous melon cultivars

with excellent results (Sauton, 1988; Kuzuya et al. 2000; Oliver et al. 2000; Kuzuya et al.

2002; Lotfi et al. 2003; Kuzuya et al. 2003; Yetisir et al. 2003).

The rate of melon haploid production is afected by genotypic factors and

environmental growth conditions of donor plants (Sauton, 1988; Cuny et al. 1992; 1993;

Yanmaz et al. 1999). Sauton (1988) studied haploid embryo production on seven melon

cultivars, belonging to five melon types, i.e. reticulatus, cantaloupensis, inodorus,

conomon and acidulus. Gynogenetic haploid embryo production among those melon

genotypes ranged from 0 to 1.7%. Cuny et al. (1993) reported significant differences in

haploid embryo production between two melon cultivars, ‘Vedrantais’ which produced

69

an average number of haploid embryos of 3.5%, whereas ‘F1.G1’ formed 1.7%. Sauton

(1988) found that ‘Arizona’ muskmelon produced the highest number of haploid embryos

(3%) if melon plants were grown during the summer season. Cuny et al. (1992) reported

the effect of season planting on haploid embryo induction. These authors found that

haploid embryo induction was greater in ‘Vedrantais’ melon fruits obtained from plants

grown in summer than those harvested in autumn.

Unlike other plant systems, such as carrot and tobacco, melon pollen is able to

tolerate high γ-irradiation doses (up to 3,600 Grays) and still germinate –in vitro as well

as in vivo- and inducing parthenocarpic haploid plant production (Cuny et al. 1992; 1993;

Yanmaz et al. 1999). However, a significant reduction in pollen tube length has been

observed using high radiation doses. This reduction was proportional to the amount of γ-

radiation used (Cuny et al. 1992; 1993; Yanmaz et al. 1999). High γ-irradiation doses can

induce an increase in the percentage of necrotic haploid embryos. The most common

gamma irradiation doses are between 250 and 350 Grays (Sauton and Dumax de Vaulx

1987; Cuny et al. 1992; 1993; Beharav and Cohen, 1995a; Yanmaz et al. 1999; Lotfi et

al. 2003; Yetisir and Sari, 2003), and for soft-X-rays the doses are between 65 and 130

kR (Kuzuya et al. 2000; 2002; 2003).

Niemirowicz-Szcztt and Kubicki (1979) demostrated that strong incompatibility

events occurred in the intergeneric and intraspecific crosses within the Cucurbitaceae

family, avoiding sexual hybridization among Cucumis melo. Thus, in vitro embryo

culture technique has been utilized to recover melon haploid plants, and to save valuable

diploid plant material through zygotic embryo culture and somatic embryogenesis.

Different diploid plant material has been recovered, such as hybrid plants obtained after

70

interspecific cross of Cucumis melo (PI140471) x C. metuliferus (PI 29190) (Norton,

1981), Cucumis metuliferus (PI 292190, PI 202681, 3503, and 701A) x C. anguria (PI

233646) (Fassuliotis and Nelson, 1988), Cucumis melo (‘Gylan’ gynoecious E6/10) x C.

metuliferus (‘Italia’) (Beharav and Cohen, 1995a), Cucumis melo (‘Cantaloup

Charentais’) x Cucumis anguria L. var. longipes (Dabauza et al. 1998), and Cucumis

melo (‘Cantaloup Charentais albino mutant’) x Cucumis myriocarpus (Bordas et al.

1998).

In summary, the potential of in vitro embryo culture has lead to reduced time to

obtain inbred melon lines. In addition, the improvement of this technique as well as the

cointegration to marker-assisted selection (MAS) (Fukino et al. 2004) may eventually

allow the transfer of disease resistance and/or other important horticultural traits from

other cucurbits into Cucumis melo species.

2.3.6 Genetic Transformation

Two main natural and artificial genetic transformation processes have been used to

obtain melon transgenic plants; Agrobacterium tumefaciens and particle gun

bombardment (Table 2.6). Succesful transformation of melon with Agrobacterium

rhizogenes has not been reported. Gaba et al. (1992) and Gonsalves et al. (1994) used

particle gun bombardment to transform melon explants and recover transgenic plants

through organogenesis. Gray et al. (1995) used the same transformation protocol and

recovered plants from embryogenic materials. Gonsalves et al. (1994) and Gray et al.

(1995) reported both that A. tumefaciens and microprojectile gene transfer produced

almost the same percentage of transgenic plants. Gray et al. (1995) produced stable

normal plants via particle bombardment while embryos from Agrobacterium-mediated

71

transformation were abnormal. Mefoxine, was used to stop Agrobacterium growth may

have caused this.

Transformation success via Agrobacterium or particle gun bombardement is

genotype-, explant source-, and in vitro culture conditions-dependent (Fang and Grumet,

1990; Dong et al. 1991; Yoshioka et al. 1992; Valles and Lasa, 1994; Gonsalves et al.

1994; Gray et al. 1995; Bordas et al. 1997; Clendennen et al. 1999; Ezura et al. 2000;

Nuñez-Palenius et al. 2002; Akasaka-Kennedy et al. 2004). The Agrobacterium strain,

vector structure, and co-cultivation with acetosyringone, have an influence on melon

transformation efficiency (Dong et al. 1991; Yoshioka et al. 1992; Valles and Lasa, 1994;

Bordas et al. 1997).

Fang and Grumet (1990) tested several factors, such as kanamycin concentration,

Agrobacterium inoculum level, length of inoculation, period of co-cultivation, and the

use of tobacco nurse cultures, on melon transformation efficiency rate. These authors

found that 75 mg.L-1 kanamycin, fresh overnight grown bacteria at a concentration of 107

– 108 bacteria.ml-1 (OD600= 0.8), 10 min of inoculation, three days of co-cultivation, and

not using tobacco nurse culture were the best conditions to attain an efficient

transformation rate. Unfortunatly, these transformation conditions were only tested in one

melon cultivar, ‘Hale’s Best Jumbo’. A similar approach was described by Dong et al.

(1991). These authors tested the sensitivity of melon cotyledons to kanamycin and

methotrexate concentrations, co-cultivation time, and different selection schemes -1. no

selection pressure, 2. explants were placed in selection medium inmediately after co-

cultivation, and 3. explants were placed under selection two weeks after co-cultivation-.

The highest transformation frequency was obtained when 75 µg.L-1 methotrexate and 100

72

mg.L-1 kanamycin, 5-6 days of co-cultivation period, and immediate selection pressure

was routinely used.

These experiments were carried out on ‘Orient Sweet, F1 Hybrid’ melon cultivar.

Vallés and Lasa (1994) reported that two days of co-cultivation with Agrobacterium

tumefaciens with cotyledons of ‘Amarillo Oro’ melon were necessary to reach an

efficient transformation process. If they used a longer period of co-cultivation, no

transgenic shoots were recovered.

Different bacterial and plant genes, which provide tolerance or resistance to several

selectable chemical agents, have been used to delay or completely inhibit the growth of

non-transformed buds and shoots of melon during the selection process. Among them,

nptII, which provides tolerance to aminoglycoside antibiotics (Fang and Grumet, 1990;

Dong et al. 1991; Yoshioka et al. 1992; Gonsalves et al. 1994; Vallés and Lasa, 1994;

Gray et al. 1995; Ayub et al. 1996; Bordas et al. 1997; Clendennen et al. 1999; Akasaka-

Kennedy et al. 2004), dhfr, which gives resistance to methotrexate (Dong et al. 1991),

and CP4syn, which offers tolerance to Glyphosate herbicide (Nuñez-Palenius et al. 2002;

2003) have been used. When typical concentrations (75-150 mg.l-1) of kanamycin are not

able to inhibit the non-transgenic bud or shoot growth (Guis et al. 1998; Dong et al.

1991), other antibiotics, which are also detoxified by the neomycin phosphotransferase

protein (NPTII), may be added to melon in vitro cultures in order to improve the selection

efficiency. Gentamycin, hygromycin, and paromomycin have been used in melon

cultures as alternative to kanamycin with excellent results (Guis et al. 1998; Nuñez-

Palenius et al. unpublished results, Ezura et al. personal communication).

73

Table 2.6 Genes transferred to melon by plant genetic transformation. Transgene Phenotypic Trait Explant used and

morphogenetic pathway

Cultivar Transformation

Method

Reference

npt-II gene Selectable marker 4 to 5-day-old cotyledons

Organogenesis

‘Hale’s Best Jumbo’ A. tumefaciens

LBA4404 strain

1

npt-II ,

Dihydrofolate

reductase, and

luciferase gene

Selectable markers

and reporter gene

Mature cotyledons

Organogenesis

‘Orient Sweet, F1

Hybrid’

A. tumefaciens

GV3111SE strain

2

CMV-coat protein

gene

Virus resistance Mature cotyledons

Organogenesis

‘ Prince’ and ‘EG360’ A. tumefaciens

LBA4404 strain

3

uidA gene GUS Reporter gene 3-day-old cotyledons

Organogenesis

‘Galia’ Particle

bombardment

4

ZYMV-coat

protein gene

Potyvirus resistance 4 to 5-day-old cotyledons

Organogenesis

‘Hale’s Best Jumbo’ A. tumefaciens

LBA4404 strain

5

uidA gene GUS Reporter gene 5-day-old cotyledons

Organogenesis

‘Amarillo Oro’ A. tumefaciens

LBA4404 strain

6

CMV-white leaf

coat protein gene

Virus resistance 3-day-old cotyledons

Organogenesis

‘Burpee Hybrid’, ‘Hale’s

Best Jumbo’, ‘Harvest

Queen’, ‘Hearts of

Gold’, and ‘Topmark’

A. tumefaciens

C58Z707 strain

7

74

Table 2.6 Continued.

Transgene Phenotypic Trait Explant used and

morphogenetic pathway

Cultivar Transformation

Method

Reference

ZYMV, WMV, and

CMV coat protein

genes

Virus resistance Leaves

Organogenesis

‘Don Luis’, ‘Galleon’,

‘Hiline’, ‘Mission’, and

‘Parental inbred’

A. tumefaciens 8

npt-II gene Selectable marker Cotyledons

Organogenesis

‘Eden Gem’ A. tumefaciens

and Particle

bombardment

9

ACC oxidase

antisense gene from

melon

Improved fruit quality 5-day-old cotyledons

Organogenesis

‘Védrantais’ A. tumefaciens

LBA4404 strain

10

HAL 1 gene Halotolerance 7-day-old cotyledons and

2-week-old leaves

Organogenesis

‘Pharo’ and ‘Amarillo

Canario’

A. tumefaciens

LBA4404 strain

11

ACC synthase

antisense gene

Improved fruit quality NRz NR A. tumefaciens 12

SAM hydrolase gene Improved fruit quality NR NR A. tumefaciens 13

ACO anti-sense gene

from melon

Improved fruit quality 10-day-old true laves

Organogenesis

‘Védrantais’ A. tumefaciens

LBA4404 strain

14

75

Table 2.6 Continued. Transgene Phenotypic Trait Explant used and

morphogenetic pathway

Cultivar Transformation

Method

Reference

SAM hydrolase

gene

Improved fruit quality NR NR A. tumefaciens 15

Ribozyme genes Potyvirus resistance 0 to 4-day-old cotyledons

Organogenesis

US Patent 5,422,259 A. tumefaciens 16

uidA and gfp genes GUS and GFP

Reporter genes

2-day-old cotyledons

Organogenesis

‘Galia’ male and female

parental lines

A. tumefaciens

ABI strain

17

ACC oxidase

antisense gene from

melon

Improved fruit quality 2-day-old cotyledons

Organogenesis

‘Galia’ male parental

line

A. tumefaciens

ABI strain

18

ACC oxidase

antisense gene from

apple

Improved fruit quality NR ‘Védrantais’ A. tumefaciens

LBA4404 strain

19

uidA and hpt genes GUS Reporter and

Selectable marker

genes

Mature seeds

Somatic embryogenesis

‘Védrantais’ and ‘Earl’s

Favourite Fuyu A’

A. tumefaciens

C58C1Rif® strain

20

1. Fang and Grumet (1990), 2. Dong et al. (1991), 3. Yoshioka et al. (1992), 4. Gaba et al. (1992), 5. Fang and Grumet (1993), 6. Vallés and Lasa (1994), 7. Gonsalves et al. (1994), 8. Clough and Hamm, (1995), 9. Gray et al. (1995), 10. Ayub et al. (1996), 11. Bordas et al. (1997), 12. Ezura et al. (1997), 13. Clendennen et al. (1999), 14. Guis et al. (2000), 15. Shellie, (2001), 16. Huttner et al. (2001), 17. Nuñez-Palenius et al. (2002), 18. Nuñez-Palenius et al. (2003), 19. Silva et al. (2004), 20. Akasa-Kennedy et al. (2004). Znot-reported.

76

Genetic transformation efficiency rate in most melon systems is normally lower

than other plant species (Fang and Grumet, 1990; Dong et al. 1991; Gaba et al. 1992;

Gonsalves et al. 1994; Bordas et al. 1997; Akasaka-Kennedy et al. 2004). Several

transformation rate efficiency values have been reported according to transformation

protocol and melon cultivar used, among them, average efficiencies such as 3-7% (Fang

and Grumet, 1990), 4-6% (Dong et al. 1991), 1% (Gaba et al. 1992), 0.0%, 0.9%, and 1%

(Gonsalves et al. 1994), 0.7-3% (Bordas et al. 1997), 2.4% (Guis et al. 2000), 10%

(Nuñez-Palenius et al, 2003), and 2.3% (Akasaka-Kennedy et al. 2004) has been

depicted. Unfortunatly, in many cases most of the recovered transgenic plants had

somaclonal variation especially ploidy changes [tetraploids (75%, Ayub et al. 1996),

octaploids, mixoploids] or had morphogenetic altered characteristics, which were

expressed in the T0 and T1 generation (Gonsalves et al. 1994).

2.3.6.1 Improvement of Disease Resistance

Cucumis melo is attacked by numerous viral, bacterial, mycoplasmal and fungal

organisms, which cause severe diseases (Zitter et al. 1998). These diseases can affect

melons at any plant developmental stage, causing enormous economic losses. According

to Zitter et al. (1998), definitive disease control is reached by using genetically resistance

melon cultivars. More than 30 viruses are able to induce disease symptoms in melon

plants. Cucumber mosaic virus (CMV), zucchini yellow mosaic virus (ZYMV) and

watermelon mosaic virus (WMV) are the most prevalent (Zitter et al. 1998; Gaba et al.

2004).

The first virus–resistant transgenic melon plants were obtained by Yoshioka et al.

(1992). These authors transferred and over-expressed the gene for CMV coat protein via

Agrobacterium tumefaciens using ‘Prince’, ‘EG360’ and ‘Sunday Aki’ melon cotyledons.

77

Those transgenic melon plants which over-expressed the CMV-CP gene, grown under

greenhouse conditions, were found to be resistant to infection after inoculation with a

low-dose of CMV (Yoshioka et al. 1993). Transgenic plants did not develop any

symptoms of disease during 46-day observation period, while control plants had mosaic

symptoms three days after inoculation. When the virus-dose was increased by ten fold,

only a delayed appearance of symptoms was observed in transgenic plants (Yoshioka et

al. 1993). After Yoshioka’s achievement, different authors were able to obtain transgenic

melon plants over-expressing the CMV-CP for other melon cultivars, such as ‘Burpee

Hybrid’, ‘Halest Best Jumbo’, and ‘Topmark’ (Gonsalves et al. 1994), and ‘Don Luis’,

‘Galleon’, ‘Hiline’, ‘Mission’, and a distinct ‘inbred line’ (Clough and Hamm, 1995). In

addition, transgenic plants over-expressing either CMV-CP for specific viral strains -

White Leaf strain- (Gonsalves et al. 1994) or multi-virus resistance –CMV, WMV, and

ZYMV- (Clough and Hamm, 1995; Fuchs et al. 1997) were described. Gonsalves et al.

(1994) found strong resistance to CMV-White Leaf strain in 5 out of 45 transgenic melon

plants. Gaba et al. (2004) stressed that CP-protection gave effective field resistance, but

not 100% protection.

Field trials were conducted to determine if transgenic plants would retard the

spread of the aphid non-transmissible strain C of CMV (Tabei et al. 1994; Clough and

Hamm, 1995; Fuchs et al. 1997; Fuchs et al. 1998). Clough and Hamm (1995) tested the

level of resistance of five melon transgenic varieties to WMV and ZYMV. Transgenic

melon plants had little or no virus infection, while more than 60% of the control plants

developed virus-symptoms. Similar results were achieved by Fuchs et al. (1997), who

evaluated transgenic melon resistance under high disease pressure, achieved by

78

mechanical inoculations, and/or natural challenge inoculations by indigenous aphid

vectors. After five different trials, more than 90% of the homozygous and 75% of the

hemizygous plants were not infected by two or three viruses whereas 99% of the wild

type melon plants had mixed virus infections. Moreover, control plants were severely

stunted (44% reduction in shoot length) and had poor fruit yield (62% loss), and most of

their fruits were unmarketable (60%) compared to transgenic melon plants.

2.3.6.2 Improvement of Tolerance to Physical Factors

Several environmental factors, such as high or low temperature, salt accumulation,

low-sun irradiance, drought, and flooding, seriously affect melon field cultivation and

production (Robinson and Decker-Walters, 1999). Only one report on transgenic melon

providing tolerance to one environmental factor has been published (Bordas et al. 1997).

The HAL1 gene, which encodes a water soluble protein and provides halotolerance in

yeast, was inserted using Agrobacterium tumefaciens-transformation protocol to ‘Pharo’

and ‘Amarillo Canario’ melon cultivars. In vitro shoots from transgenic and control

plants were evaluated for salt tolerance after 16 days of incubation on medium containing

10 g.L-1 sodium. The frequency and intensity of root formation were higher in HAL1-

positive plant populations compared to wild type plants. However, no differences in

vegetative fresh weight and number of leaves between transgenic and control plants were

scored. Moreover, greenhouse and field evaluation of transgenic plants was not reported.

2.3.6.3 Improvement of Postharvest Characteristics

According to Perishables Group Research, price, firmness and appearance are

among the top criteria for consumers when deciding to purchase melons. Appearance,

which includes color, texture, and look of any sign of damage or disease are the top

criteria for consumers to purchase melons. Customers are interested in knowing

79

nutritional and ripening information in store displays (Anonymous, 2002). Extended shelf

life in melon fruit is an important quality attribute because increase the opportunity to

commercialize melon commodities.

The first transgenic melon plants carrying genes involved in fruit ripening process

were obtained by Ayub et al. (1996). Using the Agrobacterium-mediated transformation

system and cotyledons of the Charentais type Cantaloupe melon cv. ‘Védrantais’, these

authors were able to transfer the 1-aminocyclopropane-1-carboxilic-acid oxidase gene

(ACC oxidase from melon under the control of a constitutive promoter) in antisense

orientation to reduce the level of ethylene (Ayub et al. 1996). Different ripening

parameters were evaluated in transgenic ripening improved melon fruits, such as internal

and gas space ethylene production, total soluble solids, titratable acidity, flesh pigment

content, flesh firmness, rind and flesh color, harvest maturity (timing from anthesis to full

slip), and reversion to wild type behavior by exogenous ethylene treatment (Ayub et al.

1996; Guis et al. 1997b).

Ayub et al. (1996) and Guis et al. (1997b) found that in wild type fruit attached to

the vine, the internal ethylene concentration rose at 39 days after pollination and reached

maximum (120 ppm) production at 42 days. In antisense fruit the internal ethylene

concentration on the vine remained at low levels (0.6 ppm), even at late fruit

development stages (60 days after pollination). When wild type fruits were detached from

the plant there was a significant increase in the internal ethylene concentration, producing

180 ppm 48 hours later. Detached transgenic fruit also had an increase in ethylene

production, but reached only 10 ppm 12 days after harvest. Compared to wild-type,

antisense fruit did not undergo significant rind yellowing and flesh softening at maturity

80

period. Transgenic fruit remained attached to the vine for a longer period of time (65 days

after pollination) compared to control plants (38 days after pollination). Only exogenous

ethylene treatment (50 ppm) of transgenic fruits allowed the recovery of the wild type

behavior and phenotype. There were no significant differences in carotenoid content

(flesh color) and total soluble solids (°Brix) content in wild type and transgenic fruit at

any stage of ripening.

Clendennen et al., (1999) utilized the product of the S-adenosylmethionine

hydrolase (SAMase) gene (from T3 bacteriophage) to catalyze the degradation of SAM,

the initial precursor of ethylene. Unlike the T-DNA construct used by Ayub et al. (1996),

Clendennen et al. (1999) used a fruit specific promoter (chimeric ethylene-responsive

E8/E4 promoter) aimed to overexpress the SAMase gene in two ‘American Cantalope’

lines, which were proprietary inbred lines from Harris Moran Seed Company, Inc. These

authors evaluated several postharvest fruit quality parameters, such as fruit size and

weight, firmness, mold susceptibility, external and internal color, soluble solids, harvest

maturity (timing from anthesis to full slip, measured as Heat Units), and ethylene

production, in wild type and transgenic melon fruits from plants grown under field

conditions. These authors reported that transgenic melon fruit from both lines ‘A’ and

‘B’ did not differ in horticultural traits from wild type fruits, except for the intended goal

of SAMase expression on ethylene biosynthesis and related events. In lab experiments,

transgenic fruits produced half of the ethylene amount accumulated by wild type fruits.

However, in field trials, the onset of maturity, measured on four different dates, was not

significantly delayed in transgenic fruit compared to wild type, but transgenic fruits ripe

more uniformly in the field. Firmness was also measured on transgenic and wild type

81

fruits from three different field trial locations. Significant differences were found in fruit

internal firmness between transgenic and wild type melons, but only from one location.

Clendennen et al. (1999) claimed that by expressing SAMase in a regulated manner by a

fruit-specific promoter, transgenic fruits produced less ethylene than non-transgenic fruit

resulting in a modified ripening and postharvest phenotype.

Silva et al. (2004) obtained transgenic Cantaloupe melon plants cv. Védrantais by

inserting and overexpressing ACC oxidase from apple and not from melon as Ayub et al.

(1996) protocol. These authors reported the characterization of ripening melon fruits, and

their experimental comparison between transgenic and control fruit provided very similar

results in almost all the evaluated parameters, such as harvest maturity, total soluble

solids content, rind color, and internal ethylene production, to those previously reported

by Ayub et al. (1996) and Guis et al. (1997b).

In Charentais type Cantaloupe melon (cv. Védrantais) climateric respiration,

yellowing and carotenoid content of the rind, chilling injury, and formation of the

peduncular abscission zone are events totally ethylene-dependent (Pech et al. 1999;

Flores et al. 2001a; 2001b), whereas fruit softening, volatiles synthesis, and membrane

deterioration are ethylene-partially dependent processes and display some ethylene-

independent components (Bauchot et al. 1998; Bauchot et al. 1999; Guis et al. 1999;

Flores et al. 2001a; 2001b; Flores et al. 2002). According to Guis et al. (1997b), Pech et

al. (1999), Silva et al. (2004) sugar accumulation and the increase in carotenoid content

in the flesh are ethylene-independent events. There is still some controversy about

organic acid metabolism and loss of acidity during fruit melon ripening, i.e. Guis et al.

(1997b) and Pech et al. (1999) suggested that the organic acid metabolism was ethylene-

82

independent, whereas Silva et al. (2004) recently implied that organic acid metabolism is

an ethylene-dependent regulated process. It is likely that these differences in conclusions

might be related to a different ethylene concentration reached by each transgenic fruit. In

Silva’s report transgenic fruit attained a lower internal ethylene-level (<0.09 µl.L-1) than

Guis’s results (<0.5 µl.L-1). This subtle difference in ethylene concentration could have

obvious outcomes such as at the specific levels (Srivastava, 2002; Silva et al. 2004)

All fruit quality oriented transgenic melon plants have been obtained using just

three melon cultivars, such as Védrantais and lines ‘A’ and ‘B’, belonging to one single

variety, i.e. cantaloupensis (Ayub et al. 1996; Clendennen et al. 1999; Silva et al. 2004).

Considering that there are seven commercial and horticultural important melon varieties

(cantaloupensis, reticulatus, saccharinus, inodorus, flexuosus, conomon and dudaim)

(Guis et al. 1998; Kirkbride, 1993), and hundreds of melon cultivars, much more work

could be accomplished. There still remains a need to improve in vitro regeneration and

transformation protocols (Guis et al. 1997a; Gaba et al. 2004; Akasa-Kennedy et al.

2004).

2.4 ‘Galia’ melon

2.4.1 Introduction

‘Galia’ melon is an exotic, green-fleshed, and yellow skinned -finely-netted rind

specialty hybrid muskmelon bred in Israel. ‘Galia’ is also easily recognized by the

volatile-musky aroma that the fruit is able to release (Shalit et al. 2000; Fallik et al.

2001). This muskmelon was developed for semi-arid, dry-land, and open-field and

summer season cultivation in Mediterranean areas. Turkey, Morocco and Spain are major

producers of ‘Galia’ (Karchi, Personal communication). Due its high demand within

83

European market ‘Galia’ muskmelons have recently started been cultivated in Central

American countries such as Guatemala, Honduras, Costa Rica, and Panama, and exported

to the U.S. and Europe (Table 2.7). Open-field, ‘Galia’ muskmelon yields between 35-50

t/ha of excellent-quality fruit (Karchi, 2000). Using passive ventilated greenhouses and

soilless culture in North Florida yields as high as 165 t/ha have been reported (Shaw et al.

2004). The ‘Galia’ type muskmelon world production is around 300,000-500,000 tons per

year (Reho, 2004).

‘Galia’ muskmelon is a commodity reaching dissimilar prices, from $ 1.97 to $

2.17 per fruit, mostly depending on quality characteristics and origin country (Table 2.8)

(Produce1, INC. 2005, United States Department of Agriculture [USDA] Department of

Commerce, U.S. Census Bureau, Foreign Trade Statistics, 2005). However, according to

the USDA, ‘Galia’ high-quality muskmelons can reach prices in a range from $3.0 up to

$5.0 per fruit (USDA, Department of Commerce, U.S. Census Bureau, Foreign Trade

Statistics, 2005).

2.4.2 Botany and Origin

According to Karchi (2000), ‘Galia’ muskmelon was the first Israeli melon hybrid

produced and was obtained through an extensive breeding program at the Newe Ya’ar

Research Center of the Agricultural Research Organization (A.R.O – Israel) during the

mid-1960s. ‘Galia’ has green-fleshed characteristics of ‘Ha’ Ogen’ type, which is a

smooth-skinned, sutured melon and was introduced from Hungary to Israel during the

1950’s, and used as the female parental line. ‘Galia’ has also a golden-yellow netted rind

from ‘Krymka’, Russian melon cultivar,

84

Table 2.7 ‘Galia’ Type Muskmelon Imports to U.S. 1999 2000 2001 2002 2003 TOTAL

ORIGIN (Mt) ($1,000) (Mt) ($1,000) (Mt) ($1,000) (Mt) ($1,000) (Mt) ($1,000) (Mt) ($1,000)

Brasil 363 147 0 0 0 0 0 0 7 4 370 151

Chile 2 3 16 11 0 0 0 0 1 2 19 16

Costa Rica 0 0 0 0 0 0 121 181 0 0 121 181

Dominican

Rep.

49 94 38 66 24 44 28 41 13 20 152 265

Ecuador 141 71 0 0 2 3 0 0 0 0 143 74

France 7 23 11 36 6 19 1 4 0 0 25 82

Guatemala 136 49 0 0 0 0 0 0 7 8 143 57

Honduras 0 0 0 0 0 0 272 109 142 58 414 167

Israel 905 438 95 148 169 161 53 90 11 22 1,233 859

Jamaica 0 0 0 0 0 0 4 8 0 0 4 8

Mexico 75 83 95 137 72 67 87 116 108 112 437 515

Minor Antilles 0 0 0 0 0 0 8 19 28 52 36 71

Netherlands 0 0 0 0 0 0 0 0 1 4 1 4

Panama 6 4 0 0 123 198 69 42 10 10 208 254

Spain 45 40 54 86 63 87 18 25 22 40 202 278

TOTAL 1,729 952 309 484 459 579 661 635 350 332 3,508 2,982

Source: USDA, Department of Commerce, U.S. Census Bureau, Foreign Trade Statistics (2005).

85

Table 2.8 ‘Galia’ Type Muskmelon Unit Value Imports to U.S 1999 2000 2001 2002 2003 AVERAGE

ORIGIN $/Kg $/Kg $/Kg $/Kg $/Kg $/Kg

12/1-

5/31

6/1-

11/30

12/1-

5/31

6/1-

11/30

12/1-5/31 6/1-

11/30

12/1-5/31 6/1-11/30 12/1-5/31 6/1-

11/30

12/1-

5/31

6/1-

11/30

Brasil 0.405 0 0 0 0 0 0 0 0.634 0 0.520 0

Chile 1.091 0 0.700 0 0 0 0 0 2.125 0 1.305 0

Costa Rica 0 0 0 0 0 0 1.488 0 0 0 1.488 0

Dominican Rep. 1.923 1.712 1.733 0 1.848 2.111 1.472 0 1.461 0 1.687 1.911

Ecuador 0.501 0 0 0 1.822 0 0 0 0 0 1.162 0

France 3.097 3.571 3.445 2.313 0 3.078 0 3.112 0 0 3.271 3.019

Guatemala 0.361 0 0 0 0 0 0 0 1.169 0 0.765 0

Honduras 0 0 0 0 0 0 0.401 0 0.412 0 0.407 0

Israel 1.834 0.393 1.961 1.250 2.000 0.759 2.540 1.626 1.963 2.052 2.060 1.216

Jamaica 0 0 0 0 0 0 1.750 0 0 0 1.750 0

Mexico 1.100 1.195 1.334 1.686 0.813 0.985 1.107 1.435 1.178 0.661 1.106 1.192

Minor Antilles 0 0 0 0 0 0 2.393 0 1.887 0 2.140 0

Netherlands 0 0 0 0 0 0 0 0 4.490 0 4.490 0

Panama 0.737 0 0 0 1.603 0 0.610 0 1.000 0 0.988 0

Spain 1.024 0.754 1.619 1.541 1.768 0.837 1.305 1.785 1.824 1.976 1.508 1.379

Minimum Price Guatem. Israel Chile Israel Mexico Israel Honduras Mexico Honduras Mexico

Maximum Price France France France France Israel France Israel France Netherlan

.

Israel

Source: USDA, Department of Commerce, U.S. Census Bureau, Foreign Trade Statistics (2005).

86

which was used as the male parental line (Karchi, personal communication, 2004).

‘Galia’ was named after Dr. Karchi’s daughter and released as a Hybrid F1 for

production in 1973-74 (Karchi, 2000).

‘Ha’ Ogen’ type melons are considered as members of cantaloupensis variety,

whereas ‘Krymka’ cultivar belongs to the reticulatus variety (Kirkbride, 1993; Goldman,

2002). Since ‘Galia’ is a F1 Hybrid muskmelon obtained from crossing ‘Ha’ Ogen’ and

‘Krimka’, disagreement to whether ‘Galia’ F1 is a cantaloupensis variety (Zheng and

Wolff, 2000; Staub et al. 2000; Lopez-Sese et al. 2003) or a reticulatus variety (Aharoni

et al. 1992; Aharoni et al. 1993a; 1993b; Garcia et al. 1998; Fallik et al. 2000) has

emerged.

2.4.3 Postharvest Physiology

‘Galia’ muskmelon has exceptional characteristics such as fruit quality with 13-

15% total soluble solids (TSS), bold flavor and a distinct musky aroma. The main

disadvantage of ‘Galia’ is its short storage life, since it is harvested near peak maturity for

optimum flavor. Storage is limited to two or three weeks, even when it is maintained in

low- temperature (6oC). Several pathogenic agents, such as Alternaria alternata (Fr.)

Keissler and Fusarium spp. can severely reduce the ‘Galia’ fruit storage life, causing a

rapid decay (Barkai-Golan et al. 1981; Aharoni et al. 1992; Aharoni et al. 1993a).

However, using specific fungicides, such as imazalil (2,000 µl.L-1), combined with wax

coating, ‘Galia’ fruits can be protected from decay (Aharoni et al. 1992; 1993a).

Unfortunatly, the residue level of imazalil in Israeli ‘Galia’ wax coated fruits is 3-5 µl.L-1,

more than the maximum tolerance limit (0.5 ml.L-1) imposed by most of European

87

countries (Anonymous, 1990). Therefore, new fungicides and postharvest strategies were

sought to reduce the fruit decay in ‘Galia’ muskmelon.

Other treatments including use of Sanosil-25, which is a 48% hydrogen peroxide

solution and silver salts (Aharoni et al. 1994), applying Hinokitiol (beta-thujaplicin), a

volatile oil extracted from the Japanese Hiba tree (Thujopsis dolabrata) at a concentration

of 750 ppm in the wax (Aharoni et al. 1993b), other fungicides, such as prochloraz and

orthophenylphenol (Aharoni et al. 1992), coating ‘Galia’ fruits with wax containing 2%

sodium bicarbonate (Aharoni et al. 1997), and combined treatments, such as hot water

(52oC) dipping and γ-irradiation (Barkai-Golan et al. 1994), short hot water (59oC) rinse

and rotating brushes (Fallik et al. 2000), and hot water (55oC) treatment and packing in

polyethylene bags (Halloran et al. 1999a; 1999b) The optimal treatment to reduce decay

and fulfill European import requirements, while maintaining high fruit quality after

prolongated storage and marketing simulation in ‘Galia’ type muskmelon is to place the

fruits on rotating brushes, and rinse them with ambient water followed by a hot water

rinse (HWRB) at 59oC ± 1oC for 15 s (Fallik et al. 2000).

Different strategies have been used in order to delay fruit softening in ‘Galia’

including the use of controlled atmospheres plus adding an ethylene absorbent (Aharoni

et al. 1993a), reducing the storage time during transportation by using airfreight systems,

instead of seafreight shipping (Bigalke and Huyskens-Keil, 2000), and applying

inhibitors of ethylene action, such as 1-methylcyclopropene (Ergun et al. 2005) have been

reported.

Aharoni et al. (1993a) carried out a study aimed to determine if ‘Galia’ melons

could be stored under controlled atmosphere (CA) (10% CO2 and 10% O2) plus an

88

ethylene absorbent on semi-commercial scale, in order to maintain fruit quality. Fruit

were evaluated after 14 days in CA at 6°C and additional 6 days at 20°C. These authors

found that ‘Galia’ melons stored in controlled atmosphere plus ethylene absorbent were

significantly firmer and had less decay than control treatments.

Bigalke and Huyskens-Keil (2000) studied the influence of different treatments,

such as low temperatures (5 and 8°C) and individual fruit plastic packaging, as well as

two controlled atmosphere conditions, i.e. 4% and 12% at 80% RH and 8°C for 14 days,

on postharvest characteristics of airfreight ‘Galia’ melon. External and internal fruit

quality attributes, such as color, firmness, soluble solids content, titratable acidity, and

flavor assessment by a consumer panel were evaluated. These authors did not find

statistical differences between quality attributes of bagged and non-bagged melon fruits.

Also, ‘Galia’ melons stored at 5°C maintained the highest external and internal quality

during the storage period, regarding firmness, soluble solids content, titratable acidity and

flavor assessment. Controlled atmosphere conditions tested in this study were

unfavorable to maintain excellent ‘Galia’ fruit quality characteristics.

Ergun et al. (2005) characterized the physiological responses of ‘Galia’ fruit

harvested at green (preripe) and yellow (advanced ripening) stages after treatment with 1-

methylcyclopropene (1-MCP) and further storage at 20°C. These authors found that

treatment with 1.5 µL.L-1 1-MCP delayed the climacteric peaks of respiration and

ethylene production of green fruit by 11 and 6 days, respectively, and also significantly

suppressed respiration and ethylene production maxima. Fruit softening of both

developmental stages, green and yellow, was significantly delayed after 1-MCP

treatment.

89

By delaying fruit ripening via reduction in ethylene production ‘Galia’ F1 hybrid

shelf-life might be improved.

2.4.4 Genetic Improvement by Conventional Methods and Biotechnology

‘Galia’ F1 hybrid has high tolerance to some diseases, such as vine decline and

powdery mildew race 1 (Wolff and Miller, 1998, Karchi, 2000) and elevated fruit quality

characteristics. ‘Galia’ inbred parental lines have been used in breeding programs to

develop improved hybrid with firmer fruit, and also to inbreed other melon accessions

lines for multiple virus resistance (Garcia et al. 1998; Lofti et al. 2003). Crossing

‘Ha’Ogen’ x ‘Inodorus’ type has improved shelf-life but favorable fruit quality

characteristics, such as flavor, volatiles and sugar levels, were lost (Dr. Karchi, personal

communication, 2002).

A reliable and efficient regeneration and transformation system for ‘Galia’

muskmelon has been a desired goal for more than a decade (Gaba et al. 1992). ‘Galia’

muskmelon efficient in vitro regeneration and transformation protocols are lacking (Gaba

et al. 1992; 1995; 1996; 1999; Edriss et al. 1996; Kintzios and Taravira, 1997; Galperin et

al. 2003a).

Dirks and van Buggenum (1989) described an efficient method for shoot

regeneration from leaf and cotyledon explants in ‘Accent’, ‘Galia’, ‘4215’, ‘Preco’, and

‘Viva’ melon cultivars. The best combination of plant growth regulators for shoot

induction in cotyledon explants was 1 mg/l BA, without adding IAA. For leaf explants,

there were no clear differences on shoot induction among the cultivars tested. A range of

20 to 50 shoots per explant were recorded. These authors also claimed that over 100

shoots were formed in cotyledon explants. The best results on shoot induction were

90

obtained with ‘Galia’, ‘Accent’, and ‘Preco’. Elongated shoots were rooted on hormone-

free MS, and then successfully transferred into the greenhouse.

Leshem (1989), working in the Volcani Center in Israel, reported a plant tissue

culture approach in order to study the responsive zone for organogenesis and the time for

organ induction in ‘Galia’ melon cotyledons. Calli proliferation as well as root and shoot

primordia formation was only confined to the basal side (attachment point to the stem) of

the cotyledon or its segments and never appeared on the distal cut edge. The basal

segments were always the most regenerative explants than the distal ones. The organ

induction period to develop roots required at least one day but no more than three,

whereas three days but not more than five were needed to produce shoots. According to

Leshem (1989), melon cotyledon responsiveness was apparently achieved by the

transverse cut and possibly due to accumulation of a polarly transported endogenous

growth factor (maybe IAA). Exogenous plant hormones, such as 0.2 mg.L-1 BA and 2

mg.L-1 IAA, had a fundamental role for the induction of organ formation. Leshem’s

research team, published three more articles (Leshem et al. 1990a; 1990b; 1991), where

they studied the role of exogenous cytokinins, some reserve cotyledon polypeptides and

polyamines on in vitro melon regeneration. Leshem’s team shifted from using ‘Galia’

melon cultivar to ‘Sweet’n Early’ hybrid in those published articles (Leshem et al. 1990a;

1990b; 1991). Perhaps this decision was taken because fully complete well developed

shoots were not obtained in Leshem (1989).

Leshem et al. (1994a; 1994b) used thidiazuron (TDZ), benzyladenine (BA) and

paclobutrazol (PC), and the plant hormone IAA in order to examine changes in

cotyledonary organogenetic response in ‘Galia’ F1 hybrid. They studied the effect of

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those bioregulators on temporal changes in protein profiles (mostly some reserve

cotyledonary polypeptides with molecular weights of 20-25 kDa -P20-P25-) in cultured

melon cotyledons. These authors found that TDZ exerted a cytokinin-like influence by

inducing de novo shoot proliferation and inhibition of root initiation, while PC was a

potent root-inducer, due to its ability to overcome the inhibitory effect of BA on melon

rooting. However, PC was not able to induce roots at the basal edge of the cotyledon

when TIBA (inhibitor of auxin transport) was incorporated into culture medium.

Exogenous cytokinin (2.25 mg.L-1 BA) enhanced mobilization of reserve proteins (P20-

P25) and also induced shoot regeneration in cultured cotyledons. These polypeptides

disappeared from cotyledonary tissue on day four if the media culture contained BA,

whereas a constant level of those polypeptides was maintained through the culture period

if 1.75 mg.L-1 IAA was present. Regeneration correlated well with the P20-P25

disappearance, and shoots were only induced on medium containing 2.25 mg.L-1 BA. A

delayed organogenetic regeneration and P20-P25 breakdown was observed when IAA

was present in the media. The incorporation to the medium of an inhibitor of

carboxypeptidase (PMSF) inhibited both P20-P25 disappearance and root regeneration,

but not shoot initiation.

Gaba et al. (1996) reported that ancymidol, an anti-gibberellin compound,

improved in vitro bud development in cotyledonary explants of ‘Galia’ melon. This anti-

gibberellin substance had a synergistic effect when BA was incorporated to the media.

The presence of 5 mg/l GA3 reverted that rate of regeneration induced by BA alone or

BA plus ancymidol.

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It has been suggested that cytokinin and white light operate together to enhance

polypeptide metabolism and induce shoot regeneration in ‘Galia’ cultured cotyledons

(Leshem et al. 1995; Kintzios and Taravira, 1997). Leshem et al. (1995) studied the effect

of different light intensities (PPFD 0, 3, 7, 14, 30 and 50) on shoot regeneration incidence

on medium containing 2.25 mg.L-1 BA and disappearance of P20-P25 polypeptides in

melon cotyledons. They found that BA enhanced the polypeptide loss under light

conditions (50 µmol.m2.s-1), but it failed when cotyledons were cultured in total darkness.

A reduction in white light fluency to 14 µmol.m2.s-1 was enough to observe the

polypeptide pattern as in darkness culture. The incidence of shoot regeneration was

reduced about half when cotyledons were cultivated at 14 µmol.m2.s-1 white light fluency,

and only 5% of cotyledons were responsive to shoot induction under total darkness.

These authors suggested that BA loses its shoot induction capacity in the absence of

white light, therefore, it is not able to support any growth response, even if BA

concentration is increased (2.25 mg.L-1).

Kintzios and Taravira (1997) studied several morphogenetic pathways, such as calli

induction, somatic embryogenesis and plant regeneration response, of 14 commercial

melon cultivars to two levels of photosynthetic photon flux densities (PPFD), i.e. 50 or

250 µmol.m2.s-1. ‘Galia’ was able to undergo shoot induction only when PPFD was 250

µmol.m2.s-1.

Micropropagation of ‘Galia’ F1 melon explants, using either shoot tips or axillary

buds, has been reported as well (Spetsidis et al. 1996; Edriss et al. 1996). Elevated levels

of BA (2 mg/l) were needed to reach the greatest shoot production, and the presence of

0.5 mg/l IAA did not affect that number.

93

The first report on ‘Galia’ melon genetic transformation through plant

biotechnology was described by Gaba et al. (1992; 1995). They used particle gun

bombardment to transform cotyledon explants and detect transient GUS expression.

Permanent GUS transformation was observed only after a strong selection procedure

(350 mg/l kanamycin). They were able to detect the presence of that transgene by PCR

methods, but only 1% of the explants produced transformed shoots. Gaba et al. (1999)

claimed that despite abundant shoot induction ‘Galia’ melon cotyledons (Dirks and van

Buggenum, 1989), the cultivar was recalcitrant to transformation by Agrobacterium

tumefaciens (Gaba et al. 1995; 1996).

An innovative regeneration system using the proximal zone of the hypocotyl of

‘Galia’ type melon cultivar, i.e. ‘Revigal’ has been reported (Curuk et al. 2002). This

protocol was later applied to three melon cultivars, ‘Revigal’, ‘Topmark’ and ‘Kirkagac’,

and to one cucumber cultivar, i.e. ‘Taoz’ (Curuk et al. 2003). Regeneration from

hypocotyl explants resulted in almost 100% diploid shoots, therefore, this is the first

report of regeneration from Cucumis genus producing a fully diploid plant population.

Unfortunatly, these authors did not try to regenerate the original ‘Galia’ F1 hybrid

through their system nor to transform it by means of Agrobacterium tumefaciens or

particle gun bombarment.

Galperin et al. (2003a) screened, through in vitro regeneration, 30 melon genotypes

to measure their regeneration capabilities. Those melon genotypes embraced wild

landraces, breeding lines and commercial cultivars. According to their results, 24 out of

30 genotypes did not produce any normal shoots. Only five out of six genotypes

displayed low regeneration efficiency, because a few explants developed normal shoots.

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Only ‘BU-21’ had exceptional regeneration ability. Five melon genotypes, among them

‘Galia’, were selected from the original 30 genotypes to determine the organogenetic

response on three different regeneration media, i.e. MR, HC and IK (Fang and Grumet,

1990; Blackmon and Reynolds, 1982; Moreno et al. 1985a). The regeneration efficiency

reported for ‘Galia’ melon was 0% on those three different media.

So far, there is no any report in the literature on stable genetic transformation in

‘Galia’ F1 hybrid or in any of its parental lines.

95

CHAPTER 3 EFFECT OF EXPLANT SOURCE ON REGENERATION AND TRANSFORMATION

EFFICIENCY IN ‘GALIA’ MELON (CUCUMIS MELO L.) MALE AND FEMALE PARENTAL LINES

3.1 Introduction

The Cucurbitaceae family consists of mostly frost sensitive, principally tendril-

bearing vine plants which are found in sub-tropical and tropical regions around the world

(Robinson and Decker-Walters, 1999). Melon (Cucumis melo L.) belongs to this family

and is an important horticultural produce. World melon production (Cantaloupe & others)

in 2004 was over 27 million metric tons (FAO, 2005). Traditional breeding methods in

melon have led to a considerable varietal improvement. However, strong sexual

incompatibility barriers at the interspecific and intergeneric levels have restricted

potential to readily develop new and enhanced melon cultivars (Niemirowicz-Szcztt and

Kubicki, 1979; Robinson and Decker-Walters, 1999). Using genetic engineering

strategies is feasible to overcome most of the genetic barriers among plants, which are

unsurpassable by traditional breeding techniques (Vasil, 1990; 1996; 1998; 2003).

Likewise, in order to achieve a successful commercial application from biotechnology in

melon a competent de novo regeneration system from in vitro cultures is required (Guis et

al. 1998).

In the last 25 years, more than 40 in vitro melon regeneration protocols have been

described, some using either organogenesis, somatic embryogenesis or both regeneration

pathways (Blackmon et al. 1981a; Blackmon et al. 1982; Moreno et al. 1985a; Mackay et

al. 1988; Punja et al. 1990; Chee, 1991; Shetty et al. 1992; Hosoi et al. 1994; Singh et al.

96

1996; Kintzios and Taravira, 1997; Guis et al. 1998; Guis et al. 2000; Nuñez-Palenius et

al. 2005; Stipp et al. 2001; Curuk et al. 2002a; 2002b; 2003; Galperin et al. 2003a; 2003b;

Akasa-Kennedy et al.; 2004). Several biological and physical factors influence in vitro

regeneration efficiency rate, and all have to be considered in order to develop a

reproducible and reliable melon regeneration protocol. All the systems had diverse

regeneration rates depending on genotype, culture conditions and explant source. Melon

plant regeneration has been reported from roots (Kathal et al. 1994), calli (Moreno et al.

1985b), petioles (Punja et al. 1990), hypocotyls (Moreno et al. 1985b), leaves (Yadav et

al. 1996), protoplasts (Moreno et al. 1985a), somatic embryos (Gray et al. 1993), shoot

primordia (Adelberg et al. 1999), and cotyledons (Guis et al. 1997a).

‘Galia’ muskmelon is an Israeli melon F1 hybrid with exceptional fruit quality

characteristics such as 13-15% total soluble solids (TSS), bold flavor and a distinct aroma

(Karchi, 2000). Unlike other melon types, such as Cantaloupe Charentais (Ayub et al.

1996; Akasa-Kennedy et al. 2004; Silva et al. 2004), ‘Galia’ muskmelon is not easily

cultivated in vitro and full regenerated wild type plants are especially difficult to obtain

(Leshem 1989; Leshem et al. 1994a; 1994b; Gaba et al. 1994; Gaba et al. 1996; Edriss et

al. 1996; Kintzios and Taravira 1997; Galperin et al. 2003a). Additionally, Gaba et al.

(1999) reported ‘Galia’ muskmelon to be recalcitrant to transformation by Agrobacterium

tumefaciens. Therefore, a reliable and efficient in vitro protocol for regeneration and

transformation in ‘Galia’ muskmelon has been a desired objective for more than a decade

(Gaba et al. 1992). Because different melon explants have diverse in vitro regeneration

responses, it is important to accurately assess that parameter in ‘Galia’ parental lines. The

goal of this research was to measure the source of the explant on regeneration and

97

transformation efficiency using two reporter genes (GUS and GFP) in ‘Galia’ melon male

and female parental lines.

3.2 Materials and Methods

3.2.1 Plant Material

Mature seeds of ‘Galia’ male (Cucumis melo L. var. reticulatus Ser. cv. ‘Krimka’)

and female (Cucumis melo L. var. cantaloupensis Naud. cv. ‘Noy yizre’el’) parental lines

were surface sterilized and germinated on hormone-free half-strength Murashige and

Skoog (MS) medium (Murashige and Skoog, 1962) according to Nuñez-Palenius et al.

(2005a) protocol. In vitro regeneration of both parental lines was carried out on explants

from cotyledons, hypocotyls, and non-expanded true leaves following the procedures and

plant-hormone balances reported by Nuñez-Palenius et al. (2005a), Ramirez-Malagon

and Ochoa-Alejo (1996), and Guis et al. (2000), respectively. De novo shoot regeneration

response was measured as bud forming capacity index [BFC index= (average number of

shoots per explant) X (% explants forming shoots) / 100] (Martinez-Pulido et al. 1992).

Seedling growth and plant regeneration were conducted in a growth chamber (Lab-Line

Instruments, Inc. Melrose Park, IL) under 100 µmol . m-2 . s-1 light and a 16 h photoperiod

provided by cool-white fluorescent lamps and constant 25 ± 1°C temperature.

3.2.2 Agrobacterium Inoculation and Plant Transformation

Agrobacterium tumefaciens strain ABI containing a binary vector, pMON17204,

harboring a selectable glyphosate resistance marker [CP4 syn gene encoding for 5-

enolpyruvylshikimate-3-phosphate synthase (EPSPS)], and the uidA gene encoding for β-

glucuronidase (GUS) was used (Figure 3.1a). CP4 syn gene was under control of the

Figwort Mosaic Virus (FMV) promoter (Richins et al. 1987), whereas uidA gene was

under control of the CaMV 35S promoter (35S). Agrobacterium tumefaciens strain LBA-

98

4404 containing a binary vector, pCAMBIA 2202-sGFPS65T, harboring a selectable

kanamycin-resistance marker, NPT-II gene encoding for neomycin phosphotransferase,

and a fully functional green fluorescent protein (GFP) gene was used (Figure 3.1b). Both

Agrobacterium tumefaciens strains, ABI and LBA4404, were transferred to 5 ml of

Luria-Bertani Broth (LB) medium (pH 7.5). The former was supplemented with 25 mg .

L-1 chloramphenicol, 50 mg . L-1 kanamycin, and 100 mg . L-1 spectinomycin, whereas

the second strain was supplemented with 50 mg . L-1 carbenicillin and 100 mg . L-1

chloramphenicol. Both strains were then incubated on an orbital shaker (200 rpm) for 12

hours at 25oC. Afterward, the cultures (5 ml aliquot) were transferred to 250 ml baffled

culture flask containing 50 ml liquid LB medium with the same antibiotics and

concentrations previously used, and incubated at 25oC on an orbital shaker (150 rpm) for

an additional 14-20 hours until an A600= 0.7-1.0 was reached.

Cotyledon and non-expanded leaf explants from both ‘Galia’ melon parental lines

were immersed in Agrobacterium suspension for 20-30 min with orbital shaking (50

rpm), and then were blotted three times onto sterile filter paper (Whatman No. 1) to

remove the excess bacterial suspension. Cotyledon and true leaf explants were then

cultured and de novo shoots were obtained as previously described by Nuñez-Palenius et

al. (2005) and Guis et al. (2000), respectively. Hypocotyls were inoculated with a

Agrobacterium tumefaciens suspension using a sterile syringe needle (0.72 mm gauge

and 32 mm length) following the protocol described by Ramirez-Malagon and Ochoa-

Alejo (1996).

99

Figure 3-1 Map of the T-DNA in pMON17204 and pCAMBIA 2202-sGFPS65T. (A) T-

DNA region of binary vector pMON17204. RB: right border of T-DNA, 35S: promoter for 35S RNA from Cauliflower mosaic virus, CP4syn: 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, rbcSE9: 3’end of pea rbcSE9 terminator, 35S: promoter for 35S RNA from Cauliflower mosaic virus, GUS: uidA gene, rbcSE9: 3’end of pea rbcSE9 terminator, LB: left border of T-DNA. (B) T-DNA region of binary vector pCAMBIA 2202-sGFPS65T. RB: right border of T-DNA, 35S: promoter for 35S RNA from cauliflower mosaic virus, NPT-II: neomicyn phosphotransferase gene, 35S: terminator for 35S RNA from cauliflower mosaic virus, 35S: promoter for 35S RNA from cauliflower mosaic virus, GFP: sGFPS65T (humanized, for high expression level and solubility in eukaryote cells) gene, NOS: nopaline synthase terminator, LB: left border of T-DNA.

3.2.3 Histochemical Staining for GUS Activity and GFP Detection

Histochemical localization for β-D-glucuronidase (GUS) activity in putative T0

transformant tissues was performed as described by Gama et al. (1996). Plant organ and

tissues were incubated in GUS-staining solution [50 mM sodium phosphate (pH 7.0), 0.5

mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM EDTA (pH 8.0),

0.06% (v/v) Triton X-100, 0.35 mg . mL-1 5-bromo-4-chloro-3-indolyl-β-D-

glucuronidase (X-Gluc)] at 37°C for 12-36 h. After incubation, the samples were then

CP4syn 35S

RB

GU

LB

rbcSE9 rbcSE9

NPT-II 35S

RB

GF

LB

NO35S 35S

A

B

100

transferred to 70% (v/v) ethanol to remove chlorophyll. GUS-positive tissues were

photographed with a Zeiss MC63 photomicrographic camera coupled to a DRC

stereomicroscope.

Putative GFP-positive plant tissues were examined directly to detect fluorescence

using a Zeiss Stemi SU11 fluorescence stereomicroscope having a mercury lamp and a

FITC 535 filter.

3.2.4 PCR Assay

Total DNA isolation from wild type and transgenic GUS T1 melon leaflets (0.25-

0.5 g) was performed using a modified CTAB protocol (Doyle and Doyle 1987; 1990).

The forward and reverse primers for uidA gene (GUS) were 5’-CAACGAACTGAACTG

GCAG-3’ and 5’-CATCACCACGCTTGGGTG-3’, respectively, amplifying a fragment

of 750 bp. The GUS fragments in total DNA were amplified under the following

conditions: a pre-incubation period at 94 oC for 2 minutes followed by 30 cycles of 94 oC

for 30 sec for denaturation, 55 oC for 30 sec for primer annealing, and 72 oC for 45 sec

for extension, and a final extension period at 72 oC for 5 min. The amplified PCR

products (10 µl) were subjected to electrophoresis on a 0.8% agarose gel and visualized

by UV light.

3.2.5 Experimental Design and Statistical Analysis

The experiment to measure the BFC index on three different melon explants was

carried out under a factorial design (2 x 3) with six replicates, having two levels for

genotype, and three levels for explant type. The experimental design for transformation

experiment was a factorial (2 x 3 x 2) with three replicates, having two levels for

genotype, three levels for explant type, and two levels for reporter gene used. Original

data from transformation experiment was transformed by the arcsine square root in order

101

to be analyzed for analysis of variance (ANOVA). Means were separated by Tukey’s

Studentized Range Test at P≤ 0.05 (SAS Institute, Cary, N.C.).

3.3 Results and Discussion

3.3.1 Effect of Explant Origin on Regeneration Efficiencies

Cotyledonary explants from ‘Galia’ muskmelon male and female parental line

attained the greatest BFC index compared to hypocotyl and true-leaf explants (Figure

3.2). The BFC value for cotyledons was almost 4 times greater than hypocotyls and true-

leaves. When the same explant type was used, no significant differences between male

and female BFC response were found, except for cotyledons.

Figure 3.2 Organogenetic response of ‘Galia’ muskmelon parental lines depending on

explant source. Bars with the same letter are not significantly different by Tukey’s Studentized Range Test at P≤ 0.05. BFC= bud forming capacity index (Martinez-Pulido et al. 1992).

Cotyledonary explants have been the most common plant organ used to induce de

novo regeneration through either organogenesis or somatic embryogenesis in Cucumis

melo (Blackmon et al. 1981a; 1982; Trulson and Shahin, 1986; Mackay et al. 1988;

0

1

2

3

4

5

6

7

8

Cotyledon Hypocotyl True-leaf

BFC

Male

Female

a

c c c

c

b

102

Branchard and Chateau, 1988; Dirks and Van Buggenum, 1989; Niedz et al. 1989;

Leshem et al. 1989; Chee, 1991; Tabei et al. 1991; Kageyama et al. 1991; Homma et al.

1991; Fassuliotis and Nelson, 1992; Shetty et al. 1992; Debeaujon and Branchard, 1992;

Roustan et al. 1992; Oridate et al. 1992; Gray et al. 1993; Adelberg et al. 1994; Ezura and

Oosawa, 1994b; Hosoi et al. 1994; Ficcadenti and Rotino, 1995; Yadav et al. 1996; Singh

et al. 1996; Kintzios and Taravira, 1997; Guis et al. 1997a; Stipp et al. 2001; Curuk et al.

2002a; Galperin et al. 2003a; 2003b; Akasa-Kennedy et al. 2004).The use of cotyledon as

explants for melon in vitro culture has several advantages over other plant tissues, i.e. a)

cotyledons have a quick morphogenetic response producing high number of shoots or

somatic embryos, b) cotyledons are ready to be used as explants in a short period of time

(0-5 days), and c) several cotyledonary explants (8-12) are effortlessly acquired from a

single seed (Table 3.1). Moreover, de novo shoots from cotyledon are ready to be

‘hardened’ as early as 9 weeks after the initiation of in vitro culture, whereas hypocotyl

and true-leaf systems require 23 and 17 weeks, respectively (Figure 3.3).

Overall, the cotyledon was the best explant to regenerate complete plants for both

‘Galia’ muskmelon male and female parental lines compared to hypocotyl and true-leaf

explants.

3.3.2 Plant Transformation

In order to assess the effect of source explant on transformation efficiency in

‘Galia’ male and female parental lines, two Agrobacterium tumefaciens strains, ABI and

LBA4404, harboring GUS and GFP reporter genes, respectively, were tested on

cotyledon, hypocotyl and true-leaf explants. GUS-positive tissues were detected by a

histological GUS assay (Gama et al. 1996), while GFP-positive tissues were identified by

fluorescence microscopy.

103

Table 3.1 Regeneration efficiency in ‘Galia’ muskmelon parental lines depending on explant source.

Explant source Number of explants

per seed

zBFC yPeriod (weeks)

Cotyledon 12 6.31 4-5

Hypocotyl 1 1.51 19-20

True-leaf 10 1.11 11-13

zBFC= bud forming capacity index (average number of shoots per explant) X (% explants forming shoots) / 100. (Martinez-Pulido et al. 1992). yPeriod needed to achieve complete developed shoots. Data are the average for male and female ‘Galia’ parental lines results.

Transformed cotyledon and hypocotyl explants from both ‘Galia’ male and female

parental lines produced de novo shoots and roots, which were positive for GUS and GFP

expression (Figure 3.4). Although de novo shoots were obtained, only ‘escapes’ (non

GUS- or GFP-positive shoots and roots) were observed when true-leaf explants were

used (Figures 4-5 and 4-6). It has been reported that different plant explants have diverse

susceptibility to be transformed by Agrobacterium tumefaciens (Febres et al. 2003). This

differential susceptibility has been described in melon (Bordas et al. 1997; Guis et al.

1998) cucumber (Rajagopalan and Perl-Treves, 2005), and other plant species (Tu et al.

2005). Also, the production of ‘escapes’ after Agrobacterium- or biolistic-mediated

transformation in Cucumis melo is a commonly observable result (Guis et al. 1998).

Indeed, Akasaka-Kennedy et al. (2004) have claimed that in order to reduce the problem

of high production of ‘escapes’ by melon in vitro cultures, alternative regeneration and

transformation systems have still to be obtained.

104

Figure 3.3 Time schedule for de novo shoot regeneration in hypocotyl, cotyledon and true-leaf explants of ‘Galia’ muskmelon parental lines. The numbers between the top and bottom photographs represent the number of weeks to attain from mature seeds the in vitro rooted seedlings. Hypocotyl: (a) seedlings, (b) hypocotyl injury (arrow), (c) decapitation, (d) decapitated seedlings, (e) de novo shoots, (f) in vitro seedling. Cotyledon: (a) cotyledons from 2-day-old seedlings, (b) de novo shoots, (c) in vitro seedling. True-leaf: (a) seedlings, (b) true-leaves, (c) de novo shoots, (d) in vitro seedlings.

HYPOCOTYL COTYLEDON TRUE-LEAF

b

a

c

b

c

a

d

0 0

17

9

a

b

f

e

c

d

0

23

105

A greater number of T0 GUS-positive seedlings for both ‘Galia’ male and female

parental lines was obtained when cotyledons were used as explants compared to

hypocotyls (Figure 3.5). Moreover, significant differences were observed between both

explants. Conversely, when the Agrobacterium tumefaciens strain LBA4404, containing a

binary vector pCAMBIA 2202-sGFPS65T, was used to transform both ‘Galia’ parental

lines, only female-hypocotyl explants produced a higher amount of GFP-positive

seedlings than cotyledons (Figure 3.6). It was found that 13% of hypocotyl-explants

produced transgenic shoots. ‘Galia’ male parental-hypocotyl explants had a

transformation efficiency value similar to ‘Galia’ male- and female-cotyledon explants.

The results obtained on Cucumis melo transformation efficiency in this work exceed what

it has been previously reported. In general, the genetic transformation rate efficiency in

most of melon systems is normally lower than other plant organisms (Fang and Grumet,

1990; Dong et al. 1991; Gaba et al. 1992; Gonsalves et al. 1994; Bordas et al. 1997;

Akasaka-Kennedy et al. 2004). Several transformation rate efficiency values have been

described according to transformation protocol and melon cultivar used, among them,

average efficiencies such as 3-7% (Fang and Grumet, 1990), 4-6% (Dong et al. 1991),

1% (Gaba et al. 1992), 0.0%, 0.9%, and 1% (Gonsalves et al. 1994), 0.7-3% (Bordas et

al. 1997), 2.4% (Guis et al. 2000), 10% (Nuñez-Palenius et al. 2005a), and 2.3%

(Akasaka-Kennedy et al. 2004) has been depicted.

A PCR assay was performed on total DNA isolated from T1 GUS positive plants in

order to detect the presence of uidA gene and corroborate their transgenic status. We were

able to detect the presence of a GUS-specific 750 bp fragment which belongs to the GUS

106

reporter gene, in male and female ‘Galia’ muskmelon transgenic lines but not in non-

transgenic lines (Figure 3.7).

In summary, it is possible to obtain transgenic ‘Galia’ male and female parental

plants using either cotyledon or hypocotyl explants. Moreover, cotyledonary explants

have the advantage to attain those transgenic plants in a shorter period (Figure 3.8).

Figure 3.4 Stable expression of GFP and GUS genes in T0 shoots and roots of ‘Galia’

muskmelon parental lines. A) GUS non-positive tissue, B) and C) GUS positive tissue. D) GFP non-positive tissue, E) and F) GFP positive tissue.

A

B

C

D

E

F

107

Figure 3.5 Transformation efficiency on ‘Galia’ muskmelon parental lines. GUS reporter

gene was used to transform different explant sources. Bars with the same letter are not significantly different by Tukey’s Studentized Range Test at P≤ 0.05.

Figure 3.6 Transformation efficiency on ‘Galia’ muskmelon parental lines. GFP reporter

gene was used to transform different explant sources. Bars with the same letter are not significantly different by Tukey’s Studentized Range Test at P≤ 0.05.

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Cotyledon Hypocotyl True-leaf

% o

f tra

nsfo

rmat

ion

Male

Female

a

a

b

b

c c

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

Cotyledon Hypocotyl True-leaf

% o

f tra

nsfo

rmat

ion

Male

Female

a

b b

b

c c

108

Figure 3.7 PCR assay for transgenic GUS ‘Galia’ melon male and female line plants. The

amplification product for GUS gene was 750 bp. M: 100 bp Ladder, 1: negative control (DNA from wild type melon plant), 2 and 3: DNA from ‘Galia’ male GUS positive plant, 4 and 5: DNA from ‘Galia’ female GUS positive plant, 6: positive control (DNA from GUS plasmid).

M 1 2 3 4 5 6

109

Figure 3.8 Time schedule for cotyledon and hypocotyl transformation systems in ‘Galia’

melon parental lines. A. Explant transformation B. Regeneration of transgenic shoots. C. Elongation of transgenic shoots D. Rooting E. ‘Hardening’ of seedlings.

WEEKS

Hypocotyl

B

E

C

D

A

Cotyledon

A

D

E

B

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

C

110

3.3.3 Summary

The goal of this work was to measure the source of the explant on regeneration and

transformation efficiency in ‘Galia’ melon parental lines. Cotyledon, hypocotyl and true-

leaves of both female and male ‘Galia’ parental lines were used for transformation. The

ABI strain of Agrobacterium tumefaciens, containing a construct harboring the GUS gene

under the constitutive 35S promoter, and a glyphosate tolerance gene under the

constitutive FMV promoter as selectable marker were used. The LBA4404 strain of

Agrobacterium tumefaciens carrying a plasmid vector harboring the GFP and NPTII

genes both under the constitutive 35S promoter was also used. Regeneration of ‘Galia’

melon parental line shoots was achieved from hypocotyls, true-leaves and cotyledons.

Hypocotyls were transformed according the protocol described in Ramirez-Malagon &

Ochoa-Alejo (1996). Cotyledons were transformed with a protocol previously developed

in our lab (Nuñez-Palenius et al. 2005a) and true-leaf explants were transformed using

the methodology of Guis et al. (2000). The greatest numbers of shoots were regenerated

from cotyledons. These explants also produced the highest number of GUS positive

shoots and roots. Using hypocotyls as explants gave the greatest number of GFP positive

roots and shoots. We were not able to obtain any transgenic shoots using either the GUS

or GFP constructs when true-leaves were used as explants. According to our results,

‘Galia’ melon parental lines are readily transformable with Agrobacterium tumefaciens

using the hypocotyl or cotyledon-protocol. It is possible to have full regenerated

transgenic plants in 3-4 months using the cotyledon-protocol, whereas at least 6-7 months

are needed when the hypocotyl-protocol is used.

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CHAPTER 4 TRANSFORMATION OF A MUSKMELON ‘GALIA’ HYBRID PARENTAL LINE

(CUCUMIS MELO L. VAR. RETICULATUS SER.) WITH AN ANTISENSE ACC OXIDASE GENE

4.1 Introduction

‘Galia’ muskmelon (Cucumis melo L.) is an Israeli melon F1 hybrid bred by Dr. Zvi

Karchi at the Newe Ya’ar research center of the Agricultural Research Organization

(A.R.O. - Israel) and released in 1973 (Karchi 2000). This cultivar has green-flesh

characteristics of the ‘Ha’Ogen’ melon cultivar, which was used as the female parent, and

netted rind from ‘Krymka’, which was used as the male parental line. It has exceptional

fruit quality with 13-15% total soluble solids (TSS), bold flavor and a distinct aroma,

leading to rapid adoption in both local and export markets. ‘Galia’ type melons have been

the mainstay in muskmelon sales in the European market for more than 30 years (Karchi,

2000). One disadvantage of ‘Galia’ is its storage life, which is limited to two to three

weeks. Timing of harvest is critical for ‘Galia’ fruit because in order to develop peak

flavor and aroma, the melon should be picked at maturity (Karchi, 2000). ‘Galia’ F1

hybrid tends to be extremely soft at and past peak fruit maturity. Traditional breeding

methods have helped breeders develop a strategy to obtain ‘Galia’ muskmelons with a

long shelf life; however, this approach can result in a loss of favorable fruit quality

characteristics.

Plant biotechnology has the potential to genetically transform plants and transfer

novel characteristics. In order for plant transformation to be successful a reliable plant in

vitro regeneration system must first be developed (Guis et al. 1998). Using

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Agrobacterium and particle-gun bombardment methods, several transgenes, which

provide different phenotypic characteristics, have been transferred into Cucumis melo

explants. These have included selectable marker and reporter genes (Fang and Grumet,

1990; Dong et al. 1991; Gaba et al. 1992; Vallés and Lasa, 1994; Gray et al. 1995;

Akasa-Kennedy et al. 2004), virus-resistance genes (Yoshioka et al. 1992; 1993; Fang

and Grumet, 1993; Gonsalves et al. 1994; Clough and Hamm, 1995), halotolerance-genes

(Bordas et al. 1997), and genes to improve fruit quality (Ayub et al. 1996; Ezura et al.

1997a; Clendennen et al. 1999; Guis et al. 2000; Shellie 2001; Silva et al. 2004).

Improved quality of Charentais type muskmelon has been achieved by inserting ACC

oxidase genes in antisense orientation in order to reduce fruit ethylene biosynthesis

(Ayub et al. 1996; Guis et al. 1997b; Silva et al. 2004).

Increasing the shelf life of ‘Galia’ F1 muskmelon by transformation of parental

lines is a feasible alternative. Unlike other melon types, such as Cantaloupe Charentais

(Ayub et al. 1996; Akasa-Kennedy et al. 2004; Silva et al. 2004), ‘Galia’ muskmelon is

not easily cultivated in vitro and complete regenerated wild type plants are especially

difficult to obtain (Leshem 1989; Leshem et al. 1994a; 1994b; Gaba et al. 1994; Gaba et

al. 1996; Edriss et al. 1996; Kintzios and Taravira 1997; Galperin et al. 2003a).

Moreover, Gaba et al. (1999) reported ‘Galia’ muskmelon to be recalcitrant to

transformation by Agrobacterium tumefaciens.

The goal of this research was to transform the male parental line (cv. ‘Krymka’) of

‘Galia’ muskmelon with the ACC oxidase (CMACO-1) antisense gene in an attempt to

delay fruit ripening.

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4.2 Materials and Methods

4.2.1 Plant Material

Coats of 50 male parental line ‘Galia’ (Cucumis melo L. var. reticulatus Ser.) cv.

‘Krymka’ seeds were removed, and the embryos surface sterilized in 1.2% sodium

hypochlorite (20% commercial bleach solution containing two drops of Tween 20™ per

100 ml) for 15 min in a sterile Erlenmeyer flask (50 ml), and washed three times with

sterilized distilled water. Embryos were then cultivated for 2 days in regeneration

medium (RM), which consisted of Murashige and Skoog salts (MS) (Murashige and

Skoog, 1962), supplemented with 30 g.L-1 sucrose, 0.1 g.L-1 myo-inositol, 0.001 g.L-1

thiamine-HCl, 0.05 mg.L-1 pyridoxine-HCl, 0.05 mg.L-1 nicotinic acid, 2 mg.L-1 glycine,

1 mg.L-1 benzyladenine (BA), 0.001 mg.L-1 α-Naphthaleneacetic acid (NAA), and 0.7

g.L-1 phytagar. Seedling growth and plant regeneration were conducted in a growth

chamber (Lab-Line Instruments, Inc. Melrose Park, IL) under 100 µmol . m-2 . s-1 light and

a 16 h photoperiod provided by cool-white fluorescent lamps and constant 25°C

temperature.

4.2.2 Plant Regeneration

Cotyledons from 2-day-old seedlings, grown aseptically in RM, were dissected and

cut transversely into four equal pieces, then placed with their abaxial side on the surface

of RM. Each cotyledon slice constituted an explant. Complete shoots were obtained de

novo from these cotyledon explants after four weeks of culture. Regeneration efficiency

was calculated as BFC index, BFC index= (average number of shoots per explant) X (%

explants forming shoots) / 100 (Martinez-Pulido et al., 1992). Shoots were excised from

cotyledon explants and incubated on elongation medium (EM), which consisted of MS

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salts, supplemented with 30 g.L-1 sucrose, 0.1 g.L-1 myo-inositol, 0.001 g.L-1 thiamine-

HCl, 0.05 mg.L-1 pyridoxine-HCl, 0.05 mg.L-1 nicotinic acid, 2 mg.L-1 glycine, 0.025

mg.L-1 BA and 0.8 g.L-1 phytagar, for three more weeks. Elongated shoots were then

transferred to rooting media consisting of ½ strength MS medium supplemented with 1

mg.L-1 indole-3-acetic acid (IAA). After three weeks of culture, shoots developed a

complete and normal root system. Rooted shoots were hardened using common practices

and then transferred to glasshouse. Plants were grown in an evaporative-cooled fan and

pad glasshouse, which maintained temperatures of 28oC day and 20oC night, in plastic

pots (11.3 L) filled with soil-less media and following common growing practices

recommended by Rodriguez and Cantliffe (2001). A complementary light regime was

supplied by MetalarcR lamps with a light intensity of 350 – 530 µmol . m-2 . s-1. The light

period was set to 18 hours per day.

4.2.3 Agrobacterium Inoculation and Plant Transformation

Agrobacterium tumefaciens strain ABI containing a binary vector, pCmACO1-AS,

harboring a selectable glyphosate resistance marker [CP4 syn gene encoding for 5-

enolpyruvylshikimate-3-phosphate synthase (EPSPS)], and the ACC oxidase (CMACO-

1) gene in antisense orientation was used (Figure 4.1). Both genes were under control of

the Figwort Mosaic Virus promoter (Richins et al. 1987) (Figure 4.1). Agrobacterium

was transferred to 5 ml of Luria-Bertani Broth (LB) medium (pH 7.5) supplemented with

25 mg . L-1 chloramphenicol, 50 mg . L-1 kanamycin, and 100 mg . L-1 spectinomycin,

and then incubated on an orbital shaker (200 rpm) for 12 hours at 25oC. Afterward, the

culture (5 ml aliquot) was then transferred to 250 ml baffled culture flask containing 50

ml liquid LB medium with the same antibiotics and concentrations previously used, and

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incubated at 25oC on an orbital shaker (150 rpm) for an additional 14-20 hours until an

A600= 0.7-1.0 was reached.

Cotyledonary explants were immersed in Agrobacterium suspension for 20-30 min

with orbital shaking (50 rpm), and then were blotted three times onto sterile filter paper

(Whatman No. 1) to remove the excess bacterial suspension. Explants were then cultured

and de novo shoots were obtained as previously described in Plant Regeneration section,

except that RM was supplemented with 50 µM Glyphosate and 150 mg.L-1 Timentin

(GlaxoSmithKline, Research Triangle Park, NC). Glyphosate selection was carried out

only on RM.

4.2.4 Flow Cytometry Analysis

Plant nuclei were freshly isolated from the 3rd leaf below the shoot apex of

acclimatized plants grown in a greenhouse. Briefly, plant material (4 cm2) was chopped

for three minutes (at 4oC) with a razor blade in a Petri dish containing 5 ml of

Quesenberry (1995) extraction buffer. The nuclei suspension was filtered through

Spectra/Mesh® nylon filter (60-�m mesh size) to remove cell debris. The nuclei-filtered

suspension was stained with propidium iodide by adding 1 ml of a propidium iodide

stock solution (1 mg.ml-1) to 2 ml of nuclei suspension. After gently stirring, the nuclei

mixture was incubated for 5 min and then analyzed by flow cytometry. DNA content of

the isolated plant nuclei was analyzed with a flow cytometry apparatus (FACScan. BD-

Biosciences, San Jose, Cal). It was calibrated using the 2C peak from nuclei of young

leaves of diploid plants derived from seed. A minimum of 10,000 nuclei were measured

for each sample.

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Figure 4.1 T-DNA region of binary vector pCmACO1-AS. The underlined fragment of CP4syn gene was used as a probe for Southern blot hybridization assay. RB: right border of T-DNA, FMV: figwort mosaic virus promoter, CP4syn: 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, E9: 3’end of pea rbcSE9 terminator, CMACO-1: melon ACC oxidase gene in antisense orientation, NOS: nopaline synthase terminator, LB: left border of T-DNA.

4.2.5 Detection of Transgenes

Total DNA isolation from wild type and putative transgenic melon leaflets (0.25-

0.5 g) was performed using a modified CTAB protocol (Doyle and Doyle 1987; 1990).

The forward and reverse primers for CP4 syn gene were 5’-

CGGTGCAAGCAGCCGTCCAGC-3’ and 5’-CCTTAGTGTCGGAGAGTTCG-3’,

respectively, amplifying a fragment of 1,400 bp (1.4 Kb). The forward and reverse

primers for ACC oxidase gene were 5’-GCAATTATCCGCCGTGTC-3’ and 5’-

TCTTCAAACACAAACTTGGGG-3’, respectively. These primers will produce a 503 bp

fragment from the native (genomic) ACC oxidase gene and a 378 bp product from the

transgene. The ACC oxidase and CP4 syn fragments in total DNA were amplified under

the following conditions: a pre-incubation period at 94 oC for 7 minutes followed by 40

cycles of 94 oC for 1 min for denaturation, 60 oC for 1 min for annealing, and 72 oC for 1

min for extension, and a final extension period at 72 oC for 10 min. The amplified PCR

products (25 µl) were subjected to electrophoresis on a 1% agarose gel and visualized by

UV light.

CP4syn FMFM ERB

BamH

Probe 1.4

CMACO-1 ANTISENSE NOLB

BamH

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4.2.6 Southern Blot Analysis

Total melon DNA was isolated from young leaves of wild type and transgenic T1,

TGM-AS-1 and TGM-AS-2 lines (TGM-AS stands for Transgenic Galia Male AntiSense

line), using a modified CTAB protocol from Doyle and Doyle (1987; 1990). Twenty

micrograms of this DNA were digested overnight at 37o C with BamHI and separated by

electrophoresis in a 1.3% agarose gel. Because BamHI has one cut-site within T-DNA, a

fragment bigger than 0.398 Kb is expected for each insertion event. DNA was then

denatured and transferred to N+ Hybond membrane. The nylon membrane was

prehybridized 4 h at 42o C with constant shaking at 60 rpm, in a solution containing 50%

(v/v) formamide, 5X Denhardt’s, 1% sodium dodecyl sulfate (SDS), 5X SSPE (1X SSPE

is 15 mM NaCl, 10 mM NaH2PO4.H2O and 1 mM EDTA), and 100 µg . mL-1 denatured

salmon sperm DNA. Hybridization was carried through overnight at 42oC with constant

shaking at 60 rpm, in a solution containing 50% (v/v) formamide, 5X Denhardt’s, 1%

sodium dodecyl sulfate (SDS), 5X SSPE, and 100 µg . mL-1 denatured salmon sperm

DNA plus 1 X 106 cpm.ml-1 denatured 32P-labeled 1.4 Kb PCR-product from CP4 syn

gene. All further washes were performed with constant shaking at 60 rpm. Membranes

were first washed in a solution containing 2X SSPE, 0.05% sarkosyl and 0.01% sodium

pyrophosphate at 42oC during 20 min. The second wash was performed in a solution

containing 2X SSPE, 0.05% sarkosyl and 0.01% sodium pyrophosphate at 65oC for 20

min. The third and last (fourth) washes were carried out in a solution containing 0.1 X

SSPE, 0.05% sarkosyl and 0.01% sodium pyrophosphate for 20 min at 65oC. Membranes

were exposed to Kodak BIOMAX MR Film at -80oC for 3-5 days.

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4.2.7 Segregation Analysis of Transgenes in Primary Transformants

In order to analyze the segregation pattern of transgenes, 62 randomly selected T1

seedlings of TGM-AS-1 and TGM-AS-2 transgenic lines of ‘Galia’ male parental line

were evaluated. DNA was extracted at least three times from each individual seedling

according to the modified CTAB protocol (Doyle and Doyle 1987; 1990). A PCR assay

with specific primers for CMACO-1 and CP4 syn genes was performed for each DNA

sample as described above for transgene detection.

4.3 Results and Discussion

4.3.1 Transformation Efficiency

A total of six experiments of transformation were completed, under a CR design,

using the vector pCmACO1-AS. Glyphosate selection was not completely efficient,

because several “escapes” were detected by PCR assays. In addition, these PCR assays

aided in detecting the presence of CP4 syn and CMACO-1 antisense genes (Figure 4.2).

We were able to observe the presence of 1.4, 0.5, and 0.3 kb fragments that belong to the

CP4 syn, the native ACC oxidase and the engineered ACC oxidase genes, respectively.

Using the cotyledon-protocol and the CMACO-1 construct, the individual

transformation efficiency for each experiment, assessed by PCR, ranged between 7.5%

and 12.5% (Table 4.1). Similar transformation efficiency rates (9.5%) were obtained for

muskmelon cv. ‘Krymka’ using another independent transformation system, such as the

GUS reporter gene (Figure 4.4). This transformation efficiency obtained for ‘Galia’ male

parental line is one of the highest reported for any Cucumis melo transformation protocol

(Fang and Grumet, 1990; Dong et al. 1991; Gaba et al. 1992; Gonsalves et al. 1994;

Bordas et al. 1997; Guis et al. 2000; Akasaka-Kennedy et al. 2004). This high

transformation efficiency could be the result of a combination of factors such as

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Agrobacterium tumefaciens strain, selectable marker, and construct used (Hellens and

Mullineaux 2000; Febres et al. 2003), which may have provided the right conditions to

accomplish these positive results.

Figure 4.2 PCR assay for putative transgenic ‘Galia’ muskmelon male line plants. The

amplification product for CP4syn, native ACO-1, and engineered ACO-1 genes were 1.4 Kb, 0.5 Kb, and 0.3 Kb, respectively. M: HyperLadder (Bioline), 1 through 6: putative transgenic plants, 7: DNA from positive plant, 8: DNA from negative plant, 9: PCR reaction mixture.

Transgenic explants had an epinastic response while they were growing in vitro on

Glyphosate selection (Figure 4.5). However, this abnormality was not observed once

transgenic shoots were rooted and transferred to ex vitro conditions. Glyphosate is a

strong herbicide. It is able to induce ‘stress’ responses even in Glyphosate-tolerant

M 1 2 3 4 5 6 7 8 9 M

1.4 Kb

0.5 Kb

0.3 Kb

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Table 4.1 Transformation efficiency of ‘Galia’ male parental line with CMACO-1 antisense construct.

Number of explants zBFC yPCR+ plants % of efficiency

120 0.18 12 ± 3x 10 ± 2.5

Data are the average of six different experiments, each one with 120 explants. zBFC = bud forming capacity index (average number of shoots per explant) X (% explants forming shoots) / 100. (Martinez-Pulido et al. 1992). yPCR+ = Polymerase chain reaction positive for transgenes. xMean value plus-minus standard error. plants (Saroha et al. 1998; Raymer and Grey, 2003). Also, it is well known that ethylene

is a plant hormone related to ‘stress responses’ (Klee and Clark, 2002). Therefore, it is

plausible that epinastic phenotype was induced by Glyphosate. A similar response was

observed in positive explants for β-D-glucuronidase gene (GUS) growing on Glyphosate

as well (Nunez-Palenius et al. unpublished results).

In summary, transgenic shoots were obtained by using Glyphosate as a selectable

agent and were identified by means of PCR assay. This PCR system also allows us to

identify those shoots which were non-transgenic.

4.3.2 Ploidy Level of Primary Regenerants

Melon plants regenerated through plant tissue culture methodologies are very

susceptible to increases in ploidy level while they are in vitro cultivated (Bouabdallah

and Branchard, 1986; Fassuliotis and Nelson, 1992; Ezura et al. 1992a; 1992b;

Debeaujon and Branchard, 1992; Kathal et al. 1992; Ezura and Oosawa, 1994a; Ezura et

al. 1994; Guis et al. 1998). Therefore, flow cytometry analyses were performed to

determine the ploidy level of primary regenerants. Young leaves from all in vitro

transgenic regenerated plants and also from wild-type plants obtained from seed were

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Figure 4.3 Stable expression of β-D-Glucuronidase (GUS) gene in T0 shoots (A and B)

and roots (C and D) of ‘Galia’ muskmelon male parental line.

used. This was done with the aim of discarding any triploid, aneuploid, and tetraploid or

mixoploid plants. Based on these analyses, only diploid plants were used in subsequent

experiments.

Unexpectedly, some leaves from plants obtained from seeds resulted as tetraploids.

This condition has been reported in melon plants field conditions as well (Nugent and

Ray, 1992). In Arabidopsis it has been described that ethylene and gibberellins might

have an important role in inducing the cellular endoreduplication process, which leads to

A

B

C

D

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Figure 4.4 Transgenic (left) and non-transgenic (right) in vitro ‘Galia’ male line explants.

Transgenic explants, growing on 50 µM Glyphosate, had curled leaves and shorter internodes than wild type.

an increase in ploidy level (Kondorosi et al. 2000). Figure 4.6 depicts charts for wild-type

diploid and tetraploid plants, as well as transgenic diploid and tetraploid plants. In Figure

4.6a, nuclei sample was obtained from the 3rd leaf below shoot apex (diploid), whereas

nuclei in Figure 4.6b were obtained from shoot re-growth from the plant base (4th and 5th

node distal to the cotyledonary-leaf). For subsequent experiments using flow cytometry

analysis, all leaf samples were harvested from the 3rd leaf below shoot apex. In Figure

4.7a and 4.7b, a transgenic diploid and tetraploid plant is shown. Using flow cytometry

analysis it was found that only 20% of the transgenic plants were diploid. This number of

diploid melon plants attained through in vitro culture is lower compared with results

previously reported (Guis et al. 2000; Curuk et al. 2003). This is because our system is

based on using cotyledons as explants, which is a tissue with high propensity to bear

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tetraploid cells since the mature seed stage in cucurbits (Colijn-Hooymans et al. 1994).

After an entire flow cytometry evaluation (at least seven leaves from different nodes) of

all transgenic regenerants, two completely diploid independent transgenic plants were

selected, which were named as TGM-AS-1 and TGM-AS-2. Plants that were PCR

positive and completely diploid were advanced to the next generation by selfing.

4.3.3 Southen Blot Analysis

The presence of the introduced CP4 syn gene in T1 plants, both TGM-1 and TGM-2

lines, was established by genomic Southern blot analysis (Figure 4.8). Likewise, this

assay allowed determining the copy number of the transgene insertion. The 32P-labeled

1.4 Kb PCR-product from CP4 syn gene used as a probe hybridized with a single BamHI-

released fragment of genomic DNA isolated from three individual T1 plants from TGM-1

(Figure 4.8B, lanes five, six and seven) and three individual T1 plants from TGM-2 lines

(Figure 4.8B, lanes one, two and four). These data indicated that a single copy of the T-

DNA was incorporated into the ‘Galia’ male parental line genome in TGM-AS-1 and

TGM-AS-2 lines. There was no hybridization signal in any non-PCR positive T1 plants

(Figure 4.8B, Lanes 3 and 8).

4.3.4 Transgene Inheritance in the T1 Progenies of Primary Transformants

On a population of 62 T1 plants randomly chosen from each transgenic line (TGM-

AS-1 and TGM-AS-2), DNA was extracted individually, at least three times, to

determine the transgene distribution inheritance pattern. PCR analysis of the progeny of

TGM-AS-1 and TGM-AS-2 using CP4 syn- and ACO-antisense specific primers revealed

segregation for the presence and absence of both CP4 syn and ACO-antisense fragments.

This observed distribution of the ACO-antisense and CP4 syn genes was consistent with

a 3:1 ratio (X2 value for TGM-AS-1 and TGM-AS-2 was 0.193 and 0.021, with a

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probability of 66% and 88%, respectively), which corresponded to the segregation of one

single copy insertion of T-DNA. This conclusion is supported by the Southern blot

analysis results as well

4.4.1 Conclusion

We have successfully transformed a parental line of ‘Galia’ hybrid muskmelon

with a gene of interest and obtained two completely diploid transgenic regenerants.

Analysis of T1 progenies from TGM-AS-1 and TGM-AS-2 by segregation and Southern

blot revealed that both transgenic lines had a single T-DNA insertion.

Preliminary analysis of the transgenic fruits from ACO-1 antisense male line plants

has revealed that these fruits produced less ethylene and have a lower ACC oxidase

activity in vivo than their wild type counterpart.

125

A

B

Figure 4.5 Flow cytometry analysis of propidium iodide-stained nuclei from wild type leaf tissue of ‘Galia’ male parental line. 10,000 individual nuclei were measured for each sample. (A) wild-type plant having most of nuclei (55%) at diploid level (M1). (B) wild-type plant having most of nuclei (51%) at tetraploid level (M2).

Fluorescence intensity (arbitrary units)

WT

0

800 M2 M3

M1

1 600

Num

ber

of n

ucle

i

0 200 400 600 800

Fluorescence intensity (arbitrary units)

0 200 400 600 800 1000

Num

ber

of n

ucle

i

WT

0

800

M1

M2

M3

1 600

1000

126

A

B

Figure 4.6 Flow cytometry analysis of propidium iodide-stained nuclei from transgenic leaf tissue of ‘Galia’ male parental line. 10,000 individual nuclei were measured for each sample. (A) transgenic plant having most of nuclei (64%) at diploid level (M1). (B) transgenic plant having most of nuclei (58%) at tetraploid level (M2).

Fluorescence intensity (arbitrary units)

Num

ber

of n

ucle

i

PCR+ # 2

0 200 400 600 800 0

800

M1

MM3

1 600 N

umbe

r of

nuc

lei

PCR+ # 11

0 200 400 600 800 0

1 600

800

M1

M2

M3

1000

Fluorescence intensity (arbitrary units)

1000

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A B

Figure 4.7 Southern blot hybridization assay of male muskmelon transgenic plants. (A) Total genomic DNA (20 µg) extracted from leaves of T1 plants was digested and electrophoresed according to Material and Methods section. M: HyperLadder (Bioline), WT: wild type ‘Galia’ male plant. 1 through 4: T1 plants from T0 TGM-AS-2 line. 5 through 8: T1 plants from T0 TGM-1 line. (B) Southern blot hybridization assay for BamHI-digested genomic DNA. 1, 2 and 4: PCR-positive T1 plants of T0 TGM-AS-2 line. 3: PCR-negative T1 plant of T0 TGM-AS-2 line. 5, 6 and 7: PCR-positive T1 plants of T0 TGM-AS-1 line, 8: PCR-negative T1 plant of T0 TGM-1 line.

4.5.1 Summary

‘Galia’ muskmelon (Cucumis melo L. var. reticulatus Ser.) has been recalcitrant to

transformation by Agrobacterium tumefaciens. Transformation of the ‘Galia’ male

parental line with an ACC oxidase (CMACO-1) gene in antisense orientation is described

herein. Explants were transformed using Agrobacterium tumefaciens strain ABI, which

contained a vector pCmACO1-AS plasmid, bearing an antisense gene of CMACO-1 and

the CP4 syn gene (glyphosate-tolerance). Both CMACO-1 and CP4 syn genes were

assessed by a polymerase chain reaction method. Flow cytometry analysis was performed

M WT 1 2 3 4 5 6 7 8 M

Kb 5.0 4.0 3.0 2.5 2.0

M WT 1 2 3 4 5 6 7 8 M

128

to determine plant ploidy level of primary transformants. Two completely diploid

independent transgenic plants were obtained. Southern blot and segregation analysis in

the T1 generation determined that each independent transgenic line had one single

insertion of the transgene. These transgenic muskmelon male parental lines have potential

for use in the production of ‘Galia’ F1 hybrids with improved shelf life.

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CHAPTER 5 EMBRYO-RESCUE CULTURE IN ‘GALIA’ MALE PARENTAL LINE MELON

(CUCUMIS MELO L. VAR. RETICULATUS SER.)

5.1 Introduction

As part of specific hybridization and breeding programs, crosses between distant

elite-plant genotypes sometimes fail to produce a complete embryo inside the seed, due to

embryo abortion during the process of embryogenesis. In other cases, even though the

embryo is formed, the seed fails to germinate and grow. Several embryo rescue

techniques, such as ovule, ovary and embryo culture, have been applied to save valuable

dicot and monocot plant materials (Bridgen, 1994; Brown et al. 1997; Kumlehn et al.

1997; 1998; 1999; Momotaz et al. 1998; Sukno et al. 1999; Chrungu et al. 1999;

Ramming et al. 2000; Kato et al. 2001; Ishikawa et al. 2001; Ribeiro and Giordano, 2001;

Yamada 2001; Faure et al. 2002a; 2002b; 2002c). These embryo rescue techniques have

also been useful to enhance hybridization, reduce generation time of elite germplasm, and

obtain valuable haploid plants in many crops such as onion (Martinez et al. 1997), wheat

(Xynias et al. 2001), maize (Weymann et al. 1993), potato (Eijlander et al. 1994),

sunflower (Faure et al. 2002b; 2002c), rice (Alemanno and Guiderdoni, 1994), melon

(Lotfi et al. 2003), squash (Metwally et al. 1998a; 1998b), and barley (Hoekstra et al.

1992). Not all attempts to obtain plant material through embryo rescue have been

successful; embryos generally failed to undergo complete differentiation (Dryanovska

and Ilieva, 1983; Shail and Robinson, 1987; Marcellan and Camadro, 2000; de Oliveira

et al. 2003).

130

In cucurbits, embryo rescue techniques have recovered normal seedlings from

anther, ovule, and zygotic-embryo cultures to obtain interspecific hybrids or dihaploid

lines (Lazarte and Sasser 1982; Shail and Robinson, 1987; Metwally et al. 1998a; 1998b;

Chen et al. 1997; Chen et al. 2002; Gemes-Juhasz et al. 2002; de Oliveira et al. 2003;

Sisko et al. 2003; Lotfi et al. 2003; Chen et al. 2003). Similar to other plant species,

embryo-rescue culture for cucurbits is affected by genotype (Malepszy et al. 1998;

Mackiewicz et al. 1998; Lotfi et al. 2003), formulation of media culture (Malepszy et al.

1998; de Oliveira et al. 2003 ), plant hormone-type and -dosage included into the media

(Beharav and Cohen, 1995a; 1995b; Metwally et al. 1998b), environmental factors such

as temperature and light (Metwally et al. 1998a; Gémes-Juhász et al. 2002), components

added to the media such as carbohydrate source (Metwally et al. 1998b), and embryo-

developmental stage at the time of explant isolation (Ezura et al. 1994a; 1994b; Beharav

and Cohen, 1995).

Because of these factors which affect efficiency rate of embryo culture, conditions

need to be predetermined for success with specific germplasm. This paper describes an

improved procedure for recovery of normal seedlings from wild type and transgenic

‘Galia’ muskmelon male parental line zygotic-embryos. Results are presented on the

influence of six new supplements for E-20A basic medium (Sauton and Dumax de Vaulx,

1987), five different fruit harvesting dates and two embryo-inoculation methods for plant

materials for which no information on embryo rescue exists.

5.2 Materials and Methods

5.2.1 Plant Material

Transgenic muskmelon plants with an ACC oxidase gene in antisense orientation

were obtained using the protocol previously described (Nunez-Palenius et al. 2005a).

131

Mature seeds of wild type and transgenic (ACC oxidase antisense) ‘Galia’ (Cucumis melo

L. var. reticulatus Ser.) male parental line were germinated on a mixture of 70% Terra

Lite Plug Mix (Terra Asgrow, Apopka, FL) and 30% coarse vermiculite in polystyrene

flats (cell size 2.25 cm2 and 164 cells per flat, Speedling, Bushnell, FL). Seedlings were

grown under drip fertigation in plastic pots (11.3 L) filled with soil-less media (coarse-

grade perlite) in an evaporative-cooled fan and pad glasshouse, with temperatures

maintained at 28oC day and 20oC night. Growing practices were those recommended by

Rodriguez and Cantliffe (2001). An integrated pest management program (IPM) was used

to control pests (http://www.hos.ufl.edu/protectedag/). A complementary light regime (18

h) was supplied by MetalarcR lamps (Osram Sylvania, Inc.) with a light intensity of 350 –

530 µmol . m-2 . s-1. Hermaphrodite flowers were isolated from foreign pollen with a

twist-tie band in the evening, one day before anthesis, and pollinated by hand on the next

morning (7:00-10:00 AM) using at least three male flowers. Stigmas were covered with a

gelatin capsule (Capsuline ™ size 1, Capsuline, Inc. Pompano Beach, FL) after

pollination to avoid excessive pollen dehydration.

5.2.2 Embryo Culture

Wild type and transgenic fruits were harvested at 4, 10, 17, 24 and 30 days after

pollination (DAP) in the morning (6:00 AM), and taken to the lab for subsequent rescue

procedures. The surface of the fruit was thoroughly washed with liquid detergent (5%

Liqui-Nox®), rinsed with tap water, and at room temperature. Washed fruits were then

surface-sterilized with 70% ethanol for 10 min, followed by a 40-min soak in 1.2%

sodium hypochlorite (20% commercial bleach solution containing two drops of Tween

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20™ per 100 ml) in a sterile beaker. Fruits were then rinsed three times with sterilized

distilled water under axenic conditions in a laminar-flow cabinet.

In fruits harvested 4 and 10 DAP, seeds were aseptically dissected from wild type

and transgenic fruits using a stereomicroscope. For 17, 24 and 30 DAP-fruits, seeds were

large enough to be visually dissected. Excised seeds were kept in 9% sterile sucrose

solution during the process (Ondrej et al. 2002). Embryos were excised from seeds 17, 24

and 30 DAP using a stereomicroscope. Intact seeds and embryos were cultured

immediately in 100X15-mm Petri dishes containing either E-20A (Table 5.1) or E-21

media [E-20A medium supplemented with 5% coconut water, 0.02 mg.L-1 xylose, 1

mg.L-1 glutamine, 0.25 mM putrescine, 0.01 mg.L-1 indole-3-butyric acid (IBA), and 0.01

mg.L-1 6-benzylaminopurine (BA)]. Two inoculation systems were used to culture the

embryos. In one system, immature embryos were excised only from seeds 17, 24 and 30

DAP and directly placed on either E-20A or E-21 media. In the other system (intact

seed), immature embryos from seeds 4, 10, 17, 24 and 30 DAP were not removed from

the seeds but left to develop, by directly placing the whole seed in the Petri dish with the

hilum facing the medium.

Cultures were incubated for 35 days in a growth chamber (Lab-Line Instruments,

Inc. Melrose Park, IL) in dark and constant 25±1°C temperature. After the incubation

period, plant material was transferred to maturation medium (½ strength E-21 medium

supplemented with 0.7% phytagar) and cultured between 2 to 5 more weeks under 100

µmol . m-2 . s-1 light and a 16 h photoperiod provided by cool-white fluorescent lamps and

constant 25±1°C temperature. Well-developed seedlings (cotyledonary stage) were

133

Table 5.1 Components of E-20A nutrient medium. Macroelements (mg.L-1) Microelements (mg.L-1)

KNO3 1,075.0 MnSO4 11.065

NH4NO3 619.0 ZnSO4.7H20 1.812

MgSO4.7H2O 206.0 H3BO3 1.575

CaCl2.2H2O 156.5 KI 0.345

KH2PO4 71.0 Na2Mo4.2H2O 0.094

Ca(NO3)2.4H2O 25.0 CuSO4

.5H2O 0.008

NaH2PO4.4H2O 19.0 CoCl2

.6H2O 0.008

(NH4)2SO4 17.0 Na2EDTA 37.3

KCl 3.5 FeSO4.7H2O 27.8

Organics (mg.L-1)

Plant growth regulators (mg.L-1) and

other supplements

myo-Inositol 50.300 Indole-3-acetic acid 0.01

Pyridoxine-HCl 5.500

Nicotinic acid 0.700 Sucrose (g.L) 20

Thiamine 0.600 Agar (g.L) 10

Ca-D-Pantothenate 0.500 pH 5.9

d-Biotine 0.005

Glycine 0.100

(Sauton and Dumas de Vaulx 1987).

134

transferred to soil (mixture of 70% Terra Lite Plug Mix and 30% coarse vermiculite) in

polystyrene flats of cell size 2.25 cm2 and 164 cells per flat (Speedling, Bushnell, FL),

and grown in a walking-growth chamber (250 µmol . m-2 . s-1 light and a 16 h photoperiod

provided by cool-white fluorescent lamps and constant 25±1°C temperature) until a 2-3-

true-leaf stage was reached. Seedlings were then evaluated for percent regeneration

efficiency [(No. of 2-3-true-leaf stage seedlings X 100)/No. of cultured seeds].

5.2.3 Experimental Design and Statistical Analysis

The experimental design was an incomplete factorial (2 x 2 x 2 x 5 or 3) with three

replicates, having two levels for genotype, two levels for media, two levels for

inoculation system, and five or three levels for harvesting dates. Original data was

transformed by the arcsine square root in order to be analyzed for analysis of variance

(ANOVA). Means were separated by Tukey’s Studentized Range Test at P≤ 0.05 (SAS

Institute, Cary, N.C.).

5.3 Results and Discussion

5.3.1 Embryo Development

Seeds were isolated from wild type and transgenic ‘Galia’ male parental line fruits,

which were harvested at 4, 10, 17, 24, and 30 DAP. Due to small embryo size at 4 and 10

DAP, it was not possible to dissect the embryos from seeds harvested on that stage, only

intact seeds were cultured for those dates (Figure 5.1 and 5.2). Embryos, inside of intact

seed at 4 or 10 DAP stage, underwent normal development after being cultured with the

hilum facing either E-20A or E-21 media (Figures 5.1 and 5.2). It has been reported that

culture of intact ovules, avoids embryo damage during dissection (Monnier, 1976,

Thomas, 1976, Momotaz et al., 1998). After one week of culture, embryos from intact

seeds 4 DAP became green and elongated forming the torpedo stage of development

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(Figure 5.1.c.B). After three more weeks of culture, an immature seedling with apical and

radical meristems was observed (Figure 5.1.c.E). These seedlings formed normal plants

(2-3-true-leaf stage) after the following steps: one more subculture on either E-20A or E-

21 media for two weeks then culture on maturation medium for three weeks and finally

transferred to soil. More than 12 weeks were needed to obtain complete wild type and

transgenic ‘Galia’ male parental line plants using seeds from 4 DAP fruits.

A similar development pattern was observed when intact seeds from fruits 10 DAP

were cultured on E-20A and E-21 media (Figure 5.2). However, the period to obtain full

seedlings was shorter than it was for 4 DAP-intact seeds. After one week of culture on

either E-20A or E-21 media, embryos became green and developed primordial cotyledons

(Figure 5.2.c). Two weeks later, the immature seedling had developed a normal root,

bearing root hairs, and had an apical meristem (Figure 5.2.d). At this stage, embryos

could be transferred to maturation media for further development (Figure 5.2.e).

Complete muskmelon ‘Galia’ male parental plants were obtained in 9 weeks after seeds

were dissected from 10 DAP fruits.

Embryos from fruits 17, 24 and 30 DAP were at different developmental stages and

had diverse sizes (Figures 5.3.b, 5.4.c and 5.4.f). Two inoculation systems were applied

for these embryos: excising the embryo from the seed and placing it directly on culture

media or leaving the immature embryo inside the seed (intact seed) and culturing those

seeds with the hilum facing the media (Figure 5.3.c). Using both inoculation systems,

complete and normal wild type and transgenic ‘Galia’ male parental line plants were

obtained after 4-5 weeks of in vitro culture (Figure 5.5).

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Figure 5.1 ‘Galia’ male parental line fruit at 4 DAP stage (a and b). (c) in vitro embryo

development from 4 DAP stage. A, B, C, D and E are stages after 0, 7, 15, 20 and 27 days, respectively, of in vitro culture.

5.3.2 Regeneration Efficiency

The regeneration efficiency of wild type and transgenic ‘Galia’ male muskmelon

embryos was affected by media, DAP, and inoculation system used (Table 5.2). Greater

regeneration efficiency levels were obtained when embryos or intact seeds from both

genotypes were cultured on E-21 medium than on E-20A medium (Figure 5.6 and 5.7).

These differences were significant (Table 5.3) and observed independently of DAP or

inoculation system used; i.e. wild type-30 DAP-intact seed on E-20A had a regeneration

efficiency of 50.1%, whereas the regeneration efficiency for wild-type-30 DAP-intact

seed on E-21 was more than 90% (Table 5.2).

a

b c

BA D C E

10 mm

10 mm

1 mm

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Figure 5.2 ‘Galia’ male parental line fruit at 10 DAP stage (a). In vitro embryo

development from 10 DAP stage, (b), (c), (d) and (e) are stages after 0, 7, 21 and 30 days, respectively, of in vitro culture. (b) Embryo (yellow circle) and seed coat (yellow arrow). (c) Embryo (red circle) and seed coat (red arrow). (c) Seedlings growing on elongation media.

It has been reported that more developed embryos are easier to rescue than embryos

from earlier developmental phases (Liu et al. 1993; Ondrej and Navratilova 2000).

Developed embryos may require fewer nutrients and hormones, and a lower osmotic

potential to fully develop (Ondrej and Navratilova 2000; Ondrej et al. 2002). A similar

a

b c

d e

10 mm

1 mm

5 mm

1 mm

138

pattern, regarding that older embryos are easier to rescue than younger ones, was

observed when embryos and intact seeds were obtained from fruits harvested on different

dates (Figure 5.6 and 5.7).

Figure 5.3 ‘Galia’ male parental line fruit at 17 DAP stage (a). In vitro embryo

development from 17 DAP stage, (b) yellow circle encloses the embryo, (c) inoculated seeds (without dissecting the embryo) with the hilum facing the culture medium, (d) testa removal from seeds after 15 days of in vitro culture, (e) seedlings after one week of testa removal.

b

a

c

d

10 mm

1 mm

e

139

Figure 5.4 ‘Galia’ male parental line fruit (a and b) and embryo (c) at 24 DAP stage.

‘Galia’ male parental line fruit (d and e) and embryo (f) at 30 DAP stage.

Indeed, the efficiency of this technique was greater when the time after pollination (4, 10,

17, 24 and 30 DAP) to rescue the embryos was increased (Table 5.4).

Regeneration efficiency between inoculation systems (intact seed and embryo

removed) was different (Figure 5.6 and 5.7). Firstly, unlike embryo removed system,

using the intact seed method is possible to rescue embryos from earlier stages such as 4

and 10 DAP. Secondly, the greatest amount of plants (90%) was regenerated through the

intact seed method (Figure 5.6B). Thirdly, significant differences were observed between

both inoculation systems (Table 5.5). Thus, a greater quantity of plants can be obtained in

a shorter period of time if the embryo is left inside the seed coat.

It has been reported that plant genotype can have a profound effect on the

efficiency of plant regeneration systems, and it is an important factor in establishing cell

selection and genetic transformation protocols (Machii et al. 1998; Yamada, 2001; Faure

et al. 2002c; El-Itriby et al. 2003). Therefore, plant tissue culture methods which have the

a b c

d e f

140

Figure 5.5 Normal ‘Galia’ male parental line seedlings obtained from embryo rescue,

having well-developed cotyledonary (red arrows) and true leaves (blue arrows). These plants were attained from 4, 10, 17, 24 and 30 DAP stage embryos.

capability to support sustainable growth disregarding plant genotype are considered

important tools. Similar regeneration efficiency levels on either media were attained

when wild type and transgenic ‘Galia’ male parental line embryos were cultured. Thus,

the presence of the transgene did not have any effect on regeneration efficiency values. It

might be interesting to test more GMO-melon genotypes on this improved E-21 medium.

141

Table 5.2 Regeneration efficiency of ‘Galia’ male parental line immature embryos that underwent development to form normal plants.

Media DAPz Inoculation

systemy (IS)

Wild type

(%)

Transgenic

(%)

E-20A 4 A 4.0 4.0

B NDx ND

10 A 36.9 41.5

B ND ND

17 A 53.9 45.2

B 29.6 32.7

24 A 53.1 54.0

B 34.5 44.5

30 A 50.1 51.5

B 49.7 47.1

E-21 4 A 8.8 7.3

B ND ND

10 A 48.8 53.9

B ND ND

17 A 80.6 87.1

B 54.1 53.3

24 A 90.8 90.4

B 62.1 61.4

30 A 92.3 94.0

B 66.4 69.3

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Table 5.2 Continued. Factorial treatments

Media * DAP *

Genotype NS IS *

Media x Genotype * Media x DAP *

Media x IS * Genotype x DAP *

Genotype x IS NS IS x DAP *

Media x Genotype x DAP * Media x Genotype x IS *

Genotype x DAP x IS * Media x DAP x IS *

Media x Genotype x DAP x IS *

zDAP) days after pollination. yA) intact seed, B) embryo removed from seed. xND) non-determined. NS, *Non-significant or significant, respectively, by Tukey’s Studentized Range Test at P≤ 0.05.

The described improved medium (E-21) and protocol (direct inoculation with the

hilum facing the medium) proved to be efficient in regenerating healthy seedlings from

wild type and transgenic immature ‘Galia’ male parental line embryos. One novelty of E-

21 medium is the amendment with putrescine (polyamine) to rescue embryos from

cucurbit seeds (melon). Putrescine has been used in other plant species, such as grape,

where adding 2 mM putrescine to media culture was able to significantly increase the

percentage of rescued embryos and normal plants (Ponce et al 2002a; 2002b). It is known

that polyamines are active regulators of plant growth and have the ability to interact

synergistic with plant hormones (Srivastava 2002; Bais and Ravishankar 2002; 2003).

143

Figure 5.6 Percentage of ‘Galia’ male parental line immature embryos that developed to

form normal plants. Complete seeds were directly inoculated with the hilum facing either E-20A (A) or E-21 (B) medium. Lines on bars are SE.

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

4 DAP 10 DAP 17 DAP 24 DAP 30 DAP

% re

gene

ratio

n ef

ficie

ncy

W.T.PCR+

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

4 DAP 10 DAP 17 DAP 24 DAP 30 DAP

% re

gene

ratio

n ef

ficie

ncy

W.T.PCR+

A

B

144

Figure 5.7 Percentage of ‘Galia’ male parental line immature embryos that developed to

form normal plants. Immature embryos were dissected and inoculated on either E-20A (A) or E-21 (B) medium. Lines on bars are SE.

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

17 DAP 24 DAP 30 DAP

% r

egen

erat

ion

effic

ienc

y

W.T.PCR+

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

17 DAP 24 DAP 30 DAP

% re

gene

ratio

n ef

ficie

ncy

W.T.PCR+

A

B

145

Table 5.3 Regeneration efficiency of ‘Galia’ male parental embryos cultured on two different media.

Media Regeneration efficiency (%)

E-20A 37.8 b

E-21 65.3 az

zMean separation by Tukey’s Studentized Range Test at P≤ 0.05.

Table 5.4 Regeneration efficiency of ‘Galia’ male parental embryos depending on harvesting dates.

DAP harvesting dates Regeneration efficiency (%)

4 5.5dz

10 45.2c

17 55.4b

24 63.0a

30 67.3a

zMean separation by Tukey’s Studentized Range Test at P≤ 0.05.

Table 5.5 Regeneration efficiency of ‘Galia’ male parental embryos depending on inoculation system (IS).

Inoculation system Regeneration efficiency (%)

Intact seed 70.3 az

Embryo removed 50.4 b

zMean separation by Tukey’s Studentized Range Test at P≤ 0.05.

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In cucurbits, several regeneration rates for different zygotic embryo-rescue methods

have been reported. Values such as 9% (Fassuliotis and Nelson 1988), 37.3% (Chen et al.

1997; 2002; 2003), 9.1-74% (Metwally et al. 1998a; 1998b), 59.4% (Malepszy et al.

1998), 4.5-39.0% (Mackiewicz et al. 1998), 7.1% (Gémes-Juhász et al. 2002), 27.2%

(Ondrej et al. 2002), 28% (Sisko et al. 2003), and 66.7% (de Oliveira et al. 2003) have

been described. In Cucumis melo, regeneration rates from 0.09% to 76.8% have been

obtained (Ezura et al. 1994a; Beharav and Cohen 1995a; 1995b; Lotfi et al. 2003). The

regeneration rates (90%) obtained for melon in the present study exceeds significantly

those previously reported.

Unlike other embryo rescue methods for cucurbits, some of which require double-

layer culture with two or more different media, and several subcultures in order to

regenerate healthy and complete plants (Ondrej et al. 2002), this competent embryo-

rescue technique for melon, using only one medium culture type, can be applied to save

valuable GMO hybrid-melon embryos.

5.4 Summary

In order to obtain a reliable embryo-rescue technique for wild type and transgenic

‘Galia’ muskmelon male parental line, an improved (five new supplements) nutrient

medium (named E-21) from the E-20A basic medium (Sauton and Dumax de Vaulx,

1987), an inoculation system (removing the embryo from the seed or intact seed), and the

use of different fruit harvesting dates were evaluated. Transgenic muskmelon with the

ACC oxidase gene in antisense orientation was obtained using the protocol previously

described (Nuñez-Palenius et al. 2005a). Wild type and transgenic muskmelon plants

were grown using commercial growing practices that included pruning and training to

one vertical stem and the use of soil-less media and drip fertigation. Fruits of wild type

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and transgenic (ACC oxidase gene in antisense orientation) ‘Galia’ male parental line

were harvested at 4, 10, 17, 24 and 30 days after pollination (DAP). Subsequently, seeds

were removed from fruit under sterile conditions. These seeds were used either to dissect

the embryos or placed directly with the hilum facing E-20A or E-21 medium. Seedlings

from all treatments were transferred to E-21 elongation medium, incubated five weeks,

and transferred to soil to evaluate growth. The efficiency of this technique was greater

when the time after pollination (4, 10, 17, 24 and 30 DAP) to rescue the embryos was

increased. Therefore, 30 DAP was the best time to rescue the embryos. The number of

rescued embryos using E-21 medium was greater than E-20A basic medium. We did not

find any significant differences in survival efficiency rate between wild type and

transgenic embryos. We have obtained a competent embryo-rescue technique for wild

type and transgenic ‘Galia’ male parental line, which can be applied to rescue valuable

GMO hybrid-melon embryos.

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CHAPTER 6 FRUIT RIPENING CHARACTERISTICS IN A TRANSGENIC ‘GALIA’ MALE

PARENTAL LINE MUSKMELON (CUCUMIS MELO L. VAR. RETICULATUS SER.)

6.1 Introduction

Melon fruit ripening is a genetically determined event that involves a series of changes

in color, texture, firmness, aroma, and flavor, making fruit scent and flavor appealing to

consumers (Lelievre et al. 1997; Jiang and Fu, 2000). Ethylene is known to regulate the

ripening of climacteric fruits, such as melon (Giovannoni, 2001). This plant hormone is

produced substantially and accumulates during the climacteric stage, which coincides with

the ripening process in melon fruits (Seymour and McGlasson, 1993; Giovannoni, 2001).

‘Galia’ F1 hybrid muskmelon (Cucumis melo L.) is a climacteric fruit, having a short storage

life, which is limited to two weeks or less, even when it is maintained in low-temperature

(8oC) storage (Karchi, 2000).

Since ethylene induces ripening of climacteric fruits, it is a potential target for control

of ripening (Ayub et al. 1996; Guis et al. 1997b; Clendennen et al. 1999; Silva et al. 2004).

Two regulatory enzymes in the ethylene biosynthesis pathway are ACC synthase (ACS) (EC

4.4.1.14) and ACC oxidase (ACO) (EC 1.4.3.-) (Seymour and McGlasson, 1993). RNA

antisense technology has permitted regulating the expression of specific genes involved in

tomato fruit ripening (Gray et al. 1992; Fray et al. 1993; Chen et al. 1996b). Targeting ACO

via antisense technology has been successfully applied to cantaloupe Charentais (cv.

‘Vedrantais’) melon fruits in order to reduce ethylene production, and as a consequence

improve fruit quality by delaying ripening and softening processes (Ayub et al. 1996; Guis et

149

al. 1997b; Silva et al. 2004). The reduction of ethylene production in these trangenic melon

fruits was 97-99%, compared to their wild type counterpart. However, this strong ethylene-

production inhibition did not delay, but rather arrested completely the ripening process in

those transgenic fruits. Therefore, a continuous ethylene treatment was needed in order to

restore fruit ripening, as observed by rind yellowing, fruit softening and activation of the

peduncle abscission zone (Ayub et al. 1996; Guis et al. 1997b; Silva et al. 2004). A similar

RNA antisense technology targeting ACO (CMACO-1) might be applied to ‘Galia’ male

parental line for use of ‘Galia’ F1 hybrids with improved shelf life.

Two completely diploid independent ACC oxidase antisense transgenic plants of

‘Galia’ inbred male parental line (TGM-AS-1 and TGM-AS-2) were previously obtained

(Nunez-Palenius et al. 2005a). Experiments were conducted to compare fruit quality

characteristics between transgenic ACC oxidase antisense (TGM-AS), azygous-AS,

transgenic GUS (T-GUS) and wild type (WT) fruits from plants grown in greenhouse

conditions. Physiological and biochemical fruit parameters, such as weight, length, width,

rind color, soluble solids, titratable acidity, pH, flesh thickness, firmness, ripening index,

seed cavity size, seed number, ACC oxidase activity in vivo and ethylene production were

measured during fruit development, specifically, at zero-, half- and full-slip developmental

stages.

6.2 Materials and Methods

6.2.1 Plant Material

‘Galia’ male parental line transgenic plants (Cucumis melo var. reticulatus cv.

‘Krymka’), harboring either an ACC oxidase antisense (CMACO-1) or uidA (GUS) gene,

were obtained by a cotyledon-protocol described in Nunez-Palenius et al. (2005). Two

independent diplod transgenic lines (TGM-AS-1 and TGM-AS-2) were attained with the

150

CMACO-1 construct, whereas a single independent diplod transgenic line (T-GUS) was

obtained using the GUS gene. In vitro non-transgenic-AS ‘escape’ seedlings were obtained

after Agrobacterium tumefaciens-mediated transformation. These plants were called

azygous-AS to emphasize the absence of the ACC oxidase antisense transgene.

In order to increase plant numbers, lateral branch cuttings from each T0 transgenic and

non-transgenic lines were rooted by exogenous application of Green Light™ (0.1% indole-3-

butyric acid) rooting hormone on severed stem bases. Ploidy was confirmed by flow

cytometry analysis according to the procedure described in Nuñez-Palenius et al. (2005a).

Only completely diploid plants were used to set fruit. Non-transformed WT, T-GUS, and

azygous-AS ‘Galia’ male parental plants were used as controls.

Plants were cultivated inside of an evaporative-cooled fan and pad glasshouse in

Gainesville, FL, 28oC day and 20oC night. Plants were grown in plastic pots (11.3 L) filled

with soil-less media (course-grade perlite) following common growing practices

recommended by Rodriguez and Cantliffe (2001). Complementary light was supplied by

MetalarcR lamps with a light intensity of 350-530 µmol m-2 s-1 for 18 hours per day. Training,

pruning, and fertigation of plants, as well as application of fungicides, were performed

according to those recommended by Rodriguez and Cantliffe (2001). An integrated pest

management program (IPM) was used to keep pests under control

(http://www.hos.ufl.edu/protectedag/).

Hermaphrodite flowers were self-pollinated by hand and tagged for date of pollination.

Only three fruits were kept on each plant. A sample of 10 fruit from wild type, azygous-AS

and T-GUS were harvested at 37, 42 and 50 days after pollination (DAP), which

corresponded to zero-, half- and full-slip developmental stages, respectively. TGM-AS-1 and

151

TGM-AS-2 fruits were harvested at 42, 50 and 56 DAP, which corresponded to zero-, half-

and full-slip developmental stages, respectively. Fruits harvested at full-slip stage were kept

on the vine by pantyhose (Figure 6.1).

6.2.2 Determination of Fruit Size

Harvested fruits were weighed, and measured for length and width. In order to avoid

microbial-ethylene production, fruits were gently brushed, washed in tap water (∼25°C),

dipped into 200 µL . L-1 chlorinated water for 60 sec and then air-dried for 12 h at room

temperature to pursue subsequent analysis.

6.2.3 Determination of Physical and Biochemical Characteristics

Rind color was measured with a Minolta Chroma Meter (Model CR-200 Minolta

Camera Co. Ltd Japan) using the Hue angle (°) parameter (Hurr and Huber, 2005). Due to

‘Galia’ muskmelon fruit does not develop a rind-yellowing uniform pattern, and in order to

have a more precise rind color assessment, four equidistant and independent point

measurements in the equatorial region of each melon fruit were taken.

Fruit firmness was measured as described by Jeong et al. (2002). Whole unpeeled fruits

were tested using an Instron Universal Testing Instrument (Model 4411-C8009, Canton, MA)

fitted with a flat-plate probe (31 mm diameter) and 50-kg load cell. After establishing zero

force contact between the probe and the equatorial zone of the fruit, the probe was driven

with a crosshead speed of 50 mm.min-1. The force was recorded at 2 mm deformation and

was performed at two equidistant points on the equatorial zone of each fruit.

152

Figure 6.1 Wild type and TGM-AS ‘Galia’ male parental line fruit at 50 DAP. A) Wild type

fruits have already slipped from the vine. B) Transgenic fruit remain still attached to the vine at 50 DAP.

Each fruit was cut lengthwise from the stem-scarf towards the blossom end and the

flesh size was measured in six different points (A1, A2, B1, B2, C1 and C2) in both halves as

showed in Figure 6.2. Length, width and flesh measurements were used to calculate the seed

cavity area size by means of ellipse equation (Figure 6.2). The area of the quarter that

corresponds to the measurements A1, B1 and C1 is given by:

¼ Seed Cavity Size= X1Y2 + X2Y1

2

Where (X1Y1) correspond to the coordinates of the point P in Figure 6.2, and

X2= a-A1 and Y2= b-C1 (Figure 6.2).

Seeds were collected, washed and dried according to standard seed-harvesting

procedures (Desai et al. 1997). Empty and full seeds were separated and counted for each

fruit.

A B

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Total soluble solids (TSS), titratable acidity (TA), and pH were determined, in

triplicate, from juice obtained after the mesocarpic tissue was macerated, centrifugated

(28,000xg), and filtered through Whatman paper No.1. TSS, TA, and pH were quantificated

using a digital refractometer (Abbe Mark-10480, Buffalo, N.Y.), a Fisher-395 dispenser

connected to an electrometer (Fisher 380), and a digital pH meter (model 340, Corning,

N.Y.), respectively. TA was expressed as percent malic acid equivalents. The fruit ripening

index was calculated as the quotient between TSS and TA.

Ethylene evolution and ACC oxidase activity in vivo

Twelve hours after harvest, fruits were placed in airtight plastic containers (1 fruit per

container) (3.7 L) and sealed for 2 h at 25°C. Ethylene production for each fruit was

determined by measuring the ethylene concentration in the headspace of the containers.

Sampling for ethylene was taken through a serum stopper where 1 mL of headspace was

drawn and injected into a gas chromatograph (Hewlett Packard 5890 Series II, Avondale,

Pa.) as described in Ciardi et al. (2000). The results were expressed as µL ethylene produced

per kg of tissue per hour (µL.kg-1.h-1).

ACC oxidase activity was measured in vivo in melon mesocarpic tissue based on the

conversion of exogenous ACC to ethylene (Smith et al. 1994; Amor et al. 1998). Samples of

mesocarpic tissue were taken in triplicated with a No.5 cork-borer from the equatorial region

of the fruit. After removing the peel, 1 g of tissue, was incubated for three hours at 25°C in 3

ml reaction buffer, which contained 50 mM Tris.HCl (pH 7), 100 mM sucrose, 250 µM ACC,

and 100 µM cycloheximide (Amor et al. 1998). Boiled (95°C for 10 min) mesocarpic tissue

and reaction buffer alone were used as negative controls. After the incubation period, a 1-mL

154

gas sample was taken from the head space and analyzed by gas chromatography (Ciardi et al.

2000). Negative controls always had non-detectable ACC oxidase activity.

6.2.4 Experimental Design and Statistical Analysis

A Completely Randomized Design (CRD) was used to set up the experiment. Original

data was evaluated for analysis of variance (ANOVA). Means were separated by Duncan’s

Multiple Range Test at P≤ 0.05 (SAS Institute, Cary, N.C.).

Figure 6.2 Flesh size and seed cavity size determination in ‘Galia’ male parental line fruit. Six measurements were made in both halves as indicated by A1, A2, B1, B2, C1 and C2 for flesh size.

Y2

X2

P (X1,Y1)

a

b

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6.3 Results

6.3.1 ACC Oxidase Activity In Vivo

When ‘Galia’ male parental mesocarpic tissue was incubated in the presence of

reaction buffer mixture, ACC oxidase activity was detected via quantification of ethylene

evolution. At zero-slip developmental stage, the enzyme activity in vivo was below 1 nl.g-1.h-1

for all genotypes (Figure 6.3). An increase in ACC oxidase activity was observed in WT,

azygous-AS and T-GUS mesocarpic tissue at half-slip stage. Conversely, TGM-AS-1 and

TGM-AS-2 fruit did not have that increase in ACC oxidase activity at the same

developmental stage. Indeed, expression of the ACC oxidase antisense gene in transgenic

fruits caused a reduction in ACC oxidase activity, which was four times less compared to

WT, azygous-AS and T-GUS fruit at half-slip stage (Figure 6.3). Therefore, significant

differences in ACC oxidase activity in vivo were found between ACC oxidase antisense fruit

and WT, azygous-AS and T-GUS at half-slip developmental stage (data not shown). At full-

slip stage, the lowest level of ACC oxidase activity was detected in TGM-AS-2 mesocarpic

tissue, which was significantly different to other genotypes (Figure 6.3).

6.3.2 Ethylene Production

Average ethylene production in WT, TGM-AS-1, TGM-AS-2, azygous-AS and T-GUS

fruit harvested at zero-slip stage was 7.6, 7.5, 2, 2, and 1.4 µl.kg-1.h-1, respectively (Figure

6.4). On the next developmental stage, half-slip, a substantial increase in ethylene production

was observed for WT, azygous-AS and T-GUS fruit, but not for TGM-AS-1 and TGM-AS-2

genotypes (Figure 6.4). Indeed, the ethylene production level for the ACC oxidase antisense

lines was below 7 µl.kg-1.h-1 at half-slip stage. Therefore, significant differences were

observed between ACC oxidase antisense fruit and other treatments at half-slip stage (data

not shown).

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Figure 6.3 ACC oxidase activity in vivo in WT (●), TGM-AS-1 (○), TGM-AS-2 (▼), azygous-AS (∇) and T-GUS (■) fruits. Vertical bars represent standard error of the means (n=10 fruits). Each fruit was assayed by triplicated.

TGM-AS-2 fruit always had a lower ethylene level than TGM-AS-1. A significant (P<

0.001) regression coefficient (R2=0.91) was obtained between ACC oxidase activity and

ethylene production data, supporting that both parameters are absolutely related (Figure 6.5).

Developmental stage

0 SLIP HALF SLIP FULL SLIP

AC

C o

xida

se (n

l. g-1. h-1

)

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

WT TGM-AS-1 TGM-AS-2 Azygous-AS T-GUS

157

Figure 6.4 Ethylene production from WT (●), TGM-AS-1 (○), TGM-AS-2 (▼), azygous-AS (∇) and T-GUS (■) fruits. Vertical bars represent standard error of the means (n=10 fruits).

6.3.3 Firmness, Rind Color and TSS

In order to analyze and compare more easily fruit firmness, rind color and total soluble solids

parameters to ethylene production, the firmness data, as well as rind color and total soluble

solids data, from WT, azygous-AS and T-GUS genotypes were considered as a single

average value at zero-, half- and full-slip stages, and named it as ‘WT’.

Developmental stage

0 SLIP HALF SLIP FULL SLIP

Ethy

lene

( µl. kg

-1. h-1

)

-5

0

5

10

15

20

25

30

WTTGM-AS-1TGM-AS2Azygous-AST-GUS

158

Figure 6.5 Regression analysis between ACC oxidase activity and ethylene production in ‘Galia’ male parental fruits.

Likewise, TGM-AS-1 and TGM-AS-2 data for firmness, as well as rind color and total

soluble solids data, were regarded as one single average value at zero-, half- and full-slip

stages, and named as ‘TGM-AS’.

Ethylene production data from WT, azygous-AS and T-GUS were averaged to one

single value and considered as WT. TGM-AS-1 and TGM-AS-2 ethylene production data

were regarded as TGM-AS.

Fruit softening was observed in WT and TGM-AS during the ripening process (Figure

6.6). The maximum decrease in firmness was detected in WT fruit, which had an initial

average fruit firmness value of 52 N at zero-slip stage and the final value was below 18 N at

full-slip stage. The reduction of ethylene production in TGM-AS fruits induced a delayed

y = 7.6664x - 1.2328R2 = 0.9115

-5

0

5

10

15

20

25

30

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50

ACC oxidase activity

Ethy

lene

pro

duct

ion

159

fruit softening process (Figure 6.6). The reduction of firmness in TGM-AS from half-slip

stage to full-slip stage was only 28% from WT displayed. At full-slip stage, the TGM-AS

firmness value was above 29 N. Therefore, significant differences were observed between

WT and TGM-AS fruits at full-slip developmental stage (Figure 6.6).

According to the color-chart (Konica-Minolta, Inc), green color has a higher Hue angle

(°) value than yellow. Similarly as most of muskmelon fruits, ‘Galia’ male parental fruit

turns from green to yellow rind color during the ripening process. In TGM-AS fruits, the Hue

angle (°) value was higher than WT at half- and full-slip developmental stage (Figure 6.7).

Significant differences between WT and TGM-AS treatments were observed at those

two stages (data not shown). Despite some ACC oxidase antisense transgenic fruits were last

harvested until full-slip stage, they did not reach a full golden-yellow color as WT (Figure

6.8). TSS content of WT and TGM-AS fruits increased along with ripening (Figure 6.9).

Therefore, higher TSS contents were found on fruits harvested at full-slip stage. No

significant differences on TSS accumulation were detected between WT and TGM-AS fruits

at any developmental stage. Although TGM-AS fruits remained a longer time attached to the

vine than WT fruits, they did not accumulate more TSS.

6.3.4 Mesocarpic Titratable Acidity, pH and Ripening Index

In contrast to TSS content, which had an increase along ripening, mesocarp TA level

had an important decrease, which took place simultaneously with the fruit ripening process

(Table 6.1). On zero-slip stage, the lower and greater TA values were observed for WT and

T-GUS fruit, respectively, although they were not significantly different. TGM-AS-2 TA

value was higher than WT, azygous-AS, T-GUS and TGM-AS-1 at full-slip stage, and it was

significantly different (Table 6.1). The regression coefficient (R2= 0.62, y= -0.037x + 0.45)

between TSS and TA had a significant value (P< 0.001).

160

Figure 6.6 Ethylene production from WT (●) and TGM-AS (○) fruits compared to firmness of WT (■) and TGM-AS (□) intact fruits. Vertical bars represent standard error of the means (n=30 fruits for WT and n= 20 for TGM-AS).

Similarly as TSS accumulation pattern, mesocarpic pH values of WT, azygous-AS, T-

GUS, TGM-AS-1, and TGM-AS-2 increased concomitantly with fruit ripening (Table 6-1).

No significant differences in mesocarpic pH values were found among all treatments at zero-,

half- and full-slip stages (Table 6.1). TA and pH were inversely related since they had a

significant (P< 0.001) regression coefficient and negative slope (R2= 0.80, y= -7.73x + 7.42).

Fruit ripening index increased in all treatments from zero-slip to full-slip

developmental stages (Table 6.1). In general, TGM-AS-2 fruits had a lower ripening index

than WT, azygous-AS, TGM-AS-1, and T-GUS counterpart. Significant differences were

Developmental stage

0 SLIP HALF SLIP FULL SLIP

Ethy

lene

( µl. kg

-1. h-1

)

0

5

10

15

20

25

30

Firm

ness

(N)

10

20

30

40

50

60

WT-Ethylene TGM-AS-Ethylene WT-Firmness TGM-AS-Firmness

161

Figure 6.7 Ethylene production from WT (●) and TGM-AS (○) fruits compared to rind color

of WT (■) and TGM-AS (□) intact fruits. Vertical bars represent standard error of the means (n=30 fruits for WT and n= 20 for TGM-AS).

only observed at full-slip developmental stage between TGM-AS-2 fruits and other

treatments (Table 6.1).

6.3.5 Determination of Fruit Size and Seed Number

Maximum average fruit weight, length and width of WT fruits occurred at full-slip

stage (Table 6.2). These measurements were the same as those from azygous-AS, T-GUS,

TGM-AS-1, and TGM-AS-2 counterparts at full-slip developmental stage (Table 6.2).

Minimum average fruit weight, length and width were observed at zero-slip stage, the

minimum weight parameter was found in WT fruits, whereas minimum length and width

were determined in TGM-AS-1 fruits. Similarly as maximum values, there were no

significant differences among minimum parameters from different plant genotypes

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Figure 6.8 Rind color in WT (A) and TGM-AS (B) ‘Galia’ male muskmelon fruit.

Figure 6.9 Ethylene production from WT (●) and TGM-AS (○) fruits compared to TSS content of WT (■) and TGM-AS (□) intact fruits. Vertical bars represent standard error of the means (n=30 fruits for WT and n= 20 for TGM-AS).

BA

Developmental stages

0 SLIP HALF SLIP FULL SLIP

Ethy

lene

( µl. kg

-1. h-1

)

0

5

10

15

20

25

30

TSS

%

6

8

10

12

14WT-EthyleneTGM-AS-EthyleneWT-TSSTGM-AS-TSS

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Table 6.1 Postharvest fruit characteristics of ‘Galia’ male parental line. Genotype Titratable acidity (%) pH Ripening indexz

0-Slipy Half-Slip Full-Slip 0-Slip Half-Slip Full-Slip 0-Slip Half-Slip Full-Slip

Wild type 0.15 0.106 0.076b 6.2 6.5 6.9 55.5 86.9 123.3a

Azygous-AS 0.19 0.108 0.095b 5.6 6.2 6.8 38.4 81.4 103.6b

T-GUS 0.21 0.099 0.082b 6.0 6.5 6.7 40.7 88.68 113.25ab

TGM-AS-1 0.16 0.108 0.095b 6.0 6.4 6.9 48.9 86.13 102.3b

TGM-AS-2 0.17 0.102 0.103a 6.0 6.4 6.5 46.8 88.1 93.7c

Significancex nsv ns * ns ns ns ns ns *

zFruit ripening index was calculated as the quotient between total soluble solids (TSS) and titratable acidity (TA). yDevelopmental stage. xDuncan’s Multiple Range Test at P≤0.05. vns= not-significant, * significant.

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(Table 6.2). Significant (P<0.001) regression coefficients for length-width, weight-width,

and weight-length were 0.87 (y= 0.776x + 2.6), 0.56 (y= 0.0034x + 8.8) and 0.60 (y=

0.0042x + 8.1817), respectively.

The minimum value for flesh size on all treatments was recorded in TGM-AS-1 at

zero-slip stage, but, it was not significantly different from WT, azygous-AS, T-GUS, and

TGM-AS-2 data (Table 6.3). Azygous-AS fruits had the maximum value for flesh size at

full-slip stage, although it was not significant different to other treatments (Table 6.3).

The maximum value for seed cavity size was observed in TGM-AS-2 at full-slip

stage, whereas minimum seed cavity size was detected in TGM-AS-1 at zero-slip stage.

Likewise, as previously evaluated fruit parameters (weight, length, width, and flesh size)

there were no significant differences among treatments.

The production of viable and high-vigor seeds within plant breeding programs has

a paramount importance. Therefore, full and empty seeds were evaluated on each fruit for

all genotypes. The minimum percentage of full seeds (70%) from all treatments was

determined in azygous-AS fruits at half-slip stage (Table 6.3). Consequently, the greatest

percentage of empty seeds was recorded on those fruits as well. TGM-AS-2 had the

highest full seed percentage at half-slip stage. Nevertheless, no significant differences for

full seed percentage were observed among treatments on zero- half- and full-slip

developmental stages (Table 6.3).

6.4 Discussion

Fruit ripening is a genetically determined event, and considered as a highly

complex development process (Seymour and McGlasson, 1993). Fruit in general can be

classified as either climacteric or non-climacteric on the basis of their respiration pattern

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Table 6.2 Postharvest fruit characteristics of ‘Galia’ male parental line. Genotype Weight (Kg) Length (cm) Width (cm)

0-Slipz Half-Slip Full-Slip 0-Slip Half-Slip Full-Slip 0-Slip Half-Slip Full-Slip

Wild type 0.94 1.12 1.55 12.6 13.6 15.8 12.3 12.7 14.7

Azygous-AS 1.05 0.92 1.48 13.1 11.8 14.2 12.8 11.8 13.9

T-GUS 1.10 1.21 1.37 12.8 12.0 11.50 13.0 11.7 11.4

TGM-AS-1 1.05 1.24 1.39 11.5 13.3 13.6 11.5 12.9 13.5

TGM-AS-2 1.21 1.42 1.51 13.9 12.7 14.7 13.8 12.4 14.7

Significancey nsv ns ns ns ns ns ns ns ns

zDevelopmental stage. yDuncan’s Multiple Range Test at P≤0.05. vnot-significant.

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Table 6.3 Postharvest fruit characteristics of ‘Galia’ male parental line. Genotype Flesh size (cm) Seed cavity size (cm2) Full seeds (%)

0-Slipz Half-Slip Full-Slip 0-Slip Half-Slip Full-Slip 0-Slip Half-Slip Full-Slip

Wild type 3.0 3.2 3.0 29.2 34.6 46.2 85 85 84

Azygous-AS 2.9 2.9 3.6 36.8 25.3 39.1 86 70 84

T-GUS 3.2 2.9 2.9 30.8 28.6 38.1 76 89 84

TGM-AS-1 2.7 3.1 3.0 27.0 34.5 38.1 76 78 78

TGM-AS-2 3.4 3.0 3.0 37.9 31.3 49.0 83 90 79

Significancey nsv ns ns ns ns ns ns ns ns

zDevelopmental stage. yDuncan’s Multiple Range Test at P≤0.05. vnot-significant.

167

and autocatalytic ethylene production peak during ripening (Tucker, 1993). Climacteric

fruits, such as most melon varieties, have a respiratory burst and a pronounced

autocatalytic ethylene production while the ripening process is proceeding.

The use of ACC oxidase antisense transgenic Charentais melon fruit has allowed

discriminating between ethylene-dependent and –independent processes during fruit

ripening (Guis et al. 1997b; Guis et al. 1999; Flores et al. 2001a; Flores et al. 2001b;

Silva et al. 2004). Indeed, it has been suggested that yellowing of the rind, fruit softening,

volatile production, climacteric respiration, and formation of abscission layer in the

peduncule are ethylene-dependent events, while sugar accumulation, loss of acidity and

pigmentation of the flesh are ethylene-independent processes (Guis et al. 1997b; Guis et

al. 1999; Flores et al. 2001a; Flores et al. 2001b). Recently, Silva et al. (2004) suggested

that organic acid metabolism in Cantaloupe Charentais melon fruit might not be an

ethylene-independent process. However, it can not be excluded that some of those

processes involved in melon ripening might encompass ethylene-dependent and –

independent mechanisms (Guis et al. 1999), which may be active at the same time. More

research, at molecular and physiological level, is needed to address the specific role of

ethylene on melon ripening.

The edible part of most melon fruits is the mesocarpic tissue, also commonly

known as flesh (Seymour and McGlasson, 1993). Most of the consumers prefer a melon

fruit with small seed cavity, and firm, thick and colorful flesh (Yamaguchi, 1977; Artes et

al. 1993; Goldman, 2002). Due to the insertion of certain trangenes, some secondary

unwanted and/or deleterious effects on recipient plants have occurred (Birch, 1997;

Yoshida and Shinmyo, 2000; Gomez-Lim and Litz, 2004; Halford, 2004). Therefore, in

168

order to address that possibility, several postharvest quality characteristics on transgenic

ACC oxidase antisense ‘Galia’ male parental fruit were evaluated in this study.

The highest ACC oxidase in vivo activity was observed in WT, azygous-AS and T-

GUS fruits at half-slip stage. Conversely, TGM-AS-1 and TGM-AS-2 did not have an

enzyme activity peak at half-slip stage. Smith et al. (1994) reported that the presence of

ascorbate in the reaction buffer may have deleterious effect on ACC oxidase activity in

‘Galia’ F1 hybrid fruit. These authors described that the high initial rate activity declined

rapidly after 1 h of incubation if ascorbate was present in the reaction buffer; half-life of

19 min with ascorbate. The ACC oxidase reaction buffer assay utilized in the present

study was void of ascorbate in order to circumvent this. Low levels of ethylene

production by ACC oxidase antisense melon fruit can be ascribed to a considerable

reduction in ACC oxidase activity (Ayub et al. 1996; Guis et al. 1997b; Silva et al. 2004).

Certainly, ACC oxidase activity in TGM-AS-1 and TGM-AS-2 fruit was reduced by 4.0-

and 186.7-fold, respectively, compared to WT, azygous-AS and T-GUS fruit.

External ethylene production in ‘Galia’ male muskmelon fruit had a similar pattern

as mesocarpic ACC oxidase activity during ripening. WT, azygous-AS and T-GUS fruit

displayed a rapid increase in ethylene production at half-slip stage. That ethylene peak

was not present in any of the TGM-AS lines. Interestingly, ethylene production in TGM-

AS-1 fruit was always higher than TGM-AS-2 at any date of harvest. The average

ethylene production at zero-, half- and full-slip stages by TGM-AS-1 and TGM-AS-2

was 7.71 µl.kg-1.h-1 and 0.86 µl.kg-1.h-1, respectively. These differences in ethylene

production are not related to the number of T-DNA copies inserted into the melon

genome, because both transgenic lines had a single copy of the ACC oxidase antisense

169

transgene (Nunez-Palenius et al. 2005a). Rather, T-DNA insertion position effects on

transgene expression might have played some role in that different ethylene production.

T-DNA position effects have been reported in other transgenic plants (Deblock 1993;

Zhu et al. 1999; Cotsaftis et al. 2002).

It has been reported that transgenic ACC oxidase antisense Charentais melon fruit

attached to the vine, exhibited very low internal ethylene levels: <0.5 µl.L-1 (Guis et al.

1997b) and ≤0.09 µl.L-1 (Silva et al. 2004). However, after harvesting at 38 DAP, the

internal ethylene content increased up to 4 µl.L-1 (Guis et al. 1997b). These authors

described that detachment of the fruit from the vine induces an increase on fruit ethylene

production (Guis et al. 1997; Bower et al. 2002).The value reported by Guis et al.

(1997b) on internal ethylene content in detached fruit is similar to the external ethylene

production level by TGM-AS-1 fruit separated from the vine. Even though TGM-AS-2

fruits were removed from the plant, ethylene production was 99.9% less than WT,

azygous-AS and T-GUS fruit at half-slip stage. In summary, the insertion of CMACO-1

gene in antisense orientation lead to reduced ethylene production in the ‘Galia’ male

parental fruit. The average extent of reduction compared to WT, azygous-AS, and a T-

GUS fruit was 76.9% and 99.1% in TGM-AS1 and TGM-AS-2, respectively.

Fruit softening observed during ripening is associated with textural changes that are

thought to result from modification and disassembly of the primary cell wall (Fischer and

Bennett, 1991). Fruit softening and the underlying cell wall structural changes are

complex. Softening or loss of firmness of the edible mesocarp of melon fruit may start in

the middle (around 30-45 days after anthesis, depending on cultivar) of the development

170

cycle, along with other typical changes connected with the ripening process (Lester and

Dunlap, 1985).

Among the different plant hormones which are involved in melon fruit

development, ethylene might have the main role during melon fruit softening, because

this gaseous plant hormone regulates the expression of several proteins involved in the

softening process (Rose et al., 1997, 1998). According to Guis et al. (1997), Guis et al.

(1999), Flores et al. (2001a), Flores et al. (2001b), and Silva et al. (2004), fruit softening

in melon is an ethylene-dependent event. Their ACC oxidase antisense transgenic

Cantaloupe Charentais fruits had a delayed fruit softening process, compared to wild type

counterpart. A similar response was observed in ACC oxidase antisense ‘Galia’ male

parental fruits (Figure 6.6). These fruits retained a higher firmness than WT, azygous-AS

and T-GUS fruits, mainly at full-slip developmental stage, when significant differences

were detected.

Yellowing of the rind in ‘Galia F1 hybrid fruit is induced by increased chlorophyll

degradation during the ripening process, as well as a partial decrease in total carotenoids

(Flügel and Gross 1982). Likewise, chlorophyll is degraded by the action of

chlorophyllase (EC 3.1.1.14), whose de novo synthesis is ethylene regulated (Jacob-Wilk

et al. 1999). Rind color in WT, azygous-AS, and T-GUS fruits, turned from green to

golden-yellow during ripening, whilst, ACC oxidase antisense fruit did not reach a full

golden-yellow color (Figure 6.8). It seems that lower ethylene production in CMACO-1

proves this assumption correct. The antisense transgenic ‘Galia’ male fruits did not allow

complete chlorophyll degradation, leaving partial green-patches on the rind. Analogous

results were reported in ACC oxidase antisense transgenic Cantaloupe Charentais melon

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fruits (Guis et al. 1997b; Martinez-Madrid et al. 1999; Flores et al. 2001a; Flores et al.

2001b; Martinez-Madrid et al. 2002; Silva et al. 2004).

Sweetness is one of the most important edible quality attribute of ripe melon fruits

(Yamaguchi, et al., 1977; Lester and Shellie, 1992; Artes et al. 1993). The trait for sugar

accumulation in melon is controlled by a single recessive gene, called suc (Burger et al.

2002). Therefore, sucrose accumulation is controlled through several plant hormones

such as auxins and ABA, enzymes such as sucrose phosphate synthase (EC 2.4.1.14) and

acid invertase (EC 3.2.1.26), and compartmentation processes, as well as is directly

related to fruit ripening (McCollum et al. 1988; Hubbard et al. 1989; Ofosu-Anim and

Yamaki, 1994; Lee et al. 1997; Ofosu-Anim et al. 1998; Gao et al. 1999; Gao and

Schaffer, 1999; Feusi et al. 1999; Carmi et al. 2003; Volk et al. 2003). The TSS content

of WT, azygous-AS, T-GUS, TGM-AS-1 and TGM-AS-2 mesocarp increased in average

from 7.69% to 9.89% at zero- and full-slip stage, respectively. Thus, TSS accumulation

was a simultaneous event to fruit ripening and an ethylene-independent phenomenon.

Similar results have been observed in most of sweet-type melons (McCollum et al. 1988;

Seymour and McGlasson, 1993; Guis et al. 1997b; Burger et al. 2002; Burger et al. 2003;

Silva et al. 2004; Liu et al. 2004; Ergun et al. 2005)

Although TGM-AS-1 and TGM-AS-2 remained for a longer time attached to the

vine than WT, azygous-AS and T-GUS fruits, they did not accumulate more sugars.

These results on TSS content for ACC oxidase antisense transgenic fruit are similar to

results reported by Guis et al. (1997b), but dissimilar from others (Flores et al. 2001b;

Martinez-Madrid et al. 2002; Silva et al. 2004). Guis et al. (1997b) claimed that

transgenic (ACC oxidase antisense) Cantaloupe Charentais melon fruit remained attached

172

to the vine for 10 more days than its wild type counterpart, allowing the accumulation of

more sugars, and increasing the soluble solids content from ∼11.3 (wild type) to ∼12

°Brix (antisense). However, differences between wild type and transgenic Cantaloupe

Charentais melon TSS values were not significant, since their standard errors overlap (see

Figure 3A in Guis et al. 1997b). Flores et al. (2001b) and Martinez-Madrid et al. (2002)

studied biochemical and physiological fruit characteristics of some transgenic Cantaloupe

melon plants obtained from the Guis’ (1997b) T0 original line ‘B17’. These authors found

that ACC oxidase antisense fruits accumulated significantly more sugars than wild type

fruits at 40 DAP, but not at 15, 24, 35 and 37 DAP. Silva et al. (2004) developed

transgenic (ACC oxidase antisense from apple) Cantaloupe Charentais melon which were

not ripe on 38 DAA, whereas WT fruit were ripe at ∼ 30-32 DAA. The authors concluded

that as transgenic fruit remain attached ∼5-8 more days, they were able to accumulate

more TSS. Unlike Guis’ study, transgenic fruit TSS values were significantly different

from wild type fruit counterpart.

The differences on TSS accumulation between transgenic ACC oxidase antisense

‘Galia’ male parental muskmelon and transgenic ACC oxidase antisense Cantaloupe

Charentais cv. ‘Vedrantais’ fruits (Flores et al. 2001b; Martinez-Madrid et al. 2002; Silva

et al. 2004) can be ascribed to the fact that our transgenic melons belonged to variety

reticulatus, whereas transgenic melon fruits obtained by those authors correspond to

variety cantaloupensis. As it has been reported, genetic background has a profound

effect on melon fruit sugar accumulation (Seymour and McGlasson, 1993; Stepansky et

al. 1999; Burger et al. 2002; Burger et al. 2003; Liu et al. 2004). Moreover, different

plant growing environmental conditions might have played a role on fruit-TSS buildup.

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Sugar accumulation in melon fruit is affected quantitatively by environmental and

physiological factors, such as salinity, nutrient availability, shading, cellular size in the

fruit, and available foliar area (Hubbard et al. 1990; del Amor et al. 1999; Nishizawa et

al. 2000; 2002; 2004; Kano, 2002; 2004). ACC oxidase antisense gene insertion did not

reduce or increase the fruit TSS content in any of CMACO-1 transgenic lines at fruit

maturity.

Unlike other ripe fruits, such as strawberry, pineapple and apricot, where high

organic acids content have an imperative role in developing acceptable taste for

consumers, sweet melons have a low organic acid content. Nevertheless, sensory quality

of melon fruit is also determined by organic acid levels (Sweeney et al. 1970; Yamaguchi

et al. 1977). In ‘Galia’ male parental line, TA and pH appear to be inversely related.

While TS in all treatments had a sharp decrease since the first date of harvest, pH values

started to increase steadily. Although, differences in TA and pH among all treatments

were observed, they were not significant. Similar results were obtained by Guis et al.

(1997b) and Martinez-Madrid et al. (2002). These authors reported that TA had a sharp

decrease in WT Cantaloupe Charentais melon fruit during ripening, and a similar trend

was observed in transgenic ACC oxidase antisense fruit.

Fruit ripening index calculation is commonly used as a factor to measure fruit

sensory quality, and is obtained as the quotient between the TSS content and the TA level

(Leshem et al. 1986). A similar ripening index was observed in WT, azygous-AS, T-

GUS, TGM-AS-1 and TGM-AS-2 fruits on all fruit development stages, except at full-

slip stage where significant differences were found between TGM-AS-2 and other

treatments. It has been suggested by Silva et al. (2004) that organic acid metabolism in

174

melon fruit is an ethylene-dependent process. Likewise, Flores et al. (2001b) and

Martinez-Madrid et al. (2002) reported that transgenic (ACC oxidase antisense) had a

greater ripening index than wild type Cantaloupe Charentais melon fruit. Considering that

TGM-AS-2 genotype had the lowest ethylene production level among treatments, it is

possible that this low-ethylene level in TGM-AS-2 has contributed to attain a high TA

value at full-slip stage, consequently, the lowest fruit ripening index level at full-slip

stage was observed for TGM-AS-2.

Although differences in weight, length, width, flesh size and cavity seed size were

found among WT, TGM-AS-1, TGM-AS-2, azygous-AS and T-GUS fruits, they were

not significantly different. Moreover, comparable variation in fruit weight and size has

been reported in other muskmelon cultivars (Kultur et al. 2001; Nerson et al. 2002; Liu et

al. 2004). Overall, the CMACO-1 gene single insertion in antisense orientation did not

have either a positive or negative effect on fruit size of ‘Galia’ male parental line.

Because there were not significant differences in total number of seeds, as well as

number of full seeds among treatments, it is clear that CMACO-1 gene single insertion

did not have any effect on seed development in ‘Galia’ male parental line fruit.

In summary, the results of these experiments describe that the insertion of ACC

oxidase gene in antisense orientation in ‘Galia’ male parental inbred reduced the ethylene

synthesis in TGM-AS T0 fruits. That inhibition was more evident in TGM-AS-2 than

TGM-AS-1 line. Reduced ethylene production may be a direct consequence of a low

ACC oxidase activity detected in those fruits. As a result of low ethylene production by

TGM-AS fruits, several parameters such as, yellowing of the rind, ripening index, and

fruit softening were delayed. Other traits, such as fruit size, seed development, and

175

mesocarpic total soluble solids, titratable acidity and pH were not affected by the

transgene presence. TGM-AS-1 and TGM-AS-2 T1 seeds, obtained from these T0

evaluated fruits, will be used to obtain and select future improved lines with extended

shelf-life.

6.5 Summary

‘Galia’ is a high-quality muskmelon cultivar that is grown in greenhouses or

tunnels to maximize fruit quality and yield. Maximum fruit quality and flavor are

achieved when ‘Galia’ fruits are harvested at ¾ to full-slip stage. One disadvantage of

‘Galia’ is its storage life, which is limited to two to three weeks due to rapid fruit

softening. In vitro regeneration and transformation of ‘Galia’ melon parental lines with

antisense technology, targeting enzymes involved in ethylene biosynthesis pathway, is a

feasible strategy that can be used to increase its fruit shelf-life. The male parental line of

‘Galia’ muskmelon has been transformed with two different constructs according to the

protocol described by Nunez-Palenius et al. (2005a): one plasmid was bearing the GUS

gene and another had an ACC oxidase gene (CMACO-1) in antisense orientation (Nunez-

Palenius et al. 2005a). Transgenic ACC oxidase antisense (TGM-AS), azygous-AS (PCR

negative), transgenic GUS (T-GUS) and wild type (WT) fruits, from plants grown in the

greenhouse, were harvested at zero-, half-, and full-slip developmental stage. Wild type,

azygous-AS and transgenic (both TGM-AS and T-GUS) weight, length, width, soluble

solids, pH, flesh thickness, seed cavity size, and full seed percentage parameters were not

significantly different on all fruit developmental stages. Fruit firmness from full-slip

TGM-AS was almost twice than wild type, azygous-AS and T-GUS. Ethylene production

and ACC oxidase from half-slip wild type, azygous-AS and T-GUS fruits were greater

176

than from TGM-AS fruits. TGM-AS ‘Galia’ male parental melon fruits had a delayed

fruit ripening process compared to wild type fruits.

177

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BIOGRAPHICAL SKETCH

Hector Gordon Nuñez-Palenius was born on January 19, 1962, in Guadalajara,

Jalisco state, México. He got his Master of Science degree in December, 1997 in plant

biology from the Genetic Engineering Department at the Unit of Biotechnology and Plant

Genetic Engineering in the Center of Research and Advanced Studies (CINVESTAV-

IPN. Unidad Irapuato). Afterwards, Hector G. Nuñez-Palenius started a Ph.D. program at

the University of Florida, Horticultural Sciences Department, Seed Physiology Lab. His

research focused on transformation of ‘Galia’ muskmelon to improve fruit quality,

specifically on in vitro regeneration and Agrobacterium-mediated transformation of

‘Galia’ parental lines. His future plans are to continue working in the area of

biotechnology of cucurbits, with special emphasis on genetic improvement.

.