<%BANNER%>

Transformation of 'Galia' melon to improve fruit quality

University of Florida Institutional Repository
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TRANSFORMATION OF GALIA MEL ON TO IMPROVE FRUIT QUALITY By HECTOR GORDON NUEZ-PALENIUS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Hector Gordon Nuez-Palenius

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This document is dedicated to the seven reas ons in my life, who make me wake up early all mornings, work hard in order to achi eve 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 Nuez Vargas and Salvador Federico Nuez Palenius, my Moms Janette Ann Palenius Alberi and Cons uelo Nuez Sols, my wife Nlida Contreras Snchez, my son Hector Manuel Nuez Cont reras and my daughter Consuelo Janette Nuez Contreras.

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iv ACKNOWLEDGMENTS This dissertation could not have been co mpleted 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 cons tructive 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 th eir academic guidance. I am very thankful to Dr. Donald J. Huber for his excellent advi ce 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 appreci ation 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 speci al thank to Dr. Charles Niblett for all his

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v 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 Se ttles for allowing me to use his lab to accomplish some of the plant molecular issu es, as well as to Diego Fajardo for his technical advice and untied camaraderie. I would like to make a special acknowle dgement to my friends in the Seed Physiology Lab for their help and support dur ing 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 frie nds in the Plant Molecular Lab: Dr. Mark G. Taylor, Dr. Joseph A. Ciardi, Dawn Bies, Stepha nie Maruhni, Michele Auldridge, Michele Zeigler, Anna Block, Patricia Moussatche, and especially Dr. De nise M. Tieman and Bryan Kevany for their outstan ding technical advice and frie ndship to accomplish this dissertation. I would like to show apprecia tion to my friends in the Postharvest Lab: Dr. Steve A. Sargent, James Lee, Muharem Ergun, Yasa rt 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-Mxico) and E DUCAFIN (Guanajuato-Mxico) which have supported my scholastic and living expens es during my graduate education in UF.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES...........................................................................................................xi ABSTRACT.....................................................................................................................xi v 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 Naudins categories for Cucumis melo L. are listed below:...............9 2.1.3.2 Guiss 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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vii 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 Organogenesi s .................................................................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 Toleranc e to Physical Factors................................78 2.3.6.3 Improvement of Postha rvest 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 GU S 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 MUSKM ELON 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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viii 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 Transg enes 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 an d 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 Acid ity, 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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ix LIST OF TABLES Table page 2.1 Production of various cucurbits on diffe rent 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 an d/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 ma le 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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x 6.1 Postharvest fruit char acteristics of ‘Galia’ ma le parental line...............................163 6.2 Postharvest fruit char acteristics of ‘Galia’ ma le parental line...............................165 6.3 Postharvest fruit char acteristics of ‘Galia’ ma le parental line...............................166

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xi 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 mu skmelon parental lines depending on explant source.........................................................................................................101 3.3 Time schedule for de novo shoot regeneration in hypoc otyl, cotyledon and trueleaf 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 mu skmelon parental lines. GUS reporter gene was used to transform different explant sources............................................107 3.6 Transformation efficiency on Galia mu skmelon 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 G alia muskmelon male line plants...............119 4.3 Stable expression of -D-Glucuronidase (GUS) gene in T0 shoots (A and B) and roots (C and D) of Galia mu skmelon male parental line.....................................121 4.4 Transgenic (left) and non-transgenic (right) in vitro Galia male line explants...122

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xii 4.5 Flow cytometry analysis of propidium iodide-stained nuc lei from wild type leaf tissue of ‘Galia’ male parental line........................................................................125 4.6 Flow cytometry analysis of propidium iodide-stained nuc lei 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 fru it 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 an d e) and embryo (f) at 30 DAP stage............139 5.5 Normal ‘Galia’ male parental line se edlings obtained from embryo rescue, having well-developed cotyledonary (red arro ws) 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 determin ation in ‘Galia’ male parental line fruit.........................................................................................................................1 54 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 ( ), azygousAS () 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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xiii 6.9 Ethylene production from WT () and TGM-AS () fruits compared to TSS content of WT ( ) and TGM-AS ( ) intact fruits..................................................162

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xiv Abstract of Dissertation Pres ented 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’ MEL ON TO IMPROVE FRUIT QUALITY By Hector Gordon Nuez-Palenius December 2005 Chair: Daniel J. Cantliffe Major Department: Horticultural Science Stable genetic transformation in ‘Galia’ mu skmelon 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 -glucuronidase (GUS) and green fluorescen t protein (GFP). Once this was accomplished the ACC oxidase gene (CMACO-1) in antise nse orientation was inserted into both ‘Galia’ parental lines using an Agrobacterium -mediated transformation system. By reducing ACC oxidase, it was hoped that ripe ning would be delayed thus allowing a longer time-frame to ship fruit in a firm c ondition. Three protocols using different melon explants, i.e. cotyledon, hypocotyl and true-l eaf, 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 -Naphthaleneacetic acid (NAA) as

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xv plant hormones. Efficient transformation ra tes 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 ge ne from melon (CMACO-1) in antisense orientation was used to transform ‘Galia’ ma le and female parental lines. Two (TGMAS-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 ethy lene production compared to w ild type fruits. Moreover, fruit softening was delayed in CMACO-1. The re sults of this research indicated that the insertion of an ACC oxidase ge ne in antisense orie ntation in the ‘Galia’ male parental inbred reduced ethylene synthesi s 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 nonethylene dependent traits, such as fruit si ze, seed development, and mesocarpic total soluble solids, titratable acidi ty 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 im proved lines with extended shelf-life.

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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 horticul tural groups can be identified: cantalupensis, inodorus, flexuosus, conom on, 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 initia ted 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 qua lity with 13-15% total soluble solids (TSS), bold flavor and a distinct musky aroma. Ga lia’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 Eur ope, and became a unique and special market category of muskmelon (Karchi, 2000). The ma in disadvantage of ‘Galia’ is its short storage life, since it is harv ested 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 tran sportation 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).

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2 Using traditional breeding methods and l ong-shelf life melon cultivars, breeders have been able to develop a strategy to insert gene(s) which ha ve increased ‘Galia’ muskmelon shelf-life. However, this approach has resulted in a lo ss of favorable fruit quality characteristics (Karchi, personal co mmunication). Using a plant biotechnology approach by means of plant tr ansformation is a feasible al ternative to introduce novel or native genes, which likely will increase ‘Galia’ muskmelon shelf life, without affecting its unique characteristic s. This approach was used to delay fruit ripening in Cantaloupe melon (cv. Vdrantais), by inserting the ACC ox idase 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. Ne vertheless, ‘Galia’ muskmelon has not been easily in vitro cultivated and full regenerated pl ants are especially difficult or are impossible to obtain (Leshem, 1989; Le shem 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 ‘Gal ia’ 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, th e research to find favorable conditions for successful Agrobact erium-mediated transformation will enable us to insert any gene of inte rest into this recalcitrant muskmelon cultivar, specifically with the final aim to increase its shelf-life.

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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, a nd delay the fruit ripening process. In order to achieve the objective of th is research, several experime ntal approaches were taken. These were to 1) examine the effect of seve ral plant hormones and their balances, as well as different melon explant sources on de novo shoot and root regenera tion in ‘Galia’ male and female parental lines; 2) es tablish the favorable conditions for Agrobacterium tumefaciens -mediated transformation using two re porter 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 si nce a postharvest approach.

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4 CHAPTER 2 LITERATURE REVIEW 2.1. Importance of Melon 2.1.1 Introduction The Cucurbitaceae family consists of mostly frost sensitive, principally tendrilbearing vine plants which are found in subtropical and tropical regions around the world (Robinson and Decker-Walters, 199 9). 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 tr ibes, 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 ar e exploited as medicines, such as Cucurbita andreana, which has chemical compounds (cuc urbitacins) with anticancer and antiinflamatory activities (Jay aprakasam 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 u nusual purposes as well, such as to store food (Gourd) or to be used as a sponge (Loofah) (Robinson and D ecker-Walters, 1999). The most important cultivated cucurbits –based on total production and harvestable area– around the globe are watermelon ( Citrullus lanatus Thunb.), cucumber ( Cucumis

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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 cucurb its 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 botan ical 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, deep er 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-se nsitive, 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 cucurbit s (Zitter et al. 1996). Leaves are simple, either threeor 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).

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6 Sex expression in Cucumis melo is controlled by genetic factors, as well as by environment (Wien, 1997; Robinson and Deck er-Walters, 1999). According to Wien (1997), at least four environmental factor s, such as light energy, photoperiod, water supply, and temperature, have a strong influence on sex expression. Normally, physiological conditions which favor the increas e of carbohydrates within the plant, such as low temperature, low nitrogen availa bility, short photoperi od and high moisture accessibility, promote female sex expression (Robinson and Decker-Walters, 1999). These environmental factors affect plant hormon al balance, which in turn determines sex expression. In general, gibberellins promote male flower development, whereas auxins and ethylene induce female flower production (K archi, 1970). Melon pl ants bear perfect or imperfect flowers in several combinat ions: perfect (hermaphroditic) flowers are capable of self-pollination, and imperfect fl owers are either pist illate (female) or staminate (male) (Zitter et al. 1996). Mo st melon cultivars are andromonoecious (hermaphroditic and staminate flowers presen t at the same plant), although monoecious (pistillate and staminate flowers) forms ar e found as well (Seshadri and More, 2002). Melon fruits are generally classified as an indehiscent ‘pepo’ with three ovary sections or locules. According to Robins on and Decker-Walters (1999), a ‘pepo’ is a fleshy fruit with a leathery, non-septate rind de rived from an inferior ovary. The edible flesh is derived from the placentae or meso carpic tissue (Seymour and McGlasson, 1993). Among the different parts of a melon plant, fr uit has the highest diversity in size, form, external ornamentation, and internal and ex ternal color (Kirkbride, 1993). For instance, Prin 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

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7 between 50 g and more than 15 kg (a 300-fo ld 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 (gr een, yellow, white, orange, red, gray or blend of these colors), rind texture (smooth, wart y, 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 attachme nt zone between the fr uit 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 dome stication process of melon started in Egypt over 3,000 years a go (Pangalo, 1929). From this area, melon species dispersed throughout the Middle East and Asia, where a secondary domestication and diversification development may ha ve ocurred (Kerje and Grum, 2000). 2.1.3 Classification and Cultivars According to Jeffrey (1990), melon cla ssification may be listed as follows: Class: Dycotyledoneae Subclass: Dilleniidae Superorder: Violanae Order: Cucurbitales Family: Cucurbitaceae Tribe: Melothrieae Subtribe: Cucumerinae

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8 Genus: Cucumis Subgenus: Melo Species: Cucumis melo The high polymorphism of fruits in cultivat ed melons has lent botanists to propose different infraspecific cla ssifications. An excellent, updated and complete study on Cucumis genus was undertaken by Dr. Joseph H. Ki rkbride, 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, us ing mostly living pl ants grown in the gardens of the Natural History Museum of Pa ris, proposed the first useful system of infraspecific categorization for Cucumis melo L. Naudin (1859) subdivided this species into ten groups, which were later revi sed by Munger and Robinson (1991), proposing trinomial names. However, several classifi cations have been reported (Alefeld, 1866; Cogniaux and Harms, 1924; Whitaker a nd Davis, 1962; Pangalo, 1958; Filov, 1960; Grebenš ikov, 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 s ynonymous epithets used in several publications in order to propose thei r 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.

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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 matu rity 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. Th ey 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) report ed a new categoriza tion of horticultural important melons. These authors based their classification on a pr evious biosystematic monograph of the genus Cucumis (Cucurbitaceae) reported by Kirkbride in 1993, who used morphological, cytological and macro-dist ributional 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 ri bs 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 ne tted 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.

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10 10. C. melo var. inodorus Naud. Smooth or netted surface, flesh commonly white or green, lacking the tipical musky flavor. Th ese 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 w ith red streak, white to pink flesh. On the other hand, Stepansky et al. (1999) proposed an intraspe cific 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 thei r data a “botanical-morphological” dendogram. Likewise, DNA polymorphism among the acce ssions was assessed using inter-SSR-PCR and RAPD techniques. They concluded that the molecular phylogeny agreed, broadly, with the classification of melon into two subs pecies, and it did not contradict the division into “horticultural varieties.” Recently, Liu et al. (2004) concluded af ter an extensive evaluation of 72 melon accessions belonging to six melon varieties cantaloupensis, reticulatus, inodorus, acidulus and saccharinus that accessions which were previo usly classified in the same variety by traditional taxonomy were also lo cated 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’ categ orizations have more common features than contrasting ones; therefore both are well accepted among scientists.

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11 2.1.4 Climateric and Non-climateric Fruits Fruit in general can be classified as ei ther climacteric or non-climacteric on the basis of their respiration pattern and autoca talytic ethylene pr oduction peak during ripening (Tucker, 1993, Hadfield et al. 1995). Cl imateric fruits, such as tomato, peach, avocado, apple and pear, have a respiratory bur st and a pronounced au tocatalytic ethylene production while the ripening proc ess is proceeding. Non-climateric fruits, such as bell pepper, watermelon, strawberry, grape, and ci trus, do not show evidence of an increased ethylene evolution or respirator y rise coincident with ripe ning (Seymour and McGlasson, 1993). This fruit categorization might not be completely strict for all species. Within a species there could be both climateric and nonclimateric 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; Pri n 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 performan ce of specific fruit and then extrapolate that behavior for anothe r 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 fru it growth pattern, pol lination, satisfactory double fertilization and a normal development of the ovules have to take place (Wien, 1997). Fruit growth patterns among melon cultivar s can be similar or quite diverse. In 1971 Pratt reported that both th e ‘Honey Dew’ and ‘cantaloup e’ types reached half of their total fruit growth almost at the same ti me (15 and 20 days afte r anthesis); however,

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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 we ight 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 th e abscission process is absent in some muskmelon ( reticulatus ) varieties (Sak ata 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 a ppearance of the netted pattern. A muskmelon fruit color chart has been prepared for ‘Galia ’, which categorized si x different levels of maturity: 1, very dark green; 2, green; 3, lig ht yellow with some green areas; 4, light yellow; 5, yellow; and 6, dark yellow to ora nge peel (Fallik et al 2001). On the other

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13 hand, in those melon varieties, such as inodorus, flexuosus and saccharinus where an abscission layer is not formed, other characte ristics are used to assess harvest maturity. For example according to Portela and Cantwell (1998), at commercial melon production level a variety of subtle change s in external color (green to white), peel texture (hairy to smooth), aroma at the blossom end (none to de tectable), and fruit de nsity (low to high) are used in order to assess the harvest maturity point. Fruit ripening is a genetically determined ev ent that involves a se ries of changes in color, texture, and flavor (H ashinaga et al. 1984, Keren-Keis erman et al. 2004). Flavor is a multifaceted human perception, which invo lves taste and aroma (Shewfelt, 1993). According to Tucker (1993), fruit flavor de pends on the complex in teraction of sugars, organic acids, phenolics and a wi de variety of volatile compounds. In general, the quality of melon fruit is mostly asso ciated 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 St ate 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 me lon 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 di ssimilar 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

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14 of approximately 10-14 days at cool temperatures (6-9oC) and proper humidity (90-95%) conditions (Gull, 1988). Muskmelon and netted me lons 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 a llowing a more rapidly water loss as a result of evaporation (W ebster 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 bur st of ethylene production co incides with ripening in climateric melon fruits (Pratt et al. 1 977). Kendall and Ng (1988) measured ethylene from two netted ( reticulatus variety) and three non-netted (Casaba typeinodorus variety) muskmelon cultigens and their hybrids imme diately after harvest and found that the netted muskmelon fruits synthesized consider able 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 gene rally 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 geneti c and developmental factors. Because netted fruit melons produce ethylen e during ripening, they do not require exogenous ethylene application afte r harvest (Pratt, 1971). However, inodorus fruit types may require exogenous ethylene application afte r harvest, in order to obtain a more uniform and rapid ripening, as well as better development of color, wax, and aroma (Gull,

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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, orangeor green-fleshed a nd netted rind fruit melons produce higher amounts of ethylene than greenor white-f leshed 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 Fa vourite’ 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 Fa vourite’ 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. synthesi s and/or degradation of new structural, soluble and enzymatic proteins, novel mRNAs, changes in plant hormones levels, and DNA transcription, as we ll 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; Guilln et al. 1998; Aggelis et al. 1997a; Sato -Nara et al. 1999; Flores et al. 2001a; 2001b; Villanueva et al. 2004). All thes e anabolic and catabolic events need both energy and a carbonnitrogen-framework for building blocks, whic h are supplied via resp iration. The two most

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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 ripe ning process (Bianco and Pratt, 1977; Lelivre et al. 1997; Sato-Nara, 1999). 2.2.3.2 Carbohydrate Metabolism Sweetness is the most important edible qua lity 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 th e mesocarp of ripe melon fruits. High levels of sucrose attribute fruit sw eetness in melon (McCollum et al. 1988; Hubbard et al. 1990; Burger et al. 2003; Vill anueva et al. 2004). Muskmelon fruit do not store st arch as some other fruits do (i.e. apple, and banana), therefore the fruit requires a c onstant 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 f actor which has an effect on photoassimilate translocation during fru it 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 al teration in carbon metabolism in source leaves, and in resource partitioning among the various pl ant organs because there is an increased in respiration, and a decrease in net photosynthetic rate in in fected leaves (Shalitin and Wolf, 2000; Shalitin et al. 2002). In Cucumis melo sucrose is not the only transl ocated photoassimilate carbohydrate, since galactosyl-sucrose oligosaccharides raffinose and stachyose can be found in the phloem (Mitchell et al. 1992; Gao and Schaffe r, 1999; Gao et al. 1999; Volk, et al. 2003).

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17 Mitchell et al (1992) reported maximal amount s of sucrose (60 mM), stachyose (50 mM), and raffinose (10 mM) in sap phloem measurements. Sugar continues to accumula te during fruit development (Pratt, 1971; Pratt et al. 1977). Beginning at early fruit enlargement a nd 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 contro lled by a single recessive gene, called suc (Burger et al 2002). Therefore, sucrose accumu lation is controlled through several hormones and enzymes, as well as compartm entation 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; Fe usi 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 sali nity, 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; Nishizaw a et al. 2002; Kano, 2002; Kano, 2004). Netted, muskmelon, and Honeydew fruits have similar, but not identical, patterns of sugar accumulation (Seymour and McGla sson, 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 amount s, 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 H ubbard et al. (1989), who measured the concentrations of sucrose, raffinose saccharides, glucose, fructose, and

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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, ch ito, dudaim, momordica, flexuosus, agrestis, and some non-defined varieties. Am ong the 14 genotypes classified as cantaloupensis, total sugars ranged between 40-1 00 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 ge notypes 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 (l ine 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, th e last one (PI 164320) had an unus ual 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 mo st significant component that contributed to variation in total sugars.

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19 The physiological and biochemical aspects of sucrose accumulation in melon fruit has been investigated extensively (McCollu m 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; Villanue va 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 s ynthesis 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 ar e 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 incr ease significantly (Hubbard et al. 1989; Hubbard et al. 1990; Gao et al. 1999). In a ddition, SPS activity is hi gher in sweet melon fruit compared with non-sweet genotype fr uits, 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 tw o factors: the rate of sucr ose accumulation, coupled with the duration of the accumulation peri od until abscission or harvest.” 2.2.3.3 Organic Acids Organic acids are compounds regularly f ound 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 highe r amounts of organic acids (Stepansky et al. 1999; Pitrat et al. 1999). For instance, Burger et al (2003) reported that the high-

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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 purpo ses. In the recessive condition ( so ), melon fruits have a low-organic acid attribut e. Furthermore, these authors stated that the evolution of horticultural sweet melon varie ties required the sequential se lection 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 f act that low-organic acid level is a genetically regulated feature, several envionmen tal 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 impor tant organic acids found in the flesh of different melon varieties (Leach et al. 1989; Fl ores 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 co mponent in all melon cultivars that they analized. Similarly, Flores et al. (2001b) f ound that the major organic acids found in wild type and transgenic cantaloupe melon fruit were citric and ma lic 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 fact ors, for consumer quality (Yamaguchi, 1977), and they are str ongly linked to the ripening process (Wang et al. 1996; Beaulieu and Grim, 2001). Unlike suga r accumulation in the ripe fruit, the aroma fruit caused by volatile production contin ues after harvest (Wyllie et al. 1995). The volatile profile, as well the identification of the main ‘melon odor’ substances in melon

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21 fruits have been the subject of a consider able amount of resear ch (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 a bundant 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 (K emp 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 dete ction techniques (Aubert and Bourger, 2004), such as sniffing port analysis, have shown that the volatile com pound content responsible for ‘melon aroma’ is diverse and cultivar de pendent. Indeed, Aubert and Bourger (2004) were able to differentiate sta tistically long-shelf life cultivar s 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’ mel on, which is a ‘Galia’-type melon, various volatile acetates were identified in the ri pening fruit, including nine aliphatic, four aromatic, and one compound containing a sulf ur moiety (Shalit et al. 2000). Benzyl acetate was the most abundant volatile com pound in the headspace of this cultivar,

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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 unsaturat ed esters found in muskmel on 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 pr obable that the large quantities of volatile esters also play a critical role in the in tegrated flavor of melons, and that they are necessary for the strong and characteristic fruity aroma. According to Yabumoto et al. (1978), the vol atile ester profile of ripe reticulatus variety (‘PMR-45’ and ‘Top Mark’) and inodorus variety (‘Honey Dew’ and ‘Cre nshaw’) fruit were similar, and they fit into two groups, depending on th e pattern exhibited by th e production of that compound. One group had a continuously acceler ating rate of production (ethyl esters) and another increased rapidly and th en plateaued (acetate esters). The major compounds responsible for ‘Honey Dew’ melon aroma are ethyl 2methylbutyrate, ethyl butyrate ethyl hexanoate, hexyl aceta te, 3-methylbutyl acetate, benzyl acetate, ( Z )-6-nonenyl acetate, and possibly ( E )-6-nonenol and ( Z,Z )-3,6nonadienol (Buttery et al. 1982). Horvat and Senter (1987) identifie d eight novel volatile compounds from ‘Saticoy’ melon ( reticulatus) In ‘Galia’-type melon cultivars C8 and 5080, six important aroma volatiles during ripe ning were found: ethyl acetate, isobutyl acetate, butyl acetate, 2-methylbutyl acetate, hexyl acetate, and 3-hexenyl acetate (Fallik et al. 2001).

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23 The biochemical connection between the development of aroma volatiles and free amino acid content in melon ripening fruit is well established (Y abumoto et al. 1977; Wyllie and Leach, 1992; Wyllie et al. 1995). Wa ng et al. (1996) proposed that several amino acids, such as valine, isoleucine, meth ionine, and alanine may be the precursors of the mayority of the esters f ound in melon fruit, providing the branched alkyl chain moiety, which is present in a signific ant 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 pr esence or absense of seeds in the fruit cavity can modify the final arom a volatile profile (Li et al. 2002). Likewise, the developmental fruit stage may have a strong influence on ar oma characteristics (Beaulieu and Grimm, 2001). In summary, similar to other fruits the me lon fragance is made of complex mixtures of volatiles compounds. Both th eir production and profile in me lon 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 ch aracteristic (Aubert and Bourger, 2004). 2.2.3.5 Cell Wall Degradation The plant cell wall is a dynamic struct ure, which determines cell shape and contributes to the functional specializati on of cell types (Carpita and McCann, 2000). The plant cell wall is a highly organized stru cture composed of several polysaccharides, proteins, and aromatic compounds. Likewise, the new primary cell wall comes from the cell plate during cell division, and after diffe rentiation 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 stru ctural model of the plant cell wall can be

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24 pictured as a core structure of cellulose microfibrils embedded in two coextensive networks of pectin and hemicellulose. Fruit softening observed during ripening is as sociated with textural changes that are believed to result from modification and disa ssembly 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 mel on 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 pe ctins (measured as to tal 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 c ontent 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 du ring the early ripening stages, however some PGdependent developments may contribute to overall pectin disassembly at later stages. 3. Other enzymes could be involved in that early pectin solubilization process, for instance -galactosidases and / or -galactanases. 4. Hemicellulose polymers undergo important modifications, such as changes in the degree of solubility and modifications fr om large molecular size to smaller size, and loss of specific sugars. 5. Most of the non-cellulosic neut ral sugars decrease significa ntly in the mesocarp of ripening fruits, regularly Ga lactose, Mannose and Arabinos e, whereas other neutral sugars such as Xylose might or not increase during fruit softening.

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25 6. Other enzymatic activities, such as p ectin methylesterase, which have been associated with pectin metabolis m in other fruits (Harriman et al. 1991; Tieman et al. 1992), and/or other protei ns, such as expansins, wh ich have been proposed to disrupt hydrogen bonds within the plant ce ll 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 Le Exp1 protein. Tomato fruit in which Exp1 protein accumulation was inhibited by 3% we re firmer than control fruit throughout the ripening process. Conversely, fru it overexpressing high amounts of LeExp1 protein were much softer than control fru it, even in mature green stage before the ripening event had commenced. Rose et al. (1998) and Bennett (2002) propos ed a complete model of the temporal sequence of cell wall changes, pectinase activity, and PG-m RNA expression in ripening ‘Charentais’ melon fruit at defined devel opmental stages, unfort unately they did not include the role of expansins in ce ll wall degradation in their model. Among the different plant hormones which are involved in fruit development, ethylene has the main role during melon fru it 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 pl ants to reduce ethylene production, reported that plants did not have substancial change s in pectin molecular mass observed in the wild type fruit. Moreover, exogenous ethylen e 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’ mel on, cell-wall degradation process is regulated by both ethylene-dependent and ethylene-inde pendent mechanisms. In support of a fruit softening-ethylene involvement, it was esta blished that ripening-regulated expansin

PAGE 41

26 gene(s) in tomato were influenced directly by ethylene, and the expr ession of that gene parallels the pattern of xyloglucan disassemb ly, and early fruit softening (Rose et al. 1997). The exact hormonal, molecular and enzy matic 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 us ing updated molecular and genetic techniques, such as cDNA microarrays (Fonseca et al. 2004), in order to monitor the gene expression during fruit development and ripe ning, there will be more evidence to understand and manipulate the melon fruit soft ening process. What's more, as Bennett (2002) proposed: ‘future research should focus on using genetic strate gies to assess the potential for synergistic interactions by s uppression of both hemicelluloses and pectin disassembly in ripening fruit.’ 2.2.3.6 Pigments Flesh color of melons is a nother important quality attri butes for a consumer appeal (Yamaguchi, 1977). In general, four basic and distinctive flesh colors can be observed in melon fruits: orange, light-orange or pi nk, green, and white (Watanabe et al. 1991; Goldman, 2002). According to Seymour and McGlasson (1993) the principal pigments in orangefleshed melons are: -carotene (84.7%), -carotene (6.8%), -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 di stinctive flesh colors. They found that the orange-fleshed colored melon cultivars 'Ir oquois', 'Blenheim Orange', 'Birdie Red',

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27 'Quincy' and 'Tiffany' contained about 9.2 to 18.0g/g -carotene as the major pigment, as well as a small amount of phytofluene, -carotene, -carotene and xanthophylls. They also measured pigments in light-orange-fl eshed ‘Hale’s Best’ me lon, which contained about 4.0g/g -carotene, and phytofluene; -carotene, -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 -carotene and xanthophylls. Chlorophyll and carotenoid changes in deve loping 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 c ontent 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 occu rred. In ‘PMR 45’ and ‘Chens haw’ fruits, however, there was a successive rapid decrease, which was concurrent with the ripening process. Carotenoids content increase steadily three w eeks after anthesis to high levels at full maturity. The development of orange pigmen tation 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 Li ght Emission (DLE), a nondestructive method, to study physical and chemical properties rela ted to fruit maturity in Canary melons. They found that cholorophyll and yellow pigm ents decreased with fruit development, having a high correlation with maturity i ndex (IM). Flgel and Gross (1982) studied pigment and plastid changes during ripening of the green-fleshed ‘Galia’ muskmelon

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28 fruit. They observed that the carotenoid profile in the exocar p 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 we ll as a partial decrease in total carotenoids took place. In conclusion, it seems that pigment prof ile accumulation and de gradation 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 et hylene-independent biochemical and physiological pathways throughout melon fruit ri pening (Pech et al. 1999 ; Hadfield et al. 2000; Srivastava, 2002; Silva et al. 2004), wh ich both coexist at the same time in the climacteric fruit. Likewise, besides ethylen e several plant hormones, such as indole-3acetic acid (IAA) and abscisic acid (ABA ), are involved in melon fruit ripening (Larrigaudiere et al. 1995; Dunl ap et al. 1996; Guilln et al 1998; Martnez-Madrid et al. 1999). 2.2.4.2 Biosynthesis, Perception and Effects of Ethylene Ethylene biosynthesis goes from meth ionine, through S-adenosylmethionine (SAM), then to 1-aminocyclopropane-1-carboxy lic acid (ACC), and finally to ethylene (Yang and Baur, 1969; Adams and Ya ng, 1979; Yang, 1980; Yang, 1982; Yang and Hoffman, 1984). Two regulatory enzymes in th is 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).

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29 Both ACS and ACO melon enzymes are code d 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 member s 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 transcri ptionally active. One genomic clone, named CM-ACO1 presented a coding region with four e xons 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 diffe rentially activated and expressed by several environmental and developmental fact ors (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 AC O, were able to identify in which part of the fruit the ethylene synt hesis started at the ea rly stages of ripening. They reported that the rate of ethylene pr oduction (accumulation of ACO protein) in melon fruits increased initially in the placenta l tissue, then in mesocarp tissue and finally at the rind. They also concl uded that levels of ACO mRNA and protein were low in unripe fruit stage, but became detectable in pl acental tissue at the pr e-climacteric period,

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30 and their levels increased in the mesocarp at the climacteric stage. All these results suggested that the central region of melon fr uit (placental tissue and seeds) plays a major role in the production of ethylene duri ng the early stage of fruit ripening. A RT-PCR assay was used by Lasserre et al. (1996) to det ect the differential expression of ACO melon genes ( CM-ACO1, CM-ACO2, and CM-ACO3 ). They found that these three genes were differentiall y expressed during development, ethylene treatment and wounding. CM-ACO1 was induced during fruit ripening, and also in response to wounding and ethyl ene 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. Mo reover, Bouquin et al. (1997) using the 5’untranslated region of the CM-ACO1 gene fused to the -glucuronidase (GUS) reporter gene were capable of measuring the tran scriptional activation in leaves of the CM-ACO1 gene after wounding and ethylene stimulation. Th eir results suggested that induction of CM-ACO1 gene expression occurs via two direct and independ ent signal transduction pathways in response to both stimuli. Zheng et al. (2002) studied some genetic aspects of ethylene production and its re lationship to the RFLPs of the ACC oxidase and ACC synthase genes in two melon cultivars. On e 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

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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 ch anges 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 nonclimacteric) and ‘Andes’ (known as climacteric) fruit cultivar s at different stages of maturity, finding that mRNA CMACS-1 transcripts accumulated only in the mesocarp and placentae of ‘Andes’ fruit at 50 DAP (c ommercial 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 elev ated levels in the mesocarp and placentae of both cultivars at 50 DAP, but in ‘Andes’ cultivar those transcripts were more abundant than in ‘E arl’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 abil ity 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 W u, 1992; Shibuya et al. 2002). In Cucumis melo ethylene-receptor-like homolog genes have been reported as well (Sato-

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32 Nara et al. 1999, Takahashi et al. 2002; Nukui et al. 2004; Cui et al. 2004). Sato-Nara et al. (1999) isolated and char acterized two cDNAs, which were described as putative ethylene receptors, from muskmelon using the Arabidopsis ethylene receptor genes ETR1 and ERS1 sequences. These authors measured th e expression pattern of these cDNAs during fruit enlargement and ripening by mean s of Northen blot assay, finding that both clones were expressed in a st ageand tissue-specific manne r. They named their cDNAs as Cm-ETR1 (Accession No. AF054806) and Cm-ERS1 (Accession No. AF037368). Three years later (Takahashi et al. 2002), polyclonal antibodi es against melon receptor, Cm-ERS1 were prepared in order to determin e the temporal and spatial expression pattern of Cm-ERS1 protein during melon fru it development. They reported that CmERS1 protein formed a disulphide-linked homodimer and it was present in microsomal membranes but not in soluble fractions. In a ddition, their results revealed that a posttranscriptional regulation of Cm-ERS1 expression affects stageand tissue-specific accumulation of this protein. That transition pa ttern was not cultivar-dependent because it was observed in two different melon cult ivars, i.e. ‘Fuyu A’ and ‘Natsu 4’. The cloning and characterization of two me lon ethylene receptor genes has allowed their use as molecular genetic tools in he terologous 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 sens itivity (Cui et al. 2004), maki ng transgenic plants that had a significantly extended flower longevity comp ared with the wild t ype 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

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33 japonicus plants in order to examine how and when endogenous ethylene inhibits that rhizobial infection and n odulation (Nukui et al. 2004). Endogenous ethylene in L. japonicus roots inhibits rhizobial infection at the early stag es (primary nodulation), and suggested that ethylene percep tion also assists negative feedback regulation of secondary nodule initiation. Ethylene has a profound influence on climact eric fruit ripening, and in some cases it induces biochemical and physiological change s in non-climacteric fruit (Lelivre et al. 1997; Giovannoni, 2001; Prin et al. 2002a). In re search 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 fr uit. These RFLPs were associated with flesh color, rind texture and postharvest de cay characteristics in the melon genotypes examined. Low ethylene production and greenand 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 cultivar s, such as ‘Honeybrew’ and ‘HD Green flesh’, did not accumulate any detectable ethyl ene. Likewise, Prin et al. (2002a) reported that in the non-abscission melon fruit PI 161375, exogenous ethylene failed to stimulate abscission, loss of firmness, ethylene produc tion and expression of ethylene-inducible genes. These authors obtained a recomb inant population of Charentais X PI 161375 inbred lines segregating for fruit abscissi on and ethylene producti on. Genetic analysis showed that both characters ar e controlled by two independent loci. They concluded that

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34 the non-climacteric phenotype in fruit tissues was attributable to et hylene insensitivity conferred by the recesive allelic forms from PI 161375. As it was previously pointed out, ethyle ne-dependent and ethylene-independent biochemical and physiological pathways ta ke place and coexist during melon fruit ripening. Hadfield et al. (2000) made a differential screeni ng of a ripe melon fruit cDNA library and identified 16 unique cDNAs co rresponding to mRNAs whose accumulation was estimulated by ripening. Expression of fi fteen, out of sixteen cDNAs, was ripening regulated, and twelve of them were fruit specifi c. 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 corre sponded to mRNAs whose abundance was reduced in transgenic fruit but still inducible by ethylene tr eatment. The second group of mRNAs was not significantly altered in the tr ansgenic 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 unexp ected pattern of expression, low levels of mRNA in transgenic fruit and even remain ing low after ethylene treatment. Obviously, the regulation of this third group of ge nes appears to be ethylene-independent. Pech et al. (1999) described some et hylene-dependent events in cantaloupe Charentais melon fruits and divided th em 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 othe r group included enzyme activiti es, such as galactanase, arabinosidase, -galactosidase, endo-polygalactur onase, ACC synthase (negative feedback) and ACC N-malonyltransferase. Li kewise, these authors categorized ethylene-

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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 et hylene-dependent and ethyleneindependent events provides valuable basic information, which might be used to design biochemical and/or molecular strategies w ith the aim to contro l melon fruit ripening. Ethylene regulates fruit ripening by coordi nating the expression of several genes (Lelivre et al. 1997; Aggelis et al. 1997a; Aggelis et al. 1997b; Yang and Oetiker, 1998; Jiang and Fu, 2000; Prin 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, endopolygalacturonase, galactanase, one homologue of S -adenosyl-L-homocysteine hydrolase (SAHH) and even a mRNA, which is ripening-specific, named MEL2 of unide ntified 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; Prin et al. 2002a). New insight knowledge will be available on ethylene role(s) in melon fruit development and ripening, when the mol ecular 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 coul d also be retrieved from wild and landraces melons, in order to find ripening genes to be transferred to commercial varieties.

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36 2.3 Melon Biotechnology 2.3.1 Genetic Improvement 2.3.1.1 Traditional Breeding For several thousands of years, human be igns 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 spec ific traits such as faster plant growth, larger and more nutritious seed s 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. Traditio nal breeding methods in melon have led to a consid erable varietal improvement. Strong sexual incompatibility barr iers at the interspecific and intergeneric levels have restricted the use of that ge netic potential to develop new and enhanced melon cultivars (Niemirowicz-Szcztt and K ubicki 1979; Robinson and Decker-Walters, 1999). Melon plant improvement by traditional h ybridization 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 tr aits, 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,

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37 1991). Commercial melon varieties, with sweet non-bitter and low-ac idic 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 intr aspecific crosses, using traditional breeding methods, between melon land races (Seshadr i and More, 2002) and commercial melon cultivars will produce hybrid fruit with low qua lity 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 ha ve other genetic breeding tools, besides traditional hand crossing, in order to obtain improved melon cultivars. The same results obtained from conventional breeding met hods can be developed using biotechnology strategies (Table 2.2). It is we ll 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 biot echnology’, The Conve ntion on Biological Diversity (CBD) defines biotechnology as: “any technological application that uses biological systems, living organisms, or deriva tives 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 th at are commonplace in agriculture and food production, whereas in its narrow or mol ecular sense it considers only the new DNA techniques, molecular biology and reproducti ve technological app lications, including differente technologies such as gene ma nipulation, gene transfer, DNA typing and cloning of genes, plants and animals (FAO, 2004).

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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 Intraspe cific 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 Selfpollination 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 ge netic 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, nontransfo rmed plants can be killed in culture by the toxic substance the marker ge ne codes resistance to. Within the plant biotechnology discip line, 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 accep ted that the term “plant tissue culture” refers to in vitro cultivation on nutrient me dia of any plant part, a single cell, tissue or an

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39 organ under a sterile environm ent, leading to a whole de novo regenerated plant (NunezPalenius et al. 2005a). After the milestone plant tranformation achievement by Herrera-Estrella et al. (1983), a number of technological difficulti es were surpassed, allowing the cloning and insertion of different genes a nd engineering of transgenic pl ants 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 a nd vegetables, flavor a nd fragance, c) novel production of vaccines, pharmaceuticals, and therapeutic and prophylactic proteins, d) reduced production of allergens, and e) phytor emediation activity (James and Krattiger, 1996; Vasil, 2002; 2003; Nuez-Palenius et al 2005). The first transgenic commercial plant variety in the USA was released in May 1994, when Calgene marketed its FlavrSavr delayed ripening tomato (James and Krattiger, 1996). At the present time, more than 50 transgenic crops have been approve d for commercial planting, and at least 100 more are under field trials and/or regulatory review (Vasil, 2003). The influence of plant genetic engine ering on commercial crop production is evident by the global increase of cultivated land with tran sgenic crops, also known as Genetically Modified Organisms (GMOs). All th is 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 fo r 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 tran sgenic crops is expected to increase from

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40 $450 million in 1995 to over $7 billion by 2005 (James and Krattiger, 1996; James, 2003), for instance, the total market for tran sgenic seed now exceeds $3 billion (Vasil, 2003). Throughout an eight-year period, from 19962003, 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 cont aining herbicide (Roundup) tolerance was the dominant trait used, followed by insect resistance conferred by Bacillus thuringiensis ( Bt) toxin. Global Land Area of Transgenic Crops0 10 20 30 40 50 60 70 80 90 1995199619971998199920002001200220032004M hectares Total Industrial Countries Developing Countries Figure 2.1 Global land area of cultivated transg enic 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. Neverthele ss, it seems from most recent data (2001 to 2003) that global land area cultiv ated with GMOs will be increased and it might be able to attain again similar leve ls as previous years (James, 2001; 2002; 2003).

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41 13.3 9 6.1 8.4 4.3 12.1 1.7 9.3 16.8 0 10 20 30 40 50 60 70 80 90 199619971998199920002001200220032004M hectares Increase over Previous Year Previous Year Figure 2.2 Yearly increase in global land area of transgenic crops. Numbers above bars indicate the increased amount in Mhect ares 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 h ectares) 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 incr ease in both Bt toxin maize and herbicide tolerant maize, and continued growth in he rbicide tolerant soybean (James, 2003). These transgenic plants, for the period of 2003, we re 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, herbic ide tolerance installed in soybean, maize, canola and cotton occupied 49.7 million h ectares (73%) of the global 67.7 million hectares.

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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 populati on reached 6 billion on October 12, 1999, and in view of the current huma n 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 f ood and fiber production by 2050 cannot be achieved using existing crop technology (James and Krattiger, 1996). Therefore, more research with plant molecular tools and tran sgenic 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 succe ssful transformation of melon ( Cucumis melo ) (Fang and Grumet, 1990), several valuable plant feat ures, 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, Co tton 6 PARAGUAY 1.2 Million Has. Soybean, Maize 4 BRAZIL 5.0 Million Has. Soybean

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43 Grumet 1993; Yoshioka et al. 1993; Gonsalves et al. 1994; Huttner et al. 2001), tolerance to salinity (Bordas et al. 1997) and fruit qu ality improvement (Ayub et al. 1996; Ezura et al. 1997; Shellie, 2001; Silva et al 2004; Nuez-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 applica tion was first registered in 1998 by the USDA-APHIS service (2004), th ere 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 Giffor d, 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 tran sgene of interest (due somaclonal variation and/or chromosomal rearrengements), and be cause 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 commer cial 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 orga nogenesis, 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 biologica l and physical factors influence in vitro regeneration efficiency rate, and all have to be consid ered in order to develop a reproducible and reliable melon regeneration protocol.

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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 ar e also genotype-dependent. Fo r 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 si gnificant differences in somatic embryogenic response from melon seeds among 18 commercial cultivar s. 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 othe rs did not produce any somatic embryos or the response was very low. Moreover, these au thors were able to transfer, by sexual crosses, the embryogenic regeneration response from superior to inferior responding cultivars, demo nstrating 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 varia tion in the range of somatic embryogenesis from F2 seeds. Gray et al. (1993) developed an improve d protocol for high-frequency somatic embryogenesis from melon seeds. Using the cultivar ‘Male Sterile A 147’, the authors

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45 tested several factors, such as, changes in pl ant 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 res ponse 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 Fi ccadenti and Rotino (1995), who evaluated the morphogenetic response of 11 mel on cultivars belonging to the reticulatus and inodorus genotypes. These authors found that mel on 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

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46 analysis to detect the presence of genot ypic heterogeneity within a population. These analyses, together with their experimental results, allowed the separation of seed genotypes differing up to 5% in their regene ration ability. Afterward, Molina and Nuez (1996) reported the inheritanc e 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 tw o genes, partial dominance, independent segregation and similar effects for both gene s. 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 locu s, without cytoplasmic interactions. On the other hand, Kintzios and Taravira (1997) evaluated 14 commercial melon cultivars for plant regeneration capability. On ly six cultivars responded positively to a shoot induction treatment. Similarly, Galper in et al. (2003a) sc reened 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, exhibite d very low regeneration effici ency. Only the genotype BU-21, an inbred line, had profuse re generation of multiple shoots. In summary, melon in vitro response is under genetic control, however, other factors should be taken into account for me lon 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 Earl e, 1991). A natural and spontaneous increase in the ploidy level has been observed in fieldgrowing melon plants

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47 (Nugent and Ray, 1992), nevertheless, this ploi dy increase can also been induced in muskmelon plants using chemical compounds, su ch as colchicine (Batra, 1952; Kubicki, 1962). In fact, polyploidy as a method of plan t breeding received an increase amount of attention in the late 1930s, when it was disc overed that polyploidy could be induced with colchicine treatment and the earlier dem onstration that heat treatment in early embryogeny stages could also induce a chromosome doubling (Batra, 1952). Numerous tetraploid and triploid muskme lon plants have been obtained since the 1930s (Batra, 1952; Kubicki, 1962; Nugent and Ray, 1992; Fassuliotis and Nelson, 1992; Ezura, et al. 1992a; Adelbe rg, 1993; Nugent, 1994a; Nugent 1994b; Adelberg et al. 1995; Adelberg et al. 1999). According to Ez ura et al. (1992b), Fassuliotis and Nelson (1992) and Nugent (1994b), te traploid melon plants are characterized by having large male and hermaphrodite flowers, protrudent st igmas, 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 previ ously been reported that tetraploid melon plants were highly fertile and no later in matu rity than the ordinary diploids. Moreover, Batra (1952) reported that the qua lity of melon tetraploids wa s 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 tetrap loid fruits were superior in sugar level, firmness, and had better color than diploid fruits. Howe ver, tetraploid melon plants were less productive, because they had smaller and flatte r fruits than diploids, most cultivars had

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48 low fertility, and the fruits had an increased tendency to suffer of easy cracking, therefore reducing considerably thei r 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). Desp ite triploids plants grew more vigorously than diploids, and their fruits were not as flat as tetraploids. These triploids melon plants did not have any mark etable advantage over diploids ones, because still the percentage of cracking in those triplo id 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 fr uit 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 regenerati on protocols, somaclonal variation was a common observable fact, therefore tetr aploid, octaploid, mixoploid, and aneuploid melon plants were easily recovered from in vitro cultures (Bouabdallah and Branchard, 1986; Fassuliotis and Nelson, 1992; Ezur a et al. 1992a; 1992b; Debeaujon and Branchard, 1992; Kathal et al. 1992; Ezur a and Oosawa, 1994a; 1994b; Ezura et al. 1994). According to Ezura et al. (1995), som aclonal variation could be used to obtain variants lines with low-temperature germinabilit y in melon. Changes in fatty acid patterns have been found in melon callus tissue (Halde r and Gadgil, 1984), as well changes in a repetitive DNA sequences during ca llus culture have been det ected (Grisvard et al. 1990). However, somaclonal variation has to be avoided in research, where genetic transformation is involved because genomic st ability in transgenic plants has to be maintained in order to express the inserted transgene. In addition, regeneration of melon

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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 Branchar d, 1992). Nevertheless, each morphogenetic pathway has a diffferent effect on the freque ncy 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 ca llus stage is involved in the regeneration process the likeli hood 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 (A delberg et al. 1994). Immature cotyledons produced more tetraploid regenerants than mature cotyle dons, while explants from apical meristems produced fewer or no tetraploid plants (Adelber g et al. 1994). Ezura and Oosawa (1994a; 1994b) reported th at 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 gr eater than diploid cells. These same authors reported that the ability of somatic embryos to develop into plantl ets decreased in the following order: diploid>tetraploid>octaploi d. 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.

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50 Melon plant ploidy levels can be determ ined by cytological methods, such as counting the chromosome number using root squash tips (Ezura et al. 1992a; 1992b; Kathal et al. 1992; Ezur a 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 comp licated the easy chromosome observation. Unconventional and indirect techniques have been developed in order to determine the ploidy level of regenerated melon plants (F assuliotis and Nelson, 1992; Adelberg et al. 1994; Adelberg et al. 1995). Among them, polle n grain shape and stomate length, as well as the chloroplast number in gua rd cells from stomata have been commonly used. Diploid plants have pollen grains with typical triangular-appearing shap e and are tripolar, whereas tetraploid plants produce many s quare tetraporous, r ound-monoporous or oval biporous pollen grains (Fassuliotis and Nels on, 1992; Adelberg et al. 1994; Adelberg et al. 1995). Likewise, Fassuliotis and Nelson (199 2) reported that in diploid plants the average stomate length was 22.1 2.15 m and the average number of chloroplast inside the guard cell was 9.4 1.5, while in tetraplo id plants the average stomate length was 29.1 2.15 m and the average number of chloroplas t 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, reliab le 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;

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51 Gilissen et al. 1993; Dolezel et al. 2004). Neverthelesss, the economic constraint to purchase and operate this equipmen t restricts its wide usage. In order to avoid somaclonal variation and regenerate mostly diploid plants from melon in vitro cultures, several strategies ha ve 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 mi xoploids regenerated plants wa s less than 8% after 4 years of culture. The cryopreservation of shoot prim ordia cultures at low temperatures (liquid nitrogen) using a slow prefreezing procedure has given excellent re sults as well (Niwata et al. 1991; Ogawa et al. 1997). A reliable system for transforma tion of a cantaloupe Charentais type melon leading to a majority of diploid regenerant s was developed (Guis et al. 2000). Unfortunatly, this regenerati on system did not generate completely developed transgenic shoots for other commerci al melon cultivars (Nuez-Palenius et al. 2002; Gaba, 2002, personal communi cation), again, a genetic fact or 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 so maclonal variation, in order to maintain commercial value of the new genotype. 2.3.2.3 Vitrification Woody and herbaceous explants are prone d 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 glassine ss (Paques and Boxus, 1987; Ziv, 1991).

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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 unde rgo spontaneous vitrification, even if explants are cultured on non-inductive media or c onditions (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, a nd 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 developm ent 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 th e major effect on vitrification induction on melon buds as well (Leshem et al. 1988b). Paques et al. (1987) and Ka thal et al. (1994) reported that vitrification process was an induc ible and reversable phys iological event. If the tissues are frequently subcultured, vitr ification 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, diminish ing 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 phlor idzine (Paques and Boxus, 1987; Paques et al. 1987; Ziv, 1991). I n vitro environmental conditions can also be

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53 altered including: several treatments have b een proposed: a cold treatment to the plants before in vitro culture, reducing culture room temper ature, increasing the daily dark period, increasing the containe r-environment gas exchange, and reducing the relative humidity within the flask (Paques an d Boxus, 1987; Paques et al. 1987; Ziv, 1991). 2.3.3 Regeneration by Organogenesi s 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 dive rse explants, among th em: cotyledons (from immature and quiescent seeds, and/or seedli ngs), hypocotyls, roots, leaves, protoplasts, and shoot meristems have been report ed (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 commonl y a higher regeneration frequency (above 80%) of de novo shoots using direct organogenesis co mpared 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; Nuez-Palenius et al. 2002, Galperin et al. 2003a; 2003b). Likewise, Molina and Nu ez (1995b) studied the variation in regeneration ability among and within seve ral populations of leaf, cotyledon and hypocotyl melon explants. These authors reported that in vitro clonal selection to improve the regeneration frequency from l eaf explants also raises the organogenetic response in other explant types. These resu lts suggest the presence of a partial common genetic system controlling the regenerati on frequency of all type of explants.

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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 fact ors. 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, 6benzylaminopurine (N6-benzyladenine, BA) is the most fr equently used in high levels (1 mg/l or higher) to induce bud formation. BA c oncentration is lowered (0.5 mg/l or lower) to allow shoot elongation. Elongated shoots ar e then transferred to a plant growth regulator-free medium or with low-auxin level (NAA or IAA) to induce the rooting process. If indirect regenera tion is used, twoor even a three-step method has to be utilized. First, an induction ca llus growth is stimulated on the explant by applying strong cytokinins (TDZ) and auxins (2,4-D) to th e medium culture. Sec ond, those calli, which have green nodules are transf erred to a low-level plant ho rmone-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 r ecover a whole regenerated plant. This is due to the several subcultures are required for shoot elongation and two or three mont hs are needed for rooting using the indirect method (Moreno et al. 1985a; 1985b; Kathal et al. 1986; 1994).

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55Table 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-dayold 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

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56Table 2.3 Continued Explant Source Cultivar Induction Medium (Plant Growth Regulators) Reference Cotyledons from mature seed, cotyledons and hypocotyl from 10day-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 M 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

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57Table 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 ‘Vdrantais’ 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-oldseedlings 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 Oo sawa, (1994a), 15. Fic cadenti and Rotino, (1995), 16. Yadav et al. (1996), 17. Singh et al. (1996) 18. Kintzios and Taravira, (1997), 19. Guis et al. (2000) 20. Nuez-Palenius et al. (2002), 21. Stipp et al. (2001), 22. Curuk et al. (2002a), 23. Curuk et al (2002b), 24. Galperin et al. (2003a).

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58Table 2.4 Melon regeneration thr ough indirect organogenesis. Explant Source Cultivar Callus Induction Medium (CIM ) and Shoot Induction Medium (SIM ) (Plant Growth Regulators) Reference Cotyledon-callus culture from 11 to 13day-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 cu lture from 7-dayold 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 4week-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

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59Table 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 leavesprotoplasts 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. Kath al et al. (1994), 10. Molina and Nuez, (1995b).

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60 Other plant hormones, such as gibberellin s, 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 (S hetty 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, neve rtheless, the obtained results using both types of components (plant hormones and plant additives) are either melon genotype-dependent or are not c onsistent among reported results. 2.3.3.2 Environmental Factors Environmental factors, such as light, temp erature, nature of media gelling agent, and relative humidity within flask, influence the efficien cy of regeneration method (Niedz et al. 1989; Ficcade nti and Rotino, 1995; Yadav et al. 1996; Kintzios and Taravira, 1997; Curuk et al. 2003). For instance, Niedz et al. (1989) st udied the effect of temperature (22, 25 and 29oC) and light (0, 5, 10, 30, 60 and 3,000 mol.m-2s-1) on the percentage of bud initiation in cotyledonary e xplants 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 mol.m-2s-1. Conversely, lower temperatures (22 and 25oC), darkness, and higher light intensities (60 and 3,000 mol.m.2s-1) reduced bud initiation. Similarly, Kintzios and Taravira (1997) tested two levels of light intensity, 50 and 250 mol.m.2s-1 on shoot and root induction in 14 diffe rent melon cultivars. As expected, lower light intensities induced a greater root induction in many melon cultivars. Also, higher PPFD (250 mol.m.2s-1) values adversely affect ed shoot induction from cotyledonary explants. Interestingly, Cur uk et al. (2003) recently described that

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61 regeneration from hypocotyls (proxi mal part to the cotyledons) of Cucumis species does not requiere light. High relative humidity within the fl ask culture might induce ethylene accumulation, which affects shoot regenerati on in melon cotyledons (Roustan et al. 1992). These authors added several le vels of silver nitrate (60-120 M AgNO3) into culture media to inhibit ethylene action. They we re 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 natu re of medium gelling agent also has an important role in melon regeneration (Fi ccadenti and Rotino, 1995; Yadav et al. 1996). Ficcadenti and Rotino, (1995) described that usin g agar, instead of ‘gelrite’, they were able to attain a better cotyledon organogene tic 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). So matic embryogenesis was described in melon explants before organogenesis and used to recover complete melon plants (Table 2.5) (Blackmond et al. 1981b). In general, coty ledonary tissue has been the most efficient explant for the induction of mel on somatic embryogenesis (Table 2.5). An embryogenic response in melon is a ffected by the nature of explant and genotype. Gray et al. (1993) reported significant differen ces in the frequency of

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62 embryogenic muskmelon explants when they compared two commerc ial sources of the same melon cultivar. High regeneration fr equency, up to 100%, may be attained from cotyledon explants with an av erage 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 e xplant (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 ab le to recover only 5 melon plants from hundreds of somatic embryos, Branchard and Chateau (1988) reporte d a 12% conversion rate, and Homma et al. (1991) reported a conve rsion rate from 7% up to 61%, depending on explant type. Tabei et al. (1991) concluded that cotyle dons 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 em bryos, 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 si gnificant differences in so matic 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) publishe d a complete and extensive review on somatic embryogenesis in Cucurbitaceae including Cucumis melo where they concluded that even though somatic embr yogenesis and plant recovery have been obtained from numerous plant sources including protoplasts,

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63Table 2.5 Melon regeneration th rough 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 seed s ‘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

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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 g/l 2,4-D and 0.1 g/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 ‘Vdrantais’ 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 ‘Vdrantais’ 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. Debeau jon and Branchard, (1992), 8. Oridate et al. (1992), 9. Gray et al. (1993), 10. Hos oi et al. (1994) 11. Ezura and Oosawa (1994a), 12. Gu is et al. (1997a), 13. Stipp et al. (2001), 14. Akasa-Kennedy et al. (2004).

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65 the best results were observ ed 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 re gulators) has a grea t 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 ab sence of the induction hormone. Auxins are prerequisite for inducti on of somatic embryogene sis (Tabei et al. 1991; Oridate et al. 1992; Debeaujon and Branch ard, 1993; Gray et al. 1993; Guis et al. 1997a; Nakagawa et al. 2001). In general, the most common and effici ent auxin to induce somatic embryogenesis in melon explants is 2,4-D (Oridate a nd 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 grow th (Tabei et al. 1991). Likewise, Tabei et al. (1991) reported that IAA was the most ef ficient 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

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66 culture medium in order to c ontrol 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 Ya sawa, 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 sucros e, glucose, fructose, and galactose, led to the highest rate of somatic embryogenesis. Si milarly, Gray et al. (1993) concluded that the sucrose concentration in embryo induc tion and development media had a profound effect on somatic embryogenesis, i.e. 3 % su crose produced a greater explant response than lower or higher levels of the car bohydrate. These authors found that sucrose concentration also exerted an effect on the re lative 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 enha nced the embryogenic response by almost two-fold, whereas maltose at any level tota lly 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 th e most important. In order to induce somatic embryogenesis, melon expl ants 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 Bran chard, 1992; Oridate et al. 1992, Debeaujon and Branchard, 1993; Hosoi et al. 1994; and Nakagawa et al. 2001).

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67 Somatic embryo formation has been augmented by pretreatment in a dark period, usually one or two weeks before plac ing 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 fo r this purpose, obtaining improved developed embryos by using ‘gelrite’ and/or ‘phytagel’ as a substitute for agar (Branchard and Chateau, 1988). Nevertheless, recently Akas aka-Kennedy et al. (2004) reported that somatic embryos from ‘Vedrantais’ and ‘Earl’s Favourite Fuyu A’ melon cultivars underwent development without vitr ification, if agar was used instead of ‘gelrite’. Thus different melon cultivars give diverse responses under similar in vitro conditions. Kageyama et al. (1991) and Mo reno et al. (1985a) reported that the vitrifica tion state of regenerated plants is increased if liquid cultures are used during the initial steps of somatic embryogenesis. Kageyama et al. ( 1991) reported that cons ecutive washing of somatic embryos with hormone-f ree MS medium with 0.5% ac tivated 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). Th e most common type of hybrid cultivar is produced by crossing two or more inbred line s, 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. Diploi ds can be induced by chromosome doubling agents, such as colchicine or oryzalin (Ye tisir and Sari, 2003; Lotfi et al. 2003). This

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68 approach has been used to obtain plants tole rant 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 a ndrogenesis and gynogenesis have not been successful to produce haploid melon plants. Usi ng 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 Vaul x (1987) developed an in vitro technique (commonly named embryo rescue or embryo culture) to recove r muskmelon haploid plants. These authors obtained haploid plants after pollination of hermaphrodite fl owers with irradiated (Co60 rays) pollen and in vitro culture of ovules or immature embryos. They also developed a new culture medium to allow further developm ent of these embryos in to 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 a pplied not only to indu ce and rescue haploid melon plants, but also to culture diploid em bryos as well in numerous melon cultivars with excellent results (S auton, 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 signifi cant differences in haploid embryo production between two melon cultivars, ‘Vedrantais’ which produced

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69 an average number of haploid embryos of 3.5%, whereas ‘F1.G1’ formed 1.7%. Sauton (1988) found that ‘Arizona’ mu skmelon produced the highest number of haploid embryos (3%) if melon plants were grown during th e 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 ‘Vedra ntais’ melon fruits obtained from plants grown in summer than those harvested in autumn. Unlike other plant systems, such as carro t and tobacco, melon pollen is able to tolerate high -irradiation doses (up to 3,600 Gr ays) 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 re duction in pollen tube length has been observed using high radiation doses. This re duction was proportional to the amount of radiation used (Cuny et al. 1992; 1993; Yanmaz et al. 1999). High -irradiation doses can induce an increase in the percentage of necrotic haploid embryos. The most common gamma irradiation doses ar e between 250 and 350 Grays (Sauton and Dumax de Vaulx 1987; Cuny et al. 1992; 1993; Beharav and C ohen, 1995a; Yanmaz et al. 1999; Lotfi et al. 2003; Yetisir and Sari, 2003), and for so ft-X-rays the doses are between 65 and 130 kR (Kuzuya et al. 2000; 2002; 2003). Niemirowicz-Szcztt and Kubicki (1979) de mostrated that strong incompatibility events occurred in the intergeneric and intraspecific crosses within the Cucurbitaceae family, avoiding sexual hybr idization among Cucumis melo Thus, in vitro embryo culture technique has been uti lized to recover melon haploid plants, and to save valuable diploid plant material th rough zygotic embryo culture and somatic embryogenesis. Different diploid plant material has been r ecovered, such as hybrid plants obtained after

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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 impr ovement of this tec hnique as well as the cointegration to marker-assisted selecti on (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 tran sformation 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. (1 992) 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

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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-depende nt (Fang and Grumet, 1990; Dong et al. 1991; Yoshioka et al. 1992; Valles and La sa, 1994; Gonsalves et al. 1994; Gray et al. 1995; Bordas et al. 1997; Clendennen et al. 1999; Ezura et al. 2000; Nuez-Palenius et al. 2002; Akasaka-Kennedy et al. 2004). The Agrobacterium strain, vector structure, and co-c ultivation with acetosyringone, have an influence on melon transformation efficiency (Dong et al. 1991; Yo shioka et al. 1992; Va lles and Lasa, 1994; Bordas et al. 1997). Fang and Grumet (1990) tested several fact ors, such as kanamycin concentration, Agrobacterium inoculum level, length of inocula tion, period of co-c ultivation, and the use of tobacco nurse cultures, on melon tran sformation efficiency rate. These authors found that 75 mg.L-1 kanamycin, fresh overnight grown b acteria at a concentration of 107 – 108 bacteria.ml-1 (OD600= 0.8), 10 min of inoculation, thre e days of co-cultivation, and not using tobacco nurse culture were the best conditions to attain an efficient transformation rate. Unfortunatl y, these transformation conditions were only tested in one melon cultivar, ‘Hale’s Best Jumbo’. A simila r approach was described by Dong et al. (1991). These authors tested the sensitivit y 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 cocultivation, and 3. explants were placed unde r selection two weeks after co-cultivation-. The highest transformation fr equency was obtained when 75 g.L-1 methotrexate and 100

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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. Valls 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 toleran ce 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 dur ing the selection process. Among them, nptII, which provides tolerance to aminoglycos ide antibiotics (Fang and Grumet, 1990; Dong et al. 1991; Yoshioka et al. 1992; G onsalves et al. 1994; Valls and Lasa, 1994; Gray et al. 1995; Ayub et al. 1996; Bordas et al. 1997; Clendennen et al. 1999; AkasakaKennedy et al. 2004), dhfr, which gives resistance to me thotrexate (Dong et al. 1991), and CP4syn which offers tolerance to Glyphosate he rbicide (Nuez-Palenius et al. 2002; 2003) have been used. When t ypical concentrations (75-150 mg.l-1) of kanamycin are not able to inhibit the non-transg enic bud or shoot growth (G uis et al. 1998; Dong et al. 1991), other antibiotics, which are also det oxified by the neomycin phosphotransferase protein (NPTII), may be added to melon in vitro cultures in order to improve the selection efficiency. Gentamycin, hygromycin, and pa romomycin have been used in melon cultures as alternative to kanamycin with excellent results (Gui s et al. 1998; NuezPalenius et al. unpublished results, Ezura et al. personal communication).

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73Table 2.6 Genes transferred to mel on 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

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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 ‘Vdrantais’ 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 ‘Vdrantais’ A. tumefaciens LBA4404 strain 14

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75Table 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 resist ance 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 ‘Vdrantais’ A. tumefaciens LBA4404 strain 19 uidA and hpt genes GUS Reporter and Selectable marker genes Mature seeds Somatic embryogenesis ‘Vdrantais’ 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. Valls 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. Nuez-Pal enius et al. (2002), 18. Nuez-Palenius et al. (2003), 19. Silva et al. (2004), 20. Akasa-Kennedy et al. (2004). Znot-reported.

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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. 1 997; 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% (Nuez-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 cha nges [tetraploids (75%, Ayub et al. 1996), octaploids, mixoploids] or had morphogenetic altered ch aracteristics, 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 contro l 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 pr evalent (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-expre ssed the gene for CMV coat protein via Agrobacterium tumefaciens using ‘Prince’, ‘EG360’ and ‘S unday Aki’ melon cotyledons.

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77 Those transgenic melon plants which ove r-expressed the CMV-CP gene, grown under greenhouse conditions, were found to be resistan t 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 observati on 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 obs erved in transgenic plants (Yoshioka et al. 1993). After Yoshioka’s achievement, differen t 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 e ither 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-p rotection gave effectiv e field resistance, but not 100% protection. Field trials were conducted to determine if transgenic plants would retard the spread of the aphid non-transmissible stra in 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 transgen ic 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

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78 mechanical inoculations, and/or natural ch allenge 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 wher eas 99% of the wild type melon plants had mixed virus infections Moreover, control pl ants were severely stunted (44% reduction in shoot length) and had poor fruit yiel d (62% loss), and most of their fruits were unmarketable (60%) co mpared to transgenic melon plants. 2.3.6.2 Improvement of Toleran ce to Physical Factors Several environmental factors, such as hi gh or low temperature, salt accumulation, low-sun irradiance, drought, and flooding, seri ously 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 transg enic and control plants were evaluated for salt tolerance af ter 16 days of incubation on medium containing 10 g.L-1 sodium. The frequency and intensity of root formation were higher in HAL1positive 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 Researc h, 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

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79 nutritional and ripening information in st ore 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 t ype Cantaloupe melon c v. ‘Vdrantais’, these authors were able to transfer the 1-ami nocyclopropane-1-carboxilic-acid oxidase gene (ACC oxidase from melon under the control of a constitutive promoter) in antisense orientation to reduce the level of ethyle ne (Ayub et al. 1996). Different ripening parameters were evaluated in transgenic ripe ning 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, ha rvest 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 th e 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 in crease 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

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80 period. Transgenic fruit remained attached to the vine for a longer pe riod of time (65 days after pollination) compared to control plants (38 days after pollination). Only exogenous ethylene treatment (50 ppm) of transgenic frui ts allowed the recovery of the wild type behavior and phenotype. There were no significant differenc es in carotenoid content (flesh color) and total soluble solids (Brix) c ontent in wild type a nd transgenic fruit at any stage of ripening. Clendennen et al ., (1999) utilized the product of the S-adenosylmethionine hydrolase (SAMase) gene (from T3 bacteriopha ge) to catalyze the degradation of SAM, the initial precursor of ethyl ene. Unlike the T-DNA construc t used by Ayub et al. (1996), Clendennen et al. (1999) used a fruit specific promoter (c himeric ethylene-responsive E8/E4 promoter) aimed to overexpress the SA Mase gene in two ‘American Cantalope’ lines, which were proprietary inbred lines from Harris Mora n Seed Company, Inc. These authors evaluated several postharvest fruit qua lity parameters, such as fruit size and weight, firmness, mold susceptibility, external and internal color, soluble solids, harvest maturity (timing from anthesis to full sl ip, measured as Heat Units), and ethylene production, in wild type and transgenic mel on fruits from plants grown under field conditions. These authors repo rted 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 ethyl ene biosynthesis and related ev ents. In lab experiments, transgenic fruits produced half of the ethyl ene amount accumulated by wild type fruits. However, in field trials, the onset of matur ity, 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 al so measured on transgenic and wild type

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81 fruits from three different field trial locati ons. Significant differences were found in fruit internal firmness between transgenic and wild type melons, but only from one location. Clendennen et al. (1999) clai med 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 ripe ning and postharvest phenotype. Silva et al. (2004) obtained transgenic Ca ntaloupe melon plants cv. Vdrantais by inserting and overexpressing ACC oxidase from apple and not from melon as Ayub et al. (1996) protocol. These authors reported the ch aracterization of ripe ning melon fruits, and their experimental comparison between transgen ic and control fruit provided very similar results in almost all the evaluated paramete rs, such as harvest ma turity, 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 (c v. Vdrantais) climat eric respiration, yellowing and carotenoid content of the ri nd, chilling injury, and formation of the peduncular abscission zone are events tota lly ethylene-dependent (Pech et al. 1999; Flores et al. 2001a; 2001b), wh ereas fruit softening, volatile s synthesis, and membrane deterioration are ethylene-partially depende nt processes and display some ethyleneindependent components (Bauchot et al. 1998; Bauchot et al. 1999; Guis et al. 1999; Flores et al. 2001a; 2001b; Flor es 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 even ts. There is still some controversy about organic acid metabolism and loss of acidity dur ing fruit melon ripening, i.e. Guis et al. (1997b) and Pech et al. (1999) suggested that the organic acid me tabolism was ethylene-

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82 independent, whereas Silva et al (2004) recently implied th at organic acid metabolism is an ethylene-dependent regulated process. It is likely that these differences in conclusions might be related to a different ethylene concen tration reached by each transgenic fruit. In Silva’s report transgenic fruit attained a lower internal ethylene-level (<0.09 l.L-1) than Guis’s results (<0.5 l.L-1). This subtle difference in et hylene concentration could have obvious outcomes such as at the specific le vels (Srivastava, 2002; Silva et al. 2004) All fruit quality oriented transgenic mel on plants have been obtained using just three melon cultivars, such as Vdrantais a nd 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, sacchar inus, inodorus, flexuosus, conomon and dudaim ) (Guis et al. 1998; Kirkbride, 1993), and hundred s of melon cultivars, much more work could be accomplished. There still remains a need to improve in vitro regeneration and transformation protocols (Gui s 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-fleshe d, and yellow skinned -finely-netted rind specialty hybrid muskmelon br ed in Israel. ‘Galia’ is al so 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

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83 European market ‘Galia’ musk melons have recently started been cultivated in Central American countries such as Guatemala, H onduras, Costa Rica, and Panama, and exported to the U.S. and Europe (Table 2.7). Open-f ield, ‘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 hi gh as 165 t/ha have been reported (Shaw et al. 2004). The ‘Galia’ type muskmelon worl d production is around 300,000-500,000 tons per year (Reho, 2004). ‘Galia’ muskmelon is a commodity reachi ng dissimilar prices, from $ 1.97 to $ 2.17 per fruit, mostly depending on quality char acteristics and origin country (Table 2.8) (Produce1, INC. 2005, United States Department of Agriculture [USD A] Department of Commerce, U.S. Census Bureau, Foreign Trad e 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 Commer ce, U.S. Census Bureau, Foreign Trade Statistics, 2005). 2.4.2 Botany and Origin According to Karchi (2000), ‘Galia’ musk melon was the first Israeli melon hybrid produced and was obtained through an extens ive breeding program at the Newe Ya’ar Research Center of the Agricultural Resear ch Organization (A.R.O – Israel) during the mid-1960s. ‘Galia’ has green-fle shed characteristics of ‘H a’ Ogen’ type, which is a smooth-skinned, sutured melon and was introdu ced from Hungary to Israel during the 1950’s, and used as the female parental line. ‘Galia’ has also a gol den-yellow netted rind from ‘Krymka’, Russian melon cultivar,

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84Table 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 Co mmerce, U.S. Census Bureau, Fo reign Trade Statistics (2005) .

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85Table 2.8 ‘Galia’ Type Muskmelon Unit Value Imports to U.S 1999 2000 2001 2002 2003 AVERAGE ORIGIN $/Kg $/Kg $/Kg $/Kg $/Kg $/Kg 12/15/31 6/111/30 12/15/31 6/111/30 12/1-5/31 6/111/30 12/1-5/31 6/1-11/30 12/1-5/31 6/111/30 12/15/31 6/111/30 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 Co mmerce, U.S. Census Bureau, Fo reign Trade Statistics (2005).

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86 which was used as the male parental line (Karchi, persona l communication, 2004). ‘Galia’ was named after Dr. Karchi’s daught er 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; Fa llik et al. 2000) has emerged. 2.4.3 Postharvest Physiology ‘Galia’ muskmelon has exceptional characteristics such as fruit quality with 1315% total soluble solids (TSS), bold flavor and a distinct musky aroma. The main disadvantage of ‘Galia’ is its s hort storage life, since it is harv ested near peak maturity for optimum flavor. Storage is limited to two or th ree weeks, even when it is maintained in lowtemperature (6oC). Several pathogenic agents, such as Alternaria alternata (Fr.) Keissler and Fusarium spp. can severely reduce the ‘Gal ia’ 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 l.L-1), combined with wax coating, ‘Galia’ fruits can be protected from decay (A haroni et al. 1992; 1993a). Unfortunatly, the residue level of imazalil in Israeli ‘Galia’ wax coated fruits is 3-5 l.L-1, more than the maximum tolerance limit (0.5 ml.L-1) imposed by most of European

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87 countries (Anonymous, 1990). Therefore, new f ungicides and postharvest strategies were sought to reduce the fruit decay in ‘Galia’ muskmelon. Other treatments including use of Sanos il-25, which is a 48% hydrogen peroxide solution and silver salts (Aha roni et al. 1994), applying Hi nokitiol (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), coat ing ‘Galia’ fruits w ith wax containing 2% sodium bicarbonate (Aharoni et al. 1997), and combined treatments, such as hot water (52oC) dipping and -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, wh ile maintaining high fruit quality after prolongated storage and marketing simulation in ‘Galia’ type muskme lon 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 controll ed atmospheres plus adding an ethylene absorbent (Aharoni et al. 1993a), reducing the stor age 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-met hylcyclopropene (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 controll ed atmosphere (CA) (10% CO2 and 10% O2) plus an

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88 ethylene absorbent on semi-commercial scale, in order to maintain fruit quality. Fruit were evaluated after 14 days in CA at 6C and additional 6 days at 20C. These authors found that ‘Galia’ melons stored in controlle d 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 8C) and in dividual fruit plastic packaging, as well as two controlled atmosphere conditions, i.e. 4% and 12% at 80% RH and 8C 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 we re evaluated. These authors did not find statistical differences between quality attri butes of bagged and nonbagged melon fruits. Also, ‘Galia’ melons stored at 5C maintained the highest external and internal quality during the storage period, regard ing firmness, soluble solids c ontent, titratable acidity and flavor assessment. Controlled atmosphere conditions tested in this study were unfavorable to maintain excellent ‘G alia’ fruit quality characteristics. Ergun et al. (2005) characte rized the physiological re sponses of ‘Galia’ fruit harvested at green (preripe) a nd yellow (advanced ripening) st ages after treatment with 1methylcyclopropene (1-MCP) and further st orage at 20C. These authors found that treatment with 1.5 L.L-1 1-MCP delayed the climacteric peaks of respiration and ethylene production of green fr uit by 11 and 6 days, respec tively, and also significantly suppressed respiration and ethylene produc tion maxima. Fruit softening of both developmental stages, green and yellow, was significantly delayed after 1-MCP treatment.

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89 By delaying fruit ripening via reduct ion in ethylene production ‘Galia’ F1 hybrid shelf-life might be improved. 2.4.4 Genetic Improvement by Conven tional 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 s ugar 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 transformati on protocols are lacking (Gaba et al. 1992; 1995; 1996; 1999; Edriss et al. 1996; Kintzios and Ta ravira, 1997; Galperin et al. 2003a). Dirks and van Buggenum (1989) descri bed an efficient method for shoot regeneration from leaf and cotyledon explants in ‘Accent’, ‘Galia’, ‘4215’, ‘Preco’, and ‘Viva’ melon cultivars. The best combina tion 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 i nduction among the cultivar s tested. A range of 20 to 50 shoots per explant were recorde d. These authors also claimed that over 100 shoots were formed in cotyledon explants. The best results on shoot induction were

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90 obtained with ‘Galia’, ‘Accent’, and ‘Preco’. Elongated shoots were rooted on hormonefree MS, and then successfully transferred into the greenhouse. Leshem (1989), working in the Volcani Ce nter 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. Call i proliferation as well as root and shoot primordia formation was only confined to the ba sal side (attachment poi nt to the stem) of the cotyledon or its segments and never a ppeared 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 we re needed to produce shoots. According to Leshem (1989), melon cotyledon responsiv eness was apparently achieved by the transverse cut and possibly due to accumu lation 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 arti cles (Leshem et al. 1990a; 1990b; 1991), where they studied the role of e xogenous cytokinins, some reserv e cotyledon polypeptides and polyamines on in vitro melon regeneration. Leshem’s t eam 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 obtaine d in Leshem (1989). Leshem et al. (1994a; 1994b) used thidi azuron (TDZ), benzyladenine (BA) and paclobutrazol (PC), and the plant hormone IAA in orde r to examine changes in cotyledonary organogenetic response in ‘Galia’ F1 hybrid. They studied the effect of

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91 those bioregulators on temporal changes in protein profiles (mostly some reserve cotyledonary polypeptides with molecular we ights of 20-25 kDa -P 20-P25-) in cultured melon cotyledons. These authors found that TDZ exerted a cytoki nin-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 overc ome the inhibitory effect of BA on melon rooting. However, PC was not able to induce roots at the basal e dge of the cotyledon when TIBA (inhibitor of a uxin transport) was incorpor ated into culture medium. Exogenous cytokinin (2.25 mg.L-1 BA) enhanced mobilization of reserve proteins (P20P25) and also induced shoot regeneration in cultured cotyledons. These polypeptides disappeared from cotyledonary tissue on day four if the media cu lture contained BA, whereas a constant level of those polypeptid es was maintained through the culture period if 1.75 mg.L-1 IAA was present. Regeneration co rrelated well with the P20-P25 disappearance, and shoots were onl y induced on medium containing 2.25 mg.L-1 BA. A delayed organogenetic regeneration and P 20-P25 breakdown was observed when IAA was present in the media. The incorporat ion to the medium of an inhibitor of carboxypeptidase (PMSF) inhibited both P20-P 25 disappearance and root regeneration, but not shoot initiation. Gaba et al. (1996) reported that an cymidol, an anti-gibberellin compound, improved in vitro bud development in cotyledonary expl ants of ‘Galia’ melon. This antigibberellin substance had a synergistic effect when BA was incorporated to the media. The presence of 5 mg/l GA3 reverted that rate of regene ration induced by BA alone or BA plus ancymidol.

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92 It has been suggested that cytokinin and white light operate together to enhance polypeptide metabolism and induce shoot rege neration in ‘Galia’ cultured cotyledons (Leshem et al. 1995; Kintzios and Taravira, 199 7). 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 e nhanced the polypeptide loss under light conditions (50 mol.m2.s-1), but it failed when cotyledons were cultured in total darkness. A reduction in white light fluency to 14 mol.m2.s-1 was enough to observe the polypeptide pattern as in da rkness culture. The incidence of shoot regeneration was reduced about half when cotyledons were cultivated at 14 mol.m2.s-1 white light fluency, and only 5% of cotyledons were responsiv e 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 seve ral morphogenetic pathways, such as calli induction, somatic embryogenesis and plant regeneration response, of 14 commercial melon cultivars to two levels of photosyntheti c photon flux densities (PPFD), i.e. 50 or 250 mol.m2.s-1. ‘Galia’ was able to undergo shoo t induction only when PPFD was 250 mol.m2.s-1. Micropropagation of ‘Galia’ F1 melon explants, using eith er shoot tips or axillary buds, has been reported as well (S petsidis et al. 1996; Edriss et al. 1996). Elevated levels of BA (2 mg/l) were needed to reach the gr eatest shoot production, and the presence of 0.5 mg/l IAA did not affect that number.

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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 explan ts and detect transient GUS expression. Permanent GUS transformation was observed only after a strong selection procedure (350 mg/l kanamycin). They were able to de tect 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 inducti on ‘Galia’ melon cotyledons (Dirks and van Buggenum, 1989), the cultivar was r ecalcitrant 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 repor ted (Curuk et al. 2002). This protocol was later applied to three melon cul tivars, ‘Revigal’, ‘Topmark’ and ‘Kirkagac’, and to one cucumber cultivar, i.e. ‘Taoz’ (Curuk et al. 2003). Regeneration from hypocotyl explants resulted in almost 100% di ploid 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 capabilitie s. Those melon genotypes embraced wild landraces, breeding lines and commercial cultiv ars. According to their results, 24 out of 30 genotypes did not produce any normal s hoots. Only five out of six genotypes displayed low regeneration efficiency, because a few explants developed normal shoots.

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94 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 liter ature on stable geneti c transformation in ‘Galia’ F1 hybrid or in any of its parental lines.

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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 tendrilbearing vine plants which are found in subtropical and tropical regions around the world (Robinson and Decker-Wa lters, 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 vari etal improvement. However, strong sexual incompatibility barriers at the interspecific and intergeneric leve ls have restricted potential to readily develop new and enhan ced melon cultivars (Niemirowicz-Szcztt and Kubicki, 1979; Robinson and Decker-Wa lters, 1999). Using genetic engineering strategies is feasible to ov ercome 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 appl ication 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; Hoso i et al. 1994; Singh et al.

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96 1996; Kintzios and Taravira, 1997 ; Guis et al. 1998; Guis et al. 2000; Nuez-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 biol ogical and physical factors influence in vitro regeneration efficiency rate, and all have to be considered in order to develop a reproducible and reliable melon regenerati on protocol. All the systems had diverse regeneration rates depending on genotype, cu lture conditions and e xplant source. Melon plant regeneration has been reported from root s (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 embryo s (Gray et al. 1993), shoot primordia (Adelberg et al. 1999), an d cotyledons (Guis et al. 1997a). ‘Galia’ muskmelon is an Israeli melon F1 hybrid with excepti onal fruit quality characteristics such as 13-15% total soluble solids (TSS), bold flavor and a distinct aroma (Karchi, 2000). Unlike other melon types, su ch as Cantaloupe Char entais (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 th e source of the explant on regeneration and

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97 transformation efficiency using two reporter ge nes (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 Nuez-Palenius et al. (2005a) protocol. In vitro regeneration of both parental li nes was carried out on explants from cotyledons, hypocotyls, and non-expanded true leaves following the procedures and plant-hormone balances repor ted by Nuez-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 s hoots) / 100] (MartinezPulido et al. 1992). Seedling growth and plant regeneration were conducted in a growth chamber (Lab-Line Instruments, Inc. Melrose Park, IL) under 100 mol m-2 s-1 light and a 16 h photoperiod provided by cool-white fluor escent lamps and constant 25 1C temperature. 3.2.2 Agrobacterium Inoculation and Plant Transformation Agrobacterium tumefaciens strain ABI containing a binary vector, pMON17204, harboring a selectable gl yphosate resistance marker [ CP4 syn gene encoding for 5enolpyruvylshikimate-3-phosphate synthase (EPSPS)], and the uidA gene encoding for 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-

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98 4404 containing a binary vector, pCAMBIA 2202-sGFPS65T, harboring a selectable kanamycin-resistance marker, NPTII gene encoding for neomycin phosphotransferase, and a fully functional green fluorescent prot ein (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 in cubated 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 fr om both ‘Galia’ melon parental lines were immersed in Agrobacterium suspensi on 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. Cotyle don and true leaf explants were then cultured and de novo shoots were obtained as previously described by Nuez-Palenius et al. (2005) and Guis et al. (2000), respectively. Hypocotyls were inoculated with a Agrobacterium tumefaciens suspension using a sterile sy ringe needle (0.72 mm gauge and 32 mm length) following the protocol described by Ramirez-Malagon and OchoaAlejo (1996).

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99 Figure 3-1 Map of the T-DNA in pM ON17204 and pCAMBIA 2202-sGFPS65T. (A) TDNA region of binary vector pMON17204. RB: right border of T-DNA, 35S: promoter for 35S RNA from Cauliflower mosaic virus CP4syn : 5enolpyruvylshikimate-3-phosphate syntha se (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 2202sGFPS65T. 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 cauliflowe r 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 GU S Activity and GFP Detection Histochemical localization for -D-glucuronidase (GUS) activity in putative T0 transformant tissues was performed as desc ribed by Gama et al. (1996). Plant organ and tissues were incubated in GUS-staining so lution [50 mM sodium phosphate (pH 7.0), 0.5 mM potassium ferricyanide, 0.5 mM potassi um 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-Dglucuronidase (X-Gluc)] at 37C for 12-36 h. After incubation, the samples were then CP4syn 35S RB GU LB rbcSE9 rbcSE9 NPT-II35S RB GF LB NO 35S 35S A B

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100 transferred to 70% (v/v) ethanol to remove chlorophyll. GUS-positive tissues were photographed with a Zeiss MC63 photomic rographic camera coupled to a DRC stereomicroscope. Putative GFP-positive plant tissues were ex amined directly to detect fluorescence using a Zeiss Stemi SU11 fluorescence stereo microscope 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 (0.250.5 g) was performed using a modified CT AB protocol (Doyle and Doyle 1987; 1990). The forward and reverse primers for uidA gene (GUS) were 5’-CAACGAACTGAACTG GCAG-3’ and 5’-CATCACCACGC TTGGGTG-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 l) were subjected to electrophoresis on a 0.8% agarose gel and visualized by UV light. 3.2.5 Experimental Design a nd Statistical Analysis The experiment to measure the BFC inde x on three different melon explants was carried out under a factorial design (2 x 3) with six repl icates, 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) w ith three replicates, having two levels for genotype, three levels for explant type, and tw o levels for reporter gene used. Original data from transformation experiment was tran sformed by the arcsine s quare root in order

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101 to be analyzed for analysis of varian ce (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’ musk melon 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 trueleaves. When the same explant type was us ed, 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 le tter are not significan tly 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 mo st common plant organ used to induce de novo regeneration through either organoge nesis 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 8CotyledonHypocotylTrue-leafBFC Male Female a c c c c b

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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 Taravi ra, 1997; Guis et al. 1997a; Stipp et al. 2001; Curuk et al. 2002a; Galperin et al. 2003a; 2003b; Akasa-Kenne dy et al. 2004).The use of cotyledon as explants for melon in vitro culture has several advantages ov er other plant tissues, i.e. a) cotyledons have a quick morphogenetic res ponse 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 expl ants (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 w eeks 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 eff ect of source explant on tr ansformation 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.

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103 Table 3.1 Regeneration efficiency in ‘Ga lia’ 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 num ber of shoots per explant) X (% explants forming shoots) / 100. (Marti nez-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 we re positive for GUS and GFP expression (Figure 3.4). Although de novo shoots were obtained, only ‘escapes’ (non GUSor GFP-positive shoots and roots) were observed when true-leaf explants were used (Figures 4-5 and 4-6). It has been reporte d that different plant explants have diverse susceptibility to be transformed by Agrobacterium tumefaciens (Febres et al. 2003). This differential susceptibility has been describe d 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 ‘e scapes’ after Agrobacteriumor biolistic-mediated transformation in Cucumis melo is a commonly observable result (Guis et al. 1998). Indeed, Akasaka-Kennedy et al. (2004) have cl aimed 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.

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104 Figure 3.3 Time schedule for de novo shoot regeneration in hypocotyl, cotyledon and true-leaf explants of ‘Galia’ muskmel on parental lines. The numbers between the top and bottom photographs represent th e number of weeks to attain from mature seeds the in vitro rooted seedlings. Hypoc otyl: (a) seedlings, (b) hypocotyl injury (arrow), (c) decapita tion, (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 d00 17 9 a b f e c d 0 23

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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 us ed 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’ ma le parental-hypocotyl explants had a transformation efficiency value similar to ‘Galia’ maleand 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 th an other plant organisms (Fang and Grumet, 1990; Dong et al. 1991; Gaba et al. 1992; G onsalves et al. 1994; Bordas et al. 1997; Akasaka-Kennedy et al. 2004). Seve ral transformation rate effi ciency values have been described according to transformation prot ocol 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% (Nuez-Paleni us 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-sp ecific 750 bp fragment which belongs to the GUS

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106 reporter gene, in male and female ‘Galia’ muskmelon transgenic lines but not in nontransgenic lines (Figure 3.7). In summary, it is possible to obtain transg enic ‘Galia’ male and female parental plants using either cotyledon or hypocotyl explants. Moreover, co tyledonary explants have the advantage to attain those transgen ic plants in a shor ter 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

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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 significantl y 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 significantl y different by Tukey’s Studentized Range Test at P 0.05 0.0 2.0 4.0 6.0 8.0 10.0 12.0CotyledonHypocotylTrue-leaf% of transformation Male Female a a b b c c 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0CotyledonHypocotylTrue-leaf% of transformation Male Female a b b b c c

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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). M1 23456

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109 Figure 3.8 Time schedule for cotyledon and hypoc otyl transformation systems in ‘Galia’ melon parental lines. A. Explant transf ormation B. Regeneration of transgenic shoots. C. Elongation of transgenic sh oots D. Rooting E. ‘Hardening’ of seedlings. WEEKS H yp ocot y l B E C D A Cot y ledon A D E B0 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

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110 3.3.3 Summary The goal of this work was to measure th e source of the explant on regeneration and transformation efficiency in ‘Galia’ melon parental lines. Cotyledon, hypocotyl and trueleaves of both female and male ‘Galia’ pare ntal 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-lea ves and cotyledons. Hypocotyls were transformed according the protocol described in Ramirez-Malagon & Ochoa-Alejo (1996). Cotyledons were transforme d with a protocol previously developed in our lab (Nuez-Palenius et al. 2005a) and true-leaf explants we re transformed using the methodology of Guis et al. (2000). The gr eatest numbers of shoots were regenerated from cotyledons. These explants also produced the highest number of GUS positive shoots and roots. Using hypocot yls as explants gave the greatest number of GFP positive roots and shoots. We were not able to obtai n any transgenic shoots using either the GUS or GFP constructs when true-leaves were us ed as explants. According to our results, ‘Galia’ melon parental lines ar e readily transformable with Agrobacterium tumefaciens using the hypocotyl or cotyledon-protocol. It is possible to have full regenerated transgenic plants in 3-4 mont hs using the cotyledon-protocol, whereas at least 6-7 months are needed when the hypocotyl-protocol is used.

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111 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 (Kar chi 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 bot h local and export markets. ‘Ga lia’ type melons have been the mainstay in muskmelon sales in the Europ ean market for more than 30 years (Karchi, 2000). One disadvantage of ‘Galia’ is its stor age 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 pick ed at maturity (Kar chi, 2000). ‘Galia’ F1 hybrid tends to be extremely soft at and past peak fruit maturity. Traditional breeding methods have helped breeders develop a st rategy 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 gene tically 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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112 Agrobacterium and particle-gun bombardme nt methods, several transgenes, which provide different phenotypic characteris tics, have been transferred into Cucumis melo explants. These have included selectable ma rker and reporter genes (Fang and Grumet, 1990; Dong et al. 1991; Gaba et al. 1992; Valls and Lasa, 1994; Gray et al. 1995; Akasa-Kennedy et al. 2004), virus-resistan ce 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 (A yub 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 musk melon 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; S ilva 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 ‘Gal ia’ muskmelon to be recalcitrant to transformation by Agrobacterium tumefaciens. The goal of this research was to transfor m the male parental line (cv. ‘Krymka’) of ‘Galia’ muskmelon with the ACC oxidase (CMA CO-1) antisense gene in an attempt to delay fruit ripening.

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113 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 em bryos surface sterilized in 1.2% sodium hypochlorite (20% commercial bleach so lution containing two drops of Tween 20 per 100 ml) for 15 min in a sterile Erlenmeyer fl ask (50 ml), and washed three times with sterilized distilled water. Embryos were then cultivated for 2 days in regeneration medium (RM), which consisted of Murash ige 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 -Naphthaleneacetic acid (NAA), and 0.7 g.L-1 phytagar. Seedling growth and plant re generation were conducted in a growth chamber (Lab-Line Instruments, Inc. Melrose Park, IL) under 100 mol m-2 s-1 light and a 16 h photoperiod provided by cool-white fluorescent lamps and constant 25C 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 abax ial 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= (a verage number of shoots per explant) X (% explants forming shoots) / 100 (Martinez-Pulido et al ., 1992). Shoots were excised from cotyledon explants and incubated on elongati on medium (EM), which consisted of MS

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114 salts, supplemented with 30 g.L-1 sucrose, 0.1 g.L-1 myo-inositol, 0.001 g.L-1 thiamineHCl, 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 strength MS medium supplemented with 1 mg.L-1 indole-3-acetic acid (IAA). After thr ee weeks of culture, shoots developed a complete and normal root system. Rooted s hoots were hardened using common practices and then transferred to glasshouse. Plants we re 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 mol 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 gl yphosate resistance marker [ CP4 syn gene encoding for 5enolpyruvylshikimate-3-phosphate synthase (EPSPS)], and the ACC oxidase (CMACO1) gene in antisense orientation was used (F igure 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 Br oth (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 antibioti cs and concentrations previously used, and

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115 incubated at 25oC on an orbital shaker (150 rpm) fo r 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 th e excess bacterial suspension. Explants were then cultured and de novo shoots were obtained as pr eviously described in Pl ant Regeneration section, except that RM was supplemented with 50 M Glyphosate and 150 mg.L-1 Timentin (GlaxoSmithKline, Research Triangle Park, NC). Glyphosat e selection was carried out only on RM. 4.2.4 Flow Cytometry Analysis Plant nuclei were freshly is olated 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 bl ade in a Petri dish containing 5 ml of Quesenberry (1995) extraction buffer. Th e 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 nucle i suspension. After gent ly 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. BDBiosciences, San Jose, Cal). It was calib rated using the 2C peak from nuclei of young leaves of diploid plants derived from s eed. A minimum of 10,000 nuclei were measured for each sample.

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116 Figure 4.1 T-DNA region of binary vector pC mACO1-AS. The underlined fragment of CP4syn gene was used as a probe for S outhern blot hybridization assay. RB: right border of T-DNA, FMV: figwort mosaic virus promoter, CP4syn : 5enolpyruvylshikimate-3-phosphate syntha se (EPSPS) gene, E9: 3’end of pea rbcSE9 terminator, CMACO-1 : melon ACC oxidase gene in antisense orientation, NOS: nopaline synthase term inator, LB: left border of T-DNA. 4.2.5 Detection of Transgenes Total DNA isolation from wild type and putative transgenic melon leaflets (0.250.5 g) was performed using a modified CT AB 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’, respectivel y. 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 fi nal extension period at 72 oC for 10 min. The amplified PCR products (25 l) were subjected to electrophoresis on a 1% agarose gel and visualized by UV light. CP4syn FM FMERB Bam H Probe 1.4 CMACO-1 ANTISENSE NOLB Bam H

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117 4.2.6 Southern Blot Analysis Total melon DNA was isolated from young leav es of wild type and transgenic T1, TGM-AS-1 and TGM-AS-2 lines (TGM-AS sta nds for Transgenic Galia Male AntiSense line), using a modified CTAB protocol fr om Doyle and Doyle (1987; 1990). Twenty micrograms of this DNA were digested overnight at 37o C with Bam HI and separated by electrophoresis in a 1.3% agarose gel. Because Bam HI 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 g mL-1 denatured salmon sperm DNA. Hybridization wa s carried through overnight at 42oC with constant shaking at 60 rpm, in a solution containi ng 50% (v/v) formamide, 5X Denhardt’s, 1% sodium dodecyl sulfate (S DS), 5X SSPE, and 100 g 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 w ith constant shaking at 60 rpm. Membranes were first washed in a solution containi ng 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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118 4.2.7 Segregation Analysis of Transgenes in Primary Transformants In order to analyze the segregation patte rn of transgenes, 62 randomly selected T1 seedlings of TGM-AS-1 and TGM-AS-2 transgen ic lines of ‘Galia’ male parental line were evaluated. DNA was extracted at least th ree 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 se lection 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 engineer ed 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 indepe ndent transformation system, such as the GUS reporter gene (Figure 4.4). This transfor mation efficiency obtained for ‘Galia’ male parental line is one of th e highest reported for any Cucumis melo transformation protocol (Fang and Grumet, 1990; Dong et al. 1991; Ga ba et al. 1992; Gonsalves et al. 1994; Bordas et al. 1997; Guis et al. 2000; Akasaka-Kenne dy et al. 2004). This high transformation efficiency could be the resu lt of a combination of factors such as

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119 Agrobacterium tumefaciens strain, selectable marker, a nd construct used (Hellens and Mullineaux 2000; Febres et al. 2003), which ma y 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 transgen ic 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 r ooted and transferred to ex vitro conditions. Glyphosate is a strong herbicide. It is able to induce ‘str ess’ 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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120 Table 4.1 Transformation efficiency of ‘G alia’ 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 res ponses’ (Klee and Clark, 2002). Therefore, it is plausible that epinastic phenotype was i nduced by Glyphosate. A similar response was observed in positive explants for -D-glucuronidase gene (GUS) growing on Glyphosate as well (Nunez-Palenius et al. unpublished results). In summary, transgenic shoots were obta ined by using Glyphosate as a selectable agent and were identified by m eans 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 Ne lson, 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). Th erefore, 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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121 Figure 4.3 Stable expression of -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 discardi ng any triploid, aneuploi d, and tetraploid or mixoploid plants. Based on these analyses, only diploid plants were used in subsequent experiments. Unexpectedly, some leaves from plants obt ained from seeds resulted as tetraploids. This condition has been reported in melon pl ants 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 cellu lar endoreduplication process, which leads to A B C D

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122 Figure 4.4 Transgenic (left) and non-transgenic (right) in vitro ‘Galia’ male line explants. Transgenic explants, growing on 50 M Glyphosate, had curled leaves and shorter internodes than wild type. an increase in ploidy level (K ondorosi et al. 2000). Figure 4.6 depicts charts for wild-type diploid and tetraploid plants, as well as transgenic diploid an d 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 s ubsequent 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 tetraplo id plant is shown. Using flow cytometry analysis it was found that only 20% of the tran sgenic 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, whic h is a tissue with hi gh propensity to bear

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123 tetraploid cells since the matu re seed stage in cucurbits (Colijn-Hooymans et al. 1994). After an entire flow cytometr y evaluation (at least seven leav es 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 adva nced 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 bl ot analysis (Figure 4.8). Likewise, this assay allowed determining the copy numb er of the transgene insertion. The 32P-labeled 1.4 Kb PCR-product from CP4 syn gene used as a probe hybridized with a single Bam HIreleased 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 TDNA 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 (TGMAS-1 and TGM-AS-2), DNA was extracted in dividually, at least three times, to determine the transgene distri bution inheritance pattern. PCR analysis of the progeny of TGM-AS-1 and TGM-AS-2 using CP4 synand 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-antisen se 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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124 probability of 66% and 88%, respectively), whic h corresponded to the segregation of one single copy insertion of T-DNA. This conclu sion is supported by the Southern blot analysis results as well 4.4.1 Conclusion We have successfully transformed a parent al line of ‘Galia’ hybrid muskmelon with a gene of interest a nd obtained two completely dipl oid transgenic regenerants. Analysis of T1 progenies from TGM-AS-1 and TGMAS-2 by segregation and Southern blot revealed that both transgenic lines had a single T-DNA insertion. Preliminary analysis of the transgenic fru its from ACO-1 antisense male line plants has revealed that these fru its produced less ethylene and have a lower ACC oxidase activity in vivo than their wild type counterpart.

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125 A B Figure 4.5 Flow cytometry analysis of propidi um iodide-stained nuc lei from wild type leaf tissue of ‘Galia’ male parent al line. 10,000 indivi dual nuclei were measured for each sample. (A) wild-type plant having most of nuclei (55%) at diploid level (M1). (B) wild-type plant having mo st of nuclei (51%) at tetraploid level (M2). Fluorescence intensity (arbitrary units) WT 0 800 M2 M3 M1 1 600 Number of nuclei 0 200 400 600 800 Fluorescence intensity (arbitrary units) 0 200 400 600 800 1000 Number of nuclei WT 0 800 M1 M2 M3 1 600 1000

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126 A B Figure 4.6 Flow cytometry analysis of propidi um iodide-stained nuc lei from transgenic leaf tissue of ‘Galia’ male parent al line. 10,000 indivi dual 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) Number of nuclei PCR+ # 2 0 200 400 600 800 0 800 M1 M M3 1 600 Number of nuclei PCR+ # 11 0 200 400 600 800 0 1 600 800 M1 M2 M3 1000 Fluorescence intensity (arbitrary units) 1000

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127 A B Figure 4.7 Southern blot hybridization assay of male muskmelon transgenic plants. (A) Total genomic DNA (20 g) extracted from leaves of T1 plants was digested and electrophoresed according to Material and Met hods 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 Bam HI-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, b earing 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

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128 to determine plant ploidy level of primar y transformants. Two completely diploid independent transgenic plants were obtained. Southern blot and segr egation analysis in the T1 generation determined that each i ndependent 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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129 CHAPTER 5 EMBRYO-RESCUE CULTURE IN ‘GAL IA’ MALE PARENTAL LINE MELON ( CUCUMIS MELO L. VAR. RETICULATUS SER.) 5.1 Introduction As part of specific hybridization and br eeding 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 embr yogenesis. 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 cu lture, 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; Ishi kawa et al. 2001; Ribe iro and Giordano, 2001; Yamada 2001; Faure et al. 2002a; 2002b; 2002c). These embryo rescue techniques have also been useful to enhance hybridization, re duce 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 (Eijl ander et al. 1994), sunflower (Faure et al. 2002b; 2002c), rice (Alemanno and Guiderdoni, 1994), melon (Lotfi et al. 2003), squash (M etwally et al. 1998a; 1998b), an d barley (Hoekstra et al. 1992). Not all attempts to obtain plant ma terial through embryo rescue have been successful; embryos generally failed to unde rgo complete differentiation (Dryanovska and Ilieva, 1983; Shail and Robinson, 1987; Marcellan and Camadro, 2000; de Oliveira et al. 2003).

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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 R obinson, 1987; Metwally et al. 1998a; 1998b; Chen et al. 1997; Chen et al. 2002; Gemes-J uhasz et al. 2002; de Oliveira et al. 2003; Sisko et al. 2003; Lotfi et al 2003; Chen et al. 2003). Sim ilar to other plant species, embryo-rescue culture for cu curbits is affected by genot ype (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 -dos age included into the media (Beharav and Cohen, 1995a; 1995b; Metwally et al. 1998b), environmental factors such as temperature and light (Met wally et al. 1998a; Gmes-Juh sz et al. 2002), components added to the media such as carbohydrate source (Metwally et al. 1998b), and embryodevelopmental stage at the time of explant isolation (Ezura et al. 1994a; 1994b; Beharav and Cohen, 1995). Because of these factors which affect effi ciency rate of embryo culture, conditions need to be predetermined for success with sp ecific germplasm. This paper describes an improved procedure for recovery of normal s eedlings from wild type and transgenic ‘Galia’ muskmelon male parental line z ygotic-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 A CC oxidase gene in antisense orientation were obtained using the protoc ol previously described (N unez-Palenius et al. 2005a).

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131 Mature seeds of wild type and transg enic (ACC oxidase antisense) ‘Galia’ ( Cucumis melo L. var. reticulatus Ser.) male parental line were ge rminated 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 (coarsegrade 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 comp lementary light regime (18 h) was supplied by MetalarcR lamps (Osram Sylvania, Inc.) with a light intensity of 350 – 530 mol m-2 s-1. Hermaphrodite flowers were isol ated 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 ma le 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), a nd taken to the lab for subsequent rescue procedures. The surface of th e 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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132 20 per 100 ml) in a sterile beaker. Fruits we re then rinsed three times with sterilized distilled water under axenic conditio ns in a laminar-flow cabinet. In fruits harvested 4 and 10 DAP, seeds were aseptically dissected from wild type and transgenic fruits using a stereomicros cope. For 17, 24 and 30 DAP-fruits, seeds were large enough to be visually di ssected. 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 cont aining 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 inocula tion 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 placi ng 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 251C temperature. After the incubation period, plant material was transferred to ma turation medium ( strength E-21 medium supplemented with 0.7% phytag ar) and cultured between 2 to 5 more weeks under 100 mol m-2 s-1 light and a 16 h photoperiod provided by cool-white fluorescent lamps and constant 251C temperature. Well-devel oped seedlings (cotyledonary stage) were

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133 Table 5.1 Components of E20A 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 myoInositol 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).

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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 mol m-2 s-1 light and a 16 h photoperiod provided by cool-white fluoresce nt lamps and constant 251 C temperature) until a 2-3true-leaf stage was reached. Seedlings were then evaluated for percent regeneration efficiency [(No. of 2-3-true-leaf stag e seedlings X 100)/No. of cultured seeds]. 5.2.3 Experimental Design a nd Statistical Analysis The experimental design was an incomplete f actorial (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 orde r to be analyzed for analysis of variance (ANOVA). Means were separated by T ukey’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 tr ansgenic ‘Galia’ male parental line fruits, which were harvested at 4, 10, 17, 24, and 30 DA P. 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 bei ng cultured with the hilum facing either E-20A or E-21 media (Fi gures 5.1 and 5.2). It has been reported that culture of intact ovules, avoids embryo damage du ring 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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135 (Figure 5.1.c.B). After three more weeks of cu lture, an immature seed ling 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 step s: one more subculture on either E-20A or E21 media for two weeks then cu lture 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 (Figur e 5.2). However, the period to obtain full seedlings was shorter than it was for 4 DAP-int act 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 late r, 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 medi a for further development (Figure 5.2.e). Complete muskmelon ‘Galia’ male parental pl ants 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 s eed) and culturing those seeds with the hilum facing the media (Figur e 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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136 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 i noculation system used (Table 5.2). Greater regeneration efficiency levels were obtain ed when embryos or intact seeds from both genotypes were cultured on E-21 medium th an on E-20A medium (Figure 5.6 and 5.7). These differences were significant (Table 5.3) and observed indepe ndently 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 regenerati on efficiency for wild-type-30 DAP-intact seed on E-21 was more than 90% (Table 5.2). ab cB A D C E 10 mm 10 mm 1 mm

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137 Figure 5.2 ‘Galia’ male parental li ne 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 em bryos 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 Navr atilova 2000; Ondrej et al. 2002). A similar a b c d e 10 mm 1 mm 5 mm 1 mm

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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 li ne fruit at 17 DAP stage (a). In vitro embryo development from 17 DAP stage, (b) ye llow circle encloses the embryo, (c) inoculated seeds (without dissecting th e embryo) with the hilum facing the culture medium, (d) testa remova l from seeds after 15 days of in vitro culture, (e) seedlings after one w eek of testa removal. b a c d 10 mm 1 mm e

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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 an d e) and embryo (f) at 30 DAP stage. Indeed, the efficiency of this technique wa s 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 inocul ation 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 fr om earlier stages such as 4 and 10 DAP. Secondly, the greate st amount of plants (90%) was regenerated through the intact seed method (Figure 5.6B). Thirdly, si gnificant differences were observed between both inoculation systems (Table 5.5). Thus, a gr eater quantity of plan ts can be obtained in a shorter period of time if the embryo is left inside the seed coat. It has been reported that plant geno type can have a profound effect on the efficiency of plant regeneration systems, and it is an important factor in establishing cell selection and genetic transf ormation protocols (Machii et al. 1998; Yamada, 2001; Faure et al. 2002c; El-Itriby et al. 2003). Therefore, plant tissue cu lture methods which have the a bc def

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140 Figure 5.5 Normal ‘Galia’ male parental lin e seedlings obtained from embryo rescue, having well-developed cotyledonary (re d 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 di sregarding plant genotype are considered important tools. Similar regeneration effici ency 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 a ny effect on regeneration efficiency values. It might be interesting to test more GMO-me lon genotypes on this improved E-21 medium.

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141 Table 5.2 Regeneration efficiency of ‘Galia’ ma le 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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142 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 i noculation with the hilum facing the medium) proved to be effici ent in regenerating healthy seedlings from wild type and transgenic imma ture ‘Galia’ male parental line embryos. One novelty of E21 medium is the amendment with putrescin e (polyamine) to rescue embryos from cucurbit seeds (melon). Putrescine has been us ed in other plant species, such as grape, where adding 2 mM putrescine to media cult ure was able to significantly increase the percentage of rescued embryos and normal pl ants (Ponce et al 2002a ; 2002b). It is known that polyamines are active regul ators of plant growth and ha ve the ability to interact synergistic with plant horm ones (Srivastava 2002; Bais and Ravishankar 2002; 2003).

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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 DAP10 DAP17 DAP24 DAP30 DAP% regeneration efficiency 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 DAP10 DAP17 DAP24 DAP30 DAP% regeneration efficiency W.T. PCR+ A B

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144 Figure 5.7 Percentage of ‘Galia’ male parental line immature embryos that developed to form normal plants. Immature embryo s 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 DAP24 DAP30 DAP% regeneration efficienc 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 DAP24 DAP30 DAP% regeneration efficiency W.T. PCR+ A B

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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 St udentized Range Test at P 0.05. Table 5.4 Regeneration efficiency of ‘Ga lia’ 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 St udentized Range Test at P 0.05. Table 5.5 Regeneration efficiency of ‘Ga lia’ 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 St udentized Range Test at P 0.05.

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146 In cucurbits, several regeneration rates fo r different zygotic embryo-rescue methods have been reported. Values such as 9% (F assuliotis and Nelson 1988), 37.3% (Chen et al. 1997; 2002; 2003), 9.1-74% (Metwa lly et al. 1998a; 1998b), 59.4% (Malepszy et al. 1998), 4.5-39.0% (Mackiewicz et al. 1998), 7.1% (Gmes-Juhsz 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 a nd Cohen 1995a; 1995b; Lotfi et al. 2003). The regeneration rates (90%) obtai ned for melon in the present study exceeds significantly those previously reported. Unlike other embryo rescue methods for cucurbits, some of which require doublelayer culture with two or mo re different media, and seve ral subcultures in order to regenerate healthy and complete plants (O ndrej et al. 2002), this competent embryorescue technique for melon, using only one me dium 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 wa s obtained using the protocol previously described (Nuez-Palenius et al. 2005a). Wild type and transgenic muskmelon plants were grown using commercial growing practi ces that included pr uning and training to one vertical stem and the use of soil-less medi a and drip fertigation. Fruits of wild type

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147 and transgenic (ACC oxidase gene in antisense orientation) ‘Galia’ male parental line were harvested at 4, 10, 17, 24 a nd 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 E21 elongation medium, incubated five weeks, and transferred to soil to ev aluate 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 great er 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 co mpetent embryo-rescue technique for wild type and transgenic ‘Galia’ male parental lin e, which can be applied to rescue valuable GMO hybrid-melon embryos.

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148 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 determin ed event that involves a series of changes in color, texture, firmness, aroma, and fla vor, 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 me lon (Giovannoni, 2001). This plant hormone is produced substantially and accumulates during th e climacteric stage, which coincides with the ripening process in mel on fruits (Seymour and Mc Glasson, 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, ev en 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 biosyn thesis 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 th e 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 successfu lly applied to cantal oupe Charentais (cv. ‘Vedrantais’) melon fruits in order to re duce ethylene production, and as a consequence improve fruit quality by delaying ripening and so ftening processes (Ayub et al. 1996; Guis et

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149 al. 1997b; Silva et al. 2004). The reduction of ethylene production in these trangenic melon fruits was 97-99%, compared to their wild ty pe counterpart. However, this strong ethyleneproduction inhibition did not dela y, but rather arrested comple tely the ripening process in those transgenic fruits. Therefore, a continuous ethylene treatment was needed in order to restore fruit ripening, as obser ved by rind yellowing, fruit soft ening 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 (CMA CO-1) might be applied to ‘Galia’ male parental line for use of ‘Galia’ F1 hybrids with improved shelf life. Two completely diploid inde pendent ACC oxidase antisens e transgenic plants of ‘Galia’ inbred male parental line (TGM-AS1 and TGM-AS-2) were previously obtained (Nunez-Palenius et al. 2005a). Experiment s 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 bi ochemical fruit parameters, su ch as weight, length, width, rind color, soluble solids, titratable acidity, pH, flesh thickness, firmness, ripening index, seed cavity size, seed numb er, ACC oxidase activity in vivo and ethylene production were measured during fruit development, specifically, at zero-, halfand 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 A CC oxidase antisense (CMACO-1) or uidA (GUS) gene, were obtained by a cotyledon-protocol descri bed in Nunez-Palenius et al. (2005). Two independent diplod transgenic lines (TGM-AS-1 and TGM-AS-2) were attained with the

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150 CMACO-1 construct, whereas a single inde pendent 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. Th ese 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-3butyric acid) rooting hormone on severed st em bases. Ploidy was confirmed by flow cytometry analysis according to the procedure described in Nuez-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 eva porative-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) followi ng common growing practices recommended by Rodriguez and Cantliffe ( 2001). Complementary light was supplied by MetalarcR lamps with a light intensity of 350-530 mol m-2 s-1 for 18 hours per day. Training, pruning, and fertigation of plan ts, 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.e du/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-, halfand full-slip deve lopmental stages, respectively. TGM-AS-1 and

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151 TGM-AS-2 fruits were harvested at 42, 50 a nd 56 DAP, which correspon ded to zero-, halfand 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 ( 25C), dipped into 200 L 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 ri nd-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 diamete r) and 50-kg load cell. After establishing zero force contact between the probe and the equatorial zone of th e 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.

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152 Figure 6.1 Wild type and TGM-AS ‘Galia’ male pa rental 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 meas urements 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: Seed Cavity Size= X 1 Y 2 + X 2 Y 1 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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153 Total soluble solids (TSS), titratable acidi ty (TA), and pH were determined, in triplicate, from juice obtained after the me socarpic tissue was macerated, centrifugated (28,000x g ), and filtered through Whatman paper No.1. TSS, TA, and pH we re quantificated using a digital refractometer (Abbe Mark10480, Buffalo, N.Y.), a Fisher-395 dispenser connected to an electromet er (Fisher 380), and a digita l pH meter (model 340, Corning, N.Y.), respectively. TA was expressed as per cent malic acid equivalent s. 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 concen tration in the headsp ace of the containers. Sampling for ethylene was taken through a serum stopper where 1 mL of headspace was drawn and injected into a gas chromatogra ph (Hewlett Packard 5890 Se ries II, Avondale, Pa.) as described in Ciardi et al. (2000). The results were expressed as L ethylene produced per kg of tissue per hour ( L.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 ti ssue, was incubated for three hours at 25C in 3 ml reaction buffer, whic h contained 50 mM Tris.HCl (pH 7), 100 mM sucrose, 250 M ACC, and 100 M cycloheximide (Amor et al. 1998). Boile d (95C for 10 min) mesocarpic tissue and reaction buffer alone were used as negative controls. After the incubation period, a 1-mL

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154 gas sample was taken from the head space and analyzed by gas chromatography (Ciardi et al. 2000). Negative controls always had nondetectable ACC oxidase activity. 6.2.4 Experimental Design a nd Statistical Analysis A Completely Randomized Design (CRD) was us ed to set up the e xperiment. Original data was evaluated for analysis of varian ce (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 determina tion in ‘Galia’ male parental line fruit. Six measurements were made in both halv es as indicated by A1, A2, B1, B2, C1 and C2 for flesh size. Y2X2 P (X1,Y1) a b

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155 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 ha lf-slip stage. Conversely, TGM-AS-1 and TGM-AS-2 fruit did not have that increas e in ACC oxidase activity at the same developmental stage. Indeed, expression of th e ACC oxidase antisense gene in transgenic fruits caused a reduction in ACC oxidase activit y, which was four times less compared to WT, azygous-AS and T-GUS fruit at half-slip st age (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 deve lopmental stage (data not shown). At fullslip stage, the lowest level of ACC oxidase activity was detected in TGM-AS-2 mesocarpic tissue, which was significantly differe nt to other genotypes (Figure 6.3). 6.3.2 Ethylene Production Average ethylene production in WT, TG M-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 l.kg-1.h-1, respectively (Figure 6.4). On the next developmental stage, half-slip, a s ubstantial increase in ethylene production was observed for WT, azygous-AS and T-GUS fru it, but not for TGM-AS-1 and TGM-AS-2 genotypes (Figure 6.4). Indeed, th e ethylene production level fo r the ACC oxidase antisense lines was below 7 l.kg-1.h-1 at half-slip stage. Therefore, significant differences were observed between ACC oxidase anti sense fruit and other treatmen ts at half-slip stage (data not shown).

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156 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 ethyl ene 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 SLIPHALF SLIPFULL SLIP ACC oxidase (nl.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

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157 Figure 6.4 Ethylene production from WT ( ), TGM-AS-1 (), TGM-AS-2 ( ), azygous-AS () and T-GUS ( ) fruits. Vertical bars represen t 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 da ta, as well as rind color and total soluble solids data, from WT, azygous-AS and T-GUS genotypes were consid ered as a single average value at zero-, halfand fu ll-slip stages, and named it as ‘WT’. Developmental stage 0 SLIPHALF SLIPFULL SLIP Ethylene ( l.kg-1.h-1) -5 0 5 10 15 20 25 30 WT TGM-AS-1 TGM-AS2 Azygous-AS T-GUS

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158 Figure 6.5 Regression analysis between ACC oxi dase 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 regard ed as one single average value at zero-, halfand 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 ethy lene production data were regarded as TGM-AS. Fruit softening was observed in WT and TG M-AS during the ripening process (Figure 6.6). The maximum decrease in firmness was det ected 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 1.2328 R2 = 0.9115-5 0 5 10 15 20 25 30 0.000.501.001.502.002.503.003.50ACC oxidase activityEthylene production

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159 fruit softening process (Figure 6.6). The reducti on 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, si gnificant 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 halfand full-slip de velopmental stage (Figure 6.7). Significant differences between WT and TG M-AS treatments were observed at those two stages (data not shown). Desp ite 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 in creased along with ri pening (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 a nd 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 a nd 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 ).

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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, meso carpic pH values of WT, azygous-AS, TGUS, TGM-AS-1, and TGM-AS-2 increased concom itantly with fruit ri pening (Table 6-1). No significant differences in mesocarpic pH va lues were found among all treatments at zero-, halfand full-slip stages (Tab le 6.1). TA and pH were inve rsely 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 c ounterpart. Significant differences were Developmental stage 0 SLIPHALF SLIPFULL SLIP Ethylene (l.kg-1.h-1) 0 5 10 15 20 25 30 Firmness (N) 10 20 30 40 50 60 WT-Ethylene TGM-AS-Ethylene WT-Firmness TGM-AS-Firmness

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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 st age 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 wi dth 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 fu ll-slip developmental stage (Table 6.2). Minimum average fruit weight, length and wi dth 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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162 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). B A Developmental stages 0 SLIPHALF SLIPFULL SLIP Ethylene (l.kg-1.h-1) 0 5 10 15 20 25 30 TSS % 6 8 10 12 14 WT-Ethylene TGM-AS-Ethylene WT-TSS TGM-AS-TSS

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163Table 6.1 Postharvest fruit characteris tics of ‘Galia’ male parental line. Genotype Titratable acidity (%) pH Ripening indexz 0-Slipy Half-Slip Full-Slip 0-Slip Half-Slip Fu ll-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 solid s (TSS) and titratable acidity (TA). yDevelopmental stage. xDuncan’s Multiple Range Test at P 0.05. vns= not-significant, significant.

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164 (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 significan tly 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 ot her 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 de tected in TGM-AS-1 at zero-slip stage. Likewise, as previously evalua ted fruit parameters (weight, length, width, and flesh size) there were no significant di fferences among treatments. The production of viable and high-vigor s eeds within plant breeding programs has a paramount importance. Therefore, full and em pty 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 st age (Table 6.3). Conse quently, 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. Ne vertheless, no signifi cant differences for full seed percentage were observed among treatments on zerohalfand full-slip developmental stages (Table 6.3). 6.4 Discussion Fruit ripening is a genetically determin ed event, and consid ered as a highly complex development process (Seymour and McGlasson, 1993). Fruit in general can be classified as either climacteric or non-climact eric on the basis of th eir respiration pattern

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165Table 6.2 Postharvest fruit characteris tics 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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166Table 6.3 Postharvest fruit characteris tics 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.

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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 th e ripening process is proceeding. The use of ACC oxidase antisense transgen ic Charentais melon fruit has allowed discriminating between ethylene-dependent and –independent processes during fruit ripening (Guis et al. 1997b; Gu is et al. 1999; Flores et al 2001a; Flores et al. 2001b; Silva et al. 2004). Indeed, it ha s been suggested that yellowin g of the rind, fruit softening, volatile production, climacteric respiration, and formation of abscission layer in the peduncule are ethylene-dependent events, wh ile sugar accumulation, loss of acidity and pigmentation of the flesh are ethylene-indepe ndent 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 mi ght encompass ethylene-dependent and – independent mechanisms (Guis et al. 1999), which may be activ e at the same time. More research, at molecular and physio logical 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 inserti on 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

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168 order to address that possibility, several pos tharvest quality characteristics on transgenic ACC oxidase antisense ‘Galia’ male parent al fruit were evalua ted in this study. The highest ACC oxidase in vivo activity was observed in WT, azygous-AS and TGUS 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 dele terious 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 wa s present in the reaction buffer; half-life of 19 min with ascorbate. The ACC oxidase react ion buffer assay utilized in the present study was void of ascorbate in order to ci rcumvent this. Low levels of ethylene production by ACC oxidase antisense melon fr uit 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 4.0and 186.7-fold, respectively, compared to WT, azygous-AS and T-GUS fruit. External ethylene production in ‘Galia’ ma le muskmelon fruit had a similar pattern as mesocarpic ACC oxidase activity duri ng ripening. WT, azygous-AS and T-GUS fruit displayed a rapid increase in ethylene producti on at half-slip stage. That ethylene peak was not present in any of the TGM-AS lines Interestingly, ethyle ne production in TGMAS-1 fruit was always higher than TGM-AS-2 at any date of harvest. The average ethylene production at zero-, halfand full-slip stages by TGM-AS-1 and TGM-AS-2 was 7.71 l.kg-1.h-1 and 0.86 l.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

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169 transgene (Nunez-Palenius et al. 2005a). Rather, T-DNA in sertion 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; Cots aftis 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 l.L-1 (Guis et al. 1997b) and 0.09 l.L-1 (Silva et al. 2004). However, after harvesting at 38 DAP, the internal ethylene content increased up to 4 l.L-1 (Guis et al. 1997b). These authors described that detachment of the fruit from th e vine induces an increa se 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 stag e. In summary, the insertion of CMACO-1 gene in antisense orientation lead to redu ced ethylene production in the ‘Galia’ male parental fruit. The average extent of re duction compared to WT, azygous-AS, and a TGUS fruit was 76.9% and 99.1% in TGMAS1 and TGM-AS-2, respectively. Fruit softening observed during ripening is as sociated with textural changes that are thought to result from modification and disasse mbly of the primary cell wall (Fischer and Bennett, 1991). Fruit softening and the unde rlying 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

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170 cycle, along with other typical changes connected with the ripening process (Lester and Dunlap, 1985). Among the different plant hormones wh ich are involved in melon fruit development, ethylene might have the main role during melon fruit softening, because this gaseous plant hormone regulates the expr ession of several prot eins involved in the softening process (Rose et al., 1997, 1998). Accord ing 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 ha d a delayed fruit softening pro cess, 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 firm ness than WT, azygous-AS and T-GUS fruits, mainly at full-slip devel opmental stage, when significant differences were detected. Yellowing of the rind in ‘Galia F1 hybrid fruit is induced by increased chlorophyll degradation during the ripening pr ocess, as well as a partial decrease in total carotenoids (Flgel and Gross 1982). Likewise, chlor ophyll is degraded by the action of chlorophyllase (EC 3.1.1.14), whose de novo synthe sis 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 oxida se antisense fruit did not reach a full golden-yellow color (Figure 6.8) It seems that lower ethyl ene production in CMACO-1 proves this assumption correct. The antisense tr ansgenic ‘Galia’ male fruits did not allow complete chlorophyll degradation, leaving pa rtial green-patches on the rind. Analogous results were reported in ACC oxidase antisense transgenic Cantaloupe Charentais melon

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171 fruits (Guis et al. 1997b; Martin ez-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 c ontrolled 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 compartmentati on 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 TG M-AS-2 mesocarp increased in average from 7.69% to 9.89% at zeroand full-slip st age, respectively. T hus, TSS accumulation was a simultaneous event to fruit ripeni ng 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 antise nse) Cantaloupe Charentais me lon fruit remained attached

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172 to the vine for 10 more days than its wild type counterpart, allo wing the accumulation of more sugars, and increasing the soluble solids content from 11.3 (wild type) to 12 Brix (antisense). However, differences be tween wild type and transgenic Cantaloupe Charentais melon TSS values we re not significant, since their standard errors overlap (see Figure 3A in Guis et al. 1997b). Flores et al (2001b) and Martinez-M adrid et al. (2002) studied biochemical and physiological fruit char acteristics 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 a nd 37 DAP. Silva et al. (2004) developed transgenic (ACC oxidase antisense from appl e) Cantaloupe Charenta is 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 frui t 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 tran sgenic ACC oxidase antisense Cantaloupe Charentais cv. ‘Vedrantais’ fruits (Flores et al. 2001b; Martinez-Madr id 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 (S eymour and McGlasson, 1993; Stepansky et al. 1999; Burger et al. 2002; Burger et al 2003; Liu et al. 2004). Moreover, different plant growing environmental c onditions might have played a role on fruit-TSS buildup.

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173 Sugar accumulation in melon fruit is aff ected quantitatively by environmental and physiological factors, such as salinity, nutrien t 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 antisens e gene insertion did not reduce or increase the fruit TSS content in a ny of CMACO-1 transgenic lines at fruit maturity. Unlike other ripe fruits, such as strawb erry, 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. Si milar results were obtained by Guis et al. (1997b) and Martinez-Madrid et al. (2002). These authors re ported that TA had a sharp decrease in WT Cantaloupe Charentais mel on fruit during ripening, and a similar trend was observed in transgenic ACC oxidase antisense fruit. Fruit ripening index calculation is comm only 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 i ndex was observed in WT, azygous-AS, TGUS, TGM-AS-1 and TGM-AS-2 fruits on all fruit development stages, except at fullslip stage where significant differences were found between TGM-AS-2 and other treatments. It has been suggested by Silva et al. (2004) that orga nic acid metabolism in

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174 melon fruit is an ethylene-dependent pro cess. Likewise, Flores et al. (2001b) and Martinez-Madrid et al. (2002) reported that transgenic (A CC oxidase antisense) had a greater ripening index than wild type Cantaloupe Charentais me lon fruit. Considering that TGM-AS-2 genotype had the lowest ethylen e production level among treatments, it is possible that this low-ethylene level in TG M-AS-2 has contributed to attain a high TA value at full-slip stage, consequently, the lo west fruit ripening index level at full-slip stage was observed for TGM-AS-2. Although differences in weight, length, widt h, flesh size and cavity seed size were found among WT, TGM-AS-1, TGM-AS-2, az ygous-AS and T-GUS fruits, they were not significantly different. Moreover, comparab le variation in fru it weight and size has been reported in other muskmelon cultivars (K ultur et al. 2001; Nerson et al. 2002; Liu et al. 2004). Overall, the CMACO-1 gene single in sertion 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 trea tments, it is clear that CM ACO-1 gene single insertion did not have any effect on seed developmen t in ‘Galia’ male parental line fruit. In summary, the results of these experime nts 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 lo w 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

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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 mu skmelon cultivar that is grown in greenhouses or tunnels to maximize fruit quality and yiel d. Maximum fruit qual ity and flavor are achieved when ‘Galia’ fruits are harvested at 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 us ed to increase its fruit shelf-life. The male parental line of ‘Galia’ muskmelon has been tr ansformed 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 (NunezPalenius et al. 2005a). Transgenic ACC oxidase antisense (TGM-AS), azygous-AS (PCR negative), transgenic GUS (T-GUS) and wild ty pe (WT) fruits, from plants grown in the greenhouse, were harvested at zero-, half-, a nd 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, a nd full seed percentage parameters were not significantly different on all fruit developm ental 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 fr uits were greater

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176 than from TGM-AS fruits. TGM-AS ‘Galia’ male parental melon fruits had a delayed fruit ripening process compared to wild type fruits.

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177 LIST OF REFERENCES Adams, D.O., and S.F. Yang. 1979. Ethylen e Biosynthesis Id entification of 1Aminocyclopropane-1-Carboxylic Acid as an Intermediate in the Conversion of Methionine to Ethylene. Proceedings of the National Academy of Sciences of the United States of America. 76: 170-174. Adelberg, J. 1993. Tetraploid Melon from Tissu e Culture and their Triploid Hybrids. Ph. D. Clemson University, Clemson, SC. Adelberg, J., Fujiwara, K., Kirdmanee, C ., and T. Kozai. 1999. Photoautotrophic Shoot and Root Development for Triploid Me lon. Plant Cell Tissue and Organ Culture. 57: 95-104. Adelberg, J., Nugent, P., Rhodes, B., Zha ng, X.P., and H. Skorupska. 1995. Fertility and Fruit Characters of Hybrid Triploid Melon. Breeding Science. 45: 37-43. Adelberg, J.W., Rhodes, B.B., Skorupska, H. T., and W.C. Bridges. 1994. Explant Origin Affects the Frequency of Tetraploid Plants from Tissue-Cultures of Melon. Hortscience. 29: 689-692. Aggelis, A., John, I., and D, Grierson. 1997a. Analysis of Physiol ogical and Molecular Changes in Melon ( Cucumis melo L) Varieties with Diffe rent Rates of Ripening. Journal of Experime ntal Botany. 48: 769-778. Aggelis, A., John, I., Karvouni, Z., and D. Grierson. 1997b. Characterization of Two cDNA Clones for mRNAs Expressed During Ripening of Melon ( Cucumis melo L.) Fruits. Plant Molecular Biology. 33: 313-322. Aharoni, Y., Copel, A., and E, Fallik. 1993a. Hinokitiol (Beta-Thujaplicin), for Postharvest Decay Control on Galia Mel ons. New Zealand Journal of Crop and Horticultural Science. 21: 165-169. Aharoni, Y., Copel, A., and E, Fallik. 1993b. Storing Galia Melons in a ControlledAtmosphere with Ethylene Absorb ent. Hortscience. 28: 725-726. Aharoni, Y., Copel, A., and E, Fallik. 1994. The Use of Hydrogen-Peroxide to Control Postharvest Decay on Galia Melons. A nnals of Applied Biology. 125: 189-193. Aharoni, Y., Copel, A., Davidson, H., and R. Barkaigolan. 1992. Fungicide Application in Water and in Wax for Decay Control in Galia Melons. New Zealand Journal of Crop and Horticultural Science. 20: 177-179.

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215 BIOGRAPHICAL SKETCH Hector Gordon Nuez-Palenius was bor n on January 19, 1962, in Guadalajara, Jalisco state, Mxico. He got his Master of Science degree in December, 1997 in plant biology from the Genetic Engineering Depart ment at the Unit of Biotechnology and Plant Genetic Engineering in the Center of Re search and Advanced Studies (CINVESTAVIPN. Unidad Irapuato). Afterwards, Hector G. Nuez-Palenius started a Ph.D. program at the University of Florida, Horticultural Sc iences Department, S eed Physiology Lab. His research focused on transformation of ‘Ga lia’ muskmelon to improve fruit quality, specifically on in vitro rege neration and Agrobacterium-me diated transformation of ‘Galia’ parental lines. His future plans are to continue working in the area of biotechnology of cucurbits, with spec ial emphasis on genetic improvement.


Permanent Link: http://ufdc.ufl.edu/UFE0011467/00001

Material Information

Title: Transformation of 'Galia' melon to improve fruit quality
Physical Description: Mixed Material
Language: English
Creator: Nuñez Palenius, Hector Gordon ( Dissertant )
Cantliffe, Daniel J. ( Thesis advisor )
Huber, Donald J. ( Reviewer )
Klee, Harry J. ( Reviewer )
Hopkins, Donald ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2005
Copyright Date: 2005

Subjects

Subjects / Keywords: Horticultural Science thesis, Ph. D.
Dissertations, Academic -- UF -- Horticulture

Notes

Abstract: 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 beta-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 alpha-Naphthaleneacetic acid (NAA) as 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.
Subject: ACC, Agrobacterium, ethylene, Galia, transformation
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 230 pages.
General Note: Includes vita.
Thesis: Thesis (Ph. D.)--University of Florida, 2005.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
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Material Information

Title: Transformation of 'Galia' melon to improve fruit quality
Physical Description: Mixed Material
Language: English
Creator: Nuñez Palenius, Hector Gordon ( Dissertant )
Cantliffe, Daniel J. ( Thesis advisor )
Huber, Donald J. ( Reviewer )
Klee, Harry J. ( Reviewer )
Hopkins, Donald ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2005
Copyright Date: 2005

Subjects

Subjects / Keywords: Horticultural Science thesis, Ph. D.
Dissertations, Academic -- UF -- Horticulture

Notes

Abstract: 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 beta-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 alpha-Naphthaleneacetic acid (NAA) as 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.
Subject: ACC, Agrobacterium, ethylene, Galia, transformation
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 230 pages.
General Note: Includes vita.
Thesis: Thesis (Ph. D.)--University of Florida, 2005.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0011467:00001


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TRANSFORMATION OF 'GALIA' MELON TO IMPROVE FRUIT QUALITY


By

HECTOR GORDON NUNEZ-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 Nufiez-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 Nufiez Vargas and Salvador Federico Nufiez Palenius, my
Moms Janette Ann Palenius Alberi and Consuelo Nufiez Solis, my wife Nelida Contreras
Sanchez, my son Hector Manuel Nufiez Contreras and my daughter Consuelo Janette
Nufiez Contreras.















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









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-Mexico) and EDUCAFIN (Guanajuato-Mexico) which

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
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

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

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

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

CHAPTER

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

2 LITERA TURE REVIEW .......................................................... ..............4

2.1. Im portance of M elon ........................................................................ ......... ...... 4
2 .1.1 Introduction ............... ..... ............................. ................ .. 4
2.1.2 B otany and Origin of Cucum is m elo ........................................ .................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 G uis's categories: .................................................... ............. 9
2.1.4 Climateric and Non-climateric Fruits .. ............... ................. ................... 11
2.2 Postharvest Physiology of M elon .................................................... .................. 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 In tro du ctio n ...... .... .... ...................... ................................ .. .. .. 15
2.2.3.2 Carbohydrate Metabolism ..................... .....................16
2.2.3.3 O rganic A cids......................... .. .................. .......... .... ...... .... 19
2 .2 .3 .4 V o latile s ..................................................................................... 2 0
2.2.3.5 Cell W all D egradation.................................. ........................ 23
2 .2 .3 .6 P igm ents ............................ .. .. ...................................... 2 6
2.2.4 Ethylene and Molecular Changes During Ripening................................28
2 .2 .4 .1 Introdu action .................. .. .................. ................. .. 2 8
2.2.4.2 Biosynthesis, Perception and Effects of Ethylene............................28
2 .3 M elon B biotechnology ........................................ ............................................36
2.3.1 Genetic Im provem ent ......... .... ......... ......................... .. ............... 36
2.3.1.1 T traditional B reeding ............................................ ............... .... 36
2.3.1.2 Improvement Through Genetic Engineering...................... ..37









2.3.1.3 Melon Biotechnology ........... ..... ................. ...............42
2.3.2 In vitro R generation ...................... .. .. ........................... ... ............ 43
2 .3.2 .1 G enetic C control ........................ .................... ............... .... 44
2.3.2.2 Polyploidization and Somaclonal Variation................ ......... 46
2 .3 .2 .3 V itrifi cation .................................. ................ .................. .... 5 1
2.3.3 Regeneration by Organogenesis ............... ........................................53
2.3.3.1 M edium Com position.................................... ....................... 54
2.3.3.2 Environm mental Factors .......................................... ............... 60
2.3.4 Regeneration by Somatic Embryogenesis .................................................61
2.3.4.1 M edium Com position.................................... ....................... 65
2.3.4.2 Environm mental 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' m elon ...................... .... .............. .............. ............ 82
2 .4 .1 Introdu action ............................................................82
2 .4 .2 B otany an d O rigin ........................................................... .....................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 M materials and M ethods .............................................................. .....................97
3.2.1 P lant M 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 A ssay ................................... .. ............................ 100
3.2.5 Experimental Design and Statistical Analysis..............................100
3.3 R results and D discussion .............. ... ............................. ....................... ... 101
3.3.1 Effect of Explant Origin on Regeneration Efficiencies............................101
3.3.2 Plant Transform ation ......................................................................... 102
3 .3 .3 S u m m ary .............................................................. 1 10

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

4.1 Introduction......................................................................... .... ..... 111
4.2 M materials and M ethods ......................................................... ............... 113
4.2.1 Plant M material ..................................... ...... .. ...... .. ........ .... 113
4 .2 .2 P lant R generation ............................................................. ................. ... 113
4.2.3 Agrobacterium Inoculation and Plant Transformation.............................114









4.2.4 Flow Cytometry Analysis...... ..................... ...............115
4.2.5 D election of Transgenes ........................................... .......................... 116
4.2.6 Southern Blot Analysis.............................. ...................... 117
4.2.7 Segregation Analysis of Transgenes in Primary Transformants ............118
4.3 R results and D discussion ......................................................... .............. 118
4.3.1 Transform ation Efficiency.............................................. .................. 118
4.3.2 Ploidy Level of Prim ary 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 C onclu sion ...... .. ............... ........................................... ............ 124
4 .5 .1 S u m m ary ............................................................................................. 12 7

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

5.1 Introduction.................................................... ........................... ....... 129
5.2 M materials and M ethods ............................................... ............................ 130
5 .2 .1 P lant M material ............................................... .. .. ...... .. ................130
5.2.2 Em bryo Culture ................................................. .... ................. 131
5.2.3 Experimental Design and Statistical Analysis.................... .......... 134
5.3 R results and D iscu ssion .......................................................................... .. .... 134
5.3.1 Em bryo D evelopm ent......................................... .......................... 134
5.3.2 R generation Efficiency ................................... ..................................... 136
5.4 Sum m ary ..................................... ................................ ..........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 M materials and M ethods .............................................. ............................. 149
6.2.1 Plant M 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 A CC Oxidase Activity In Vivo ..................................... ............... 155
6.3.2 Ethylene Production ........................................... .......................... 155
6.3.3 Firm ness, 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 D iscu ssio n ........................ .. ..............................
6.5 Summary ............... ......... ................... 175

LIST O F R EFEREN CE S ......... .............................. .............................. ............... 177

B IO G R A PH IC A L SK E T C H ........................................ ............................................215



viii
















LIST OF TABLES


Table pge

2.1 Production of various cucurbits on different countries in 2004 (1,000 metric
ton s) ............................................................. . .5

2.2 Similarities between conventional and biotechnological methods for melon plant
im prove ent .................................................................................................. .......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 'G alia' Type M uskm elon Im ports 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
ex p lant sou rce......................................................................................... 10 3

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

5.1 Com ponents of E-20A nutrient m edium ...............................................................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 m edia. .................................................................... 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









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
















LIST OF FIGURES


Figure pge

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.
(2 004). .............................................................................. 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
ex p lan t sou rce ........................................................................................ 10 1

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 To shoots and roots of'Galia'
m u skm elon 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'
m elon 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 P-D-Glucuronidase (GUS) gene in To 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









4.5 Flow cytometry analysis of propidium iodide-stained nuclei from wild type leaf
tissue of 'G alia' m ale parental line. ................................................. ...............125

4.6 Flow cytometry analysis of propidium iodide-stained nuclei from transgenic leaf
tissue of 'G alia' m ale 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
develop ent from 4 D AP 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 norm al plants. ........................ .... ................ .. ...... ... ............... 143

5.7 Percentage of 'Galia' male parental line immature embryos that developed to
form norm al 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-A S (=) and T-GU S (m) fruits.............................................. ...............156

6.4 Ethylene production from WT (*), TGM-AS-1 ()), TGM-AS-2 (->), azygous-
A S (=) and T-GU S (m) fruits. ..... ...................................................................... 157

6.5 Regression analysis between ACC oxidase activity and ethylene production in
'G alia' m ale parental fruits. ........................................... ............. ...............158

6.6 Ethylene production from WT (*) and TGM-AS ()) fruits compared to firmness
of WT (m) and TGM-AS (o) intact fruits....................... .......................160

6.7 Ethylene production from WT (*) and TGM-AS ()) fruits compared to rind
color of WT (m) and TGM-AS (o) intact fruits................................. ...............161

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









6.9 Ethylene production from WT (*) and TGM-AS ()) fruits compared to TSS
content of WT (m) and TGM-AS (o) intact fruits .................................................162















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 Nufiez-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

P-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 mgL-1 benzyladenine (BA) and 0.001 mg'L1 ca-Naphthaleneacetic acid (NAA) as









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) To 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 To evaluated fruits, will be

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














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 (6-8C).

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).









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. Vedrantais), 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.









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.














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










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 modem cultivars with shortened intemodes, 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 bore 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 unbranchedd).









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

pistillatee 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,

Perin 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









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 occurred (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









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;

GrebenSikov, 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.









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. 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.









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; Perin 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,









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 reticulatuss) 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









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









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

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

of 2-5 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 100 C and 85-90% relative humidity up to three or four

weeks, but lower temperatures of 6 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 reticulatuss 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,









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; Guillen 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









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; Lelievre 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).









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









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 andflexuosus 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.









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-









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 environmental 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









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 reticulatuss

varieties) and Honey Dew inodoruss 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,









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 reticulatuss). 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).









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 majority 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









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 substantial 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 P-galactosidases and / or P-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.









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
another were overexpressed in the LeExp protein. Tomato fruit in which Expl
protein accumulation was inhibited by 3% were firmer than control fruit throughout
the ripening process. Conversely, fruit overexpressing high amounts of LeExp
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 substantial 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 expansion









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: P-carotene (84.7%), 6-carotene (6.8%), a-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',









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

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

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

about 4.0-tlg/g P-carotene, and phytofluene; a-carotene, C-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 P-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). Flugel and Gross (1982) studied

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









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; Guillen et al. 1998; Martinez-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-l-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 formerly

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).









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-AC02 and CM-AC03, were only interrupted by two introns, at

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

CM-AC03 relative to CM-ACO1 was 75%. In contrast, the degree of DNA homology of

CM-AC02 relative to both CM-ACO1 and CM-AC03 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,









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-AC02, and CM-AC03). 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-AC02 was detectable at low

levels in etiolated hypocotyls, whereas CM-AC03 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-AC03 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 P-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









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-









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-ERS] (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-ERS 1 protein during melon fruit development. They reported that Cm-

ERS 1 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-ERS 1 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









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 (Lelievre et al.

1997; Giovannoni, 2001; Perin 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, Perin 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









the non-climacteric phenotype in fruit tissues was attributable to ethylene insensitivity

conferred by the receive 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, ca-

arabinosidase, P-galactosidase, endo-polygalacturonase, ACC synthase (negative

feedback) and ACC N-malonyltransferase. Likewise, these authors categorized ethylene-









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

(Lelievre et al. 1997; Aggelis et al. 1997a; Aggelis et al. 1997b; Yang and Oetiker, 1998;

Jiang and Fu, 2000; Perin 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;

Perin 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.









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 transmitted 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,









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

different technologies such as gene manipulation, gene transfer, DNA typing and

cloning of genes, plants and animals (FAO, 2004).









Table 2.2 Similarities between conventional and biotechnological methods for melon
plant improvement.
Steps in Breeding Conventional Methods Biotechnological Methods


1. Collection and Genetic analysis of Cloning of valuable gene
evaluation of genetic valuable traits
resources
2. Generation of variation Intraspecific crosses Genetic transformation,
embryo culture, somatic cell
fusion, polyploidy, anther
culture, and somaclonal
variation
3. Selection of desirable Growth and evaluation PCR, selectable markers,
variants DNA markers, etc.


4. Production of fixed Self-pollination Self-pollination and haploid
lines production by gynogenesis


5. Seed production Growth in controlled DNA markers to test seed
conditions 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









organ under a sterile environment, leading to a whole de novo regenerated plant (Nunez-

Palenius et al. 2005a).

After the milestone plant transformation 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; Nufiez-Palenius et al. 2005). The first transgenic commercial

plant variety in the USA was released in May 1994, when Calgene marketed its Flavr-

SavrTM 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









$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

90
80
-*- Total
70 --- Industrial Countries
S60 -A- Developing Countries
5 50 -
S40 -
30
20 -
10 -
0

1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

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 substantial

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).











90
13.3
80
9
70 I Increase
6.1 over
S60 8.4 Previous
50 -4.3 Year
| 12.1 El Previous
40 168 Year
M 16.8
30
20 9.3
10 1.7
0-
1996 1997 1998 1999 2000 2001 2002 2003 2004
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.

























2ARGENTINA ".c ". 10 AUSTRALIA
16.2 Million Has. A n I -< on c 0.2 Million Has.




9 URUGUAY 4 BRAZIL A
0.3 Million Has. 5.0 Million Has.
Soybean, Maize Soybean

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 substantially 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









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; Nufiez-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 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 (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.









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 reticulatuss, 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 Fi 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 different 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









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









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 Fi 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 haploidd 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









(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









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









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 different 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.









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 |tm 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 |tm 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;









Gilissen et al. 1993; Dolezel et al. 2004). Nevertheless, 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). Unfortunately, this regeneration system did not generate completely

developed transgenic shoots for other commercial melon cultivars (Nufiez-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).









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









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; Nufiez-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.









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/1 or higher) to induce bud formation. BA concentration is lowered (0.5 mg/1 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).














Table 2.3 Melon regeneration (shoots, roots and/or complete plants) through direct organogenesis.
Explant Source Cultivar Induction Medium Reference
(Plant Growth Regulators)
Cotyledon and primary leaf 'Bananna', 'Dixie Jumbo', Planters BR-1 medium: 0. mg/l NOA, 20 mg/1 2iP, and 0.1 mg/1 1


Hypocotyl from 11 to 13-day-old
seedlings

Leaf (0.3-0.5 cm) from 14-day-old
seedlings
Cotyledons


Cotyledons from mature seeds, and
cotyledons and leaves from 5 to 7-day-
old seedlings

Cotyledons from 4-day-old seedlings


Cotyledons from 4-day-old seedlings

Cotyledons from 9 to 10-day-old
seedlings


Jumbo', 'Morgan', 'Cavaillon Red
Flesh', and 'Saticoy Hybrid'


'Amarillo Oro'


'Pusa Sharbati'

'Halest Best', 'Iroquois', and
'Perlita'

'Accent', 'Galia', '4215', 'Preco',
and 'Viva'


'Superstart', 'Hearts of Gold',
'Hale's Best Jumbo' and 'Goldstart'


Not reported


CCC
HC medium: 0.05 mg/1 NOA, 10 mg/1 2iP, and 0.1 mg/1
CCC


4.5 mg/1 IAA


0.22 mg/1 BA and 0.2 mg/1 2iP

NAA and BA


1 mg/1 BA


0.88 mg/1 IAA and 1.13 mg/l BA


0.2 mg/l BA 7

1 mg/l 8


'Topmark'














Table 2.3 Continued
Explant Source

Cotyledons from mature seed,
cotyledons and hypocotyl from 10-
day-old seedlings, and leaf segment
and petioles from 3-weeks-old
seedlings

Cotyledons from 8-day-old seedlings


Cotyledons from 2-day-old seedlings

Cotyledons from 7-day-old seedlings

Cotyledons from immature seeds

Cotyledons from mature seeds

Cotyledons from 4-day-old seedlings

3-4 cm diam expanded leaves


Cotyledons from 7-day-old seedlings


Cultivar

'Earl's Favorite Harukei No.3'





'Charentais' and 'Gulfstream'


'Sunday Aki'

Five Inbred lines from Teziers

'Miniloup', 'L-14', and 'B-Line'

'Prince'and 'Andes'

11 genotypes

'Hale's Best Jumbo' and 'Ananas El
Dokki'

'Pusa Madhuras'


Induction Medium
(Plant Growth Regulators)
0.01 mg/1 2,4-D or 1 mg/1 IAA and 0.1 mg/1 BA





1.12 mg/1 BA for 'Charentais' and 1.12 mg/1 BA and 1.75
mg/1 IAA for 'Gulfstream'

1 mg/1 BA, 50-200 pM salycilic acid and 10 mM proline

0.1 mg/1 NAA and 0.5 mg/1 BA

2.25 mg/1 BA

1 mg/1 BA

0.63 mg/1 BA and 0.26 mg/1 ABA

0.87 mg/1 IAA, 1.13 mg/1 BA and 0.026 ABA


0.22 mg/1 BA


Reference

9














Table 2.3 Continued
Explant Source


Cotyledons from 2-week-old seedlings

Leaves from 10 day-old seedlings

Cotyledons from 2-day-old seedlings


Cotyledons from mature seeds

Proximal zone of the Hypocotyl from
4-day-old seedlings

Cotyledons from 4 to 5-day-old-
seedlings


Cultivar


14 cultivars

'VWdrantais'


'Galia' male and female parental
lines


'Yellow Queen', 'Yellow King',
and 'Hybrid AF-222'
'Revigal'


Some Turkish cultivars: 'Hasanbey'
I, 'Yuva', 'Kirkagac 637',
'Topatan', 'Kuscular' and 'Ananas'


Induction Medium
(Plant Growth Regulators)
Embryogenesis: 1.98 mg/1 2,4-D and 4.99 mg/1
Organogenesis: 0.01 mg/1 2,4-D and 0.059 mg/1 BA
0.22 mg/1 BA and 0.33 mg/ 2iP


0.001 mg/1 NAA and 1 mg/1 BA


1 mg/1 BA

1 mg/1 BA


Medium and plant growth regulators from reference 6


Cotyledons from mature seeds Thirty melon genotypes 1 mg/1 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.
Nufiez-Palenius et al. (2002), 21. Stipp et al. (2001), 22. Curuk et al. (2002a), 23. Curuk et al. (2002b), 24. Galperin et al. (2003a).


Reference












Table 2.4 Melon regeneration through indirect organogenesis.
Explant Source Cultivar Callus Induction Medium (CIM) and Shoot Reference
Induction Medium (SIM)
(Plant Growth Regulators)


Cotyledon-callus culture from 11 to 13-
day-old seedlings

Hypocotyl-callus culture from 7-day-
old seedlings

Cotyledon-callus from 7 to 9-day-old
seedlings

Cotyledon-callus from 11 to 13-day-old
seedlings


Cotyledon-protoplasts from 2-week-old
seedlings



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


'Amarillo Oro'


'Pusa Sharbati'


'Charentais T',
'Doublon', 'CM 17 187',
and 'Piboule'
15 cultivars belonging to
cantaloupensis, inodorus
and reticulatus varieties

'Hong-Xin-cui'




'Cantaloupe PMR'


CIM: 1.5 mg/1 IAA and 6.0 mg/l KIN
SIM: 0.Olmg/l NAA and 0.1 mg/1 BA

CIM: 1.0 mg/1 IAA and 0.5 mg/l KIN
SIM: 0.5 mg/1 BA and 0.5 mg/1 2ip

CIM: 2 mg/1 IAA and 2 mg/1 KIN
SIM: no plant growth regulators

CIM: 6.0 mg/1 KIN and 1.5 mg/1 IAA
SIM: Same as CIM


Protoplast Culture Medium and CIM: 0.5 mg/1
2,4-D, 0.5 mg/1 Zeatin and 0.5 mg/1 BA
SIM: 0.3 mg/1 2,4-D, 1.0 mg/1 Zeatin and 0.5
mg/1 BA

CIM: 5.0 mg/l NAA and 2.5 mg/1 BA
SIM: no plant growth regulators


Cotyledon-protoplasts from 2-week-old 'Charentais'
seedlings


Protoplast Culture Medium and CIM: 0.05 mg/1
2,4-D and 0.5 mg/1 BA. SIM: 2 mg/1 BA












Table 2.4 Continued
Explant Source


Cultivar


Callus Induction Medium (CIM) and Shoot
Induction Medium (SIM)
(Plant Growth Regulators)


Cotyledon-protoplasts from 12-day-old 'Charentais T' and Fl Protoplast Culture Medium and CIM: 0.75 mg/1 BA 8
seedlings and fully expanded leaves- hybrid cv. 'Preco' SIM: 1.0 mg/1 2,4-D and 0.1 mg/1 BA
protoplasts from 3-week-old seedlings

Root-callus culture from 21-day-old 'Pusa Sharbati' CIM: 0.61 mg/l 2iP and 0.68 mg/l BA 9
seedlings SIM: 0.22 mg/1 BA

Cotyledon and Hypocotyl from 11-13 'Charentais' CIM: 2.5 mg/l NAA and 1 mg/l BA 10
day-old-seedlings SIM: 0.01 mg/1 NAA and 6 mg/1 Kin
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).


Reference









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 290C) and light (0, 5, 10, 30, 60 and 3,000 [tmolm-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 290C under a range of

light intensity of 5 to 30 [tmolm-2s-1. Conversely, lower temperatures (22 and 25C),

darkness, and higher light intensities (60 and 3,000 [tmolm.2s-1) reduced bud initiation.

Similarly, Kintzios and Taravira (1997) tested two levels of light intensity, 50 and 250

[tmolm.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 [tmolm.2s-1) values adversely affected shoot induction from

cotyledonary explants. Interestingly, Curuk et al. (2003) recently described that









regeneration from hypocotyls proximall part to the cotyledons) of Cucumis species does

not require 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 [LM 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









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,












Table 2.5 Melon regeneration through somatic embryogenesis.
Explant Source Cultivar Induction Medium (IM) and Development Reference
Medium (DM)
(Plant Growth Regulators)


Cotyledons from 5-day-old
seedlings

Expanded cotyledons


Cotyledons from mature seeds
and cotyledons and hypocotyls
from 10-day-old seedlings, leaves
and petioles from 3-week-old
plantlets

Cotyledons and hypocotyls from
mature seeds

Hypocotyls from mature seeds


Cotyledons from 1-day-old
seedlings

Protoplasts from 12-day-old
cotyledons


'Hale's Best No.36' and
'Rocky Ford'

'Charentais T'


'Earl's Favorite Harukei No.
3'




'Green Pearl' and 'Earl's
Favourite'

'Earl's Favourite'


'Earl's Favourite Haru 1'


'Charentais T' and Fl
hybrid 'Preco'


IM: 1.0 mg/1 2,4-D and 0.5 mg/l BA
DM: 0.5 mg/1 2,4-D and 0.25 mg/l BA

IM: 4.52 mg/1 2,4-D and 0.44 mg/l BA
DM: no hormones

IM: 2.0 mg/1 2,4,-D or 25 mg/1 IAA
DM: no hormones




IM: 4 mg/1 2,4-D, 2 mg/1 NAA, and 0.1 mg/1 BA
DM: no hormones

IM: 1 mg/1 2,4-D, 1 to 4 mg/1 NAA, and 0.1 mg/1
BA
DM: no hormones

IM: 1 mg/1 2,4-D, 1 mg/l NAA, and 0.1 mg/1 BA
DM: no hormones

IM: 1 mg/1 2,4-D and 0.1 mg/1 BA
DM: no hormones













Table 2.5 Continued
Explant Source


Cotyledons and hypocotyls from
mature seeds


Cotyledons from mature seeds


Mature seeds


Hypocotyls from imbibed seeds


Cotyledons from mature seeds


Cotyledons from mature seeds

Mature seeds


Cultivar


18 cultivars belonging to
reticulatus, inodorus,
makuwa and intermediate
type between reticulatus
and cantaloupensis varieties


52 cultivars


'Earl's Favourite'


'Prince' and 'Sunday Aki'


'Vedrantais'


'Yellow Queen' and
'Yellow King'
'Vedrantais' and 'Earl's
Favoutire Fuyu A'


Induction Medium (IM) and Development
Medium (DM)
(Plant Growth Regulators)
IM: 3.0 mg/1 2,4-D and 0.1 mg/1 BA
DM: no hormones


IM: 5 mg/1 2,4-D and 0.1 mg/1 TDZ
DM: no hormones

IM: 4 /tg/l 2,4-D and 0.1 [[g/l BA
DM: no hormones

IM: 1 mg/1 2,4-D, 2 mg/1 NAA and 0.1 mg/1 BA
DM: no hormones

IM: 2.2 mg/1 2,4-D and 0.11 mg/1 BA
DM: no hormones

IM: 5.0 mg/1 2,4-D and 0.075 mg/1 TDZ
DM: no hormones
IM: 2.0 mg/1 2,4-D and 0.1 mg/1 BA
DM: no hormones


Reference


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).









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 mgL-1) was supplemented into the









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).









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 Fi 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









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 y-

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 affected 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









an average number of haploid embryos of 3.5%, whereas 'F .Gl' 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 y-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 y-

radiation used (Cuny et al. 1992; 1993; Yanmaz et al. 1999). High y-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) demonstrated 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









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









transformation were abnormal. Mefoxine, was used to stop Agrobacterium growth may

have caused this.

Transformation success via Agrobacterium or particle gun bombardment 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;

Nufiez-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 mgL-1 kanamycin, fresh overnight grown bacteria at a concentration of 107

- 108 bacteriaml-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. Unfortunately, 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 lggL-1 methotrexate and 100









mgL-1 kanamycin, 5-6 days of co-cultivation period, and immediate selection pressure

was routinely used.

These experiments were carried out on 'Orient Sweet, F Hybrid' melon cultivar.

Valles 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,

nptll, which provides tolerance to aminoglycoside antibiotics (Fang and Grumet, 1990;

Dong et al. 1991; Yoshioka et al. 1992; Gonsalves et al. 1994; Valles 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 (Nufiez-Palenius et al. 2002;

2003) have been used. When typical concentrations (75-150 mg1-1) 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; Nufiez-

Palenius et al. unpublished results, Ezura et al. personal communication).












Table 2.6 Genes transferred to melon by plant genetic transformation.
Transgene Phenotypic Trait Explant used and

morphogenetic pathway
npt-II gene Selectable marker 4 to 5-day-old cotyledons

Organogenesis
npt-II, Selectable markers Mature cotyledons
Dihydrofolate and reporter gene Organogenesis


reductase, and
luciferase gene
CMV-coat protein

gene
uidA gene


ZYMV-coat

protein gene
uidA gene


CMV-white leaf
coat protein gene


Virus resistance


GUS Reporter gene


Potyvirus resistance


GUS Reporter gene


Virus resistance


Mature cotyledons
Organogenesis
3-day-old cotyledons
Organogenesis
4 to 5-day-old cotyledons
Organogenesis
5-day-old cotyledons
Organogenesis
3-day-old cotyledons
Organogenesis


Cultivar


'Hale's Best Jumbo'


'Orient Sweet, F 1
Hybrid'




'Prince' and 'EG360'


'Galia'


'Hale's Best Jumbo'


'Amarillo Oro'


'Burpee Hybrid', 'Hale's
Best Jumbo', 'Harvest
Queen', 'Hearts of
Gold', and 'Topmark'


Transformation
Method

A. tumefaciens
LBA4404 strain
A. tumefaciens
GV3111SE strain




A. tumefaciens
LBA4404 strain
Particle
bombardment
A. tumefaciens
LBA4404 strain
A. tumefaciens
LBA4404 strain

A. tumefaciens
C58Z707 strain


Reference


1


2













Table 2.6 Continued.
Transgene


ZYMV, WMV, and
CMV coat protein
genes
npt-II gene




ACC oxidase
antisense gene from
melon
HAL 1 gene




ACC synthase
antisense gene
SAM hydrolase gene
ACO anti-sense gene
from melon


Phenotypic Trait


Virus resistance




Selectable marker




Improved fruit quality




Halotolerance




Improved fruit quality


Improved fruit quality
Improved fruit quality


Explant used and

morphogenetic pathway

Leaves

Organogenesis


Cotyledons

Organogenesis


5-day-old cotyledons

Organogenesis


7-day-old cotyledons and
2-week-old leaves

Organogenesis
NRz


NR
10-day-old true laves

Organogenesis


Cultivar


'Don Luis', 'Galleon',
'Hiline', 'Mission', and
'Parental inbred'
'Eden Gem'




'Vedrantais'




'Pharo' and 'Amarillo
Canario'


NR


NR
'Vedrantais'


Transformation

Method

A. tumefaciens




A. tumefaciens
and Particle
bombardment
A. tumefaciens
LBA4404 strain


A. tumefaciens
LBA4404 strain


A. tumefaciens


A. tumefaciens
A. tumefaciens
LBA4404 strain


Reference












Table 2.6 Continued.
Transgene


SAM hydrolase
gene
Ribozyme genes


uidA and gfp genes


ACC oxidase
antisense gene from
melon
ACC oxidase
antisense gene from
apple
uidA and hpt genes


Phenotypic Trait


Improved fruit quality


Potyvirus resistance


GUS and GFP
Reporter genes
Improved fruit quality




Improved fruit quality




GUS Reporter and
Selectable marker
genes


Explant used and
morphogenetic pathway

NR


0 to 4-day-old cotyledons
Organogenesis
2-day-old cotyledons
Organogenesis
2-day-old cotyledons
Organogenesis


NR




Mature seeds
Somatic embryogenesis


Cultivar


NR


US Patent 5,422,259


'Galia' male and female
parental lines
'Galia' male parental
line


'Vedrantais'




'Vedrantais' and 'Earl's
Favourite Fuyu A'


Transformation
Method

A. tumefaciens


A. tumefaciens


A. tumefaciens
ABI strain
A. tumefaciens
ABI strain


A. tumefaciens
LBA4404 strain


A. tumefaciens

C58C1Rif strain


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. Valles 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. Nufiez-Palenius et al. (2002), 18.
Nufiez-Palenius et al. (2003), 19. Silva et al. (2004), 20. Akasa-Kennedy et al. (2004).
Znot-reported.


Reference









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%

(Nufiez-Palenius et al, 2003), and 2.3% (Akasaka-Kennedy et al. 2004) has been

depicted. Unfortunately, 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 To 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.









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









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 HAL] 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 gL-1 sodium. The frequency and intensity of root formation were higher in HALl-

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









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. 'Vedrantais', 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









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









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. Vedrantais 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. Vedrantais) 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-









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 [tlL1) than

Guis's results (<0.5 [tlL1). 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 Vedrantais 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









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)

(Producel, 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,













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
Chile

Costa Rica
Dominican

Rep.
Ecuador
France
Guatemala

Honduras
Israel
Jamaica
Mexico

Minor Antilles
Netherlands
Panama

Spain

TOTAL


363
2

0
49


141
7
136
0
905

0
75
0
0
6
45


147
3

0
94


71

23
49
0
438
0
83
0
0

4
40


0

11
0
0
95
0
95
0
0

0
54


2

6
0
0
169
0
72
0
0

123
63


3

19
0
0
161
0
67
0
0

198
87


0

0
121
28


0

1
0
272
53

4
87
8
0
69
18


0

0
181
41


370
19
121
152


0

0
7
142
11
0
108
28
1

10
22


143
25
143

414
1,233
4
437

36
1
208
202


151
16
181
265


74
82
57

167
859
8
515

71
4
254
278


Source: USDA, Department of Commerce, U.S. Census Bureau, Foreign Trade Statistics (2005).


1,729 952 309 484 459 579 661 635 350 332 3,508 2,982















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- 6/1- 12/1- 6/1- 12/1-5/31 6/1- 12/1-5/31 6/1-11/30 12/1-5/31 6/1- 12/1- 6/1-
5/31 11/30 5/31 11/30 11/30 11/30 5/31 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).