Proceedings of Cucurbitaceae 2004 the 8Th EUCARPIA

European Association for Research on Plant Breeding

Progress in Cucurbit Genetics and Breeding Research

Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding

July 1217, 2004 Olomouc, Czech Republic

A. Lebeda and H.S. Paris

Editors

Palacký University in Olomouc Czech Republic 2004 1 Editors: Ale Lebeda and Harry S. Paris Title: Progress in Cucurbit Genetics and Breeding Research Subtitle: Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding Olomouc, Czech Republic, 1217 July, 2004 Organizing institution: Palacký University, Faculty of Science, Department of Botany, lechtitelù 11, 783 71 Olomouc, Czech Republic Executive redactor: Ale Lebeda Technical redactor: Pavel Rajtr Front and back cover: Designed by Eva Køístková, Ale Lebeda, Pavel Rajtr Front cover: Echinocystis lobata (Michx) Torrey et A.Gray (Cucurbitaceae), native in the North America, introduced and domesticated in some areas of the Cent- ral- and South-Eastern Europe, sparingly cultivated in home gardens as a fast growing ornamental plant, covering fences and alcoves (Flora of the Czech Republic, Part 2. Academia, Praha, 1990, p. 450). Plant from the herbal (location Horní Pìna (discrict Jindøichùv Hradec), South Bohemia, Czech Re- public, 1993). Back cover: Cucumber called Citruli. Mattioli, P.A.: Herbal (1596). Number of pages: 558 Publisher: Palacký University in Olomouc Olomouc 2004, Czech Republic Print: JOLA, v.o.s., Bezruèova 53, 798 41 Kostelec na Hané, Czech Republic, tel., fax: +420-582 373 683, E-mail: [email protected] First edition

ISBN 80-86636-04-6

2 Motto:

Science sans conscience n´est que ruine de l´ âme François Rabelais (1495-1553)

Science without conscience is just a ruin of the soul François Rabelais (1495-1553)

3 4 Contents

Foreword 13

Acknowledgements 15

Introduction (A. Lebeda) 17

Scientific contributions I. General aspects, culture and management J. Moravec, A. Lebeda and E. Køístková (Czech Republic) History of growing and breeding of cucurbitaceous vegetables in Czech Lands 21

J. Luný, A. Lebeda and E. Køístková (Czech Republic) A dedication to Franz Frimmel, a Czech leader of cucurbit breeding 39

L. Nowaczyk and P. Nowaczyk (Poland) Cucumber fruit size and seed yield affected by growth regulators 45

N. Biriukova and E. Maslovskaya (Russia) The influence of cultivation conditions on parthenocarpy of cucumber 51

G. Baysal, R. Tipirdamaz and Y. Ekmekci (Turkey) Effects of salinity on some physiological parameters in three cultivars of cucumber (Cucumis sativus) 57

M. Edelstein, M. Ben-Hur, R. Cohen, Y. Burger and I. Ravina (Israel) Comparison of grafted and non-grafted melon plants under excess of boron and salinity stress 63

A.M. Granero, J.M.G. Sanz, J.L.M. Vidal, A.G. Frenich, A.R. Serrano and F.J.E. González (Spain) Sugars and volatiles composition of nectar of zucchini flowers 69

J.M.G. Sanz, A.R. Serrano and A.M. Granero (Spain) Pollination of zucchini culture by bumblebees: Advance of results of quality production 75

II. Germplasm

V.S. Seshadri and T.A. More (India) History and antiquity of cucurbits in India 81

5 W. van Dooijeweert (the Netherlands) The status of the cucumber (Cucumis sativus) collection of CGN 91

V. Vinter, A. Køístková, A. Lebeda and E. Køístková (Czech Republic) Descriptor lists for genetic resources of the genus Cucumis and cultivated species of the genus Cucurbita 95

H. Pavlikaki, C. Ponce Navarro and N. Fanourakis (Greece) Genetic relationships of different Greek landraces of cucumber (Cucumis sativus) as assessed by RAPDs 101

T.C. Andres (USA) Diversity in tropical pumpkin (Cucurbita moschata): a review of infraspecific classifications 107

T.C. Andres (USA) Diversity in tropical pumpkin (Cucurbita moschata): cultivar origin and history 113

E. Køístková, A. Køístková and V. Vinter (Czech Republic) Morphological variation of cultivated Cucurbita species 119

M.L. Gomez-Guillamón, E. Moriones, M.S. Luis-Arteaga, V. Carnide, A. Börner, N. Sari, K. Abak and J.M. Alvarez (Spain, Portugal, Germany, Turkey) Management, conservation and valorization on genetic resources of Cucumis melo and wild relatives 129

V. Carnide, S. Martins, F.J. Vences, L.E. Sáenz de Miera and M.R. Barroso (Portugal, Spain) Evaluation of Portuguese melon landraces conserved on farm by morphological traits and RAPDs 135

M.R. Barroso, S. Martins, F.J. Vences, L.E. Sáenz de Miera and V. Carnide (Portugal, Spain) Comparative analysis of melon landraces from South Portugal using RAPD markers 143

Y. Burger, Y. Yeselson, U. Saar, H.S. Paris, N. Katzir, Y. Tadmor and A.A. Schaffer (Israel) Screening of melon (Cucumis melo) germplasm for consistently high sucrose content and for high ascorbic acid content 151

M.A. Queiroz, M.L. Silva, L.M. Silveira, R.C.S. Dias, M.A.J.F. Ferreira, S.R.R. Ramos, R.L. Romão, J.G.A. Assis, F.F. Souza and M.C.C.L. Moura (Brazil) Pre-breeding in the watermelon germplasm bank of the Northeast of Brazil 157

6 M. Koutsika-Sotiriou, E. Traka-Mavrona, A.L. Tsivelikas, G. Mpardas, A. Mpeis and E. Klonari (Greece) Use of genetic resources in a dual approach toward selecting improved scion/rootstock grafting combinations of melon (Cucumis melo) on Cucurbita spp. 163

A. López-Sesé and J.E. Staub (USA) Effects of seed maturation and temperature in germination of squash accessions: implications for gene flow 169

M. Sugiyama, K. Sugiyama, T. Ohara, M. Morishita and Y. Sakata (Japan) Characteristics and inheritance of a high hermaphroditic flower- bearing accession of watermelon (Citrullus lanatus) 175

Y. Tadmor, N. Katzir, S. King, A. Levi, A. Davis and J. Hirschberg (Israel, USA) Fruit coloration in watermelon: lessons from the tomato 181

III. Diseases and pests, disease resistance J.F. Chen, G. Moriarty and M. Jahn (China, USA) Some disease resistance tests in Cucumis hystrix and its progenies from interspecific hybridization with cucumber 189

T. Montoro, S. Sánchez-Campos, R. Camero, C.F. Marco, P. Corel- la and M.L. Gómez-Guillamón (Spain) Searching for resistance to cucumber vein yellowing virus in Cucumis melo 197

C. Mallor, J.M. Álvarez and M. Luis-Arteaga (Spain) Behaviour of Cucumis melo Cantaloup Haogen against melon necrotic spot virus (MNSV) 203

J. Garcia-Mas, M. Morales, H. van Leeuwen, A. Monfort, P. Puigdo- menech, P. Arús, C. Nieto, M.A. Aranda, C. Dogimont, G. Orjeda, M. Caboche and A. Bendahmane (Spain, France) 209 A physical map covering the Nsv locus in melon

B.S. Daryono, S. Somowiyarjo and K.T. Natsuaki (Japan, Indonesia) Detection of resistant melons to the Indonesian isolate of KGMMV 213

J.D. McCreight (USA) Progress in breeding melon for resistance to lettuce infectious yellows virus 219

N. Guner and T.C. Wehner (USA) Resistance to a severe strain of zucchini yellow mosaic virus in watermelon 223

7 J. Svoboda and J. Polák (Czech Republic) Preliminary evaluation of squash cultivars for resistance to a Czech isolate of zucchini yellow mosaic virus 231

M. Pachner and T. Lelley (Austria) Different genes for resistance to zucchini yellow mosaic virus (ZYMV) in Cucurbita moschata 237

H. Olczak-Woltman, M. Bakowska, M. Schollenberger and K. Niemirowicz-Szczytt (Poland) Cucumber screening for resistance to angular leaf spot 245

D.L. Hopkins (USA) Characteristics of resistance to Acidovorax avenae subsp. citrul- lii in the Citrullus lanatus accessions PI 482279 and PI 494817 251

D. Kenigsbuch, D. Taler, M. Galperin, I. Benjamin and Y. Cohen (Israel) Plant eR genes encoding for glyoxalate aminotransferase enzymes confer resistance against downy mildew in melon 257

A. Lebeda and J. Urban (Czech Republic) Disease impact and pathogenicity variation in Czech populations of Pseudoperonospora cubensis 267

J. Urban and A. Lebeda (Czech Republic) Differential sensitivity to fungicides in Czech populations of Pseu- doperonospora cubensis 275

A. Lebeda and B. Sedláková (Czech Republic) Disease impact and pathogenicity variation in Czech populations of cucurbit powdery mildews 281

B. Sedláková and A. Lebeda (Czech Republic) Variation in sensitivity to fungicides in Czech populations of cucurbit powdery mildews 289

T. Montoro, M. Salinas, J. Capel, M. Gómez-Guillamón and R. Lozano (Spain) Genetic variability in Sphaerotheca fusca as determined by AFLPs: the case of race 2 as a causal agent of powdery mildew in melon 295

R. Song, G. Gusmini and T.C. Wehner (China, USA) A summary of eleven preliminary studies of greenhouse and field testing methods for resistance to gummy stem blight in watermelon 301

L. Perchepied, C. Dogimont and M. Pitrat (France) Genetic analysis of resistance to Fusarium oxysporum f.sp. melonis race 1.2 in melon 307

8 R. Cohen, Y. Burger, C. Horev, A. Porat, U. Saar and M. Edelstein (Israel) Reduction of Monosporascus wilt incidence using different Ga- lia-type melons grafted onto Cucurbita rootstock 313

M. Grinberg, V. Soroker, E. Palevsky, I. Shomer and R. Perl-Treves (Israel) Response of cucumber to the broad mite (Polyphagotarsonemus latus) 319

J. Pauquet, E. Burget, L. Hagen, V. Chovelon, A. Le Menn, N. Valot, S. Desloire, M. Caboche, P. Rousselle, M. Pitrat, A. Bendahmane and C. Dogimont (France) Map-based cloning of the Vat gene from melon conferring resistance to both aphid colonization and aphid transmission of several viruses 325

IV. Breeding and genetics K. Bartoszak (Poland) Performance of pickling cucumber cultivars presently on the Polish National List 333

U. Klosinska and E.U. Kozik (Poland) Suitability of new cucumber F hybrids for open-field cultivation 337 1

J. Berenji and D. Papp (Serbia, Hungary) The effect of ethephon on the sex expression of naked seeded oil pumpkin 341

N. Ercan, M. Temirkaynak, F. ªensoy and A.S. ªensoy (Turkey) Evaluation of some inbred lines of summer squash for plant, flower, fruit and seed properties 345

P. Gómez, A. Peñaranda, D. Garrido and M. Jamilena (Spain) Evaluation of flower abscission and sex expression in different cultivars of zucchini squash (Cucurbita pepo) 347

L. Perchepied, C. Périn, N. Giovinazzo, D. Besombes, C. Dogimont and M. Pitrat (France) Susceptibility to sulfur dusting and inheritance in melon 353

B. Kowalczyk (Poland) Distinctness, uniformity and stability testing of cucumber cultivars in Poland 359

Z. Sun, R.L. Lower and J.E. Staub (USA) Generation means analysis of parthenocarpic characters in a processing cucumber (Cucumis sativus) population 365

J.E. Zalapa, J.E. Staub and J.D. McCreight (USA) Genetic analysis of branching in melon (Cucumis melo) 373

9 J.E. Staub, J.E. Zalapa, M.K. Paris and J.D. McCreight (USA) Selection for lateral branch number in melon (Cucumis melo) 381

H.S. Paris, A. Hanan and F. Baumkoler (Israel) Assortment of five gene loci in Cucurbita pepo 389

L. Wessel-Beaver, H.E. Cuevas and T.C. Andres (USA) Genetic compatibility between Cucurbita moschata and C. argyrosperma 393

M.D. Robbins and J.E. Staub (USA) Strategies for selection of multiple quantitatively inherited yield components in cucumber 401

V. Tissue culture, biotechnology, molecular genetics and mapping

J. Sztangret, J. Wronka, T. Galecka, A. Korzeniewska and K. Niemirowicz-Szczytt (Poland) Cucumber (Cucumis sativus) haploids developed from parthenocarpic hybrids 411

D. Skálová, A. Lebeda and B. Navrátilová (Czech Republic) Embryo and ovule cultures in Cucumis species and their utilization in interspecific hybridization 415

H. Ezura and Y. Akasaka-Kennedy (Japan) Somatic embryogenesis in a model cultivar, PI 161375 (Cucumis melo subsp. agrestis), of melon 431

E. Kiss-Bába, S. Pánczél, V. Zarka, Gy.D. Bisztray and I. Velich (Hungary) Regeneration ability of some Hungarian melon varieties 437

J. Gajdová, A. Lebeda and B. Navrátilová (Czech Republic) Protoplast cultures of Cucumis and Cucurbita spp. 441

G. Bartoszewski, S. Malepszy and M.J. Havey (Poland) Mosaic (MSC) cucumbers regenerated from independent cell cultures possess different mitochondrial rearrangements 455

S. Curuk, S. Cetiner, C. Elman, X. Xia, Y. Wang, A. Yeheskel, L. Zil- berstein, R. Perl-Treves, A.A. Watad and V. Gaba (Turkey, Israel) Wounding by vortexing with carborundum facilitates Agrobacte- rium-mediated transformation of melon (Cucumis melo) 459

A. Atarés, B. Garcia-Sogo, B. Pineda, P. Ellul and V. Moreno (Spain) Transformation of melon via PEG-induced direct DNA uptake into protoplasts 465

10 Z. Gao, M. Petreikov, Y. Burger, S. Shen and A.A. Schaffer (Israel) Stachyose to sucrose metabolism in sweet melon (Cucumis melo) fruit mesocarp during the sucrose accumulation stage 471

S-M. Chung and J.E. Staub (USA) Consensus chloroplast primer analysis: A molecular tool for evolutionary studies in Cucurbitaceae 477

Y. Brotman, I. Kovalski, C. Dogimont, M. Pitrat, N. Katzir and R. Perl-Treves (Israel, France) Molecular mapping of the melon Fom-1/Prv locus 485

Y. Yariv, V. Portnoy, Y. Burger, Y. Benyamini, E. Lewinsohn, Y. Tadmor, U. Ravid, R. White, J. Giovannoni, A.A. Schaffer and N. Katzir (Israel, USA) Isolation and characterization of fruit-related genes in melon (Cucumis melo) using SSH and macroarray techniques 491

I. Eduardo, P. Arús and A.J. Monforte (Spain) Genetics of fruit quality in melon. Verification of QTLs involved in fruit shape with near-isogenic lines (NILs) 499

N. Fukino, M. Kuzuya, M. Kunihisa and S. Matsumoto (Japan) Characterization of simple sequence repeats (SSRs) and development of SSR markers in melon (Cucumis melo) 503

A. Zraidi and T. Lelley (Austria) Genetic map for pumpkin Cucurbita pepo using random amplified polymorphic DNA markers 507

A. Levi, C.E. Thomas, J. Thies, A. Simmons, Y. Xu, X. Zhang, O.U.K. Reddy, Y. Tadmor, N. Katzir, T. Trebitsh, S. King, A. Davis, J. Fauve and T. Wehner (USA, China, Israel, France) Developing a genetic linkage map for watermelon: polymorphism, segregation and distribution of markers 515

P.A. Rajagopalan, T. Saraf-Levy, A. Lizhe and R. Perl-Treves (Israel, China) Increased femaleness in transgenic cucumbers that overexpress an ethylene receptor 525

Authors index 533

List of participants 537

11 12 Foreword

This book contains full-length contributed papers of all lectures and most of the posters presented at Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. The papers cover a wide range of topics and have been arranged according to subject matter. The date of issue of this book, 12 July 2004, is the first day of the conference. This follows the tradition begun at Cucurbitaceae 96, the 6th EUCARPIA Meeting on Cucurbit Genetics and Breeding, that was held in Malaga, Spain. We edited the original contributed manuscripts and if we have made or overloo- ked some errors, we apologize. The format is similar to the compact but easy-to-comprehend format of the Proce- edings of Cucurbitaceae 2000, the 7th EUCARPIA Meeting on Cucurbit Genetics and Breeding. We thank all contributing authors for their cooperation in preparing their manuscripts according to the format.

30 April 2004 Ale Lebeda Harry S. Paris Editors

13 14 Acknowledgements

The Organizing Committee thanks the following institutions, organizations and companies for their financial and material support to this meeting:

Moravoseed Ltd., Breeding and Seed Company, Mikulov-Mulov, the Czech Republic Olympus C&S Ltd., Praha, the Czech Republic Palacký University in Olomouc, Olomouc, the Czech Republic Ing. B.Holman, Cucumber Breeding and Seed Production, Bzenec, the Czech Republic Mendel Museum, Abbey of St. Thomas, Brno, the Czech Republic Municipality of the town of Olomouc, the Czech Republic Vinselekt Michlovský Ltd., Rakvice, the Czech Republic Agricultural Company, Sedlec u Mikulova, the Czech Republic School Agricultural Enterprice, Lednice, the Czech Republic Ing. B. Palièka, Ph.D., Mutìnice, the Czech Republic

Editors highly appreciate an excellent cooperation with Pavel Rajtr and Josef Kejzlar from the printing company JOLA in Kostelec na Hané, the Czech Republic. A. Lebeda expresses his sincere thanks to Dr. I. Dolealová, Mgr. J. Gajdová, Mrs. J. Hýbnerová, Dr. E. Køístková, Dr. B. Navrátilová, Mgr. I. Petrelová, Dr. M. Sedláøo- vá, Mgr. D. Skálová, Mrs. I. Vaculová, Mrs. D. Vondráková and Mr. V. Vetièka for their generous help and excellent cooperation during the preparation of the Cucurbi- taceae 2004 meeting and this book.

15 16 Introduction

Ladies and getlemen, dear colleagues and friends,

On behalf of the Organizing Committee of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding, I would like to welcome all of you to the Czech Republic and the historical city of Olomouc. This meeting is the second EUCAR- PIA Cucurbitaceae meeting organized on the territory of the former Czechoslovakia, but is the first in the independent Czech Republic. The first meeting was organized in Slova- kia in July 1977 at Bratislava, now the capitol of the Slovak Republic. The main organi- zer of this meeting was Assoc. Prof. Jan Luný, Ph.D., at that time head of Department of Plant Breeding at Faculty of Horticulture in Lednice na Moravì, Agriculture University in Brno. Professor J. Luný was my teacher on plant breeding during my studies of horticulture in the beginning of the 1970s. Personally I am very happy that after nearly 30 years I have the opportunity to continue his good work and organize this meeting again in historical Czech Lands, recent Czech Republic and in Olomouc. However, this meeting is not the first EUCARPIA meeting organized in this city. In 1985 Olomouc hosted the EUCARPIA meeting on the genetics and breeding of carrot and other root vegetables (organized again by Prof. J. Luný), and in 1999 I had the chance to organize a EUCAR- PIA meeting on Leafy Vegetables Leafy Vegetables 99`. From this brief historical excursion it is evident that Olomouc can be considered without any hesitation as a city of vegetable crops research and breeding. In my introduction I would like to use and renew some of the ideas given in my introduction speach five years ago. The historical city of Olomouc is situated in the region of Central Moravia. The main part of this region is formed by the fertile and wonderful Haná lowlands. The name Haná was first used by Jan Blahoslav and Jan Amos Comenius who reproduced it on a map of Moravia in 1627. Historically the city of Olomouc is considered as a capitol of Central Moravia and a prominent centre of education and culture. It is well known, that advances in culture and science in Czech Lands have been and are still closely related to the developments of higher education and the Universi- ty in Olomouc. In 1573 the city was granted university rights. The University in Olo- mouc is the second oldest university in the Czech Lands (after Charles University in Prague established in 1348). The era of the old University is associated with the names of numerous outstanding scientists, i.e. Gregor Johann Mendel, the founder of genetics, Jan Svatopluk Presl and Jan Nestler, the naturalists, and Jakub Kresa, the mathematici- an (the Euclid of the West), born in 1648 in the village Smrice near Olomouc. The recent name of Palacký University is from historician Frantiek Palacký, known as the father of the Czech nation. At present, the University has seven faculties, over 1000 teaching staff and more than 15000 students. The main venue of Cucurbitaceae 2004, the monumental complex of baroque buildings named Konvikt, is very closely linked to the reach history of the city Olomouc, of the Jesuit Boarding School (Jesuit Seminary, Jesuit College) and the University. The history of this building is going back to the Medieval time and later on to the beginning of 16th century. The building in recent state was finished in 1708 and used for long period by the Olomouc University. Unfortunately, in the end of 18th century the building of the former Jesuit College was handed over to the military to be used as a barrack unit. However, after the revolutio- nary changes in 1989, the University received back this building, and after extensive

17 reconstruction and revitalization was completed in 2002, and used for their original academic purpose. In recent status a building has extraordinary historic and artistic value, and is used as the Art Centre of Palacký University (five art departments of the Philosophical and Pedagogical Faculties are located here) and representative conference halls are used for national and international venues such as Cucurbitaceae 2004. Olomouc is well known not only as a university city, but also as a scientific centre. Research related to natural sciences, plant biology, breeding and growing of vegetable crops has a long tradition in this area. In January 1951 the Research Institute of Vegetable Crops was established here. This institute had a substantial influence on the development of vegetable crop growing and breeding in this country over the next four decades. However, the dramatic political and economic changes after November 1989 resulted in its closure in 1994, and the buildings and facilities were given to Faculty of Science of Palacký Univer- sity, which established here a Biocentre, focusing on biology and plant sciences. On the campus are located other related institutions such as a Scientific Park and Innovative Cen- tre of Palacký University, and the Gene Bank of Research Institute of Crop Production in Prague, and State Phytosanitary Administration. In Olomouc is also located Institute of Experimental Botany of the Czech Academy of Sciences which is closely linked with the University. All these institutions are focused on science and/or the application of science in agricultural and horticultural practice. The former Research and Breeding Institute of Vegetable Crops in Olomouc-Holice made substantial contributions to the development of cucumber breeding and production at least in this country. Some historical relationships, most important activities and names related to this development are summarized in our two contributions in this book. We are trying to continue these activities, mostly focused on cucurbit genetic resources, plant pathology and resistance breeding, tissue culture and some aspects of biotechnology. Our contribution to progress in some of these activities is directly linked to international contacts and cooperation. From the contents of this book it is evident that enormous progress has been made in various branches of Cucurbitaceae research and breeding. The scientific and professional part of this meeting is based on more than 80 oral and poster contributions, and 76 are published. From the programme and the contents of the book it is evident that there is some balance between pure science, genetics and breeding, including practical applications of the results. We can see two basic aspects: 1) Application of interdisciplinary, bio- technological and molecular approaches in research and breeding of cucurbits; and 2) In- creasing international and intercontinental cooperation in cucurbit research. Thus creating quite a new situation and a very good foundation for the future. We are very happy that our University and our country will host such gathering of scientists and breeders from 28 countries and four continents. Cucurbitaceae 2004 is part of a series of cucurbit genetics and breeding conferences begun in Europe in the 1970s and in the U.S.A. in the 1980s. These conferences have been held on a regular basis, in even-numbered years and alternately on both sides of the Atlantic Ocean, beginning with Poland in 1992, then Texas, Spain, California, Israel, Florida and now we have the privilege to organize and host this 2004 conference here in the Czech Republic. We will do our best to make Cucurbitaceae 2004 a most informative and enjoyable experience for all participants. We believe that our conference will take place in the spirit of cooperation and friendship, and that everybody will enjoy this conference, the historical city of Olomouc, Moravia and the Czech Republic. We hope that you will find Cucurbitaceae 2004 to be a most memo- rable occasion. Ale Lebeda

18 Scientific contributions

I. General aspects, culture and management

19 20 History of growing and breeding of cucurbitaceous vegetables in Czech Lands

J. Moravec1, A. Lebeda2 and E. Køístková3 1Sienkiewiczova 1, 772 00 Olomouc, Czech Republic 2Palacký University in Olomouc, Faculty of Science, Department of Botany, lechti- telù 11, 783 71 OlomoucHolice, Czech Republic; e-mail: [email protected] 3Research Institute of Crop Production, Division of Genetics and Plant Breeding, Department of Gene Bank, Workplace Olomouc, lechtitelù 11, 783 71 OlomoucHolice, Czech Republic; e-mail: [email protected]

Summary

Among cucurbitaceous vegetables, cucumbers (Cucumis sativus L.) are the most important in the Czech Republic, with the history of cultivation from the 9th century. Cucurbita maxima Duchesne and C. pepo L. are cultivated both in bigger production areas and home gardens for processing and fresh market. The cultivation of melons, Cucumis melo L., and watermelons, Citrullus lanatus (Thunb.) Matsum et Nakai, is restricted to the warmest regions of southern Moravia; landraces are known from the first half of the 19th century, and the first original cultivars were selected from them in the beginning of 20th century. Cucurbit breeding intensified beginning in the 1960s, first by state breeding stations, and after the political changes of 1989 by private companies. Registered local cultivars are listed. Genetic resources of cucurbitaceous vegetables are maintained by the Gene Bank at Olomouc, of the Research Institute of Crop Production in Praha.

Keywords: Cucurbitaceae, cucumber, melon, watermelon, squash, pumpkin, gourd, Czechoslo- vakia, Czech Republic, archeology, breeding, genetic resources, growing, processing industry

Introduction

The most important cucurbitaceous vegetable crops belong to the genera Cucumis L., Cucurbita L. and Citrullus L. (Rubatzky and Yamaguchi, 1997). The history of their cultivation and use on Czech Lands (part of former Czechoslovakia, recently the Czech Republic, including Bohemia, Moravia and Silesia) is shorter than in tropical and subtropical regions of the New and Old Worlds, but they have gained a solid position among vegetable species. Among them, cucumbers (Cucumis sativus L.) are the most important cucurbitaceous crop, commonly cultivated for industrial and home processing as pickles and for the production of salad cucumbers. Cucurbita maxima Duchesne and C. pepo L. are cultivated in both, bigger production areas and home gardens, for processing and fresh market. The cultivation of melons, Cucumis melo L., and watermelons, Citrullus lanatus (Thunb.) Matsum. et Nakai, is restricted to the warmest regions of southern Moravia; melons and watermelons are mostly imported, and watermelons especially are very popular as a refreshing sweet juicy vegetable during the summer.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 21 General historical overview of the growing and breeding of cucurbitaceous vegetables in the Czech Lands Each species has its own history of introduction, cultivation, and breeding which are described below under crop-specific headings. The cultivation of cucurbitaceous vegetables in Czech Lands in the 9th century, during the epoch of the Great Moravian Empire, is documented archeologically by cucumber seeds originating from Mikulèi- ce (Southern Moravia) (Dr. L. Poláèek, pers. commun., 2004). The introduction and distribution of new plant species in the Middle Ages was through numerous abbeys and castle gardens. We did not search data on cucurbitaceous vegetables from hand- written herbals. Agriculture in the Czech Lands, although well advanced by the 13th century, underwent its most significant development in the 14th century, during the reign of the Czech king and Holy Roman emperor Charles IV (Kubaèák, 2003). There is published documentation on the cultivation of cucurbitaceous vegetables in Czech Lands beginning in this period, as Charles IV initiated the compilation of two plant encyclopaedias, Bohemáø and Prepurský rukopis. The first one, Bohemáø, includes in 1,000 verses the Czech names of plants. The second one, Prepurský rukopis is in Czech and Latin and is an independent encyclopaedia of botany and on plant use in medicine. The first Czech-language printed herbal, by Jan Èerný (Johannes Niger de Praga), was published in Nürnberg (Germany) in 1517 (Èerný, 1981). Jan Èerný based his work on text and illustrations of the Herbarius Moguntinus, published in 1484 by Peter Schöffer. The herbarium of the Italian physician Pierre Andrea Mattioli (15011577), who was from 1554 to 1568 the physician of the emperor Ferdinand I and later of Maxmi- lian II in Prague, was published in 1554 (second edition published in 1558, written in Prague and Komotau). The second Czech edition of this herbal was printed in 1596. This book was translated and substantially supplemented with much botanical, medicinal, cultural, and culinary information by Adam Huber from Rysenbach and by Daniel Adam from Veleslavín (Domin, 1945). Thus it can provide us with information on cucurbitaceous vegetables in Czech Lands during the late 16th century. This Czech version of Mattioli´s herbal mentiones cucumbers, melons, pumpkins/squash, and watermelons (Mathioli, 1998). Cucurbit cultivation, mainly cucumbers, spread during the 16th and 17th centuries, but became common place only in the 19th century (Moravec, 1959). By the beginning of the 19th century, vegetable seeds were marketed. The seed catalogue of the Prague merchant J.F. Konvalina from 1811 contains imports almost exclusively. Five cucumbers are offered Dlouhé bílé aneb hadinné (Long white or snake), Sprosté aneb obyèejné rané bílé (Common early white), Dlouhaté zelené (Long green), Sprosté obyèejné zelené (Common green), Malé zelené k nakládání (Small green for pickling), and two melons, Melouny holandské (Dutch melons), Kan- talupy holandské (Dutch cantaloups); no squash or pumpkins were offered. Local cultivars were developed during the 19th century, originating as selections by gardeners. To this development substantially contributed the Moravian and Sile- sian Agricultural Society and their section for plant breeding, where J.G. Mendel pla- yed an important role (Albano and Wallace, 2002; Orel, 2003). Later, methods of mass and individual selection were employed. In 1918, Czech Lands and Slovakia were

22 united to form Czechoslovakia, but the first cultivars developed remained localized in their distribution. The first companies and organizations specializing in breeding were founded in the 1920s and 1930s (e.g. the Mendeleum in Lednice na Moravì), but the extent of vegetable breeding was very limited (Podeva, 1959). During the Second World War, more than 200 German vegetable cultivars were introduced to the Czech Lands and they were maintained. After 1945 some of them were improved and added to the Czechoslovak list of allowed (registered) cultivars. The official list of cucurbitaceous vegetables grown for markets in 1948 is given in Table 1. After 1948, when the whole of industry and agriculture were completely nationa- lized, private garden companies disappeared and some private estates were transformed into state breeding stations. For example, the Station at MìlníkMlazice was created from the former gardening company owned by the skillful gardener and breeder, J. Vyskoèil. He bred there flower and vegetable cultivars, including the cucumbers Jedineèná and Unikát. In 1951, the Vegetable Research Institute at Olomouc was founded, and a network of breeding stations was developed. While cucumber breeding was located primarily at the breeding stations in Bohemia and Moravia, the melon and watermelon breeding was located in Slovakia. A system of cultivar testing was organized by the government through the State Checking and Controlling Institute and suitable cultivars were listed in the List of Allowed Cultivars. From the 1950s through the 1980s, the production of cucurbits increased. Gherkins were field-produced for the processing industry, and salad cucumbers were grown in both, fields and greenhouses. The return to democracy in Czechoslovakia from 17 November 1989 brought pro- found changes to vegetable production and breeding. The activity of the Vegetable Research and Breeding Institute at Olomouc ended on 31 May 1994 (Lebeda, 1994); similarly, several breeding stations quit their activities, and some of them were priva- tized. Presently, the breeding of cucurbits is conducted mainly by the companies SEMO Ltd. in Smrice, Moravoseed Ltd. in Mikulov, and Cucumber Breeding Company of Dipl. Ing. B. Holman in Bzenec. The seed market is also open to companies from abroad. All cultivars are mentioned in the List of Cultivars Inscribed in the State Book of Cultivars. In the 1990s, large-scale vegetable production was reduced and concentrated in a small number of companies. While in 1982 the production of cucumbers and gherkins was 158,551 Mt (metric tons), in 2002 it was 42,200 Mt (source F.A.O.). As the consumption of vegetables has increased in the Czech Republic, importation plays an important role. The Czech population is more open to new vegetables. For example, C. pepo zucchini, oil-seed pumpkins, spaghetti squash and others have become very popular. The consumption of imported melons has increased too. Some other species, such as Cucumis metuliferus (kiwano), Luffa sp., Lagenaria sp. and Cyc- lanthera sp. are cultivated by hobby gardeners as novelties.

Genetic resources of the cucurbitaceous vegetables in the Czech Republic

The collections of vegetable genetic resources were placed in 1951 in the then newly established Research Institute of Vegetable Growing and Breeding at Olomouc. Maintenance of the germplasm collections by the Institute was ceased at the end of

23 Table 1. Cucurbit cultivars on the Czechoslovak plant variety list from the 1948 (Anonymous, 1948)

Species/Breeding level Cultivar name-in Czech Cultivar name-free version in English

Cucurbita maxima Km Centýøová zelená Green Centner Km Centýøová lutá Yellow Centner Km Melounová obøí síovaná Netted Giant Melon Cucurbita pepo Km Vegetable marrow (chøestová) Vegetable Marrow (asparagus like) Cucumis melo Km Americký ananasový èervený American Pineapple Red Km Berlínský síovaný Berliner Netted Km Blenheimský oranový Blenheim Orange Km Cantaloup tourský Cantaloupe from Tours Km Turkestanský Turkestanic Citrullus lanatus Km Kropenatý Spotted Km Ruský raný Russian Early Km Zelený Green Cucumis sativus - field cucumbers O Hanácké nakladaèky Gherkins from Haná O Podrabského mìlnické nakladaèky Gherkins from Podrabský of Mìlník Kr Bzenecké nakladaèky Gherkins from Bzenec Km Èínské hadovité Chinese snake-like Km Lánské hoøèièné Sinapisms from Lány Km Delikates Delikates Km Hroznovité Racemose Kr Mìlnické nakladaèky Gherkins from Mìlník Kr Mladoboleslavské salátnice Salad Cucumbers from Mladá Boleslav Km Polodlouhé úrodné Half-long Productives Kr Znojemské nakládaèky Gherkin from Znojmo Cucumis sativus - greenhouse cucumbers Km Nejlepí ze vech Best of All Km Spotresisting Spotresisting Cucumis sativus - forcing cucumbers (for hot beds, cold beds) O Landovského From Landovský Km Konkurent Konkurent Km Produkta Produkta Km Schützova reforma Reform from Schütz

Km maintained as a strain; Kr local cultivar, landrace; O improved (bred).

24 1993, to be replaced by the newly established Gene Bank at Olomouc, under the aegis of the Research Institute of Crop Production (RICP) in PrahaRuzynì. Collections maintained by the Gene Bank at Olomouc include more than 10,000 accessions of about 430 species (Køístková and Lebeda, 1995). The Cucurbitaceae make up a large part of this collection, with about 1,600 accessions potentially available. Of these 1,600 accessions, 900 are of Cucumis species, 650 are of Cucurbita species, and 50 are of other cucurbit genera (Køístková, 2002). Passport data are available on the web site www.vurv.cz (part databases, EVIGEZ).

Growing and breeding of cucurbitaceous crops special part

Cucumbers

Early history, centres of cultivation and processing

Cucumbers (Cucumis sativus L.) are the most popular cucurbitaceous vegetable in central Europe. From their centre of origin in India, cucumbers reached ancient Greece via Asia Minor by the 6th or 5th century B.C.E. (Jirásek, 1958). In the Byzan- tine Empire cucumber was known in the 4th or 5th century C.E. (Sinskaja, 1969). With expansion of the Roman Empire they were transported across the Alps northward to central Europe via Roman roads and the trails of Great Moravia (Kvìt, 2003). It is possible that during the Roman occupation cucumbers first arrived within the territory of the Czech Lands. So cucumeres in the book Capitulare de villis (from around the year 812) of Charles Magnus (768814) probably refers to true cucumbers directly descended from those introduced by the Romans. However, there is also the conjecture that Slavonic people obtained cucumbers from the By- zantines (Sinskaja, 1969). Archaeological findings of cucumber seeds in Mikulèice (Southern Moravia) prove that they were cultivated within the territory of Great Moravia (Grand-Moravian Kingdom) from 830906 C.E.; this territory extended along the Morava river, i.e. recent Moravia (Czech Republic), Northern Austria, Hungary and Western Slovakia (Dr. L. Poláèek, pers. commun., 2004). This Kingdom had close ties and trade with other parts of Europe (Kvìt, 2003). The cucumber varieties similar to the recent ones were probably transported to Central Europe in the Medieval Age from the Byzantine Kingdom, where they were exclusively known under the name anguria. From this word originates probably the Slavonic name agurka and also the Czech name okurka (Jirásek, 1958). Merchants from Venice, who had contacts with Arabians and Constantinople, visited local fairs in Mikulèice and probably brought cucumbers with them (Moravec, 1959). Cucum- ber seeds dating from the period of the 10th to the 12th centuries were discovered in Praha Kaprová (Bohemia) and Pøerov (Central Moravia) (Dr. L. Poláèek, pers. commun., 2004). In the Medieval Age, the distribution of cucumbers was aided by the abbey in Louka near Znojmo (Abbas lucensis) in South Moravia, founded in 1059 by Jitka (Judita), a wife of the Czech prince Bøetislav I. In the epoch of the abbot Sebastian Freytag, elected in 1573, cucumbers were grown in the abbey garden there. Another

25 written source says that cucumbers were introduced to Znojmo from Hungary in 1579 as a medicament against pests. Cucumber seeds dating from the 14th and 15th centuries have been found in Mora- via and Silesia (Olomouc, Pøerov, Uherský Brod, Opava), and Bohemia (Plzeò, Most and Prague-Malostranské Square). Cucumbers are mentioned in the Czech version of Matthioli from 1562. It shows two forms of cucumbers, the form Citruli, which look like small pickling cucumbers, and the snake cucumbers, called Cucumeres anguini, which look like C. melo var. flexuosus. Besides other medical recommendations, it was written, that the excessive consumption of cucumbers can cause serious putrefac- tive fevers putridae febres (Mathioli, 1998).

Cucumber processing

The town of Znojmo in Southern Moravia and the surrounding area were famous for both the growing and preservation of cucumbers (gherkins). In 1628, the fermented cucumbers from Znojmo were served as a delicacy at a dinner organized for the occasion of the Land Assembly, in the presence of Emperor Ferdinand II. Later, the town of Znojmo became the centre of cucumber growing and marketing. Mr. A. F. Petrtill used to sell fresh and fermented cucumbers. In the 1863, Mrs. Tekla Henesch from Znojmo obtained a licence for the production of preserved cucumbers. In 1885, the area of cucumber growing around Znojmo reached 183 ha, expanding to more than 800 ha by 1900. The yield was 11 Mt per ha and the gain was 377 zla- tých (goldens) per ha. The gherkins from Znojmo became a synonym for a pickling cucumber of a special fruit shape and size and it was popular also abroad, in Germa- ny, under the name of Znaimer Essigurken. Gherkins of a bigger size were preserved during the summer by lactic acid fermentation in a salt solution (4-5% solution of salt NaCl) with addition of vine leaves, horse radish, onion, estragon and dill, at 25°C in wooden drums for four weeks. From the first half of the 20th century, small-sized gherkins preserved as pickles in sweet- sour solution became more popular. In 1958, a big cucumber processing factory was built in Znojmo. During the 1970s, it annually produced about 13 000 Mt of pickled cucumbers for local use and export to Western Europe.

Gherkin cultivation and breeding from the 19th century until 1945

In the 19th century, cucumbers became a popular summer vegetable, commonly cultivated in home gardens and having several areas of mass production both in South Moravia, around the town Znojmo and, since the beginning of the 20th century, around Bzenec. Cucumber production in the Czech Lands (Bohemia) was concentrated around the towns of Mìlník, Vetaty, Døísy, Mladá Boleslav, Kolín, Kutná Hora, Hradec Králo- vé, Podboøany and Litomìøice (Fig. 1). The first local landraces of cucumbers originated in the territory of their first cultivation, South Moravia, and they were called Znojemská nakladaèka. In Central Moravia

26 14Opava 15 Pøerov 16 Smrice 17 Svijanský Újezd 18 Valtice 19 Veltrusy 20 idovice 7 Litomìøice 8 Lysá nad Labem 9 Mìlník Mlazice 10 Mikulov 11 Mikulèice 12 Mladá Boleslav 13 Most 1 Bzenec 2 Dobrá Voda 3 Kromìøí 4Kutná Hora 5 Lednice na Moravì 6 Libochovice nad Ohøí Sites of cucurbit breeding and growing in the Czech Republic. Figure 1.

27 were grown Olomoucká nakladaèka and Kojetínská nakladaèka. The first cultivars were bred during the first third of the 20th century. The breeding of cucumber was conducted in the Land Institute for Improvement of Plants in Pøerov (Central Moravia) in the 1920s. Hanácká nakladaèka was bred from landraces cultivated in the area around Kromìøí. In Bohemia, Mìlnické nakladaèky and Kutnohorské nakladaèky were developed. Beginning 1938, the breeder and gardener Josef Podrabský of Kralupy nad Vltavou improved the Podrabského mìlnická nakladaèka. The resulting Mìlnické nakladaèky is inscribed in the State Book of Cultivars until the present day. The breeder and farmer (gardener) Jaroslav Pour in Dobrá Voda near Hoøice v Podkrkonoí bred Bílské nakladaèky from a landrace grown in the village Bílsko. Since the 1920s, cucumber breeding has been conducted in Lednice na Moravì and Valtice (Southern Moravia) by Frantiek Frimmel (18881957) (Luný et al., this volume), who strongly accelerated and stimulated cucumber breeding. Using the results of his own research and observations on plant and flower biology with respect to yield parameters, he developed, in the 1930s, Lednické nakladaèky and Znojemské na- kladaèky from local landraces. Before the Second World War, he formulated the most important principles of cucumber breeding methodologies.

Gherkin breeding after 1945 In 1955, Dr. Frimmel, working at the Research Institute of Vegetable Breeding at Olomouc, initiated a breeding project with the goal of developing F hybrid cucum- 1 bers (Luný et al., this volume). Hybrid seeds were produced by removing the staminate flowers by hand from the female parent Znojemská nakladaèka. The male parent, Podrabského mìlnická nakladaèka was used as the sole source of pollen and pollination was performed by honey-bees in the greenhouse. As he did not have access to maternal lines from Asia as did breeders from the Netherlands, his hybrids were not commercially successful. In 1967 Václav Oveèka (Oveèka, 1955, 1964) bred Znojmia, later re-named as Palava (Table 2). Cucumber breeding has been conducted since 1967 by B. Holman at the Vege- table Breeding Station in Smrice (near the town of Prostìjov) (Luný and Holman, 1973; Lebeda, 1987). The breeding process has been supported by infrastructure and technical equipment (greenhouses, plastic tunnels and growth chambers), biotechno- logical approaches (mutation breeding, in-vitro tissue cultures), screening for diseases resistance (response to pathogens), developed by the phytopathological laboratory of A. Lebeda (Lebeda, 1986), and an increasing collection of cucumber genetic resources and related wild Cucumis species (Lebeda, 1988, 1996). Research and breeding for resistance to Cucumber mosaic virus (CMV) has been conducted with specialists of the Research Institute of Vegetable Growing and Breeding and Institute of Experi- mental Botany of the Czechoslovak Academy of Sciences in Olomouc (P. Havránek, F.J. Novák, J. Betlach and M. Havránková). The breeding was based on a wide range of genotypes that included cultivars, landraces and related wild species. A number of F hybrids have been created using gynoecious material as the female parent (Table 2). 1 Since the 1960s, cucumber breeding has also been conducted by E. Troníèková Pekárková at the Research Institute of Crop Production in PrahaRuzynì, in coopera-

28 Table 2. Cultivars of cucurbitaceous vegetables bred in the Czech Lands and/or former Czechoslovakia (Anonymous, 1958-1998; 1999-2003)

Species/Cv. name* Year** Author Institution, place of breeding Cucumis sativus - pickling cucumbers Mìlnické 1946 present J. Podrabský Kralupy nakladaèky 1,2,3,4 Znojemské 1946 1954F. Frimmel Valtice nakladaèky 1,2,4 Bílské 1952 1991 J. Pour st. Dobrá Voda nakladaèky 1,2,3,4 Znojemské 3 1954 1968 F. Frimmel Valtice Palava (Znojmia) 1,3,4 1967 1991 V. Oveèka Valtice Primela H 1979 1989 B. Holman, F. Mach, Smrice, Valtice J. Sobotka, K. Janota Triga H 1979 1985 E. Troníèková, S. Cucová Praha Ruzynì Lyra H 1982 1998 E. Troníèková, S. Cucová, Praha Ruzynì, Lysá nad Labem Z. Jech, M. Svaèinová Valta H 1982 present B. Holman, F. Mach, Smrice, Valtice K. Janota Vega H 1985 1991 E. Troníèková, Praha Ruzynì, Lysá nad Labem M. Svaèinová, Z. Jech Ada H 1985 1991 B. Holman, F. Mach, Smrice, Valtice K. Janota Dana H 1987 present B. Holman, K.Janota, Smrice, Valtice M. Havránková Petra H 1988 1998 E. Pekárková, A. Procházková, Praha Ruzynì, Lysá nad Labem M. Svaèinová, Z. Jech Nora H 1989 present B. Holman, K.Janota, Smrice, Valtice M. Havránková Hana H 1991 present B. Holman, F. Mach, K. Janota, Smrice, Valtice M. Havránková Korona H 1991 present E. Pekárková, A. Procházková, Praha Ruzynì, Lysá nad Labem M. Svaèinová Regina H 1991 present B. Holman, K.Janota, Smrice, Valtice M. Havránková Admira H 1993 present B. Holman, K.Janota, Smrice, Valtice M. Havránková Alena H 1993 present E. Pekárková, M. Svaèinová Praha Ruzynì, Lysá nad Labem Fatima H 1993 present B. Holman, K.Janota, Smrice, Valtice M. Havránková Partena H 1993 present E. Pekárková, M. Svaèinová Praha Ruzynì, Lysá nad Labem Blanka H 1996 present J. Práil, J. Hrubanová SEMO Ltd. Smrice Charlotte H 1998 present J. Práil, J. Hrubanová SEMO Ltd. Smrice Mira H 1998 present J. Horal Moravoseed Ltd. Mikulov Ornello H 1998 present J. Práil, J. Hrubanová SEMO Ltd. Smrice Bohdana H 2000 present B. Holman B. Holman Cucumber Breeding

29 Desdemona H 2000 present J. Horal Moravoseed Ltd. Mikulov Ela H 2000 present J. Horal Moravoseed Ltd. Mikulov Dalila H 2001 present J. Horal Moravoseed Ltd. Mikulov Elisabet H 2001 present J. Práil, J. Hrubanová SEMO Ltd. Smrice Jitka H 2001 present B. Holman B. Holman Cucumber Breeding Klárka H 2001 present SEMPRA Ltd. Praha Lada H 2001 present B. Holman B. Holman Cucumber Breeding Altaj H 2002 present J. Horal Moravoseed Ltd. Mikulov Everest H 2002 present J. Horal Moravoseed Ltd. Mikulov Romana H 2002 present B. Holman B. Holman Cucumber Breeding Twigy H 2002 present J. Horal Moravoseed Ltd. Mikulov Partner H 2003 present J. Horal Moravoseed Ltd. Mikulov Cucumis sativus - salad outdoor cucumbers Èínské hadovité (1941) Author unknown Sibøina 1952 1965 Delikates 1,4 (1941) Author unknown Veltrusy 1952 1983 Mladoboleslavské 1952 1978 J. Netuil, p. Zita Semèice salátnice 1,2,3,4 Fénix 1,4 1972 1983 F. Mach, B. Holman Smrice Laura H 1980 1993 B. Holman, F. Mach Smrice StelaP H 1982 present B. Holman, F. Mach Linda Mix H 1989 present B. Holman, K.Janota, Smrice, Valtice M. Havránková LivieP H 1993 present B. Holman, K.Janota, SEMO Ltd. Smrice, Seva Flora M. Havránková Ltd. Valtice PerseusP H 1993 present B. Holman, K.Janota, SEMO Ltd. Smrice, Seva Flora M. Havránková Ltd. Valtice Natalie H 2001 present B. Holman B. Holman Cucumber Breeding LiliP H 1998 present J. Práil, J. Hrubanová SEMO Smrice CheerP H 2002 present J. Práil, J. Hrubanová SEMO Smrice Obelix H 2003 present J. Horal Moravoseed Ltd. Mikulov Cucumis sativus - salad cucumbers for hot and cold beds Landovského 1946 1956 F. Landovský, J. Landovský Sibøina paøenitní okurka 2 Jedineèná 1,2,3,4 1952 1982 J. Vyskoèil Mìlník Mlazice idovická produkta 1,3,4 1956 present E. Dobiá idovice Reforma (zlepená) 1,2,4 1957 1977 V. Machurek Kvetoslavov (Slovak Republic) Cucumis sativus - greenhouse salad cucumbers Nejlepí ze vech 1,4 (1941) S. Bene Libochovice 1952 1978 Spotresisting 1,3 (1941) J. Vyskoèil Mìlník Mlazice 1952 1970 Unikát 1,4 1958 1983 J. Vyskoèil Mìlník Mlazice Leda H 1978 1989 M. Prùdek Lednice na Moravì Marta H 1983 present V. Machurek Kvetoslavov (Slovak Republic) MinisprintP H 1997 present J. Práil SEMO Ltd. Smrice Superstar H 1997 present J. Práil SEMO Ltd. Smrice

30 Vista H 1997 present J. Práil SEMO Ltd. Smrice PaladinkaP H 1999 present J. Práil SEMO Ltd. Smrice BabyP H 2002 present J. Práil SEMO Ltd. Smrice FormuleP H 2002 present J. Práil SEMO Ltd. Smrice Sherpa H 2002 present J. Horal Moravoseed Ltd. Mikulov Jogger H 2003 present J. Horal Moravoseed Ltd. Mikulov Cucurbita maxima pumpkins, gourds Veltruská obrovská 1,3 1952 present J. Vlk Veltrusy Goliá 1 1969 present S. Bene Libochovice nad Ohøí Cucurbita pepo pumpkins, squashes Kveta 1 1962 present F. Pùlkrábek, K. Michálek Kvetoslavov (Slovak Republic) Diamant F 1972 1996 E. Troníèková Praha - Ruzynì 2 Opavská (f. oleifera) 1999 present J. Havel, M. Judlová- Hájková OSEVA - PRO Ltd. workplace Opava Bìtka H 2002 present J. Práil, J. Hrubanová SEMO Smrice Delikates 2002 present J. Práil, J. Hrubanová SEMO Smrice Goldline H 2002 present J. Práil, J. Hrubanová SEMO Smrice Startgreen H 2002 present J. Práil, J. Hrubanová SEMO Smrice Apetit (f. oleifera) 2003 present J. Práil, J. Hrubanová SEMO Smrice Goldena 2003 present J. Horal Moravoseed Ltd. Mikulov Nefertiti 2003 present J. Horal Moravoseed Ltd. Mikulov Olga (f. oleifera) 2003 present J. Horal Moravoseed Ltd. Mikulov Tapir 2003 present J. Horal Moravoseed Ltd. Mikulov Cucumis melo - melons Togo krajový 1 1950 1964 maintained by breeding station in Topolniky (Slovak Republic) Lednický 1952 1972 F. Frimmel Lednice na Moravì Solartur 1 1961 present L. Venény, J. Odehnal, Solary (Slovak Republic) M. Bartalo, L. Rákoczi Oran 1 1971 1996 L. Venény, L. Rákoczi Solary (Slovak Republic) Solar H 1982 1996 L. Rákoczi, I. Csibrányiová, Solary (Slovak Republic) J. Odehnal Nektar H 1991 present M. Bartalo, L. Rákoczi, Solary (Slovak Republic) I. Csibrányiová Citrullus lanatus watermelons Dunaj 1,3 1962 1996 L. Venény, J. Odehnal, Solary (Slovak Republic) M. Bartalo, L. Rákoczi Lajko H 1970 1979 L. Venény, L. Rákoczi Solary (Slovak Republic) Melko H 1970 1991 L. Venény, L. Rákoczi Solary (Slovak Republic) Lajko II H 1978 present J. Odehnal, L. Rákoczi, Solary (Slovak Republic) I. Csibrányiová Vital H 1989 present L. Rákoczi, I. Csibrányiová, Solary (Slovak Republic) M. Bartalo Rapid H 1991 2002 Solary (Slovak Republic)

* Maintained in the genebank in: 1 Czech Republic (RICP), 2 The Netherlands (CGN), 3 Rus- sia (VIR), 4 USA (USDA); ** Registration restriction; H F hybrid, F used temporarily 1 2 P Suitable also for the cultivation in the plastic tunnels

31 tion with the Breeding Station in Lysá nad Labem. Hybrid cultivars have been created by a trihybrid methodology using hermaphroditic lines (Troníèková and Procház- ková, 1984). A list of extant Czech cucumber cultivars is presented in Table 2; recent cucumber cultivars bred by private companies are included in this list.

Salad cucumbers for field cultivation

The landrace Bzenecká salátnice has been used in the Southern Moravia as a fresh vegetable for salads and also for preservation (fermentation) in salt solution. The landrace Èernavská salátnice has been cultivated around the town of Mladá Boleslav (Central Bohemia). Its populations from the villages of Nìmèice and Libi- chov were improved by J. Netuil since 1927 and concurrently by the Union of Vege- table Growers in Mladá Boleslav since 1935 and distributed under the name of Mla- doboleslavská salátnice. It was maintained until 1978 in the Research Institute of the Beet by Mr. Zita. The landrace Hajnavka was developed from the snake cucumbers (Chinese cucumber) from China. The landrace Vetatská has been cultivated around the village of Vetaty (Central Bohemia). The landrace Branické hadov- ky cultivated by the gardeners in PragueBráník has been of local importance. The foreign Delikates was officially registered and maintained from 1941 until 1983 and was very popular. Its young fruits were processed as pickles, and the bigger fruits were consumed fresh in salads. The breeding of field salad cucumbers has been conducted exclusively by the Vegetable Breeding Station in Smrice since the 1960s, as an initiative of J. Homola, F. Mach and B. Holman. Later, the breeding of salad cucumbers in Smrice was conducted under identical conditions as described for gherkins. The list of all cultivars bred here is summarized in Table 2, including the modern cultivars created after 1989 at private breeding companies.

Cucumbers for hot and cold beds

The production of salad cucumbers in forcing frames in the Czech Republic was high during the first half of the 20th century and all approaches, that is early cultivation in hot beds, medium early cultivation and late cultivation in cold frames, have been used. Local gardeners used their own local cultivars that were well-adapted for their own climatic conditions and cultivation practices (Podeva, 1959). In PragueLibeò, a local cultivar, Libeòská trní was cultivated. Another local Prague cultivar, Branické paøenitní okurky, grown since the 1870s, has been improved since 1929 by F. Landovský at the Horticultural Research Institute in Prùho- nice near Prague. The breeding process has been conducted since 1938 by his brother J. Landovský. Landovského paøenitní okurka was officially registered until 1956. In 1956, idovická produkta was released by E. Dobiá in idovice near Roud- nice nad Labem (Central Bohemia) and until now it is registered in the State Book of Cultivars. Jedineèná, released by J. Vyskoèil in MìlníkMlazice in 1952, was used and registered until 1982.

32 The cultivation of cucumbers in hot beds and cold frames needed a lot of manual work and from the 1970s it was replaced by growing in plastic tunnels (Mareèek, 1976).

Greenhouse cucumbers

Cucumbers were first cultivated in greenhouses of the castle gardens; later this experience was transferred to production gardens near bigger towns. After the Second World War, many special greenhouses, later with hydroponics, were constructed. This trend ended by the end of the 1980s, because of economic reasons (high inputs of energy under the climatic conditions of Bohemia and Moravia), with local production having been replaced by importation. Similarly, the breeding of greenhouse cucumbers was not as advanced when as the breeding of field ones. First, the English and later the German cultivars were used. The English Spotresisting was maintained at the Breeding Station in ibøina (West from Prague) from 1941 to 1970. The German Nejlepí ze vech, bred by the company Weigelt in Erfurt, was maintained from 1941 to 1978 by S. Bene. Unikát, the first Czech cultivar, was released in 1958 by J. Vyskoèil in Mìlník Mlazice and the first Czech hybrid cultivar, Leda F was released in 1978 by M. Prù- 1 dek in Lednice na Moravì. The hybrid parthenocarpic salad cucumber, Marta F was 1 released in 1983 in Slovakia and is still registered also in the Czech Republic (Table 2). Recent cultivars bred after 1989 by private companies are listed in the Table 2.

Pumpkins and squash

Cucurbits of the genus Cucurbita are of New World origin. Pumpkins and squash of Cucurbita maxima Duchesne and C. pepo L. were documented in Czech Lands in the Czech translation of the Matthioli herbal from 1562. Matthioli listed several kinds of cucurbits and/or gourds, one of which was described as a local and well-known gourd with white flowers; from the description, the bottle gourd (Lagenaria sp.) can be identified. Another kind, named as Indian (supposedly from India, in fact from America) or foreign gourds, called at that time in the Czech Lands Turkish cabbage are of the genus Cucurbita. The first Czech written and printed herbal of Èerný mentions in the chapter CCCXCIIII. one cucurbitaceous species called our home cucurbits with its Latin name of Cucurbita and German one of Kürbsz (Èerný, 1517), but without plant descriptions. Plant on the accompanying drawing can be identified as bottle- gourd (Lagenaria sp.), and also the Linnaeus name Cucurbita lagenaria L. was used for Lagenaria siceraria (Molina) Standl. (Jeffrey, 2001) C. maxima is traditionally grown in home gardens and it is processed in a sweet juice in home kitchens and partly industrially processed. C. pepo, especially of the Zucchini Group, has had increasing popularity since 1980s. The C. maxima Veltruská obrovská, was released in 1952 by J. Vlk at the Bree- ding Station in Veltrusy (north of Prague), and Goliá was bred by S. Bene at the Breeding Station in Libochovice nad Ohøí (Table 2). E. von Tschermak-Seysenegg, born on the 15th of November 1871 in Vienna (Rucken-

33 bauer, 1999), was one of the plant scientists who rediscovered Mendels classic paper in genetics (Tschermak, 1900). Tschermark contributed substantially to the improvement of C. pepo pumpkins carrying the mutant characteristic of lack of a lignified seed coat (Ts- chermak, 1934), cultivated in Austria and Moravia. He provided the seed of populations having bush habit to F. Frimmel in Lednice na Moravì (Luný et al., this volume). By the beginning of the 1950s, F. Frimmel had combined bush habit with hull-less seeds in a new cultivar. Unfortunately, this new cultivar was not accepted by the oil processing industry because other cheaper raw materials were available. Kveta, a vegetable marrow, was released in Slovakia in 1962 (Luný et al., 1988). In 1972, the zucchini Diamant F2 was registered (Table 2). Recent Czech cultivars are summarized in the Table 2.

Melons and watermelons

The centre of diversity and perhaps of the origin of the principal melons, Cucumis melo L., of world commerce, i.e. the Inodorus, Cantalupensis, and Reticulatus Groups, is in Western or Central Asia (Schwanitz, 1967; Jeffrey, 1980; Ladizinsky, 1998). Melons have been in cultivation for a long time, from approximately 2000 B.C.E. in Egypt, Mesopotamia, eastern Iran and China, and from 1000 B.C.E. in India. The first cultivated melons were probably non-sweet forms, known as chate or adzhur in Egypt (Pitrat et al., 2000). These were probably transported later to the ancient Greece and Roman Empires. However, it is difficult to identify the Hippokrat sikkura, Dioskorides pepon or Plinius melopepo as melons, cucumbers or watermelons (Jirásek, 1958). The true edible sweet melons appeared in the Mediterranean basin in the fifth century and probably were introduced to this area from Near East (Anatolia) (Sinskaja, 1969). In the 13th century Marco Polo mentioned the excellent melons in Chiva, a place in the Near East. The melon group Cantalupensis was probably transported from Armenia to Eu- rope by Catholic monks. Melons were cultivated in the abbey of Cantaluppi in Italy and by the end of 15th century they were transported to France (Jirásek, 1958). Our knowledge of melon (Cucumis melo) transport from the area of its origin to the Czech Lands as compared to that on cucumber is very limited. The 14th century and the period of the Czech king and Roman Emperor Charles IV brought widespread development of cultivation of new crops (Kubaèák, 2003). Cul- tivation of melons was in 14th and 15th centuries very advanced in the Czech Lands and famous in Central Europe (Domin, 1945). Melons are reported in the first Czech written and printed herbal of Èerný from 1517 (Èerný, 1981). The Latin name of melons is Melopepones in the Czech translation of the Matthioli herbal of 1562. It is mentioned there, that besides some positive medicinal properties, the enormous consumption of melons reverses the blood into water and cause fever, and that Emperor Albrecht II and the Czech king Rudolf, after an enormous consumption of melons, suffered a red illness and terminal fever and died (Mathioli, 1998). The frequent cultivation of melons in the Czech Lands is mentioned by Bohuslav Balbín in the second half of the 17th century (Jirásek, 1958). The commercial growing of melons in the Czech Republic is limited to the warmest region of South Moravia. In the 1930s, melons were produced by the machine producing (engineering) company Wiesner in the town of Chrudim, in greenhouses heated directly from the factory by its waste heat. In 1952, F. Frimmel released in

34 Lednice na Moravì the melon Lednický (Table 2) (Luný et al., this volume). The watermelon, Citrullus lanatus (Thunb.) Matsumura & Nakai, was probably derived from local forms in Central Asia (Sinskaja, 1969). According to a second interpretation, the watermelon was derived from the indigenous African (Egypt, Algeria) populations of C. colocynthis L. (Sinskaja, 1969; Bates and Robinson, 1995). Despite early watermelon cultivation in Egypt and perhaps in the Near East and India, historical accounts are sparse until the sixteenth century and it is not sure if they were known in the ancient Greek and Roman Empires. The distribution of watermelons to the Mediterranean basin was performed by Arabians in the 11th and 12th centuries. Water- melons were transported to the area around the Black Sea by Tatars. Watermelons are referred to Angurie in the Matthioli herbal from 1562. Watermelons have been and are very popular in the Czech Lands and they are frequently imported from Slovakia, Hungary and Balkan countries. It is cultivated sparingly in home gardens. In the period of the former Czechoslovakia (19181992), the melon and watermelon breeding was located at the Breeding Station in Solary in Southern Slovakia. Several cultivars from this period are registered until now in the Czech Republic (Table 2). Modern Czech cultivars have not yet been bred.

Luffa, lagenaria and other minor cucurbitaceous vegetables

The cultivation of Luffa sp. was introduced in the 1920s by the owner and direc- tor of the famous shoe factory, Tomá Baa, in South Moravia. During a short period between the two world wars, he used the sponge of mature fruits for shoe production. After 1945, this production ceased. Other cucurbitaceous vegetables, such as Lagenaria sp. (bottle gourd), Momordica charantia (bitter melon or balsam pear), Cucumis metuliferus (kiwano), and Cyclanthera pedata (caihua or wild cucumber), are cultivated occasionally in hobby gardens as novelties.

Second author´s note

Some historical remarks from the 20th century are without any references and are personal recollections of the first author, Dipl. Ing. Jiøí Moravec (b. 1923), who devoted his life to vegetable growing, breeding and genetic resources. From 1952 to 1990, he worked in the Vegetable Research Institute in Olomouc, and most of this period he served as head of the Vegetable Gene Bank. He lives in Olomouc, provides consul- tancy on traditional vegetable varieties to curators of collections and growers, and supports them by his enthusiasm, optimism and élan vital until the present time.

35 Figure 2. Dipl. Ing. Jiøí Moravec. Ph.D. (Spring 2003).

Acknowledgements

This work was partly supported by grants: QD 1357; MSM 153100010; National Programme on Plant Genetic Resources Conservation and Utilization in the Czech Republic (E-79/01-3106-0200).

References

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36 Jeffrey, C. 2001. Cucurbita. In: Hanelt, P. and Institute of Plant Genetics and Crop Plant Research (Ed.), Mansfelds Encyclopedia of Agricultural and Horticultural Crops (Except Ornamentals). Springer, Berlin, pp. 1541-1552. Jirásek, V. 1958. Rostliny na naem stole (Plants on Our Table). Orbis, Prague, Czechoslovakia, 625 pp. (in Czech) Køístková, E. 2002. The Czech national collection of cucurbitaceous vegetables. In: Díez, M.J., Pico, B. and Nuez, F. (Comp.), Cucurbit Genetic Resources in Europe, Report of Ad hoc meeting, Adana, Turkey, 19 January 2002, IPGRI, Rome, Italy, pp. 18-29. Køístková, E. and Lebeda, A. 1995. Genetic resources of vegetable crops from the family Cucurbita- ceae (Genové zdroje zelenin èeledi Cucurbitaceae). Zahradnictví (Hortic. Sci., Prague), 22: 123-128. Kubaèák, A. 2003. Contribution of Czech Lands to European Agriculture. Ministry of Agricultu- re of the Czech Republic, Praha, Czech Republic. Kvìt, R. 2003. Due krajiny Staré stezky v promìnách vìkù (Spirit of the Land Ancient Trails in the Passage of Time). Academia, Praha, Czech Republic. (in Czech, English Summary) Ladizinsky, G. 1998. Plant Evolution under Domestication. Kluwer Academic Press, Dordrecht, the Netherlands. Lebeda, A. (Ed.) 1986. Metody testování rezistence zelenin vùèi rostlinným patogenùm (Methods of Testing Vegetable Crops for Resistance to Plant Pathogens). VHJ Sempra, VÚZ Olomouc, Czechoslovakia. (in Czech) Lebeda, A. (Ed.) 1987. lechtitelská stanice Smrice Èelechovice na Hané (Plant Breeding Station Smrice Èelechovice na Hané). VHJ Sempra, Praha, Czechoslovakia. (in Czech, English summary) Lebeda, A. 1988. Výzkum rezistence genových zdrojù zelenin /Úvod, salát, okurky/ (Resistance research of vegetable genetic resources /Introduction, lettuce, cucumbers/). Záhradníctvo, 13: 352-354. Lebeda, A. 1994. Zamylení nad ukonèením èinnosti Výzkumného a lechtitelského ústavu zeli- náøského v Olomouci (Thinking about closing of activity of Vegetable Research and Breeding Institute in Olomouc). Záhradníctvo, 19: 141-143. Lebeda, A. 1996. Výzkum rezistence genových zdrojù zahradních rostlin modelový systém Lactuca spp. Bremia lactucae (Research on resistance of genetic resources of horticultural crops a model relationship between Lactuca spp. and Bremia lactucae). Zahradnictví (Hort. Sci., Pra- gue), 23: 63-70. Luný, J. and Holman, B. 1973. The contemporary breeding of field cucumbers (Cucumis sativus L.) in Czechoslovakia. Eucarpia Meeting, Transactions of the Meeting. Hannover, Germany, pp. 1-9. Luný, J. Paszko, J. and Vako, . 1988. A contribution to Cucurbita breeding in Czechoslova- kia. Proceedings of the Eucarpia Meeting on Cucurbit Genetics and Breeding. INRA, Avignon Montfavet, France, pp. 187-191. Mareèek, F. (Ed.) 1976. Trní zelináøství (Vegetable Growing for Market). Státní zemìdìlské nakladatelství (State Agricultural Publishing House), Praha, Czechoslovakia. (in Czech) Mathioli, P.O. 1998. Herbáø neboli bylináø, pøetisk z roku 1562. (Herbarium, reprint from 1562). Dobra & Fontána, Olomouc, Czech Republic 1998, pp. 350-359. (in Czech) Moravec, J. 1959. Rostliny z èeledi tykvovitých (Vegetables from the family Cucurbitaceae). In: Podeva, J. (Ed.), Encyclopaedia of Vegetable Production, Part II, Czechoslovak Academy of Agricultural Science, Prague, Czechoslovakia, pp. 290-399. (in Czech) Moravec, J. 2000. Zeleniny a koøeninové rostliny (Vegetables and spice plants). In: Collective of authors. Almanac of the Czech and Moravian plant breeding. Bohemian-Moravian Association of Plant Breeding and Seed Production, Prague, Czech Republic, pp. 62-71. (in Czech) Orel, V. 2003. Gregor Mendel a poèátky genetiky (Gregor Mendel and Beginning of Genetics). Academia, Praha, Czech Republic. (in Czech) Oveèka, V. 1955. Novì vylechtìné odrùdy okurek a póru (Newly bred cultivars of cucumbers and leek). Sborník Za socialistické zemìdìlství, Praha, 5: 1162-1167. (in Czech) Oveèka, V. 1964. Studium vlivu samoopylení (incuchtu) u polních okurek Znojemských nakla- daèek (Cucumis sativus L.) (Studies of the influence of self-pollination /inzucht/ on the field cucumbers Znojemské nakladaèky). ÚVTI Sborník Rostlinná výroba (Praha), 10: 1053- 1062. (in Czech) Pitrat, M., Hanelt, P. and Hammer, K. 2000. Some comments on infraspecific classification of cultivars of melon. In: Katzir, N. and Paris, H.S. (Eds.), Proceedings of Cucurbitaceae 2000. Acta Hort., 510: 29-36.

37 Podeva, J. (Ed.) 1959. Encyklopedie zelináøství, Èást Speciální, II (Encyclopaedia of Vegetable Growing, Part Special, II). Vydala Èeskoslovenská akademie zemìdìlských vìd, ve Státním zemìdìlském nakladatelství Praha (Published by the Czechoslovak Academy of Agricultural Science, State Agricultural Publishing House), Praha, Czechoslovakia. (in Czech) Poláèek, L. 2004. Personnel information from the Archaeological Institute of the Czech Acade- my of Sciences, Brno, Czech Republic, E-mail from 22 January 2004.([email protected].) Rubatzky, V.E. and Yamaguchi, M. 1997. World Vegetables. Principles, Production, and Nutritive Values. Second Edition. Chapman & Hall, New York. Ruckenbauer, P. 1999. E. v. Tschermak-Seysenegg his role in practical plant breeding after the rediscovery of the Mendelian laws in 1900. Acta univ. agric. Et silvic. Mendel. Brun. (Brno), XLVII, No. 4: 31-36. Schwanitz, F. 1967. Die Evolution der Kulturpflanze. BLV Bayerischer Landwirtschaftsverlag, GmbH, München, Germany. Sinskaja, E.N. 1969. Historická geografie kulturních rostlin (Historical Geography of Cultivated Plants). Academia, Praha, Czechoslovakia. (in Czech) Troníèková, E. and Procházková, A. 1984. Flowering and fruiting biology with hermaphrodite pickling cucumber and its utilization in the breeding of hybrids. I. Glasshouse conditions. Sborník ÚVTIZ, Zahradnictví (ÚVTIZ Proceedings, Gardening, Prague), 11: 277-285. (in Czech) Tschermak, E. 1900. Über künstliche Kreuzung bei Pisum sativum. Berichte der Deutschen Bota- nischen Gesellschaft 18: 232-239. Tschermak, v. E. 1934. Der schalenlose Kürbis als Ölfrucht. Deutsche landwirt. Presse, 6(8): 94-96.

38 A dedication to Franz Frimmel, a Czech leader of cucurbit breeding

J. Luný1, A. Lebeda2 and E. Køístková3 1Za Potou 3, 772 00 Olomouc, Czech Republic 2Palacký University in Olomouc, Faculty of Science, Department of Botany, lechti- telù 11, 783 71 Olomouc-Holice, Czech Republic; e-mail: [email protected] 3Research Institute of Crop Production, Division of Genetics and Plant Breeding, Department of Gene Bank, Workplace Olomouc, lechtitelù 11, 783 71 Olomouc-Ho- lice, Czech Republic; e-mail: [email protected]

Summary

Professor Franz Frimmel (18881957), successor of Prof. E. v. TschermakSeysenegg, significantly promoted and accelerated the development of plant genetics and breeding in the first half of the 20th century in the territory of Czech Lands. He developed a number of cultivars, breeding methods and approaches, and authored a number of scientific publications. He also developed, introduced and explored new methods for breeding of cucumbers, melons and pumpkins.

Keywords: Cucurbitaceae, cucumber, melon, squash, pumpkin, gourd, Czechoslovakia, breeding methods, genetic resources, F hybrids 1

Introduction

Motto: It is good to remember those who are no longer among us and whose contributions we may continue. By these words we remember an excellent specialist, scientist and the real perso- nality in plant breeding, Professor PhDr. Franz Frimmel (18881957) (Tschermak-Seysenegg, 1958; Betlach and Floss, 1967). Dr. F. Frimmel belongs to the gallery of immortal personalities of the 20th century who significantly promoted and accelerated the development of plant genetics and breeding, that includes Erwin Baur (18751933), Karel Hrubý (19101962), Hans Kappert (18901976), Hermann Kuckuck (19031984), Theodor Roemer (18831951), Kurt Rümker (18591940), Hans Stubbe (19021989), Nikolaj Ivanoviè Vavilov (18871942) and others. Although he was born in Vienna (recent Austria), Dr. Frimmel spent his whole professional life in Moravia (presently Czech Republic, formerly Czechoslovakia) and partly in Slovakia, and from this point of view he was a naturalized citizen of the former Czechoslovakia. By his enthusiasm, selfless unique activities and erudite approaches in research, plant breeding and practice, he influenced the agricultural and horticultural development in the whole of Central Europe during the first half of the 20th century (Luný, 1969). He was the developer of numerous plant cultivars, plant breeding methods and approaches, and authored a number of scientific publications. His book Praxis der Planzenzüchtung (Practice of Plant Breeding), issued in 1951, can be used as a breeder vademécum (breeder´s textbook) even at the present time.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 39 Figure 1. F. Frimmel in the 50th of the Figure 2. Memorial of F. Frimmel at Men- 20th century at the Vegetable Research In- deleum in Lednice na Moravì. stitute in Olomouc.

It develops publication from Dr. H. Kappert on plant genetics and breeding Die ve- erbungswissenchaftlichen Grundlagen der Züchtung (Scientific Basis of Breeding) from the 1948. Both books were published by the company Paul Parey (Berlin and Hamburg, Germany). Part of Dr. Frimmel´s research activities was devoted to the cultivated Cucurbita- ceae. Cucumbers (Cucumis sativus L.) were his favourite subject of research and breeding. In the 1920s and 1930s he introduced, together with Dr. Albert Stumer, new methodologies of clonal and resistance breeding of grapes in the region around the town of Znojmo (Southern Moravia). This region is famous for cucumber production and processing of the inimitable pickled sour-sweet gherkins Znojmia (Moravec et al., 2004, this issue). Dr. Frimmel soon recognized the importance of this vegetable and during the 1930s he introduced Lednicko-Znojemské nakladaèky (Gherkins from Lednice and Znojmo). This cultivar was later known under the name of Zno- jemská (Moravec et al., 2004, this issue). The breeding efforts of Dr. Frimmel were later continued by V. Oveèka, who bred Znojmia (later re-named to Palava) (Mo- ravec et al., 2004, this issue). Through the Benary Co. (Erfurt, Germany), Dr. Frimmel was the first to introduce F hybrids of tomatoes to the European market; he intended to do the same with cu- 1 cumbers. His idea was to take advantage of the male pre-stage, i.e. the period after

40 Figure 3. View on the building and glasshouse in Mendeleum (Lednice na Moravì) where F. Frimmel spent the most important part of his professional life (photo from 1950s). opening of the first staminate flower and before the opening of the pistillate one. The early occurring male (staminate) flowers were removed from the female parental line by hand. The plants were then allowed to be pollinated by bees or bumble-bees in the presence of the male parental line. This methodology was later successfully utilized by other breeders. Mrs. Z. Nováková continued in these hybridization experiments at the Vegetable Research Institute in Olomouc. Dr. Frimmel created an early maturing, high yielding, flavourful, aromatic melon (Cucumis melo L.) Lednický, known in German as Eisgruber köstliche (Moravec et al., 2004, this issue). Breeding and selection was based on a large collection of melon landraces originating mostly from Turkmenistan (former Soviet Union) and involved complex

Figure 4. Melon cultivar Led- nický bred by F. Frimmel in Mendeleum.

41 crosses and progeny testing. Dr. Frimmel recognized advantages of hybrids issued from crosses between geographically distant parents. For the creation of F hybrid melons 1 he also developed and utilized methodology of signal features i.e. phenotypic markers. Recessive characteristics were incorporated into breeding lines to be used as female parents. This methodology was also used by his successors. The melon and Lednic- ký itself was later used by other breeders as a parental component. He also developed and used another breeding methodology, referred to locally as blind crossing, which was quite useful when it was not possible to select plants neither before flowering nor at a stage of young fruits. A portion of the seeds from each lines used for crossing was not sown; after testing the F progeny of each line, 1 remnant seeds of the lines producing the best F s were then sown for further selecti- 1 on. This method was successful in the breeding of naked-seed pumpkins (Cucurbi- ta pepo L.) with bush growth habit; using a source population with half-naked seeds provided by Prof. E. Tschermak (Tschemark-Seysenegg, 1934; Frimmel, 1957). We commemorate the professional efforts of Dr. F. Frimmel and to reminisce that it was exactly 90 years ago, in 1914, that he joined the Mendel Institute (later Mende- leum) in Lednice na Moravì (South Moravia), as the assistant of Prof. E. v. Tscher- mak-Seysenegg (Ruckenbauer, 1999).

2nd Author´s Note Prof. Dipl. Ing. Jan Luný, Ph.D. (born in 1926), devoted his professional life to the plant breeding. In the years 19631991 he was a staff member at the Mendel University of Agriculture and Forestry in Brno (Faculty of Horticulture in Lednice na Moravì), and from 1975 through 1991 served as head of the Department of Plant Breeding.

Literature related to the breeding of cucurbits published by Dr. F. Frimmel

Frimmel, F. 1927. Remontatní schopnosti okurek (Remontant abilities of cucumbers). Èeskoslo- venský zemìdìlec, Prague, IX, 36: 551-553. (in Czech) Frimmel, F. and Lauche, K. 1940. Neue Vege der Gürkenzüchtung. Obst und Gemüsebau, 86. (in German) Frimmel, F. 1942. Züchtungsfragen bei Kürbisgewächse. Obst und Gemüsebau, 88. (in German) Frimmel, F. 1943. Die züchterische Bedeutung der Remontierfähigkeit. Leistungssteigerung in Gartenbau. H.I., Wiesbaden, 1: 41-56. (in German) Frimmel, F. 1951. Die Praxis der Pflanzenzüchtung. Paul Parey, Berlin. (in German) Frimmel, F. 1957. Pøíspìvek ke lechtìní okurek nakladaèek (Cucumis sativus) (Contribution to the cucumber breeding /Cucumis sativus /). Bulletin ÈSAZV, Výzkumný ústav zelináøský, Olo- mouc, 1: 7-15. (in Czech)

References

Betlach, J. and Floss, K. 1967. Vzpomínka na prof. Dr. Frantika Frimmela (Commemorate on prof. Dr. Frantiek Frimmel; including a list of 18 most important Frimmel´s papers relating to the plant breeding). Genetika a lechtìní (Genetics and Plant Breeding, Prague), 3 (XL): 235- 238. (in Czech) Frimmel, F. 1957. Pøíspìvek ke lechtìní okurek nakladaèek (Cucumis sativus) (Contribution to the cucumber breeding (Cucumis sativus)). Bulletin ÈSAZV, Výzkumný ústav zelináøský, Olo- mouc, 1: 7-15. (in Czech)

42 Luný, J. 1969. The 80th anniversary of birth of the late professor Franz Frimmel-Traisenau, Ph.D. A prominent European pioneer in the heterosis plant breeding. Folia Mendeliana, Musei Moraviae, No. 4: 45-46. Moravec, J., Lebeda, A. and Køístková, E. 2004. Growing and breeding of cucurbitaceous vegetables in the Czech lands. In: Lebeda, A. and Paris, H.S. (Eds.), Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), (this volume). Ruckenbauer, P. 1999. E. v. Tschermak-Seysenegg his role in practical plant breeding after the rediscovery of the Mendelian laws in 1900. Acta univ. agric. et silvic. Mendel. Brun. (Brno), XLVII, No. 4: 31-36. TschermakSeysenegg, E. 1934. Der schalenlose Kürbis als Ölfrucht. Deutsche landwirt. Presse, 6., 8., pp. 94-96. (in German) Tschermak-Seysenegg, E. v. 1958. Nachruf Franz Frimmel v. Traisenau. Z. Pflanzenzüchtg., 39: 121-126.

43 44 Cucumber fruit size and seed yield affected by growth regulators

L. Nowaczyk and P. Nowaczyk University of Technology and Agriculture, Department of Genetics and Plant Bree- ding, Bydgoszcz, Poland

Summary

Gibberellic acid (GA3) was used to induce male flowers on a parthenocarpic, gynoecious hybrid cucumber (Cucumis sativus) Polonez F . GA3 treatment resulted in increased fruit volume and 1 weight. This was accompanied by a considerable increase in fruit seed yield which was attribu- table to a statistically non-significant increase in the number of seeds per fruit combined with a similar increase in seed size. During blooming and pollination, plants were treated with 2,4-dichlorophenoxyacetic acid (2,4 D) in concentrations of 0.005% and 0.01%. This treatment did not reduce fertility. No bi-embryonic seeds were found.

Keywords: Cucumis sativus, gibberellic acid (GA3), 2,4-dichlorophenoxyacetic acid (2,4 D), fertility

Introduction Gynoecious, parthenocarpic genotypes are of particular significance in the production of cucumbers, Cucumis sativus L. They give high fruit crops with no necessity of flower pollination. However, they are more difficult to reproduce, since they produce few, if any, male flowers, and even if male flowers are present they appear during early blooming only. To obtain a bigger number of male flowers as a source of pollen, gibberellic acid is applied to the plants. Treatment of young plants with a solution of this growth regulator can induce the initiation of sufficient male flowers over a long duration. Similar effects can be observed when the young plants are treated with AgNO (Lower and Edwards, 1986). However, silver nitrate reduces the pro- 3 duction of female flowers (Mibus et al., 2000; Stankovic and Prodanovic, 2002). The opposite effect, the reduction of male flower number, can be induced with ethephon (Sitaram et al., 1989; Korzeniewska et al., 2000). In Cucurbita pepo L., the number of female flowers was increased by treatments with ethephon and auxins (Mancini and Calabrese, 1999). Gibberellic acid can induce changes in plant growth habit and fruit growth (No- waczyk and Nowaczyk, 2002). Gibberellic acid application can affect fertility, increasing seed yield, which seems to be interesting from a practical point of view. Con- trary effects were observed when flowers of pepper and tomato were treated with 2,4D (Nowaczyk and Nowaczyk, 1996, 2000), leading us to investigate the effect of this auxin on cucumbers, with and without application of GA3.

The plant material was the parthenocarpic gynoecious cucumber Polonez F . Plants 1 were not treated or treated three times with a solution of gibberellic acid (GA3) of 1.5 g/l water; whole plants were sprayed for the first time when they had developed three leaves and then again three and six weeks later. Plants not treated or treated with gibberellic acid were either untreated or treated with 2,4-dichlorophenoxyacetic acid (2,4D); those treated with 2,4D received either a 0.005 % or 0.01% solution. Thus, there were altogether six treatment combinations, including the untreated control (C) plants. Treat- ment with 2,4D was by a single immersion of the female flowers in the 2,4D solution with pollination conducted by direct application of pollen from anthers of male flowers induced with GA3. These operations were carried out so that two or three fruits would set on one plant. There were eight plants in each of the six treatment combinations. Mature fruits were picked from the plants and after four weeks of storage they were subject to volume and weight measurement. The seeds together with the jelly-like mass was removed from the fruits and weighed, then fermented to separate the seeds. The seeds were then rinsed with water, dried, weighed, and counted. The last of the features mentioned was considered as the fertility criterion. The results were analyzed statistically, and values of the smallest significant difference were determined by Students t-test at P = 0.05.

Results and discussion The mean weight of the fruit varied considerably. Nonetheless, a clear trend was observed toward increased fruit weight when the plants were treated with gibberellic acid (Fig. 1). The increase was statistically significant in two of the three comparisons.

Figure 1. Mean fruit weight of plants treated or not treated with GA3.

46 Similarly, a clear trend was observed toward increased fruit volume when the plants were treated with GA3, but for fruits set from flowers treated with 0.005% 2,4D, this difference was not statistically significant. This effect of GA3 on fruit volume of cucumber is reminiscent of its effect on Sultanina table grape (Retamales et al., 1995), on which an application of GA3 at the 4 mm fruit diameter stage improved berry size.

Figure 2. Mean fruit volume of plants treated or not treated with GA3.

Assuming that changes in fertility should be accompanied by changes in the weight of the jelly surrounding the seeds, we decided to measure this property. The weight of jelly removed from fruits of plants treated with GA3 was almost double that of plants that were not treated with either growth regulator (Fig. 3). A similar relationship was observed for GA3-treated and GA3-untreated plants that were treated with 0.01% 2,4 D.

Figure 3. Mean jelly weight of plants treated or not treated with GA3.

47 From the practical point of view, the weight of seeds from the fruit is obviously of significant importance, since it determines the effectiveness of seed production. Un- der the conditions of this experiment, differences between extreme values reached 70% (Fig. 4). Higher yield (weight) of seeds was characteristic of fruit coming from plants treated with GA3.

Figure 4. Mean yield (total weight) of seeds per fruit of plants treated or not treated with GA3.

The number of seeds per non-treated fruit was typical of that of salad cucumbers and similar to that observed by Jankulovski et al. (1997) in an experiment with F 1 seed production. While treatment of plants with gibberellic acid did seem to favor an increase in the number of seeds per fruit (Fig. 5), this increase was statistically non- significant.

Figure 5. Mean number of seeds per fruit of plants treated or not treated with GA3.

48 In cucumber, as shown by the present results, 2,4 D, used at the concentrations we have indicated, did not reduce fertility. In pepper and tomato, 2,4 D applied in a similar manner reduced fertility but did have a favourable effect toward induction of additional embryos. The cucumber seeds collected in our experiment were tested for presence of additional embryos. All of them contained single embryos. The exceptional activity of 2,4 D in polyembrony induction was reported first by Haccius (1955) in Eranthis hiemalis L. We did not observe polyembryony in this experiment with cucumbers.

References Haccius, B. 1955. Experimentally induced twinning in plants. Nature, 176: 355-356. Jankulovski, D., Cirkova, G.M., Martinovski, D. and Sokolovski, S. 1997. Production of hybrid seeds of cucumber (Cucumis sativus L) in greenhouses. Sel. i Sem., 4 (1/2): 171-175. Korzeniewska, A., Ga³ecka, T. and Niemirowicz-Szczytt, K. 2000. Ethephon treatment on a monoecious cucumber accession for hybrid seed production. Acta Hort., 510: 269-271. Lower, R.L. and Edwards, M.D. 1986. Cucumber breeding. In: Basset, M.J. (Ed.), Breeding Vegetable Crops. Avi Publishing Company, Inc. Westport, Connecticut, pp. 173-207. Mancini, L. and Calabrese, N. 1999. Effect of growth regulators on flower differentiation and yield in zucchini (Cucurbita pepo L.) grown in protected cultivation. Acta Hort., 492: 265- 272. Mibus, H., Vural, I. and Tatlioglu, T. 2000. Investigation of sex expression of Cucumis sativus by grafting and cooper application. Acta Hort., 510: 211-218. Nowaczyk, P. and Nowaczyk, L. 1996. The influence of growth regulators on the frequency of polyembryony in pepper (Capsicum annuum L.). J. Appl. Genet., 37A: 204-207. Nowaczyk, L. and Nowaczyk, P. 2000. The fertility changes in tomato under growth regulators treatment. Acta Physiol. Plant., 23: 309-311. Nowaczyk, L. and Nowaczyk, P. 2002. Changes in size and fertility of cucumber (Cucumis sativus L.) as a result of treatment with growth regulators. In: Maynard, D.N. (Ed.), Cucurbitace- ae 2002. ASHS Press, Alexandria, VA, USA, pp. 330-335. Retamales, J., Bangerth, F., Cooper, T. and Callejas, R. 1995. Effects of CPPU and GA3 on fruit quality of sultania table grape. Acta Hort., 394: 149-158. Sitaram, A., Habib, F. and Kulkarni, G.N. 1989. Effect of growth regulators on seed production and quality in hybrid cucumber (Cucumis sativus L.). Seed Res., 17: 6-10. Stankovic, L. and Prodanovic, S. 2002. Silver nitrate effects on sex expression in cucumber. Acta Hort., 579: 203-206.

49 50 The influence of cultivation conditions on parthenocarpy of cucumber

N. Biriukova and E. Maslovskaya All-Russian Research Institute for Vegetable Crops, Novomitishinskyi Prospect 82, Mitishi, Moscow Region, Russia 141018; e-mail: [email protected]

Summary

Experiments were conducted, with winter and spring sowings, in glasshouses and a plastic greenhouse with the goal of comparing cucumber hybrids and their parents for yield and parthenocarpic tendency. Parthenocarpic tendency of cucumber plants grown in the plastic greenhouse was stronger than that of cucumber plants grown in glasshouses. This might be explained by the fact that the germplasm had been selected in the spring plastic greenhouse and hence was not as well adapted to the conditions of winter and spring cultivation in the glasshouse. In the glasshouses the yield level was higher, while in the plastic greenhouse it was more constant. In the majority of the cases, the yield of the hybrids was higher than that of their parental lines. The results indicate that the hybrids and parental lines of cucumber have differing reactions to differing conditions of cultivation, as expressed by differences in parthenocarpic tendency and yield.

Keywords: Cucumis sativus, plastic greenhouses, glasshouses, heterosis, parthenocarpy, yield

Introduction Parthenocarpy, the ability to set fruit without pollination, is of economic value in some horticultural crops. Research on the cultivation of parthenocarpic long-fruited greenhouse cucumbers (Cucumis sativus L.) was initiated in Europe early in the 20th century. The parthenocarpic cucumbers Telegraph, Grosse, Schlangen, Duke of Edinburgh, and others were commercially distributed at that time (Gustafson, 1942). The occurrence of parthenocarpy in cucumber is, to a considerable degree, defined by a complex of environmental factors that influences growth and development (Gustafson, 1942), including soil fertility, light, and temperature. Yakimovich (1935) observed the formation of parthenocarpic fruits of short-climbing varieties of cucumber in drought years. Nitsch (1952) recorded parthenocarpy in cucumber under short days and low night temperatures. Zhivnitskaya and Guseva (1978) and Guseva (1986) observed a higher percentage of parthenocarpy in plants of cucumber in plastic greenhouses, possibly connected with the high level of ultraviolet radiation. According to the data of Strelnikova (1984), parthenocarpic tendency is higher in spring crops. Differences in spectral composition of insolation can affect parthenocarpic response differently among cultivars (Alkuvejti, 1966). Generally, it is known that cucumber cultivars differ in parthenocarpic tendency (Strong, 1932). Herewith is a description of work conducted at the All-Russian Research Institute for Vegetable Crops toward the creation of parthenocarpic hybrids of cucumber for spring-summer crops in plastic greenhouses.

The present investigations included comparisons of the behavior of parental and hybrid breeding material of the All-Russian Research Institute for Vegetable Crops under different conditions, plastic greenhouses and glasshouses of spring-summer crops (1998-1999) and glasshouses of winter-spring crop rotation (1999-2000). Studies were conducted with cucumber parental lines and hybrids, selected from 1995 to 1997 for spring-summer crop rotation in plastic greenhouses, in conditions of the Moscow suburbs. The germplasm was evaluated according to the following parameters: dates of basic phenological phases of cucumber development and duration of the fruiting period; yield (early and total) as weight and quantity of fruits; parthenocarpy as according to Rogova (1975) on the 20 basal nodes, with 8 counted plants in a sample.

Table 1. Phenological phases of plants in different periods of cultivation

Spring-summer Spring-summer Winter-spring crop crop crop No. Phenophase Glasshouse Plastic house Glasshouse

1. Sowing April 6 May 5 December 17 2. Transplanting April 27 May 25 January 13 3. Beginning of flowering May 17 June 4January 29 4. Beginning of production From May 27 From June 14 From February 14 5. Sowing to start of production 51 days 42 days 57 days 6. End of crop August 25 August 25 July 14 7. Production duration 90 days 72 days 153 days

Results and discussion From the analysis of the course of phenological phases of cucumber under the various conditions (Table 1), it was obvious that there is lengthening of the vegetative period from sowing until the start of fruit production, in glasshouses for spring-summer crop rotation by 9 days and by 15 days in winter as compared with plastic greenhouses. Under conditions of the winter-spring crop, the period from the beginning of flowering until the beginning of fruit production is 5 days longer than in spring. Table 2 shows percentages of parthenocarpy of cucumber depending on conditions of cultivation. No. 129 slightly surpassed the parental lines in plastic greenhouses (79%). F No. 126 reached an intermediate value in spring glasshouses, while F 1 1 No. 129 was at the parental level with a low value. Other hybrids F had intermediate 1 parthenocarpic tendencies in comparison with parental lines, both in glasshouses and plastic greenhouses. Parental lines of hybrids No. 127 and No. 128 were characterized by high parthenocarpy in spring glasshouses (93% and 72%) and in plastic greenhouses (88% and 98%). These two lines have a special importance for further sele-

52 Table 2. Percent parthenocarpy of cucumber depending on conditions of cultivation

Spring-summer crop rotation Winter-spring crop rotation No of hybrid Character Glass greenhouse Plastic greenhouse Glass greenhouse

F F R R F R R F R R 1 1 1 2 1 1 2 1 1 2

P 55 26 0 91 92 92 41 12 41 125 A 37 14 0 69 60 92 40 11 41 B 67 55 0 75 65 100 98 94100

P 45 53 23 79 77 72 20 35 46 126 A 23 16 14 51 58 53 20 35 40 B 51 30 60 67 76 72 100 100 88

P 65 93 25 75 88 77 35 39 65 127 A 41 64 14 55 53 68 33 37 54 B 63 69 56 74 75 91 95 96 83

P 20 72 0 90 98 92 52 0 41 128 A 16 33 0 73 64 92 52 0 41 B 82 47 0 82 65 100 100 100 100

P 25 23 50 79 69 72 48 46 40 129 A 17 14 22 70 53 50 48 40 38 B 67 60 45 89 78 78 100 88 95

F = hybrid F ; R = mother lines; R = father lines; P = parthenocarpy coefficient; 1 1 1 2 A = % reproduction without pollination; B = % reproduction after pollination. ction both in plastic and in glasshouses. Parthenocarpy of all samples was lower in the winter-spring crop than in the spring-summer crop. Parthenocarpy was not observed in the common male parent line of hybrids No. 125 and No. 128 under spring glasshouse conditions, but in the plastic greenhouse this line was characterized by high parthenocarpy (92%). According to Alkuvejti (1966), it is possible to assume that these changes in plants take place due to the elevated night temperatures and to the prevalence of the long-wave part of the spectrum. In plastic greenhouses, night temperatures are lower and the short-wave part of the spectrum is typical for them. On the contrary, the maternal parent line of hybrid No. 128, in conditions of spring-summer crop, had 72% parthenocarpy but in winter-spring 0%. Apparently, the maternal line of hybrid No. 128 belongs to the long-day type; possibly, its parthenocarpic potential is not expressed in short days.

53 In characterizing parthenocarpic level of hybrids, it can be stated that in spring glasshouses the highest value was recorded for hybrids No. 125 and No. 127 (55 and 65%, respectively), and in plastic greenhouses for No. 125 and No. 128 (91 and 90%, respectively). We can conclude from the data that parthenocarpy of cucumber in plastic greenhouses is higher than in glasshouses. It is perhaps because the transplanted samples were selected in a spring plastic greenhouse in a later sowing and were not adapted to cultivation conditions in the early season, although parthenocarpy would be expected to appear more strongly expressed under short days and low light intensity. In order to have comparative data for times of cultivation, productivity was speci- fied for the first 48 days of production for all samples. Yields of cucumber hybrids and their parental lines were higher in plastic greenhouses (Table 3). In most cases, the yields of the hybrids exceeded those of their parental lines, as they bore more and larger fruits. In plastic greenhouses, the hybrids No. 126, 127 and 128 (8.06, 9.39, 6.78 kg/m2, respectively) had the highest yields. Only in hybrid No. 129 both early and late yield were below parental lines at all times of cultivation. Hybrids No. 125, 126, 127 and 128 (4.63, 4.95, 4.60, 4.55 kg/m2 accordingly) were characterized by the highest early yield in spring glasshouses. In the plastic greenhouse, the early yield of hybrids No 125, 127 and 128 surpassed the best parent by 88, 193 and 165%, respectively. From the data, it seems likely that the higher yields in the plastic greenhouse resulted from the higher parthenocarpic tendency in the spring. In samples of winter-spring crop rotation, yields were lower than at the later time of cultivation because plants were not adapted for such conditions. Similar to results received in conditions of spring-summer crop rotation, in winter-spring rotation the greatest yield was produced by hybrid No. 126 (1.18 kg/m2 in early and 3.71kg/m2 in total yield). Under glasshouse conditions of the winter-spring crop, the plants developed aty- pically, having short laterals (mean of 4.4 cm) early (January-February) on 4-5 internodes, and female flowering was augmented, with clusters of up to 6-7 ovaries observed, in all lines. Almost in all hybrid samples and their parental lines we observed that, in the case of multiple-flowering pistillate plants, the majority of the ovaries aborted, as the plants were not able to develop more than one or two fruits at each node. In the winter-spring crop, the plants of the parental forms of all hybrids were of weak growth, had extended and thinner main stalks and lateral sprouts, and fewer and smaller leaves. Later in the season (March - April), with increased light exposure, the lateral sprouts became of normal length and the top lateral sprouts gave flowers and fruits typical to of the plant type. All of these changes in vegetative growth can be attributed to reactions of cucumber plants to insufficient light exposure and short days early in the season. The reaction of plants to low incident radiation was reflected in the decrease in the rate of biomass accumulation, plant size reduction including all its organs, and in morphogenesis. In conditions of early planting (beginning of January), plants which belong to a spring ecotype were delayed in apical meristem activity, resulting in small growth sprouts (Strelnikova, 1984). We also noticed this phenomenon on the bottom lateral sprouts in all samples planted in January.

54 NM 2 %% kg/m NM 2 kg/m %% NM = father lines; Spring-summer crop rotation Winter-spring crop rotation 2 2 1.20 6 80 0.15 0.94 3 940.26 1.001.20 6 76 6 80 0.15 1.00 0.94 3 100 940.26100 6 76 %% kg/m Productivity Productivity Productivity 0 0 - - E.P. B.P. G.P. B.P. E.P. B.P. G.P B.P. E.P. B.P. G.P B.P. 4.63193 6.802.33 135 100 24115 5.05 100 3.741884.95 7.96 20 138 3093.60 99 8.58 100 132 341172.15 1.99 6.50 100 0.86 100 19 2.584.60 172 148 24107 114 100 2.974.05 2.86 2.73 3.78 6.98 198 100 14117 105 1092.58 100 6.40 8.06 15 0.50 6.68 100 28 121 99 100 1004.55 121 30 1.50 127 33 173.58 31 4.92 10086 6.63 8.55 100 118 156 19385 115 2.55 7 7.43 1.18 9.39 1.15 1.26 100 100 27 99 183 199 100 4.401.98 128 3.71 27 3.6849 39 16 1012.58 3.13 108 100 5.43 124 158 3.71 265824.05 1.18 17 19 1.06 6.78 1.06 100 100 123 16 90 133 241 24 6.60 2.81 132 3.32 6.63 100 100 29 100 1.61 138 0.80 9 124 51 4.70 11 17 100 15 98 4.42 0.84 100 131 156 116 30 71 0.10 2.4 323 3.19 10 88 2.16 100 16 100 255 216 2.53 141 15 0.50 2.2 10 0.2280 0.72 109 15 1.00 6.27 27 0.80 100 2.7 6 3 3 27 196 139 119 127 0.10 1.52 4 100 8 100 3.66 167 100 0.72 20 98 3 119 = mother lines; R 1 ; R 1 1 2 1 2 1 2 1 2 1 2 1 1 1 1 1 F F F F F R R R R R The productivity of cucumber hybrids and their initial lines depending on cultivation conditions Generation Glasshouses Plastic greenhouse Glasshouses = hybrid F 1 E.P. = E.P. Early productivity (1 month fructification); = G.P. general productivity (for 48 days of fructification); = %B.P. percentage to best parent; N = number of fruits on 1 plant; M = average weight of a fruit (g). No Table Table 3. Hybrid 125 R 126 R 127 R 128 R 129F R

55 Conclusions

Based on these observations, it is apparent that the parental lines and hybrids differ genetically in their response to cultural conditions, as expressed in parthenocarpic tendency and yield. Hybrid No. 126 had high yield uniformity in the spring-summer crop and under glass as well as plastic, and had high productivity as a winter-spring crop. This research approach toward breeding parthenocarpic cucumbers allowed the selection of germplasm adaptable to various conditions of cultivation, and has resulted in the release of the F hybrid Ryabinushka. 1

References

Alkuvejti, A.I.S.D. 1966. (Development, growth and organogenesis of cucumber (Cucumis sativus L.) cultivars under various light conditions). Internal report of the Thesis for the Acqui- sition of the title Candidate of Biological Sciences, Moscow State University, Moscow (In Russian). Guseva, L.I. 1986. (Methods of the selection of tomato and cucumber for industrial technologies). Internal report of the Thesis for the Acquisition of the title Doctor of Agricultural Sciences, The Moldavian Institute of Irrigated Agriculture and Vegetable Growing, Tiraspol (In Russian). Gustafson, F.G. 1942. Parthenocarpic and normal fruits compared as to percentage of selling and size. Botanical Gar., 102. Nitsch, J. 1952. Plant hormones in the development of fruits. Quarter. Rev. Biol., 27: 33-57. Rogova, N.T. 1975. (Specialities of biology of fruit formation and the methods of selection of greenhouse parthenocarpic types of cucumber). Internal report of the thesis for the acquisition of the title Candidate of Agricultural Sciences, All-Russian Research Institute for Vegetables, Moscow (In Russian). Strelnikova, T.R., Mashtakova, A.K. and Guseva, L.I. 1984. (Selection of heterosis hybrids of cucumber). Shtinitsa, Kishinev, 210 pp. (In Russian). Strong, W.J. 1932. Parthenocarpy in the cucumber. Scientific Agric., 12: 665-669. Tarakanova, S.I. 1978. (Specialities of formation of assimilation apparatus and yield in parthenocarpic cultivars and hybrids of cucumber in winter-spring crop rotation). In: (Biological principles of increased productivity of agricultural crops). All-Russian Research Institute for Vegetable Crops, Moscow, pp. 122-125. (In Russian) Tiedjens, V.A. 1928. The relation of environment to shape of fruit in Cucumis sativus and its bearing on the genetic potentialities of the plants. J. Agric. Res., 36: 804. Yakimovich, A.D. 1935. (Biology of cucumber (Cucumis sativus L.) flowering). In: (Results of selection of vegetable crops at the Gribovskaya Station). Selkhozgiz, Moscow, pp. 118-133. (in Russian) Zhivnitskaya, M.D. and Guseva, L.I. 1978. (Original material for the selection of parthenocarpy inclining cultivars of cucumber intended for mechanized harvest). Shtinitsa, Kishinev, pp. 186- 187. (Abstract, in Russian)

56 Effects of salinity on some physiological parameters in three cultivars of cucumber (Cucumis sativus)

G. Baysal1, R. Tipirdamaz2 and Y. Ekmekci2 1Gazi University, Science and Arts Faculty, Biology Department, Ankara, Turkey; e-mail: [email protected] 2Hacettepe University, Science Faculty, Biology Department, 06532Beytepe-Ankara,Turkey

Summary

In this study, effects of salinity on some physiological parameters of cucumber (Cucumis sativus L.) cultivars (Çengelköy, Anadolu F1, Beith Alpha) were investigated. Three cultivars of cucumber were grown under the controlled conditions in perlit culture and irrigated with Hoagland nutrient solution for a period of 7 days. After this period, seedlings were treated with 0, 50, 100, 150 and 200 mM NaCl solutions for 15 days and some physiological parameters [fresh and dry weight and number of leaves, inorganic ions, relative water content (RWC), total chlorophyll and carotenoid contents] were determined. In general, the applied salinity affected all of these considered parameters. High NaCl concentration caused a reduction in fresh and dry weight of all cultivars. These changes were associated with a decrease in RWC and total chlorophyll content. Increasing NaCl concentration induced Na+ and Cl- accumulation in all cultivars, but K+ concentration was increased in only Çengelköy. Ca++ concentration was also decreased in all cultivars compared to their controls. Because Çengelköy has lower Na+ and Cl-, higher K+ concentrations, RWC and total chlorophyll content compared to the other cultivars, it was classified as a tolerant cultivar. This response of Çengelköy to salinity could be related with their osmotic adjustment.

Keywords: Cucumis sativus, fresh and dry weight, ion concentration, relative water content, salinity, total chlorophyll and carotenoid contents

Introduction

Abiotic stress can be identified as an environmental factor which limits crops productivity or destroys their biomass (Grime, 1979). Salinity is one of the major abiotic stresses which can be caused by limited rainfall, high evaporation, saline irrigation, water and poor water managements especially in arid and semiarid regions of the world. Plants are stressed in saline soils in these ways; low osmotic potential of soil solution(water stress) (1), nutrient imbalance by depression in in uptake and shoot transport (2), toxic effects of specific ions mainly Na+ and Cl- (3) and combination of these factor (4) (Ashraf, 1994; Marschner, 1995). Selection and breeding of cultivars are more permanent and complementary solutions to minimize the deleterious effects of salinity so that we can produce more economic yield under salinity conditions (Epstein et al., 1980; Fooland, 1996). The selection and breeding techniques are mostly based on differences in agronomic characters which include the integration of the physiological mechanisms conferring salinity tolerance and represent the combined genetic and environmental effects on plant growth (Noble et al., 1984; Ashraf, 1994; Shan- non, 1998; Ashraf, 2002). To uncover the variability in salinity tolerance among genotypes

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 57 efficient screening techniques are necessary (Nieman and Shannon, 1976; Shannon, 1979). Some of the parameters that have been used to screen plants for salt tolerance were; yield and plant height, leaf area and number, visual symptoms (e.g. leaf injury), relative growth rate, accumulation of compatible solutes such as prolin, glycine betain and inorganic ions such as sodium, potassium, calcium and chloride (Cramer et al.,1990; He and Cramer, 1992; Noble and Rogers, 1992; Franco et al., 1993; Munns, 1993). The aim of this study was to determine the effects of salinity on different cucumber cultivars and to classify the cultivars according to their salt tolerance using some physiological parameters (fresh and dry weight of leaves, RWC, total chlorophyll and inorganic ions contents).

Material and methods

Three cucumber (Cucumis sativus L.) cultivars (Anadolu F1, Çengelköy, Beith-alpha) were used as plant material. They were grown under the controlled growth conditions (25±2°C temperature, 50-60% relative humudity, 16h light /8h dark photoperiod and 100 µmol.m-2s-1 light intensity) in perlit culture. The seeds were irrigated with Hoa- gland nutrient solution for a period of 7 days. After this period, the cucumber seedlings at the cotyledon stage were treated with 0, 50, 100, 150, 200 mM NaCl solutions. To avoid osmotic shock, NaCl concentrations were increased gradually by 50 mM every day until the desired concentration was reached. After 15 days (exclusive of the addition ones) of salt treatment, the plants were harvested and analyzed to measure the physiological parameters. Each experiment was carried out with three replications. Fresh weight (FW) and dry weight(DW) (after drying at 70°C for 48 h) of the leaves were determined. Leaf relative water content (RWC) was measured according to Smart and Bingham (1978). Total chlorophyll and carotenoid contents were determined using the method of Lichtenthaler (1987). The mineral ions were determined in extracts prepared according to Prakash and Prathapasenan (1988). Na+, K+ and Ca++ were measured by using Ephendorf Flame Fotometer. Cl- was measured by using Cotlove Cloridometer. The differences between the treatments as well as the three species were tested using SPSS statistical programme. Variance analysis of the results was performed and compared with least significant differences (LSD) at 5% level.

Results and discussion

All used NaCl concentrations caused a significant reduction of all the measured parameters, except leaf number, in all cultivars. FW of cucumber leaves decreased with an increasing in NaCl concentration. The reduction was not significant in Çengelköy at 50 and 100 mM NaCl; but for the other cultivars, it was more significant at all salt treatments. The DW also was affected by NaCl treatment. Based on these growth parameters, Çengelköy was less affected than the other cultivars and showed less reduction in FW and DW of the leaves due to salinity (Fig. 1).

58 NaCl Concentrations (mM)

Figure 1. Effect of increasing NaCl concentration on fresh and dry weight of three cucumber cultivars leaves. Values are means (±S.E.) of three replicates.

Decline in vegetative growth in cucumbers with increasing salinity stress was reported by Chartzoulakis (1990). Abd-Alla et al. (1992) reported that the reductions observed in plant height, leaf area and numbers in response to salinity were also reflected in fresh and dry weight of cucumber plants. Salt treatment caused a significant decrease in RWC in all cultivars. In general, RWC decrease with increasing NaCl concentration. When compared to the control, the relative decrease was higher for Beith Alpha than that was from 80% in the control to 51% in 200 mM NaCl treatment. On the other hand, decreasing in RWC was lower in Çengelköy with 73% for the control and 51% at 200 mM NaCl (Fig. 2). According to Katerji et al. (1997) the decrease in RWC indicated a loss of turgor that resulted in limited water avability for cell extension prosses. Ghoulam et al. (2002) reported that salt treatment induced a reduction in leaves RWC, and the growth inhibition in less tolerant cultivar could be related to decrease of RWC provoked by the salt treatment for sugar beet.

Figure 2. RWC of leaves of three cucumber cultivars submitted to increasing NaCl concentrations. Values are means (±S.E.) of three replicates.

59 The presence of NaCl in the rooting medium induced a significant increase in Na+ and Cl- concentrations in the leaves (Fig. 3A and B). All cultivars accumulate Na+ and Cl- ions in the leaves but Çengelköy accumulated less ions than the others. Al- though K+ concentration of Beta Alpha and Anadolu F1 leaves gradually decreased in response to salinity, it increased in Çengelköy with increasing NaCl content. But this effect was found to be significant at only 200 mM NaCl concentrations (Fig. 3C). High K+ and low Na+ accumulation were associated with the salt tolerance of Çengel- köy. Ca++ concentration of all cultivars significantly decreased with NaCl treatments compared to their controls (Fig. 3D). Similar results were observed for melon seedlings (Botia et al., 1998; Carvajal et al., 1998). Wyn Jones and Gorhan (1983) suggested that many solutes could be used in osmotic adjustment including inorganic ions, such as Na+, K+ and Cl-. Villora et al. (1997) reported that high levels of Na+ had significant effects upon K+ for zucchini plants.

NaCl Concentrations (mM)

Figure 3. Effect of increasing NaCl concentration on Na+, Cl-, K+ and Ca++ and ion contents of three cucumber cultivars leaves. Values are means (±S.E.) of three replicates.

Total chlorophyll content decreased with the increase in salinity in all cultivars (Fig. 4). The carotenoid content was increased in Çengelköy and Anadolu F1 but decreased

60 in Beith Alpha by the salt treatment. Sudhakar et al. (1991) reported that the reduction in chlorophyll might be due to enhancement of chlorophyllase activity at higher salinity levels. The decrease of chlorophyll in presence of salt has already been described by Le Dily et al. (1993).

NaCl Concentrations (mM)

Figure 4. Effect of increasing NaCl concentration on chlorophyll and carotenoid contents of three cucumber cultivars leaves. Values are means (±S.E.) of three replicates.

Based on the experimental results it was concluded that, all of the considered parameters were affected by salinity with a varietal difference. Because Çengelköy has lower Na+ and Cl-, higher K+ concentrations, RWC and total chlorophyll content than the other cultivars, it was found to be tolerant cultivar to salinity. This response of Çengelköy to salinity could be related with their osmotic adjustment.

References

Abd-Alla, A.M., Abou-Hadid, A.F. and Jones, R.A.1992. Salinity stress alters the vegetative and reproductive growth of cucumber plants. Acta Hort., 323: 411-421. Ashraf, M. 1994. Breeding for salinity tolerance in plants. Crit. Rev. Plant Sci., 13: 17-42. Ashraf, M. 2002. Salt tolerance of cotton: some new advances. Crit. Rev. Plant Sci., 21: 1-30. Botia, P., Carvajal, M., Cerda, A. and Martinez, V. 1998. Response of eight Cucumis melo cultivars to salinity during germination and vegetative growth. Agronomie, 18: 503-513. Carvajal, M., del Amor, F.M., Fernandez-Ballester, G., Martinez, V. and Cerda, A. 1998. Time course of solute accumulation and water relations in muskmelon plants exposed to salt during different growth stages. Plant Sci., 138: 103-112. Chartzoulakis, K.S. 1990. Effects of saline irrigation water on germination, growth and yield of greenhouse cucumber. Acta Hort., 287: 327-334. Cramer, G.R., Epstein, E. and Lauchli, A. 1990. Effects of sodium, potassium and calcium on salt-stressed barley. Physiol. Plant., 80: 83-88. Epstein, E., Norlyn, J.D., Rush, D.W., Kingsbury, R.W., Kelly, D.B., Gunningham, G.A. and Wrona, A.F. 1980. Saline cultures of crops: A genetic approach. Science, 210: 399-404. Fooland, M.R. 1996. Genetic analysis of salt tolerance during vegetative growth in tomato, Lyco- persicon esculentum Mill. Plant Breeding, 115: 245-250.

61 Franco, J.A., Esteban, C. and Rodriguez, C. 1993. Effects of salinity on various growth stages of muskmelon cv. Revigal. J. Hort. Sci., 68: 899-904. Ghoulam, C., Foursy, A. and Fares, K. 2002. Effects of salt stress on growth, inorganic ions and proline accumulation in relation to osmotic adjustment in five sugar beet cultivars. Envir. Exp. Bot., 47: 39-50. Grime, J.P. 1979. Plant Strategies and Vegetation Process. Wiley, New York. He, T. and Cramer, G.R. 1992. Growth and mineral nutrition of six rapid-cycling Brassica species in response to seawater salinity. Plant Soil, 139: 285-294. Katerji, N., van Hoorn, J.W., Hamdy, A., Mastrorilli, M. and Mou Karzel, E. 1997. Osmotic adjustment of sugar beets in response to soil salinity and its influence on stomatal conductance, growth and yield. Agric. Water Manag., 34: 57-69. Le Dily, F., Billard, J.P., Le Saos, J. and Huault, C. 1993. Effects of NaCl and gabaculine on chlorophyll and proline levels during growth of radish cotyledons. Plant Physiol. Biochem., 31: 303-310. Lichtenthaler, H.K. 1987. Chlorophylls and carotenoids, the pigments of photosynthetic biome- mranes. Methods Enzymol., 148: 350-382. Marschner, H. 1995. Mineral Nutrition of Higher Plants. Academic Press, London, pp. 657-680. Munns, R. 1993. Physiological processes limiting plant growth in saline soils: some dogmas and hypotheses. Plant Cell Environ., 16: 15-24. Nieman, R.H. and Shannon, M.C. 1976. Screening plants for salinity tolerance. In: Wright, M.J. (Ed.), Proc. Workshop on Plant Adaptation to Mineral Stress in Problem Soils. Beltsville, MD, USA. Noble, C.L., Halloran, G.M. and West, D.W. 1984. Identification and selection for salt tolerance in lucerne (Medicago sativa L.). Aust. J. Agric. Res., 35: 239-252. Noble, C.L. and Rogers, M.E. 1992. Arguments for the use of physiological criteria for improving the salt tolerance in crops. Plant Soil, 146: 99-107. Prakash, L. and Prathapasenan, G. 1988. Effects of NaCl salinity and putrescine on shoot growth, tissue ion concentration and yield of rice (Oryza sativa L. var. GR-3). J. Agron. Crop Sci., 160: 325-334. Shannon, M.C. 1979. In quest of rapid screening techniques for plant salt tolerance. Hort Sci., 14: 587-589. Shannon, M.C. 1998. Adaptation of plants to salinity. Adv. Agron., 60: 75-119. Smart, R.E. and Bingham, G.E. 1978. Rapid estimates of relative water content. Plant Physiol., 53: 258-260. Sudhakar, C., Reddy, P.S. and Veeranjaneyulu, K. 1991. Changes in Respiration. Its allied enzymes, pigment composition, chlorophyllase and Hill reaction activity of horsegram seedlings under salt stress. Indian J. Plant Physiol., 34: 171-177. Villora, G., Pulgar, G., Moreno, D.A. and Romero, L. 1997. Effect of salinity treatments on nutrient concentration in zucchini plants (Cucurbita pepo L. var. moschata). Aust. J. Exp. Ag- ric., 37: 605-608. Wyn Jones, R.G. and Gorhan, J. 1983. Osmoregulation. In: Lange, O.L., Nobel, P.S., Osmond, C.B. and Ziegler, H. (Eds), Physiological Plant Ecology. III. Responses to the Chemical and Biological Environment (Encyclopedia of Plant Physiology, New Series. Vol.12C.) P. 35. Springer- Verlag, Berlin-Heidelberg-New York.

62 Comparison of grafted and non-grafted melon plants under excess of boron and salinity stress

M. Edelstein1, M. Ben-Hur2, R. Cohen1, Y. Burger1 and I. Ravina3 1Department of Vegetable Crops, Agricultural Research Organization, Newe Yaar Research Center, P. O. Box 1021, Ramat Yishay 30-095, Israel; e-mail: [email protected] 2Department of Environmental and Physical Chemistry, Agricultural Research Orga- nization, P.O.B. 6, Bet Dagan 50-250, Israel 3Department of Agricultural Engineering, Technion, Haifa 32-000, Israel

Summary

Production of melons (Cucumis melo) can be limited by excess of boron and salinity. A greenhouse study was conducted in order to compare the responses of grafted and non-grafted melon plants to combinations of high levels of boron and salinity. Boron levels were 0.25, 0.8, 2.5, 5.0, 10.0 mg l-1 and salinity levels were 1.8 and 4.6 dS m-1. Foliar injury caused by boron was more severe in the non-grafted than in the grafted plants. Likewise, boron accumulation in leaf tissue from non-grafted plants was higher than in grafted plants. High salinity led to decreased boron accumulation in the leaves. Fruit yield was decreased only at a boron concentration of 10 mg l- 1, and the decrease in grafted plants was smaller than that in non-grafted plants. A negative correlation was found between boron accumulation in leaves and fruit yield. The results showed that melon plants grafted on Cucurbita rootstock are more tolerant than non-grafted ones to high boron concentrations, and this can probably be explained by the decrease in boron accumulation caused by the rootstock.

Keywords: Cucumis melo, grafting, boron tolerance, foliar injury

Introduction

Boron is a minor element that is essential to plant growth. In many cases natural soil boron levels are insufficient, and boron is therefore added as fertilizer (Gupta et al., 1985). Boron in soil and in irrigation water can reach concentrations that are toxic to plants (Keren and Bingham, 1985; Tsadilas, 1997), and this is of great concern in arid regions where saline soils and saline water can be prevalent. Municipal and other wastewater effluents used for irrigation are also possible sources of excess boron in agricultural systems (Tsadilas, 1997). Salinity is a yield-limiting factor in legume crops; increasing salinity decreases vegetative growth and yield in melon plants (Nerson and Paris, 1984; Franco et al., 1997). Plant tolerance to boron and salinity differs widely among species, and to some extent among cultivars within a species (Marschner, 1998). El-Sheikh et al. (1971) reported a 50% decrease in vegetative growth of cucumber, squash, muskmelon and corn, in solutions containing 6,12, 12 and 16 mg/l of boron, respectively, with cucumber being the most sensitive and corn the least sensitive to boron. The damage to plants caused by salinity and boron has been attributed mainly to the excessive ac-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 63 cumulation of Cl-, Na+ and B in their leaves. In general grafting plants may increase their tolerance to low temperatures and salinity (Rivero et al., 2003). Little information is available concerning the combined effects of salinity and boron. The goal of the present study was to investigate the growth and yield responses of melons to the stress caused by combinations of boron and salt in the irrigation water, in comparison with the responses of grafted plants grown under the same conditions.

Materials and methods

Plant material and culture conditions Seedlings of the melon (Cucumis melo L.) scion Arava were grafted (Edelstein et al., 1999) onto the commercial Cucurbita maxima Duchesne × Cucurbita moschata Duchesne rootstock TZ 148. The experiment was conducted in a heated greenhouse at the Newe Yaar Research Center in northern Israel. Seedlings of grafted and non- grafted plants were transplanted on 4 February 2002 in 10-liter pots containing Per- lite no. 2 (Agrical, HaBonim, Israel). The combined effects of boron and salinity were investigated by combining five boron concentrations with two salinity levels in irrigation water. The completely randomized experimental design consisted of two water qualities (final EC, 1.8 dS m-1 with SAR, 4.0; and EC, 4.6 dS m-1 with SAR, 6.0) each with boron concentrations of 0.25, 0.8, 2.5, 5.0, and 10.0 mg l-1. Boron was added to the irrigation water as boric acid (H BO ). Nitrogen, phosphorus and potassium ferti- 3 3 lizers (enriched with micronutrients) were applied through the irrigation system at levels used in local commercial cultivation. Plants were irrigated five times a day. Fruits were harvested when fully mature (full slip) during April May 2002. The yield (number and weight) from each pot was recorded. Total soluble solids (TSS) were evaluated for each fruit, with a digital refractometer (Atago Co., Tokyo, Japan). At the end of the experiment the plants were harvested and plant dry matter was analyzed for boron.

Results and discussion

Boron toxicity symptoms appeared first on the old leaves at the base of the plant. The symptoms (slight marginal leaf chlorosis) first appeared in the non-grafted plants in the 10 mg l-1 treatment within 17 days after planting, and became more severe later in the season. In general the symptoms were more severe in the non-grafted plants than in the grafted ones. The saline treatments produced less fruits than the non-saline treatments (Table 1). These results are consistent with those of Shannon and Francois (1978), who reported that salinity caused a decrease in fruit numbers; mean fruit weight was not significantly affected by boron or salinity. In the present study, at EC 1.8 fruit yield was reduced only at a boron level of 10 mg l-1 boron, whilst the yield of the grafted plants was almost double that of the non-grafted ones (Table 1). At EC 4.6 the fruit yield was lower, and the effect of boron on the yield was moderate; a significant interaction was found between boron and salinity. The same behavior was reported in beans (Sternberg et al., 2001) and tomato (Ben-Gal and Shani, 2002), when they were expo-

64 Table 1. Effects of boron and salinity on yields of grafted and non-grafted melon plants

Boron (mg/l) Fruit no. Fruit wt Fruit yield TSSx (per 5 plants) (g) (g per 5 plants)

Non-grafted EC 1.8 0.25 13 856 11135 10.7 0.80 15 923 13845 11.0 2.50 14 1019 14265 10.5 5.00 11 1016 11175 10.6 10.00 5 781 3905 9.1 Grafted EC 1.8 0.25 13 1053 12635 10.7 0.80 11 1148 12630 10.9 2.50 13 898 11680 11.0 5.00 15 807 12105 10.8 10.00 10 741 7410 9.3 Non-grafted EC 4.6 0.25 10 887 8870 10.4 0.80 10 717 7170 10.9 2.50 11 810 8915 11.0 5.00 10 710 7100 11.0 10.00 9 663 5970 11.5 Grafted EC 4.6 0.25 10 689 6890 11.0 0.08 8 794 6355 11.0 2.50 9 655 5895 10.5 5.00 7 702 4915 10.8 10.00 8 711 5690 10.5

Significancey B ** ns *** ns S *** ns *** ns Gnsnsnsns B x S ns ns ** ns B x G ns ns ns ns S x G ns ns ns ns B x S x G ns ns ns ns x TSS = Total soluble solids; y B = boron; S = salinity; G = grafted. ns, *, **, *** = Nonsignificant, or significant at P £ 0.05, 0.01 or 0.001, respectively. sed to excess boron and salinity. A small (non-significant) decrease in total soluble solids (TSS) was found at a boron concentration of 10 mg l-1 when plants were irrigated with non-saline water (Table 1).

65 Table 2. Absorbed boron in leaves (of the lower third of the plant) of grafted and non-grafted melons, as affected by boron concentration and salinity in irrigation water

Boron (mg l-1) EC 1.8 EC 4.6 in irrigation water Non- Non- Grafted grafted Grafted grafted

0.25 275 250 242 192 0.80 171 262 201 248 2.50 666 1031 407 655 5.00 1129 1859 1167 1676 10.00 1846 2827 1085 2222

Figure 1. Fruit yield of grafted and non-grafted melon plants as a function of boron absorption in leaves of the lower third of the plant.

66 Boron analysis confirmed that leaf injury symptoms were due primarily to boron toxicity. Table 2 shows the boron absorption in leaves of the lower third of grafted and non-grafted melon plants; in general increasing the boron concentration in the irrigation water increased the concentration of boron accumulated in the leaves. This finding is in agreement with those of Ben-Gal and Shani (2002) and Sternberg et al. (2001); grafted plants absorbed less boron in the leaves than non-grafted ones. It is assumed that grafted plants develop various mechanisms to avoid physiological damage caused by the excessive accumulation of boron in their leaves. One such mechanism could be the exclusion of boron by the roots of the rootstock. In the present study, increasing the salinity (EC 4.6) decreased the boron absorption in both grafted and non-grafted plants (Table 2), which is consistent with earlier findings of Ben-Gal and Shani in tomatoes (2002). In the present study, at EC 1.8 a negative correlation was found between leaf concentration of boron and fruit yield, in both grafted and non-grafted plants, but at EC 4.6 no correlation was found (Fig. 1). It is obvious that boron had a major effect on fruit yield when salinity was low, but that at a high salinity level, salinity alone affected the yield.

References

Ben-Gal, A. and Shani, U. 2002. Yield, transpiration and growth of tomatoes under combined excess boron and salinity stress. Plant Soil, 247: 211-221. Edelstein, M., Cohen, R., Shreiber, S. Pivonia, S. and Shtienberg, D. 1999. Integrated management of sudden wilt in melons caused by Monosporascus cannonballus using grafting and reduced rates of methyl bromide. Plant Dis., 83: 1142-1145. El-Sheikh, A.M., Ulrich, A., Awad, S.K. and Mawardy, A.E. 1971. Boron tolerance of squash, melon, cucumber, and corn. J. Amer. Soc. Hort. Sci., 96: 536-537. Franco, J.A., Fernandez, J.A. and Banion, S. 1997. Relationship between the effects of salinity on seedling leaf area and fruit yield of six muskmelon cultivars. HortSci., 32: 642-644. Gupta, U.C., James, Y.W., Campbell, C.A., Leyshon, A.J. and Nicholaichuk, W. 1985. Boron toxicity and deficiency: A review. Can. J. Soil Sci., 65: 381-409. Keren, R. and Bingham, F.T. 1985. Boron in water, soil and plants. In: Stuart, R. (Ed.), Advances in Soil Science Vol. 1. Springer-Verlag, New York, pp. 229-276. Marschner, H. 1998. Mineral Nutrition of Higher Plants 2nd ed. Academic Press, New York. Nerson, H. and Paris, H.S. 1984. Effects of salinity on germination, seedling growth, and yield of melons. Irrig. Sci., 5: 265-273. Rivero, R.M., Ruiz, J.M. and Romero, L. 2003. Role of grafting in horticultural plants under stress conditions. Food Agric. Environ., 1: 70-74. Shannon, M. and Francois, L. 1978. Salt tolerance of three muskmelon cultivars. J. Amer. Soc. Hort. Sci., 103: 127-130. Sternberg, P.D., Ulery, A.L. and Villa, C.M. 2001. Salinity and boron effects on growth and yield of tepary and kidney beans. HortSci., 36: 1269-1272. Tsadilas, C.D. 1997. Soil contamination with boron due to irrigation with treated municipal waste water. In: Bell, R.W. and Rerkasem, B. (Eds.), Boron in Soil and Plants. Kluwer Academic Publishers, Dordrecht, pp. 265-270.

67 68 Sugars and volatiles composition of nectar of zucchini flowers

A.M. Granero1, J.M.G. Sanz1, J.L.M. Vidal2, A.G. Frenich2, A.R. Serrano3 and F.J. E. González2 1CIFA La Mojonera, Consejería de Agricultura y Pesca, Junta de Andalucía, Aut. Del Mediterráneo, Sal. 420, Paraje S. Nicolas, 04745 La Mojonera, Almeria, Spain; e-mail: [email protected] 2Department of Analytical Chemistry, University of Almería, 04120 Almería, Spain 3AgroBío, S.L., Crtra. Nac. 340, Km.419, La Mojonera, Almería, Spain

Summary

Zucchini nectar have been analysed looking for scent main compounds and sugars. Major compounds of nectar scent have identified as monoterpenes, benzenoids and some fatty acid derivatives. The principal aroma molecules found were 1,4-dimethoxybenzene, 1,2,4-trimethoxybenzene, Linalool, Eucalyptol, a-(+)-Pinene, Myrcene and Ocimene. Nectar sugar analysis results showed that its main compounds were Sucrose, with more than 90 % of the total sugars, Glucose and Fructose. The results of both analysis are discussed from the point of view of pollinators, honey making and floral origin markers.

Keywords: Cucurbita pepo, nectar, volatiles, sugars, pollinator attractions

Introduction

Nectar is more than a mixture of sugars in solution. The sugars, their concentrations, and their relative amounts seem all relevant to pollination, pollinators and plant systematics (Kevan, 2003). The ratio sucrose to hexoses (Sucrose: [Glucose + Fructo- se]), the most commonly encountered sugars in nectars, has become a standard measure for describing nectars and inferring aspects of pollination biology (Baker and Baker, 1983). Pollinators are attracted to flowers by a set of clues, including pollen, nectar and aromas (Dobson et al., 1990), among others. Very few works have been dedicated to a whole assessment of pollinator attraction, such as nectar sugar and volatiles composition. The nectar aroma composition have been neglected so far, despites its relevance from the point of view of honeys origin (Guyot et al., 1998; Radovic et al. 2001; Perez et al., 2002; Soria et al. 2004).

Material and methods

Plant material Cucurbita pepo (L.) cultivar Tosca (Clause Iberica, S.L.), was cultured under plastic greenhouse conditions in the experimental farm CIFA La Mojonera (Almeria-Spain). The culture was carried out in perlite sacs irrigated and nourished by drip irrigation. Sampling of nectar flowers was made in October 2003, when the blooming and crop

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 69 was at a maximum level. Sampling collection took place during anthesis hours, between 7.00 a.m. and noon time.

Nectar sampling Nectar samples were extracted from the nectaries of male and female flowers, using a micropipette, 80 ml were placed directly in Headspace (HS) vials for volatile compounds analysis, fitted with a Teflon-lined septum and the total volume of a nectary was collected for sugar characterisation. Samples were transported to the laboratory at 4°C for immediate analysis of volatile compounds. Samples for sugar analysis were transported to the laboratory and kept frozen at 20°C until being processed. Volati- les were sampled at 60°C during 10 minutes. The fibre was then immediately inserted into the injector port of the gas chromatograph during 9 minutes at 250°C.

Analysis of volatiles Reference standards of current volatiles presents in nectar of Cucurbits (Metcalf et al., 1991; Peterson et al., 1994; Metcalf et al.,1998), benzene, toluene, o-xylene, m-xylene, p-xylene, ethylbenzene, a-(+)-pinene, myrcene, R-limonene, eucalyptol, ocymene, linalool, hydroquinone dimethylether, p-anisaldehyde, cinnamaldehyde, indole, cinnamyl alcohol, dibuthyl phtalate, eugenol and 1,2,4-trimethoxybenzeneand internal standard (IS), p-xylene-d10, were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Tokyo Kasei (Nihonbashi, Tokyo, Japan). A reference standard solution was prepared for each compound using acetone as solvent at 200 mg ml-1 concentration and a multicompound working standard solution (2 mg ml-1 concentration) was prepared from the above by appropriate dilution with acetone and stored under refrigeration (4°C). The analysis was performed by low pressure gas chromatography-tandem mass spectrometry (HS-SPME-LP-GC-MS/MS) (GC Varian 3800 with Electronic Flow Con- trol (EFC) and fitted with a Saturn 2000 ion-trap mass spectrometer, Varian Instru- ments, Sunnyvale, CA, USA ) by previously coupling Headspace with solid phase microextraction, on line with the chromatographic system (HS-SPME-LP-GC-MS/MS). The HS-SPME step was carried out using a heated carrousel connected to a Varian 8200 autosampler holding the syringe of the SPME unit, which was equipped with a 65 mm polydimethylsiloxane-divinylbenzene (PDMS-DVB) fibre (Supelco, Bellefon- te, PA, USA). Fibres were conditioned prior use according to suppliers prescriptions. A fused silica untreated capillary column 2 m x 0.25 mm i.d. from Supelco (Bellefon- te, PA, USA) was used as guard column connected to a Rapid-MS (WCOT fused silica CP-Sil 8 CB low bleed of 10 m x 0.53 mm i.d. x 0.25 mm film thickness) analytical column from Varian Instruments (Sunnyvale, CA, USA) for high speed analysis. The mass spectrometer was operated in Electron Impact (EI). The controlling computer system had an EI-MS/MS library specially created for the target analytes under our experimental conditions. Other EI-MS libraries were also available. The mass spectrometer was calibrated weekly with perfluorotributyamine. Helium (99.999%) at a flow rate of 1 ml min-1 was used as carrier and collision gas. Volatilisation of compounds in the HS vials was achieved by setting them at 60°C in the termostatised carrousel of SPME unit. Then the fibre is exposed to the HS during 10 min in order to complete the adsorption of compounds. Fibre is injected into the injection port of the GC, which was set at 250°C in splitless mode, the desorption of

70 analytes from the fibre took 9 min, enough to desorbs and transfer the analytes to the analytical column. The initial column temperature was set at 35°C during injection, 9 min hold, then increased at 1°C min-1 to 55° C, at 3°C min-1 to 65°C, and finally raised to 300°C at 100°C min-1 that was held for 5 min. The ion-trap mass spectrometer was operated in EI-MS/MS. The transfer line, ma- nifold and trap temperatures were 280, 50 and 200°C, respectively. The identification of the target compounds was based on the retention time windows (RTW), which is defined as the retention time of the analytes obtained from the analysis of 10 spiked samples at the concentration equivalent to the second calibra- tion level, ± 3 times their standard deviation (Table 1). The confirmation of previously identified compounds is performed by matching the MS/MS spectra obtained in the sample with those stored in the MS/MS library created in the same experimental conditions. The library of reference spectra is checked daily by matching it with the results of a spiked sample included in each batch of samples as quality control sample.

Sugars analysis An aliquot of each sample was diluted in MilliQ grade water and subjected to Liquid Chromatography in a SugarPack1TM column (300mm x 6,5 mm i.d.) by HPLC technique with the help of a Waters system (Waters 1515 Isocratic HPLC Pump) equipped with a Waters Refractive Index Detector 2414 according to the following conditions: 0,5 ml/min flow, H O mobile phase, 90°C column temperature, and 30°C detector tem- 2 perature. The sugars were identified by their retention times in comparison with pure chemicals purchased from Sigma, Fluka or Supelco. Reference standards used were: Sucrose, D -(+)- Glucose, D -(+)- Glucose, Maltoheptose, Maltohexaose, Malto- pentaose, Maltotetraose, Maltotriose, Stachyose, D-(+)-Melezitose, D -(+)- Raffinose, Isomaltotriose. Sugar amount quantification from samples was carried out by injection of known concentrations of standards of pure chemicals and the help of the Bree- ze software provided by the Waters system.

Results and discussion The nectar scent of the Cucurbita pepo, either in male flowers as in female, contained mainly monoterpenes, benzenoids and some fatty acid derivatives. The fragrance of the male flowers nectar, consisted mainly on 1,4-dimethoxybenzene (35.1%), 1,2,4- trimethoxybenzene (19.2%), Linalool (13.6%), Eucalyptol (12.6%), a-(+)-Pinene (5.2%), Myrcene (5.2%), Ocimene (5.1%). The major components in nectar of female flower were 1,4-dimethoxybenzene (25.1%), 1,2,4-trimethoxybenzene (16.2%), Eucalyptol (9.6%), Myrcene (4.2%), Linalool (3.6%), a-(+)-Pinene (3.2%). Between themselves, the aroma concentrate showed other distinguishable compounds that were detected at low percentages (below 2.0%), Table 1. It can be seen that no significant differences were found in the composition of volatile compound between male and female nectar samples, but the concentration of volatiles in nectar from male is greater than female. These differences can be explained by the distinct morphology of the nectaries. Ne- ctaries of Cucurbita pepo present sexual dimorphism (Nepi and Pacini, 1993). Thus,

71 male flower nectary forms a enclosed round channel below anther column, only open by three opercula. Female flower nectary is present below stigma and form and open receptacle. Nectar volume dynamics mirrors the different morphological source of both nectaries, being female volume normally higher than male (Roldan et al., 2002). Su- gar composition of male and female nectar are described in Table 2.

Table 1. Percent data range for volatile composition of 12 nectar samples

Number Compound RTW (min) Male Female min max min max 1 Benzene 0.53-0.55 1.2 1.8 0.2 0.8 2 Toluene 0.75-0.79 1.4 1.7 0.7 1.6 3 Ethylbenzene 1.21-1.24 1.2 1.5 0.3 0.7 4 m-Xylene 1.28-1.35 1.3 1.9 1.2 1.5 5 p-Xylene 1.30-1.37 1.1 1.5 0.8 1.9 6 o-Xylene 1.46-1.51 1.4 1.6 1.3 1.5 7 α-(+)Pinene 1.97-2.01 2.1 8.3 2.4 4.0 8 Myrcene 3.24-3.29 2.4 9.0 1.5 6.9 9 R-(+)-Limonene 4.29-4.34 1.8 2.0 0.2 1.8 10 Eucalyptol 4.28-4.31 8.9 16.3 6.3 12.9 11 Ocimene 4.86-4.91 1.9 8.3 1.3 2.0 12 Linalool 8.58-8.63 2.3 4.9 2.8 4.4 13 1,4-dimethoxybenzene 10.85-10.91 22.4 47.8 16.8 33.4 14 p-Anisaldehyde 17.48-17.52 0.0 0.7 0.5 1.2 15 Cinnamaldehyde 19.14-19.20 1.7 2.0 1.3 1.9 16 Indole 21.29-21.34 1.4 1.6 0.0 1.1 17 Cinnamyl alcohol 23.49-23.53 1.5 1.8 0.5 1.3 18 Dibuthyl Phthalate 24.56-24.60 0.0 0.7 1.5 1.8 19 Eugenol 24.56-24.60 1.2 1.9 1.3 1.7 20 1,2,4-Trimethoxybenzene 25.84-25.89 10.5 27.9 9.8 22.6

Table 2. Means and Standard deviations of sugar concentration and Sugar ratio of male and female nectars of October samples. Means followed by different letters indicated statistically significant differences at 95% level by LSD comparison test

Sugar Male Female

Sucrose (g/l) 288.1a ± 22.9 207.1b ± 23.7 Glucose (g/l) 32.8 ± 1.5 24.8 ± 4.8 Fructose (g/l) 24.8 ± 1.2 21.5 ± 2.9 Ratio 5.0 ± 0.5 4.5 ± 1.0

72 Zucchini nectar belongs to the Sucrose-dominant type, with sugar ratio between 4.5 and 5.0, which is in accordance with the results obtained by Nepi and Pacini (1993), and Roldan et al. (2002). ANOVA results indicated that Sucrose differences between male and female are statistically significant (p-value = 0.0027; N= 8), but none of the other sugars, neither Sugar ratio, have statistically significant differences. The sugar concentration trends presented here are not constant along the culture (data not shown), which is an indication of the photosynthetic origin of nectar. According to Fahn (2000), the origin of secreted nectar is the phloem sap. This means that individual and seasonal variations can be expected. Sugar ratios indicated that honeybees and bumblebees are adequate pollinators for this culture (Baker and Baker, 1983). The potential of zucchini nectar for honey making should be stressed, taking into account the great amount of sucrose always present in them, representing more than 90% of the sugars. Two major components of the nectar aroma, namely, 1,4-dimethoxybenzene (35.1- 25.1%) and 1,2,4-trimethoxybenzene (19.2-16.2%) could be used as chemical markers for floral honey origin.

Acknowledgements

Authors acknowledge the financial support to Programa Regional de Investigación Agroalimentaria y Pesquera (I+D) and to INIA (projects PIA-03-032 and RTA03-087).

References

Baker, H.G. and Baker, I. 1983. A brief historical review of the chemistry of floral nectars. In: Bentley, B. and Elias, T. (Eds.), The Biology of the Nectaries. Columbia University Press, New York, pp: 126-152. Dobson, H.E.M., Bergström, G. and Groth, I. 1990. Differences in fragrance chemistry between flower parts of Rosa rugosa Thumb. (Rosaceae). Israel J. Bot., 39: 143-156. Fahn, A. 2000. Structure and function of secretory cells. Adv. Bot. Res., 31: 37-75. Guyot, C., Bouseta, A., Scheirman, V.V. and Collin, S. 1998. Floral origin markers of chestnut and lime tree honeys. J. Agric. Food Chem., 46: 625-633. Kevan, P.G. 2003. The modern science of ambrosiology: in honour of Herbert and Irene Baker. Plant Syst. Evol., 238: 1-5. Metcalf, L.R. and Lampman, R.L. 1991. Evolution of diabroticite rootworm beetle (Chrysomeli- dae) receptors for Cucurbita blossom volatiles. Appl. Biol. Sci., 88: 1869-1872. Metcalf, L.R., Lampman, R.L. and Lewis, A.P. 1998. Comparative Kairomonal chemical ecology of Diabrocitice Beetles (Coleoptera: Chrysomelidae: Galerucinae: Luperine: Diabroticina) in a reconstituted tallgrass prairie ecosystem. Ecology and Behavior, 91: 881-890. Nepi, M. and Pacini, E. 1993. Pollination, pollen viability and pistil receptivity in Cucurbita pepo. Ann. Bot., 72: 527-536. Perez, R.A., Sanchez-Brunete, C., Calvo, R.M. and Tadeo, J.L. 2002. Analysis of volatiles from Spanish honeys by solid-phase microextraction and gas chromatography-mass spectrometry. J. Agric. Food Chem., 50: 2633-2637. Peterson, J.K., Horvat, R.J. and Elsey, K.D. 1994. Squash leaf glandular trichome volatiles: identification and influence on behaviour of female pickleworm moth. J. Chem. Ecol., 20: 2099- 2109. Radovic, B.S., Carere, M., Mangia, A., Musci, M., Gerbole, M. and Anklam, E. 2001. Contribu- tion of dynamic headspace GC-MS analysis of aroma compounds to authenticity testing. Food Chem., 72: 511-520.

73 Roldán-Serrano, A.S., Guerra-Sanz, J.M. and Ortuño-Izquierdo, M.J. 2002. Flower attractiveness to Bumble-bees (Bombus terrestris L.) in Zucchini (Cucurbita pepo L.). In: Maynard, D.N. (Ed.), Cucurbitaceae 2002. ASHS Press, Alexandria, pp. 343-348. Soria, A.C., González, M., de Lorenzo, C., Martínez-Castro, I. and Sanz, J. 2004. Characteriza- tion of artisanal honeys from Madrid (Central Spain) on the basis of their melissopalynologi- cal, physicochemical and volatile composition data. Food Chem., 85: 121-130.

74 Pollination of zucchini culture by bumblebees: Advance of results of quality production

J.M.G. Sanz1, A.R. Serrano2, A.M. Granero1 1CIFA La Mojonera, Consejería de Agricultura y Pesca, Junta de Andalucía, Aut. Del Mediterráneo, Sal. 420, Paraje S. Nicolas, 04745 La Mojonera, Almeria, Spain; e-mail: [email protected] 2AgroBío, S.L., Crtra. Nac. 340, Km.419, La Mojonera,Almería, Spain

Summary

A research on pollination by bumblebees of zucchini has been carried out under plastic greenhouse conditons. Advances of results of a campaign show that pollination by bumblebees treatments was more productive in terms of commercial fruits than control by parthenocarpy induction. These first results are encouraging the use of natural pollinators to improve the quality production of zucchini.

Keywords: Cucurbita pepo, zucchini, bumblebees, parthenocarpy induction

Introduction

Zucchini culture is very popular in extra-early horticulture crops in Southern Spain, specially under plastic greenhouse conditions. Its culture needs a lot of man power due to parthenocarpic artificial induction procedures that use the farmers (Rylski and Aloni, 1991; Robinson and Reiners, 1999). This practice produces very often deformation of fruits (Rylski and Aloni, 1991). An alternative system to this would be the use of pollinators. Very few works have been done on the assessment of pollination of zucchini by bumblebees, the more used pollinators in greenhouses for convenien- ce. Thus, an exploratory research have been carried out to study the effect of bumblebees pollination on the quality production of zucchini.

Material and methods Four commercial varieties of zucchini (Cucurbita pepo L.) have been used: Tosca (Clause Iberica, S.L.), Consul (Seminis Seed Corp.), Chapin (Fito S.L.) and Balboa (Ramiro Arnedo, S.L.). Those varieties were sown in August 2003 on perlite hole- trays and transplanted to perlite sacs ten days after nascence. The greenhouse used has 1000 m2 of culture surface, under of 3 coats plastic. Irrigation and mineral nouris- hment was carried out by drip automatic system. The greenhouse was divided into three places by vertical antithrips nets, giving place to three plots of approximately 330 m2. Each plot was used to held one pollination treatment. Thus, treatment 1 had one commercial bumblebees nest along the culture.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 75 Treatment 2 had two commercial bumblebees nests, and control did no have any bumblebees and their plants were treated as customary in the region by parthenocarpy inducers (plant growth regulators). Four repetitions of each variety were grown in each plot, at 0.99 plant/m2 of density. Quality yield was measured by cuttings fruits more than 20 cm long. From each variety and treatment the following traits were collected: Number of normal and abnormal fruits, and weight. Data analysis was performed with the help of Statgrahic 4.0 software.

Results and discussion

Figure 1 shows the results of commercial yield per variety along the four months of culture. Commercial yield do not include abnormal fruits. ANOVA results indicated that variety had statistically significant effect on commercial production(p-value = 0.0000; N= 168), being Tosca different from the other three at 95% confidence level. Pollination treatment affected commercial yield as is shown in Fig. 2. ANOVA results indicated that Control treatment behaved differently from pollination treatments (1 and 2), attaining statistically significant differences (p-value = 0.0000; N= 168).

15 b a a 10 a

(kg/plant) 5

0 Commercial production balboa chapin consul tosca Varieties Figure 1. Means and standard errors per variety.

0,8

0,6 a b 0,4 a

(kg/plant) 0,2

0 Commercial production Sin 2ab 1ab Pollination treatments Figure 2. Commercial production per treatment. Pollination treatments: Sin (parthenocarpy induction) 1 ab (1 bumblebees nest) 2 ab (2 bumblebees nest)

76 Finally, the number of normal and abnormal fruits compared by treatements is displayed in Figure 3. ANOVA results pointed out that Control treatment was statistically significant different from the other two treatments (p-value = 0.0000; N= 168). We can conclude that pollination treatements had better commercial yield than control and some assessment on the use of bumblebees pollinators for zucchini culture have been obtained and these will be used in further research.

100 b b 80 a 60 a 40 b b

Commercial and 20 0 non-commercial fruits (%) Sin 2ab 1ab Pollination treatments Figure 3. Percentages of commercial and non-commercial fruits by treatments.

Pollination treatments: Sin (parthenocarpy induction) 1 ab (1 bumblebees nest) 2 ab (2 bumblebees nest)

Acknowledgements

This research has been supported by Grants INIA-RTA-03-087 and PIA-03-032.

References

Rylski, I. and Aloni, B. 1991. Parthenocarpic fruit set and development in cucurbitaceae and solanaceae under protected cultivation in mild winter climate. Acta Hort., 287: 117-126 Robinson, R.W. and Reiners, S. 1999. Parthenocarpy in summer squash. HortScience, 34: 715-717.

77 78 Scientific contributions

II. Germplasm

79 80 History and antiquity of cucurbits in India

V.S. Seshadri and T.A. More College of Agriculture, Dhule-424 004, Maharashtra, India; e-mail: [email protected]

Summary

Cucurbits are a highly evolutionary group and they are known to be vegetables for human consumption from the remote age of civilization. The ancient Indian civilization was basically dependent upon and intimately related with the forests and flora. Sanskrit prose, scriptures, epics, poetical works like Vedas, Upanishads, Ramayana, Mahabharata, Brahmanas, Puranas, medical treatises, etc. dating back to the ages before Christian era, mention several kinds of cucurbits. Ethnobotany of old world cucurbits reflect their relationship in social and religious life in several ethnic groups. In India, cucurbits present a large spectrum of vegetables of both indigenous and introduced ones (from very early times), adapted to wide ranging climatic conditions and notably indigenous technology of growing cucurbits on river beds got developed. World cultivated taxa consist of 36 species and 18 genera while, in India 18 species and 11 genera are cultivated. India is a home of good number of cucurbits, like cucumber, Luffa gourds, Indian squash (Praecitrullus fistulosus) and ivy gourd (Coccinia indica) having primary gene center and melon and watermelon, having a secondary center of diversification. Others like Trichosanthes, Benincasa, Momordi- ca, Lagenaria and Cucurbita have wide distribution. Historical evidence though fragmentary, has to be delved from the original texts and manuscripts. Besides, more archaeobotanical studies seem to be necessary.

Keywords: cucurbits, Benincasa, Citrullus, Coccinia, Cucumis, Cucurbita, Lagenaria, Luffa, Momordica, Trichosanthes, taxonomy, archaeobotany, ethnobotany, origin, growing, utilization

Introduction

Cucurbitaceae is essentially a family of tropical plants and for this reason, it is believed to be a relatively old one. There had been a long and intimate association of humankind with the plants of this family and Jeffrey (1980a) estimated the relationship as of longest standing (ca 15,000 years B.P.) of greatest economic importance. The family is notable for comparatively large number of species known only in cultivation.

History

Cucurbits are a highly evolutionary group and they are known to be vegetables for human consumption from the remote age of civilization. Even in the pre-historic civilization of Negrittos and then Protoaustraloids (called Nisada in Sanskrit classic language literature), the use of fruits and vegetables like brinjal or eggplant called vettingara, a cucurbit called alabu probably bottle gourd, the watermelon called kalinga, banana, etc. has been noted by philological studies of Jean Prezyluski, Jules Bosch and Sylvan Levi (Om Prakash, 1961). Absence of written records about the beginning of Agriculture in pre-historic In- dia, has led to the reliance on the archaeobotanical evidence wherever available. Three

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 81 types of archaeological sites occur in India viz., Neolithic (7500-6500 B.C.), Neoli- thic-Chalcolithic and Chalcolithic (2295-1300 B.C.). The general picture is gleaned from 100 odd morphotypes obtained from nearly 170 sites including Mesolithic (non- food producing cultures), as well as Neolithic, Chalcolithic, Harappan, Megalithic and early historical cultures (all food producers with varying degrees of agro-pastrolism). Important seeds/grains finds from archaeological sites of Indian sub-continent (including Pakistan, Bangladesh, Srilanka and Myanmar) are, inter alia some cucurbits like Cucumis type, melon, colocynth, etc. (Kajale, 1991). The ancient Indian civilization was basically aranyaka primarily dependent upon and intimately related with the forests and flora. Sanskrit prose, scriptures and poetical works like Vedas, Upanishads (esoteric scriptures), epics like Ramayana and Mahabharata, Brahmanas (ritual texts), Aranyakas (text for forest dwellers) dating back to the Ages before the Christian era, mention several kinds of cucurbits. These suggest many kinds of associations of man, like some plants/trees visualized as of sacred origin, some plants or parts thereof liked by Gods and Goddesses like fruits of urvaruka (Momordica dioica ?) mentioned in Yoginitantrum, etc. (Sensarma, 1998). Cucumber, supposed to be a native of India was known to Rigvedic (ca 3700 B.C.) Indian (Aiyar, 1956). The bottle gourd and its cooking, has been mentioned in Yajur Veda. The Puranas (dated to be between the Ages of Vedas and classical literature), contain detailed ethnobotanical information, like on alabu (Lagenaria siceraria), karkotaka (Momordica dioica), kusmanda (Benincasa cerifera), patola (Trichosanthes dioica), and trapusa (Cucumis sativus) in Matsya Purana. Besides, others recorded in other Puranas were indravaruni (Citrullus colocynthis), karavella (Momordica charantia), ksirakasaka (Cucumis sativus), kundru and bimba (Coccinia indica) and tarumunja (Citrullus vulgaris) (Sensarma, 1998). Ethnobotany of old world cucurbits reflect their important relationship in social and religious life in several ethnic groups, like magic rituals (Momordica charantia), ceremonies (Luffa acutangula and Momordica balsamina), charms (Lagenaria leu- cantha) and worship (Cucumis sativus, Cucurbita sp.) (Mehra, 1980). Early Buddhist works (around 600 B.C.) like Uttaradhyana Sutra, Prajapana Sut- ra and Jatakas record cucumber, bottle gourd, etc. were recommended by Lord Bud- dha himself. From the period 300 B.C. to 75 A.D. comprising Maurya and Sunga eras, Kautilyas Arthasastra (around 400 B.C.) and Patanjalis Mahabhashya refer to extensive use of vegetables and consider man as Sakabhajin i.e. consumer of vegetables. Medical treatises like Caraka Samhita dated to be around 600 B.C. (Ray and Gupta, 1980) and Susruta Samhita probably of 3rd to 4th century A.D. (Ray et al., 1980), published by Indian National Science Academy in English, record several cucurbits like bryony and colocynth, having therapeutical properties against ailments. Gupta period (ca 300 A.D. to 750 A.D.) believed to be a prosperous one in India, recorded in Angavijja a long list of vegetarian preparations. Some information of this period can be gleaned from the Sanskrit plays of Kalidasa like Mrcchakatika and Brahat Samhita by Varaha Mihira (ca 500 A.D.). But the first connected account of food habits comes from Chinese travellers, Fahien, Yuan Chwang and Itsing. Further down the centuries, central Asian Moghul invasions have brought in several vegetables and fruits to India during 15th to 18th centuries. Ain-i-Akbari (Ano- nymous, 1873), gives detailed account of vegetables and among them are patol (probably

82 Trichosanthes dioica), chichinda or snake gourd (Trichosanthes anguina), petha or ash or wax gourd (Benincasa cerifera) and karela or bitter gourd (Momordica charantia). There were several varieties of muskmelon locally grown and obtained from Afghanistan and Iran later in the season, extending its availability from April to November. Also colonizing traders from Britain, Portugal, France, etc. introduced several vegetables from new World during the same period.

Adaptation India thus, has a rich tradition of vegetable growing from ancient times and cucurbits especially, present a large spectrum of vegetables of both indigenous and introduced kinds (from very early times) adapted to wide ranging climatic conditions and notably indigenous technology of growing cucurbits on river beds got developed (Seshad- ri and Chatterjee, 2000). An unique system of cultivation of cucurbits in India on riverbeds is practised from Ganges and Yamuna in north India to Pamba riverbeds in Kerala in south-west India. It is a kind of vegetable forcing under sub-marginal conditions of cultivation on river sand. After the cessation of south-west monsoon in October and consequent inundation of riverbeds, the cucurbits are grown in trenches and pits from November to May even to July in some parts of north-west India. It is an indigenous technology (Diara cultivation), developed to utilize the long tap root system of cucurbits which can reach the subterranean moisture levels underneath the sandy riverbeds. Further in north and north-western India, cucurbits when sown in November are able to withstand the cool temperature of winter months up to February next, indicating that the sandy riverbeds get less cooled than the garden soil during that period. It is essentially a mixed cropping system with phased sowing of different cucurbits to reach the markets successively with bottle gourd, summer squash, muskmelon, watermelon and lastly pumpkin. In eastern U.P., Bihar and West Bengal pointed gourd is grown on riverbeds while in Kerala bitter gourd and pumpkin are raised. This system practised in meandering riverbeds in far flung and inaccessible regions, constitutes 60 per cent of total cucurbit area of the country, which may be around 0.50 million hectares. Unfortunately, mixed cropping system of several cucurbits and absence of clear land records due to changing course of river systems, exact statistics of area and production are not available. It is significant that Kautilyas Arthasast- ra (around 400 B.C.) records riverbed cultivation of cucurbits between rows of ve- tiver (Vetiveria zizanoides L.) (Nene, 2000). Moghals also promoted riverbed cultivation, since there is still some kind of ethnic association of Muslims with it. One interesting feature is that this system acts as a repository of native germplasm of cucurbits adapted to the forcing technology. Unfortunately this system has not yet received technological upgradation which acts as a blessing in disguise, preventing genetic erosion of native variability in these crops (More et al., 1998).

83 Cultivated taxa

Cucurbitaceae is a moderately large family of about 825 species and 117 genera (Jeffrey, 1980a), distributed in the warmer regions of the world, in south and southeast Asia, tropical Africa, and central and south America. In India ca 100 species and 36 genera have been recorded by Chakravarty (1982). World cultivated taxa consist of 36 species and 18 genera, while in India 18 species and 11 genera are cultivated. Cultivated taxa in India comprise both native indigenous and introduced ones from other centers of diversity from very early times. Hindustani centre of diversity of Zeven and de Wet (1982) namely India, is the home of a good number of cucurbits. Among these, having primary gene centre here, are cucumber (Cucumis sativus), Luffa gourds (L. acutangula and L. cylindrica), Indian squash (Praecitrullus fistulosus Pang.) and ivy gourd (Coccinia grandis syn. C. indica). However, there are other cucurbits which have considerable genetic diversity in this centre - to be reckoned as a secondary one - are, melon (Cucumis melo) and watermelon (Citrullus lanatus) both originally from tropical Africa. Bitter gourd (Momordica charantia), snake gourd (Trichosanthes cucumerina var. anguina), pointed gourd (T. dioica) and wax or ash gourd (Beninca- sa hispida) all belonging to Indo-Malaysian region, bottle gourd (Lagenaria siceraria) from Africa and pumpkin (Cucurbita moschata) from central America, have wide distribution in India. Brief notes on these taxa are given below.

Benincasa Savi. Benincasa is a monotypic genus (Chakravarty, 1982) having only one species B. hispida (Thunb.) Cogn. (syn. B. cerifera Savi.) nowhere found in wild, but widely distributed in tropical and sub-tropical regions of south and east Asia. Fruits are called wax or ash or white gourd because outer rind of fruits is hairy when young and develops waxy bloom which ensures its resistance to spoilage caused by microorganisms. Fruits can be stored without refrigeration for as long as one year. In India and Cuba, fruits are used in confectionary like candy prepared by cooking the fruit cubes/slices in syrup. In China, it is used in soup and is called Chinese preserving melon signifying a vegetable of ancient China. It has spread to Japan, central America and Caribbean islands. Its antiquity in India is known from its mention in Matsya Purana (Sensarma, 1989) and medical treatises - Caraka Samhita and Susruta samhita, as Sanskrit equivalent kusmanda (Ray et al., 1980; Ray and Gupta, 1980).

Citrullus Schrad. From four species, two occur in India (Chakravarty, 1982). Distributed in western Asia, eastern Mediterranean, and tropical and south Africa. Citrullus colocynthis (L.) Schrad. in India, it is mostly used in Ayurvedic medicine system and colocynth is credited to be a native of Hindustani centre (Zeven and de Wet, 1982). Citrullus lanatus (Thunb.) Matsumura & Nakai (2n=22) (syn. C. vulgaris Schrad.) watermelon, is widely cultivated for the dessert fruit quality. This species is native to African centre of diversity (Zeven and de Wet, 1982), more specifically to Kalahari region of south Africa and known as Tsama race. Watermelon is known as kalinga to Protoaustraloids of pre-historic times. In

84 archaeological excavations, colocynth was found in Pirak (Baluchistan presently in Pakistan) (ca 30232205 B.C.) and watermelon was known to be grown during Indus Valley civilization (Kajale, 1991). Colocynth is known as indravaruni in medical treatise Caraka Samhita (l.c.) and Puranas. Watermelon is quoted as tarumunja in Puranas. In view of extensive variability and ancient cultivation, Hindustani centre is considered a secondary centre of diversity of watermelon (Zeven and de Wet, 1982).

Coccinia Wight & Arn. There are 30 species of Coccinia mostly confined to tropical Africa and one in India (Jeffrey, 1980a) viz. Coccinia grandis (L.) Voigt (2n=24) (syn. C. indica Wight and Arn., Cephalandra indica Naud.) or ivy gourd. It is a typical dioecious species (with sex chromosomes of X and Y) and is extensively distributed in India, Sri Lan- ka, Myanmar, Malaysia, China, Japan and Africa. It is a fruit of ancient times and was identified from Hulas in Saharanpur (Uttar Pradesh) (ca 3028-1200 B.C.) belonging to Indus-Saraswati phase (Mehra, 2002). The Sanskrit equivalent is bimba (or bimbi) familiarly compared with womans lips for the soft and red colour, referred to in Mahabharata and Kalidasas plays. It is known as bimbi in Caraka Samhita (l.c.) and kundru in Puranas (Sensarma, 1989). A parthenocarpic (female clone) line has been identified, which can set fruits without pollination from male clone.

Cucumis L. There are about 25 species mostly in equatorial and south Africa and only 6 in India. An important genus with two distinct and non-crossable chromosome groups viz., x = 7 (C. sativus - the common cucumber) and x = 12 (C. melo, the melon or muskmelon) consisting of dessert sweet fruits and distinct non-sweet or non-dessert fruits mostly land races, the latter being extensively grown in India. Under the x = 7 group are, C. sativus L. (and its feral associate C. hardwickii), C. hystrix Chakra. (syn. C. muriculatus Chakra.), C. sativus var. sikkimensis and C. setosus Cogn. It has been open to discussion whether to consider C. hardwickii, as a feral form of C. sativus or its progenitor (Bates et al., 1995). Under the x = 12 group there are Cucumis melo and its botanical varieties C. melo var. agrestis Naud. and C. melo var. melo or C. melo var. cultus Kurz., and non-dessert forms like C. melo var. momordica Duthie & Fuller and C. melo var. utilissimus Duthie & Fuller (Jeffrey, 1980b; Chakravarty, 1982). Other species found in India is C. prophetarum. There has been some confusion about the identity of C. callosus (Roettl.) Cogn. and C. trigonus Roxb. There are several perennial, xerophytic, dioecious species of varying ploidy levels of Cucumis (2n = 48, 72, etc.) in Africa, but no x = 7 chromosome species (allied to C. sativus) has so far been recorded in Africa. During 4rd and 3rd millennia B.C. (of Chalcolithic period) several plant communities flourished in Balakot of Baluchistan (presently in Pakistan). Among them were melon or gourd, Cucumis spp. which were found cultivated. Several artifacts would suggest that Indus Valley cultures were familiar with several fruits and a few vestiges of seeds comparable to those of melons were found from Mohan-ja-Daro and Harappa (Vats, 1940; Vishnu Mittre, 1974). Also recorded in Indus-Saraswati phase (2500-2300 B.C.) in Rojidi of Gujarat, are cucumber and melons (Mehra, 2002). Unfortunately seeds of C. sativus and C. melo are not clearly distinguishable in archaelogical remains of very early times.

85 Cucumber is mentioned in Rigveda under the name urvaruka in the prayer viz., May I be liberated from death like urvaruka fruit from the stalk (Aiyar, 1956). There are several Sanskrit names for cucumber lamba (in Susruta Samhita), ksirakasaka and trapusa in Puranas and cidibita in Arthasastra. Greek historians record cucumber in India around 400 B.C., when Greek Commander Alexander the Great invaded north-western India (presently Pakistan). Similarly melon (C. melo) is referred to as panduphala or karkaru in the medical treatises. Zeven and de Wet (1982) assigned Hindustani centre as the primary gene centre for C. sativus, while secondary centre of diversification of C. melo has been noted here. Collating the observations on genetical divergence, cytogenetical aspects and isozyme variation, Seshadri and More (1996) believe that non-dessert forms like Phoot or snapmelon (C. melo var. momordica), tar or kakri or long or serpent melon (C. melo var. utilissimus) and other land races like vellarikkai of Tamil Nadu used like a salad cucumber; budam kay and nakka dosa kay of Andhra Pradesh used as cooked cucumber; vellari and chavathakkai of Kerala used as ripened cucumber fruit; chibur or chibud of coastal Maharashtra, etc. (all monoecious), form a distinct group. They represent one phase of secondary diversification probably much earlier than Moghul introductions of sweet dessert types from central Asia. These two groups represent the opposite ends of a polymorphic spectrum. A tropical taxon C. melo originally from equatorial Africa, responded to diversification in sub-tropical regions of south-west and central Asia where worlds sweetest melons are now grown. The non-dessert (non-sweet) characters got manifested under conscious selection under high temperature tropical conditions of central and south India. It has not been possible to explain how two cucumber like vegetables, one true cucumber (C. sativus) native of India and another of non-dessert melons (C. melo) used like cucumber got domesticated simultaneously in India itself and without any natural interspecific hybrids due to their non crossability (Seshadri and More, 1996). The domestication of each of the four cultivated taxa viz., C. sativus, C. melo, C. anguria (West Indian Gherkin) and C. metuliferus (African horned cucumber) - the latter two of African distribution only - apparently occurred independently.

Cucurbita L. A New World genus (2n = 40) distributed in Central and South America. Among the 21 species recorded, only four C. moschata Duch. ex Poir., C. maxima Duch., C. pepo. L. and C. ficifolia Bouche. (Chakravarty, 1982), are recorded in India. Fifth one, C. mixta Pang. (later renamed as C. argyrosperma) is mostly confined to Latin America. Even though confined to New World regions, some Cucurbita sp. have been introduced into India in very early times. There had been a mix-up in identification of the species found in India as C. maxima or C. moschata. In Atharva Veda, pumpkin has been mentioned as a charm against diseases. Also later in Buddhistic Jatakas (600 B.C.), pumpkin has been recorded as under cultivation (Aiyar, 1956). C. pepo and C. ficifolia were very later introductions and the latter called fig leaved gourd is grown in north-east India (Meghalaya). Winter squash (C. maxima) along with maize and beans were the foundation diet of pre-Columbian civilizations (Aztec, Inca and Maya) in central and south America

86 (Whitaker and Bemis, 1975). The origin and domestication of Cucurbita species have been reviewed recently by Merrick (1995), confining herself to new World regions. However, in India pumpkin (C. moschata) because of its very early introduction, has adapted and even diversified giving wide range of varieties. Compared to other Cucurbita species, C. moschata is more adapted to warmer regions like India, while C. maxima, C. ficifolia and C. pepo are comparatively cold tolerant.

Lagenaria Ser. Originally considered as monotypic genus, but 6 species have now been recognized in Africa, but only one species has been found and cultivated in India (Chakra- varty, 1982), viz. L. siceraria (Mol.) Standl. (2n = 22) (syn. L. vulgaris Ser., L. leu- cantha Duch.) bottle gourd. The bottle gourd is an ancient vegetable of India recorded in pre-historic civilizations as alabu by Protoaustraloids and later in Ya- jurveda. Further in the Puranas, it carries the Sanskrit equivalent tumbi, besides alabu. In the medical treatises, Caraka Samhita and Susruta Samhita, it has been mentioned for its medicinal properties as alabu and iksvaku. Lagenaria has been credited with wide bi-hemispheric and pre-Columbian distribution. Archaelogical remains of its shell have been identified in Spirit caves of Thailand (10,000 - 6000 B.C.), in Mexico (7000 - 5000 B.C.), in Peru (4000 - 3000 B.C.), in Egyptian tombs (3500- 3000 B.C.) and in China (500 A.D.). Some estimate that human utilization of Lage- naria was at least 15,000 years old in New World and 12,000 years in Old World, while there is a view that Lagenaria was independently domesticated in two worlds (Heiser, 1979). He considered it of African origin (agreed to by Whitaker) even though there was no decisive enough evidence. Purseglove (1974) regards Lagenaria as the oldest crop cultivated in the tropics. There is a fascinating account by Heiser (1979) of its several uses like in making of musical instruments viz., Zithers in Africa and Sitar in India. Even in early Indian scriptures, the sage Naradas veena (stringed in- strument) or Goddess Saraswatis veena or kamandalu (water pitcher) used by her- mits might have been made from bottle gourd shells. In Africa, there are species like L. sphaerica, L. bicornuta Chakra., L. abyssinica, L. guineensis, L. rufa (Zeven and de Wet, 1982), but in India, however, there is wide variability of seed characters within L. siceraria itself. Essentially a monoecious species, an andromonoecious line has been isolated at NDUAT, Kumarganj (Faizabad), India, recently.

Luffa L. A cosmopolitan genus of 9 species of which 7 are in India. The cultivated species L. acutangula (L.) Roxb. (2n = 26) (syn. L. acutangula var. amara, L. hermaphrodita Singh & Bhandari) is the familiar ridge or ribbed gourd, with a wide distribution in Myanmar, Sri Lanka, Malaysia, Indonesia and South Africa. Another cultivated species is L. cylindrica (L.). Roem. (2n = 26) (syn. L. aegyptiaca Mill.) - smooth or sponge gourd. Other wild relatives are L. graveolens Roxb. (var. longistyla) occurring in India, Indonesia and Australia, and L. echinata Roxb. a dioecious perennial recorded in India and Africa. Others are L. tuberosa Roxb. distributed in peninsular India and tropical Africa and L. umbellata Roem. is being endemic in Kerala (south-west India) (Arora and Nayar, 1984). L. operculata is confined to central and south Ame- rica (Bates et al., 1995). Luffa is essentially an Old World genus with long history of

87 cultivation in tropical countries of Asia and Africa. The name Luffa or Loofah is of Arabic origin, because sponge characteristic has been described in Egyptian wri- tings and the epithet Szkua in China, refers to dish cloth or towel gourd in early Chinese literature. Wild populations of L. cylindrica var. leiocarpa are spread over from Myanmar to Philippines. In ancient India kosataki is the Sanskrit equivalent in Arthasastra (around 400 B.C.), dharmaragava in the medical treatise Caraka Samhita and in Susruta Sa- mhita, recorded as ervaruka and kratavedhana. From sponge gourd, fibrous sponges of mature fruits are used in womens footwear and scrubbers, industrial filters, etc. South and south-east Asia is the centre of diversity, with the possibility of having originated in India-Hindustani centre (Zeven and de Wet, 1982).

Momordica L. It is cosmopolitan genus of 60 species with 7 occurring in India and others in China, south-east Asia, Polynesia and tropical Africa. Nearly 23 species have been recorded in Africa alone. In India species recorded are, M. balsamina L. with Indo-Malayan, Chinese and African distribution; M. charantia L. (2n=22), the bitter gourd, with extensive Indo-Malayan and south-east Asian distribution; M. cochinchinensis (Lour.) Spreng. dioecious and tuberous rooted sweet gourd of Assam (India) with Pacific and soth- east Asian distribution besides China, and M. dioica (2n = 28) Roxb. again dioecious, perennial tuberous rooted, kakrol in Bihar and Orissa or kartoli in western Maharashtra, mainly a tribal vegetable, distributed in India, Myanmar, Srilanka and China (Chakravarty, 1982). Other species are M. denudata Clarke, M. macrophylla Gage and M. subangulata Blume (Jeffrey, 1980b). The Sanskrit epithets for bitter gourd are karuvella and karavellika in Pura- nas and medical treatises Caraka and Susruta Samhitas, karkataki (M. cochinchinensis Spreng.) and karkotaka (M. dioica) are mentioned in these medical treatises and Puranas. Hypoglycaemic properties (viz. reducing blood sugar levels) of bitter gourd are well known. Although nativity of bitter gourd is uncertain (Zeven and de Wet, 1982), eastern India and southern China are possible centres of domestication. A gynoecious line has been isolated in monoecious bitter gourd at Indian Institute of Vegetable Research, Varanasi, India.

Trichosanthes L. Principally Indo-Malayan genus of about 44 species, 22 are in India (Chakravarty, 1982). The two cultivated species are T. cucumerina L. var. anguina (2n = 22) the common snake gourd and T. dioica Roxb. (2n = 22) - the pointed gourd, a dioecious and vegetatively propagated one. Other wild related species of India are T. tricuspi- data Lour. (syn. T. bracteata Voigt and T. palmata Roxb.) (2n = 66), T. wallichiana (Ser.) Wight (syn. T. multiloba Clarke and T. khasiana Kundu), T. anamalaiensis Bedd., T. rubriflos Thorel, T. villosa Blume, T. cordata Roxb., T. truncata Clarke, T. integri- folia Thwaites, T. nervifolia L., T. lobata Roxb. (syn. T. villosula Cogn. and T. per- rottetiana Cogn.) and T. ovigera Blume (syn. T. himalensis Clarke) (Jeffrey, 1980b). The compound trichosanthin, believed to have anti-HIV activity, has been isolated from T. kirilowii var. japonica roots (Bates et al., 1995).

88 The centre of origin of Trichosanthes is not precisely known, but most authors agree that Indo-Malayan region would be the centre of large diversity. However, in the case of pointed gourd, Indian origin is the most probable one. The pointed gourd is mentioned as patola in Matsya Purana and in Caraka Samhita.

Minor Cucurbits Praecitrullus fistulosus (Pang.).: Indian squash (tinda) (2n = 24) indigenous to north-west India and Pakistan, is a recent and partial domesticate, because of low genetic variation. It is neither related to muskmelon nor to watermelon. Introduced from central America: Sechium edule (Jack.) Sw.: It is called chow-chow or chayote mostly grown in northeast India under humid condition. Cyclanthera pedata (L.) Schrad.: It is grown in western Himalayas. Introduced from China: Hodgsonia heteroclita (L.) Hook & Thoms.: It is grown in Meghalaya (north-east India) (Arora and Hardas, 1977).

Summing up, cucurbits are the ancient group of vegetables of India, steeped in antiquity. There is a rich heritage of variability handed down through centuries of human selection. Historical evidence though fragmentary, has to be delved into, from the original texts and manuscripts for more details. Besides, more archaeobotanical studies seem to be necessary.

References

Aiyar, A.K.Y. 1956. The antiquity of some field and forest flora of India. Bangalore Printing & Publishing Co., Ltd., Bangalore. Anonymous. 1873. Ain-i-Akbari - English translation by H. Blochman, Ed. S. L. Groomer Aa- diesh Book Depot., Delhi. Arora, R.K. and Hardas, M.W. 1977. Hodgsonia heteroclita - oil rich cucurbit. J. Bombay Nat. His. Soc., 74: 559-561. Arora, R.K. and Nayar, E.R. 1984. Wild relatives of crop plants in India. NBPGR Sci. Monogra- ph No. 7, I.C.A.R., New Delhi. Bates, D.M. and Robinson, R.W. 1995. Cucumbers, melons and watermelons. In: Smartt, J. and Simmonds, N.W. (Eds.), Evolution of Crop Plants, Second Edition. Longmans, Singapore, pp. 89-96. Bates, D.M., Merrick, L.C. and Robinson, R.W. 1995. Minor cucurbits. In: Smartt, J. and Sim- monds, N.W. (Eds.), Evolution of Crop Plants, Second Edition. Longmans, Singapore, pp. 105-111. Chakravarty, H.L. 1982. Fascicles of Flora of India. 11. Cucurbitaceae. Botanical Survey of India, Calcutta, India. Heiser, C.B. 1979. The Gourd Book. University of Oklahoma Press, Norman, OK. Jeffrey, C. 1980a. A review of Cucurbitaceae. Bot. J. Linn. Soc., 81: 233-247. Jeffrey, C. 1980b. Further notes on Cucurbitaceae. V. The Cucurbitaceae of Indian sub-continent. Kew Bull., 34: 789-809. Kajale, M.D. 1991. Current status on Indian palaeoethnobotany. In: Renfrew, J.M. (Ed.), New Light on Early Farming. Recent Developments in Palaeonobotany. Edinburgh, pp. 155-189. Mehra, K.L. 1980. Ethnobotany of old World Cucurbits. In: Conference on the Biology and Chemistry of Cucurbitaceae, August 3-6, 1980. Cornell University Press, Ithaca, N.Y., U.S.A., p. 9.

89 Mehra, K.L. 2002. Agricultural foundation of Indus-Saraswati civilization. In: Nene, Y.L. and Choudhary, S.L. (Eds.), Agricultural Heritage of India. Asian-Agri-History Foundation, Se- cunderabad, pp. 1-21. Merrick, L.C. 1995. Squashes, pumpkins and gourds. In: Smartt, J. and Simmonds, N.W. (Eds.), Evolution of Crop Plants, Second Edition. Longmans, Singapore, pp. 97-104. More, T.A., Seshadri, V.S. and Misra, J.P. 1998. Cultivation in riverbeds. In: Nayar, N.M. and More, T.A. (Eds.), Cucurbits. Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi, pp. 205-210. Nene, Y.L. 2000. Orientation to ancient and medieval history of agriculture with special reference to India. In: Proc. of Summer School, Udaipur, India, June 1999, pp. 1-15. Om Prakash. 1961. Food and Drinks in Ancient India (from earliest times to c. 1200 A.D.). Munshiram Manoharlal Oriental Booksellers & Publishers, Delhi. Purseglove, J.W. 1974. Tropical Crops Dicotyledons. Longmans, London. Ray, P. and Gupta, H.N. 1980. Caraka Samhita Scientific synopsis. Indian National Science Academy, New Delhi. Ray, P., Gupta, H.N. and Roy, M. 1980. Susruta Samhita Scientific synopsis. Indian National Science Academy, New Delhi. Sensarma, P. 1989. Plants in Indian Puranas-an Ethnobotanical Investigation. Naya Prokash, Cal- cutta, India. Sensarma, P. 1998. Conservation in Ancient India. Centre for Indigenous Knowledge on Indian Bioresources, India. Seshadri, V.S. and Chatterjee, S.S. 2000. The history and adaptation of some introduced vegetable crops in India. Asian-Agri-History, 4: 175-202. Seshadri, V.S. and More, T.A. 1996. Some considerations on the diversification of muskmelons in India. In: Gómez-Guillamón, M.L., Soria, C., Cuartero, J., Torés, J.A. and Fernández-Muñoz, R. (Eds.), Cucurbits Towards 2000. Proc. of VIth Eucarpia Meeting on Cucurbit Genetics and Breeding, Malaga, Spain, May 28-30, 1996, pp. 112-119. Vats, M.S. 1940. Excavations at Harappa. Centre for Indigenous Knowledge on Indian Bioresou- rces. Delhi, India. Vishnu Mittre. 1974. Paleobotanical evidence in India. In: Hutchinson, J. (Ed.), Evolutionary Studies in World Crops. Cambridge University Press, Cambridge. Whitaker, T.W. and Bemis, W.P. 1975. Origin and evolution of the cultivated Cucurbita. Bull. Torrey Bot. Club, 102: 362-368. Zeven, A.C. and de Wet, J.M.J. 1982. Dictionary of cultivated plants and their regions of diversity. Centre for Agricultural Publishing & Documentation, Wageningen.

90 The status of the cucumber (Cucumis sativus) collection of CGN

W. van Dooijeweert Plant Research International, Centre for Genetic Resources the Netherlands (CGN), P.O.Box 16, Wageningen, The Netherlands; website: http://www.cgn.wur.nl

Summary

The paper summarize the genetic resources of cucumber maintained in the CGN. The collection consists of 922 accessions originating from 63 countries. The cucumber collection is assessed for 19 morphological characters and most of the characterisation data are searchable and can be downloaded from the CGN website. The accessions of cucumber have been distributed and used mainly for research purposes.

Keywords: cucumber, genebank, genetic resources, characterization, utilization

Introduction

The Centre for Genetic Resources, the Netherlands (CGN) holds the mandate to conserve and promote the utilisation of plant and animal genetic resources in the Netherlands. It was established in 1985. CGN has focused on a limited number of collections, for which it attempts to maintain high quality seed, which is readily available to bona fide users. CGN strives to increase knowledge over its germplasm relevant to its users. All parties which use its germplasm for breeding, research or cultivation and which have access to facilities needed to attain these objectives qualify as bona fide users. The complete CGN collection holds about 23.000 accessions of 20 horticultural and agricultural crops (Soest and Bouke- ma, 1995; http://www.cgn.wur.nl).

The CGN cucumber collection

The cucumber (Cucumis sativus) collection originates from the former Institute for Horticultural Plant Breeding (IVT). This collection was a working collection for their breeding work. The material has been characterized for morphological characters. The collection includes mainly old cultivars received from Dutch and foreign seed companies and genebanks. CGN adopted the collection in 1992 (Groot and Boukema, 1997). The quality and quantity of the seeds of this collection were assessed and accessions meeting the CGN standards have been given CGN accession numbers. The collection was rationalised by rejecting duplicates and hybrids. Passport data are available and searchable from the CGN website, but are not complete. The cucumber collection holds only accessions of the cultivated Cucumis sativus. By January 2004 the collection consists of 922 accessions. An overview of the collection is given in Tables 1 and 2.

Group No. of accessions Cucumis sativus group Cucumber 359 Cucumis sativus group Gherkin 350 Cucumis sativus var. hardwickii (Royle) Gabaev 1 Cucumis sativus 212 Total 922

Table 2. Number of cucumber accessions per country of origin

Country No. of Country No. of accessions accessions Afghanistan 3 Lebanon 1 Albania 1 Lithuania 1 Argentina 1 Mauritius 3 Australia 4 Moldova 2 Austria 2 Nepal 4 Bangladesh 1 Netherlands 159 Bulgaria 3 Nigeria 1 Brazil 4 Pakistan 11 Canada 5 Papua New Guinea 2 China 42 Poland 27 Croatia 1 Romania 5 Cyprus 1 Russian Federation 34 Czechoslovakia 8 South Africa 2 Czech Republic 1 Spain 1 Denmark 24 Sri Lanka 3 Egypt 19 Surinam 2 Ethiopia 1 Sweden 14 Far East 2 Switzerland 1 France 9 Syria 1 Germany 42 Taiwan 9 Georgia 1 Tajikistan 1 Greece 1 Thailand 5 Hungary 21 Turkey 10 India 43 Ukrainian SSR 10 Indonesia 15 United Kingdom 14 Iran 13 USA 116 Iraq 1 USSR 35 Israel 9 Uzbekistan 18 Italy 1 Vietnam 5 Japan 65 Yugoslavia 4 Kirghiztan 3 Zaire 2 Korea 2 unknown 70 Total 922

92 Regeneration

Material of which the quality and quantity was not enough is regenerated. Regenera- tion takes place in insect free glasshouses on rockwool. Per accessions ten plants are regenerated. Two stems per plant are grown along ropes. For seed production, so-called chain pollination is carried out by hand. Per accession plant 1 is crossed with plant 2, plant 2 with plant 3, etc. Per plant 2 to 4 ripe fruits are harvested. Biological control is used to keep the crop healthy. The Dutch breeding companies assist in the regeneration.

Sample viability

Sample viability is determined in germination tests. The CGN standards for viability are a germination percentage of at least 80%. In general the germination percentage should be at least 80% if samples are to be included in the collection. Five different types of samples for storage are distinguished: user samples (25 seeds), germination samples (200), regeneration sample (100), safety duplication sample (100 seeds) and a residual sample.

Storage

The seeds are dried until a seed moisture content of about 5% is reached. The seeds are packed in laminated aluminium foil bags and stored at 20°C. CGN has both long- and medium-term storage facilities. The seed-storage facilities of the CGN consist of the following compartments: l 2 deep freezer compartments (20°C) total 80 m2, l 1 cooler compartment (+4°C) of 33 m2, l 1 dryer compartment (+15°C, RH 15%) of 12 m2, l 1 working compartment of 61 m2, l 1 storage compartment of 6 m2. The numbered boxes are grouped by crop and placed on numbered shelves in the storage rooms. The location of storage (box and shelve) is recorded in the CGN information system.

Safety duplication

About 95% of the collection is duplicated at the Genetic Resources Unit of HRI Wellesbourne, United Kingdom.

Characterization/evaluation The cucumber collection is characterized for 19 morphological characters. So far, no evaluation data have been recorded. Most of the characterization data are sear-

93 chable and can be downloaded from the CGN website. On-line users can select a given number of accessions with the broadest representation of types and origin using the core selector on the website.

Collecting missions

In 1997 and in 1999 multicrop collection missions to Uzbekistan and Kirghiztan took place. These two missions resulted in 18 new Cucumis sativus accessions.

Utilization

Users have to sign a Material Transfer Agreement and return the results obtained with the CGN material. Most of the accessions distributed in the last three years were used for research purposes. Results will be made available via the website. If required an embargo period can be obtained.

References

CGN website: http://www.cgn.wur.nl Groot, E.C. de and Boukema, I.W. 1997. Economisch belangrijke vruchtgroenten geconserveerd (Economical important fruit vegetables conserved). Prophyta, 51(2): 14-16. Soest, L.J.M. van and Boukema, I.W. (Eds.). 1995. Diversiteit in de Nederlandse Genenbank. Een overzicht van de CGN collecties (Diversity in the Dutch genebank. An overview of the CGN collection). Centrum voor Genetische Bronnen Nederland (CGN). Centrum voor Planten- veredelings - en Reproductieonderzoek (CPRO-DLO), Wageningen. 126 pp.

94 Descriptor lists for genetic resources of the genus Cucumis and cultivated species of the genus Cucurbita

V. Vinter1, A. Køístková2, A. Lebeda1 and E. Køístková3 1Palacký University in Olomouc, Faculty of Science, Department of Botany, lech- titelù 11, 783 71 Olomouc-Holice, Czech Republic; e-mail: [email protected] 2Secondary School, Tomkova 45, 779 00 Olomouc-Hejèín, Czech Republic 3Research Institute of Crop Production, Division of Genetics and Plant Breeding, Department of Gene Bank, Workplace Olomouc, lechtitelù 11, 783 71 Olomouc -Holice, Czech Republic; e-mail: [email protected]

Summary

Lists of morphological descriptors for genetic resources of the genus Cucumis and of the cultivated species of the genus Cucurbita were created within the framework of the Czech National Program of Conservation and Utilization of Plant Genetic Resources. Both are bilingual, in Czech and English. First parts of descriptors give the overview of taxonomy, biology, morphology, karyology, and biochemistry of both genera and standards for plant genetic resources regeneration and evaluation. Tables consist of 65 descriptors for Cucumis and 75 descriptors for Cucurbita and, when necessary, they are supported by illustrations. Descriptor lists provide the tools for determination and characterization of Cucumis and Cucurbita species and for a discrimination of their infraspecific variation. Descriptor lists are used for genetic resources within framework of the Czech national system EVIGEZ and can be used also by the international gene bank community within the framework of the Cucurbitaceae Working Group ECP/GR.

Keywords: Cucurbitaceae, cucurbits, cucumber, melon, squash, gourd, Cucumis melo, Cucumis sativus, wild Cucumis species, Cucurbita argyrosperma, Cucurbita ficifolia, Cucurbita maxima, Cucurbita moschata, Cucurbita pepo, morphology, germplasm, data, descriptors, infraspecific variation, interspecific variation

Introduction The Czech Republic (former Czechoslovakia) participates in international activities aimed at the protection of biodiversity. In 1994, the Ministry of Agriculture of the Czech Republic undertook the project National Programme of Conservation and Utilization of Genetic Resources of Cultivated Plants. The Research Institute of Crop Production in PrahaRuzynì (RICP) is the national coordinator of this programme. The Gene Bank at Olomouc, under the aegis of the RICP, is responsible for conservation and documentation of the genetic diversity of the species of vegetables, medicinal, aromatic and spice plant species traditionally grown in the Czech Repub- lic. Collections maintained by the Gene Bank at Olomouc include more than 10,000 accessions of about 430 plant species The collection of cucurbitaceous genetic resources, which consists of approximately 1600 accessions, is one of the most extensive ones (Køístková and Lebeda, 1995). The Czech collection of Cucumis spp. genetic resources includes about 900 accessions of cultivated C. sativus and C. melo and 90 accessions of wild species (C.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 95 africanus, C. anguria, C. heptadactylus, C. myriocarpus, C. prophetarum, C. zeyheri) (Køístková, 2002). Included within the collection of nearly 600 Cucurbita spp. accessions are all original Czech landraces and cultivars as well as local cultivars from many other countries, introduced mostly from U.S.A. gene banks. The collection includes about 550 accessions of the cultivated species C. argyrosperma, C. ficifolia, C. maxima, C. moschata and C. pepo and also the wild taxa C. ecuadorensis, C. foetidissima, C. pepo subsp. fraterna, C. pepo subsp. texana, C. lundelliana, and C. okeechobeensis subsp. martinezii (Køístková, 2002). Passport data on accessions are available on the web site http://www.vurv.cz (part of databases, EVIGEZ). Morphological data obtained during observation of some of the accessions did not always coincide with the descriptions of species presented in monographs. Appa- rently, the morphological range of variation of some of the accessions need to be re- considered. Similarly, the interpretation of scientific results, including plant-pathogen interactions, should be based on exact taxonomic determinations and morphological descriptions of the plant material. International cooperation of European gene banks is promoted by the IPGRI (In- ternational Plant Genetic Resources Institute) within the framework of the European Cooperation Program on Plant Genetic Resources (ECP/GR) and its working groups. The most important tasks of the Cucurbitaceae Working Group were formulated during its informal meeting in Adana (Turkey) in 2002 (Díez et al., 2002). The creation of descriptors, that is, systems of descriptive data, has fundamental importance, as it improves the quality and flow of information on the various taxa and facilitates their utilization. International descriptors for genetic resources of Cucumis spp. and Cucurbita spp. have not as yet been worked out.

Descriptor lists

Lists of morphological descriptors for genetic resources of the genus Cucumis and of cultivated species of the genus Cucurbita were created within the framework of the Czech National Program of Conservation and Utilization of Plant Genetic Resou- rces. Both are bilingual, in Czech and English. The creation of descriptor lists was based on study of plant genetic resources in the RICP Gene Bank at OlomoucHoli- ce, available literary data (monographs, descriptors), and author experience and knowledge of plant biology, taxonomy, anatomy and morphology. First parts of descriptors give the overview of taxonomy, biology, morphology, karyology, and biochemistry of both genera and standards for plant genetic resources regeneration and evaluation. The terminology of botanical morphology and illustrations are based on recent valid official sources. Explanations of descriptors are followed by examples of corresponding Cucumis and/or Cucurbita species with literature cited or by accession number of genetic resources. About one-third of the descriptors are supported by figures. Descriptor lists are terminologically exact and at the same time highly understandable. Highly discriminating descriptors are marked with an asterisk. The letter S indicates a species characterizing descriptor, state of descriptor marked with a letter I discri- minates an infraspecific variation, and letters in brackets are of a secondary significance.

96 The format for the creation of these descriptor lists follows the rules given of the National Council for Plant Genetic Resources of the Czech Republic. The descriptors will be appended to the central documentation system of plant genetic resources EVIGEZ and can be also used by the international gene bank community within the framework of the Cucurbitaceae Working Group ECP/GR.

Descriptor list for genetic resources of Cucumis

The set of morphological descriptors for the genus Cucumis contains 65 descriptors and 20 of them are supported with figures (Køístková et al., 2003b). A part of the table and figures from this descriptor are given by the Table 1 and Fig. 1. It was developed on the basis of study of genetic resources at the RICP Gene Bank at Olo- mouc, the Cucumis spp. monograph of Kirkbride (1983), the descriptor lists published by IBPGR (Esquinas-Alcazar and Gulick, 1983), the descriptor list and codes for Cucumis spp. of the North Central Regional Plant Introduction Station at Ames (U.S.A),

Table 1. Example of the morphological descriptors for Cucumis spp. (Køístková et al., 2003b)

Number Descriptor name Value Descriptor state Explanation Figure Note 1. Morphological descriptors 1.2. Stem 1.2.1.* S Stem indumentum 0 glabrous at 1 with breakaway C. sacleuxii botanical trichomes (Kirkbride, 1993) maturity 2 with nonbreakaway C. sativus trichomes 3 with aculei C. aculeatus (Kirkbride, 1993) 2. Biological features 2.1. Reproductive strategy 2.1.4. * I Reproductive strategy Figure 2.1.4. 1 monoecious male and female flowers on the same plant 2 andromonoecious male and hermaphroditic flowers on the same plant 3 gynomonoecious female and hermaphroditic flowers on the same plant 4dioecious male and female flowers on separate plants 5 androecious male flowers only (male line) 6 gynoecious female flowers only (female line) 7 polygamous male, female and hermaphroditic (trimonoecious) flowers on the same plant

97 Figure 1. Illustration for morphological descriptors of Cucumis spp. (Køístková et al., 2003b). the descriptor for Cucumis sativus L. published by VIR in Leningrad (USSR), and the UPOV Guidelines for conducting of tests for distinctness, homogeneity and stability of C. sativus and C. melo.

Descriptor list for genetic resources of cultivated Cucurbita species

The set of morphological descriptors for genetic resources of cultivated species of the genus Cucurbita contains 75 descriptors, of which 22 of them are supported with illustrations (Køístková et al., 2004 in press). It was developed on the basis of morphological assessment of the genetic resources of the cultivated Cucurbita species (Køístková et al., 2003a; Køístková et al., this volume), the monograph of Lira-Saade (1995), available international descriptors published by IBPGR (Esquinas-Alcazar and Gulick, 1983), the descriptor list and codes for Cucurbita spp. used by Plant Introdu- ction Stations at Ames, Griffin, Geneva and Pullman (U.S.A.), and the descriptor for Cucurbita spp. published by VIR in Leningrad (Russian Federation) and the UPOV Guidelines.

Acknowledgements

This work was supported by the Ministry of Agriculture of the Czech Republic through the National Program of Conservation and Utilization of Cultivated Plants (Grant E-97/01-3160-0200) and by the Ministry of Education of the Czech Republic through grants MSM 153100010 and FRV 38/2004 Visual aids to practical exercises of selected botanical subjects; anatomical atlas of vascular plants. Morphologi- cal assessments of Cucurbita spp. plants have been performed within the framework of the student Secondary school research activity in 2002 and 2003.

98 References

Díez, M.J., Pico, B. and Nuez, F. 2002. Discussion and recommendations. In: Díez, M.J., Pico, B. and Nuez, F. (Comp.), Cucurbit Genetic Resources in Europe, Report of Ad hoc meeting. Adana (Turkey), 19 January 2002. IPGRI, Rome, pp. 1-6. Esquinas-Alcazar, J.T. and Gulick, P.J. 1983. Genetic resources of Cucurbitaceae - a global report. IBPGR, Rome, 100 pp. Kirkbride, J.H., Jr. 1993. Biosystematic monograph of the genus Cucumis (Cucurbitaceae). Par- kway Publ., Boone, North Carolina, 159 pp. Køístková, E. 2002. The Czech national collection of cucurbitaceous vegetables. In: Díez, M.J., Pico, B. and Nuez F. (Comp.), Cucurbit Genetic Resources in Europe, Report of Ad hoc meeting. Adana (Turkey), 19 January 2002. IPGRI, Rome, pp. 1829. Køístková, E., Køístková, A., Vinter, V. and Lebeda, A. 2004. Genetic resources of cultivated Cucurbita species (C. argyrosperma, C. ficifolia, C. maxima, C. moschata, C. pepo) and their morphological description (English Czech version). Hort. Sci. (Prague) (in press). Køístková, E., Køístková, A., Vinter, V. and Losík, J. 2003a. Morphological variation of cultivated Cucurbita species. In: Benediková, D. (Ed.), Proc. 3th Seminary on Evaluation of Plant Genetic Resources, Pieany (Slovakia), 27-28 May 2003, pp. 51-57. (in Czech) Køístková, E. and Lebeda, A. 1995. Genetic resources of vegetable crops from the family Cucur- bitaceae. Hort. Sci. (Prague), 22: 123-128. (in Czech, English summary) Køístková, E., Lebeda, A., Vinter, V. and Blahouek, O. 2003b. Genetic resources of the genus Cucumis and their morphological description (English-Czech version). Hort. Sci. (Prague), 30: 14-42. Lira-Saade, R. 1995. Estudios taxonómicos y ecogeográficos de las Cucurbitaceae latinoamericanas de importancia económica. Systematics and Ecogeographic Studies on Crop Genepools. No. 9, International Plant Genetic Resources Institute, Rome, Italy, 281 pp. (in Spanish)

99 100 Genetic relationships of different Greek landraces of cucumber (Cucumis sativus) as assessed by RAPDs

H. Pavlikaki, C. Ponce Navarro and N. Fanourakis Technological Education Institute, School of Agricultural Technology, Laboratory of Genetics and Plant Breeding, Heraklion Crete, 71500 Greece

Summary

The aim of this study was to identify existing variation and the genetic relationships among cucumber landraces in Greece. DNA sequence polymorphisms determined by RAPD analysis have been applied in a number of plant taxa to detect genetic diversity and relationships. RAPD molecular marker analysis was applied for the following 8 traditional varieties of cucumber: Knos- sos, Kalivia, Kos2, Ikaria, Samos, Crete1, Crete2, and Crete3. Fourteen of the 20 primers used showed consistently reproducible and simple amplification products. Assaying the variation of the accessions yielded 42 RAPD markers, 33 of which were polymorphic. Cluster analysis and the resulted dendrogram indicated two main groupings which could be associated with the most commercial varieties and the geographic origin of the accessions. Genetic distance measures among them and the possible genetic interrelationships are discussed.

Keywords: cucumber, geographic origin, molecular marker, polymorphism, variation, genetic distance

Introduction

Various approaches ranging from morphology to molecular techniques have been used to infer patterns of diversity and relationships among plant genomes. A number of molecular markers including restriction fragment length polymorphism (RFLPs) and random amplified polymorphic DNAs (RAPDs) have been used to study genomes in Cucumis species (Kner and Staub, 1992; Staub and Meglic, 1993). RAPD genetic analysis is a popular method used for the determination of phylogenetic relationships and genetic variations in several plant species. Its application to cucumber and related species is only recently attempted. Poeter et al. (1992) gave one of the first reports on a methodology for RAPD analysis in cucumber by testing various DNA extraction methods and the reproducibility of the RAPD analysis. Their data indicated that RAPD patterns were not significantly affected by tissue age, infection of plants by some diseases and presence or absence of fruits. The characterization of molecular markers (isozymes, RFLPs, RAPDs) for germplasm evaluation in cucumber was also attempted by constructing genetic maps (Staub et al., 1996). They described different parts of the cucumber genetic organization indicating also that the level of polymorphism in cucumber was relatively low in their germplasm with clustering of molecular markers on genetic maps. The applicability of RAPDs to detect genetic variability at the intravarietal level and to determine genetic relationships among Cucumis melo genotypes has been also reported (Garcia-Rodriguez et al., 1996). Their results indicated that although the genetic distance values suggest low variability in the species level, enough poly-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 101 morphism was detected to cluster together genotypes in the same varietal type. They also suggested that RAPDs are better suited than RFLPs in detecting intravarietal polymorphism. Lopez-Seze et al. (2002) tried also to assess intra- and intervarietal variation for evaluation of large collections of melon germplasm using RAPD and SSR markers. The relatively high level of polymorphism detected by RAPD markers and SSR loci helped in discriminating accessions. Cluster analysis and genetic distance estimates resulted in similar and consistent groupings of the accessions studied. The objective of this study was a first attempt to detect the genetic diversity of traditional Greek cucumber varieties which have not been described previously. De- tection of genetic diversity will help to identify and protect traditional varieties and may provide useful information for the genetic improvement of commercial varieties.

Material and methods

Cucumber landraces Eight traditional Greek cucumber varieties of different geographical origin were tested for genetic variation. The seeds were obtained from the Gene Bank of Thessa- loniki and the Plant Breeding Laboratory of TEI of Crete (Table 1, Fig. 1). The varieties Knossos and Kalivia are well known commercial varieties for more than 100 years with almost the same morphological, disease resistance, and fruit characteristics. The other six accessions (Crete1, Crete2, Crete3, Kos2, Ikaria and Samos) are locally cultivated landraces collected from the respective islands of Greece.

Figure 1. Geographical origin of the Greek cucumber varieties analysed.

102 DNA extraction and RAPD amplification Total genomic DNA was extracted from leaf tissue by CTAB method (Hulbert and Bennetzen, 1991). All PCR reactions were carried out in a final volume of 25µl. The final concentration of each component in the reaction mixture was 25pmol of single decamer primer (OPA1, OPA2, , OPA20) (Operon Technologies), 1.5u Taq polymerase (Minotech, Crete, Greece), 50mM KCl, 10mM Tris-HCl pH9.0, 1.5mM MgCl , 2 0.1% Triton, 0.25mM dNTPs and 100ng of total genomic DNA. Samples were denatured at 94°C (5 min), followed by 45 cycles of 94°C (1 min), 36°C (1 min), and 72°C (1 min). Amplified products were separated by electrophoresis on a 1% agarose gel in 1X TAE. Reproducibility was assessed by running PCR reactions from at least two different DNA extractions of the same variety for each primer.

Molecular markers All plants were scored for presence/absence of RAPD fragments and the resulting data were entered into a molecular data matrix (1=presence and 0=absence for a DNA band of the same size). Statistics of genetic variation (Nei, 1978; Nei and Li, 1979) (Neis gene diversity, genetic distance, and polymorphism) were obtained from the phenotypic frequencies of RAPD markers. Cluster analysis was performed on the genetic distance matrix by using UPGMA method to determine the relationships among accessions (dendrograms). Statistics and cluster analysis were performed by using the computer program POPGENE (Yeh et al., 1997) and PHYLIP version 3.6.

Results and discussion

Twenty decamer primers were used in the present study to amplify genomic DNA of the 8 cucumber varieties (Table 1, Fig. 1). Fourteen of these primers showed consistently reproducible and simple amplification products. Assaying RAPD variation in the accessions of cucumber varieties with these primers yielded 42 bands which ranged in size from approximately 400 to 2500 bp. Thirty three of these amplified fragments were polymorphic (78.57% of the fragments detected) among the populations examined. The mean number of bands per primer was 3. Identified variation at the 33 polymorphic RAPD loci was used to assess genetic differences among the 8 Greek cucumber variants. An average of 21.6% of the markers analyzed were present in every individual and 20.4% were absent in any one accession. Cluster analysis (UPGMA) to these RAPD markers resulted in the construction of a dendrogram (Fig. 2). Two main branches are included, the one containing three of the Cretan varieties and the other containing all the others. Cluster groupings could be associated with cucumber commercial varieties and the geographical origin of the landraces. Cretan landraces grouped separately from the Aegean islands landraces. The genetic distance between the two commercial varieties, Knossos and Kalivia, was expected to be higher than the one detected (0.0139) since they come from different and distant geographic regions (Table 2). The fact that they were the most extensively cultivated varieties in all over Greece for more than 100 years could probably explain these results as it could suggest the existence of a genetic flow between them. The smallest genetic distance was detected among the varieties Kos2, Ikaria

103 and Samos. The islands of Kos, Ikaria, and Samos are closely located in the Aegean Sea sharing everyday communication especially between Samos and Ikaria. While there is no other information about these 3 landraces they could have the same origin with a genetic drift from one island to the other.

Table 1. Greek varieties of cucumber assessed for genetic diversity by RAPDs

Name# Accession Geographical origin RAPD banding number* morphotype@

Present Absent

Kalivia - Kalivia (Attiki-Sterea Ellada) 1428 Knossos - Knossos (Heraklio-Crete) 15 27 Crete1 2966 Pachia Ammos (Lasithi-Crete) 28 14 Crete2 2967 Lagadia (Lasithi-Crete) 27 15 Crete3 2968 Agia Irini (Heraklio-Crete) 36 6 Kos2 2963 Mastichari (Kos island) 19 23 Ikaria 2964 Raches (Ikaria island) 19 23 Samos 2962 Pandrosos (Samos island) 15 27

Average 21.6 20.4

# Code number given in Figure 1 * Gene Bank Thessaloniki (Greece) @ Of 42 analyzed bands, present or absent of all individuals

Table 2. Genetic distance measures among the 8 varieties as determined by 42 RAPD markers by using Neis distance measure

Kalivia Knossos Crete1 Crete2 Crete3 Kos2 Ikaria Samos

Kalivia 0.0000 0.0139 0.0241 0.0314 0.0370 0.0144 0.0204 0.0187 Knossos 0.0139 0.0000 0.0253 0.0241 0.0380 0.0160 0.0175 0.0156 Crete1 0.0241 0.0253 0.0000 0.0160 0.0124 0.0123 0.0134 0.0216 Crete2 0.03140.0241 0.0160 0.0000 0.0136 0.02040.0096 0.0169 Crete3 0.0370 0.0380 0.01240.0136 0.0000 0.02040.02420.0267 Kos2 0.01440.0160 0.0123 0.02040.02040.0000 0.0105 0.0120 Ikaria 0.02040.0175 0.01340.0096 0.02420.0105 0.0000 0.0063 Samos 0.0187 0.0156 0.0216 0.0169 0.0267 0.0120 0.0063 0.0000

Regarding the varieties Crete1, Crete2, Crete3, and Knossos, the observed genetic distances are higher than expected taking into account that they have the same geographical origin. The varieties Crete1, Crete2, and Crete3 are local landraces with

104 low commercial cultivation and nothing is known about their origin. The most cultivated commercially is the variety Knossos. It is likely that locally practiced selection and the isolation of small areas of cultivation could have resulted in the absence of genetic flow between them.

Figure 2. Cluster analysis of 8 Greek varieties grouped by UPGMA using genetic distances as estimated by 42 RAPD bands (Neis distance).

The applicability of RAPDs to detect genetic variability and relationships has been attempted in species of Cucurbitaceae. Staub et al. (1996) reported on the methodology for RAPD analysis, indicating its reproducible results independently of the incidence of several cultural conditions on the plant. Bernet et al. (2003) tried also to answer the question of using molecular markers to assess distinctness of varieties of cucumber. The comparison of 36 varieties between a morphological and a molecular characterization indicated that varieties considered uniform for morphological traits proved not to be so for molecular markers. Our results give a preliminary indication of agreement with the above for similar molecular characterization, however it remains to be confirmed in our next steps by more extensive analyses of the reference, and reveal the range of genetic variation and relationships of the Greek varieties.

References

Bernet, P.G., Bramardi, S., Calvache, D., Carbonell, A.E. and Asins, M.J. 2003. Applicability of molecular markers in the context of protection of new varieties of cucumber. Plant Breeding, 122: 146-152. Garcia-Rodriguez, E., Alvarez, J.I. and Lozano, R. 1996. RAPDs as markers to determining genetic relationships among Cucumis melo genotypes. In: Gómez-Guillamón, M.L., Soria, C., Cuartero, J., Torés, J.A. and Fernández-Muñoz, R. (Eds.), Cucurbits Towards 2000. Procee- dings of the VIth Eucarpia Meeting on Cucurbit Genetics and Breeding. Málaga, Spain, pp. 187-193. Horejsi, T. and Staub, J.E. 1999. Genetic variation in Cucumber (Cucumis sativus L.) as assessed by random amplified polymorphic DNA. Genet. Res. Crop Evol., 46: 337-350. Hulbert, S.H. and Bennetzen, J. 1991. Recombination at the Rp1 locus of maize. Mol. Gen Ge- net., 226: 377-382.

105 Knerr, L.D. and Staub, J.E. 1992. Inheritance and linkage relationships of isozyme loci in cucumber (Cucumis sativus L.). Theor. Appl. Genet., 84: 217-224. Lopez-Seze, A., Staub, J.E., Katzir, N. and Gomez-Guillamon, M.L. 2002. Estimation of between and within accession variation in selected Spanish melon germplasm using RAPD and SSR markers. Euphytica, 127: 41-51. Nei, M. 1978. Estimation of average heterozygosity and genetic distance from small number of individuals. Genetics, 89: 583-590. Nei, M. and Li, W.H. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA, 76: 5269-5273. Poeter, K., Fruth, R. and Staub, J. 1992. RAPD analysis in cucumber map construction and breeding. In: Doruchowski, R.W., Kozik, E. and Niemirowicz-Szczytt, K (Eds.), Fifth EUCARPIA Cucurbi- taceae Symosium. Research Institute of Vegetable Crops, Skierniewice, Poland, pp. 11-21. Staub, J.E. and Meglic, V. 1993. Molecular genetic markers and their legal relevance for cultigen discrimination: A case study in cucumber. Hort. Technol., 3: 291-300. Staub, J, Serquen, F. and Bacher, J. 1996. Genetic map construction and map merging in cucumber. In: Gómez-Guillamón, M.L., Soria, C., Cuartero, J., Torés, J.A. and Fernández-Muñoz, R. (Eds.), Cucurbits Towards 2000. Proceedings of the VIth Eucarpia Meeting on Cucurbit Gene- tics and Breeding. Málaga, Spain, pp. 163-171. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingeyl, S.V. 1990. DNA polymorphisms amplified by arbitary primers are useful genetic markers. Nucleic Acids Res., 18: 6531-6535. Wolf, K. and Morgan, R.M. 1998. PCR markers distinguish Plantago major subspecies. Theor. Appl. Genet., 96: 282-286. Yeh, F.C., Yang, R.C., Boiley, T., Ye, Z.-H. and Mao, J.X. 1997. POPGENE, the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Center, Uni- versity of Alberta, Canada.

106 Diversity in tropical pumpkin (Cucurbita moschata): a review of infraspecific classifications

T.C. Andres The Cucurbit Network, 5440 Netherland Ave., D24, Bronx, New York, USA; e-mail: [email protected].

Summary

Cucurbita moschata Duchesne is a highly polymorphic domesticate for which have been presented various infraspecific classifications based on fruit shape, geographic origins, and other characteristics. While there are over 120 named cultivars in North America and Europe, the center of diversity lies in the American tropics in the form of innumerable unnamed landraces. These landraces have not been adequately described and do not readily fit into a scheme of cultivar- groups. One cultivated field may contain a landrace with variable fruits that fit into more than one market type or are intermediate between two market types. Furthermore, most geographically defined groups, such as Japonica, are not unique to their eponymous regions, but can be found in the center of the diversity of the species. Until a worldwide, thorough, comparative phenotypic and molecular survey of the species is done to show whether there are distinct groups within C. moschata, no formal taxonomic infraspecific classification should be used. Informal classifications of market types at the regional level are useful, however, in organizing and communicating some of the diversity. For example, names of market types available in the U.S.A. are butternut squash, winter crookneck squash, cheese pumpkin, and calabaza pumpkin.

Keywords: cultivars, landraces, butternut squash, calabaza pumpkin

Introduction

Cucurbita moschata Duchesne is a highly polymorphic species. For some time after Duchesne first named and described it in 1786 (Paris, 2000), botanists struggled with its circumscription. At times C. moschata was lumped under Cucurbita pepo L. or Cucurbita maxima Duchesne, and there were a number of instances in which C. moschata cultivars were misidentified as belonging to other species. In 1930, C. argyrosperma Huber (= C. mixta Pangalo) was split off from C. moschata (Pangalo, 1930). Today C. moschata is recognized as a good species with only partial incompatibility with its closest related species (Whitaker and Davis, 1962; Wilson, 1990; Wessel-Beaver, 2000). By far the greatest diversity in C. moschata occurs among the innumerable landraces that are grown in the American tropics. The objectives of this paper are to tabulate a list of the named cultivars of C. moschata, discuss their morphological diversity and uses, compare various proposed infraspecific classifications of this species, and discuss the difficulties involved in presenting an infraspecific classification that would be useful as well as reflective of genetic relationships.

Over 120 names are applied to cultigens of C. moschata that are grown in the U.S.A., Canada, Europe, North Africa, and the Middle East (Table 1). Whealy (1999) listed 40 non-hybrid cultivars in the United States and Canada. This is less than one-fourth the number of non-hybrid cultivars of C. pepo and less than half that of C. maxima. However, this may not be an accurate indication of the genetic diversity existing within each of these three species. Numerous landraces, i.e. unnamed cultivars, are found in all three species, but especially in C. moschata. In the tropics where C. moschata is primarily grown, there are few named cultivars. The best germplasm collections of C. moschata landraces are in major genebanks in Mexico and Costa Rica, which cover those countries as well as Central America and most of the West Indies. The innumerable South American landraces are poorly known outside of their geographic localities. Many of these landraces are short-day plants that require a long growing season and therefore cannot be cultivated at higher latitudes.

Infraspecific classification

The complexity and comprehensiveness of infraspecific classification schemes for Cucurbita moschata have tended to increase progressively over the past 150 years (Table 2). Taxonomists relied initially on germplasm derived from a limited geographic range and classified it according to size and shape of the fruits. With time, considerably more germplasm was collected, evaluated, and compared for these and other characteristics, leading to a bi-level classification to subspecies and botanical varieties presented by Russian scientists (Filov, 1966; Fursa and Filov, 1982). A detailed rationale underlying each of these classifications is beyond the scope of this review, but a few examples will serve to illustrate the historical development of this effort to organize such vast diversity into a reasonably defensible classification. Naudin (1856) defined three groups, but admitted that he was not very familiar with the species since it generally required a warmer climate than he had in France. He noticed intermediates between the three groups, which included the equivalent of butternut and winter crookneck (Courge berbere), oblong-shaped fruits (grande Cour- ge pleine), and round, very large fruits (Courge muscade des Marseillais). Alefeld (1866), working in Germany, expanded upon Naudin and defined eight groups by suffixing a Latin epithet to the species name, viz. C. moschata abbreviata and C. moschata macro- carpa. Castetter (1925) and Whitaker and Davis (1962) both proposed three horticultural groups based on fruit morphology. Castetter misidentified some of the cultivars to species, for example, Kentucky Field is a C. moschata that he misclassified as C. pepo. His Cushaw group included cultivars of the species C. argyrosperma, which had not yet been fully described. The cushaws of both species share the feature of a solid neck, that is, the portion of the fruit nearer the pedicel with no seed cavity, which is culinarily useful. Castetters Miscellaneous group included Chirimen which is reported to have come from Japan. Whitaker and Davis (1962) split Castetters Cushaw or necked group into two groups, the Crook-necks, which includes Canada Crook- neck, and the Bell-shape, which is synonymous with butternuts.

108 Table 1. Alphabetical list of C. moschata cultivar names from recent seed catalogs and from seed saver organizations in North America and Europe*

C. moschata Aehobag, Aizu Gokuwase, Alagold (African Squash), Argenta, Argonaut, Atlas, Avalon, Baby Buternut, Barbara, Betternut, Borinquen, Buckskin, Bugle, Burpee Butterbush, Butterbowl, Butterboy, Butternut Patriot, Butternut Ponca, Butternut Supreme, Cala- baza Segualca, Calhoun, Canada Crookneck, Canadai Mezoides, Canesi, Cangold, Carrizo (Sonora/Sinaloa Border), Chieftan, Chirimen, Choctaw Sweet Potato, Creole, Dickin- son Field, Dulce de Horno, Early Butternut, Eastern Butternut, El Dorado, Estribo, Fairytale, French Cocoanut, Futsu Kurokawa, Futtsu Black, Galeux des Antilles, Ge- fleckter Fagtoong, Genoppter Fagtoong, Golden Cushaw (Mammoth Golden Cushaw), Golden Papaya, Grey, Guarijio Segualco, Hamdan, Hayato, Hercules, Hopi Tan, Ka- shiphal, Kentucky Field, Kikuza, Kogigu, La Estrella, La Primera, La Segunda, Lan- dreth Cheese, Libbys Select, Long Island Cheese (Cutchoque Flat Cheese), Long Ne- apolitan (Bedouin Squash, Carpet Bag Squash, Piena di Napoli, Pleine de Naples, Port- manteau Squash, Porte-manteau), Longfellow, Longue de Nice, Magdalena Big Che- ese, Martinica, Matilde, Mayo Segualca, Mediterranean Giant, Menina Brasileira, Menina Verde, Metermoschata, Middle Rio Conchos, Milk Pumpkin, Mirepoix Musk Squash, Musquée du Maroc, Musquee de Provençe (Muscade de Provence, Muscat de Pro- vence), Naples Squash (Lunga di Napoli), Neck Pumpkin (Winter Crookneck), Nicklows Delight, Nigerian Local, Old-Fashioned Tennessee Vining, Orient, Paw Paw, Peraoro, Phoenix, Piedras Verdes Segualca, Pilgrim, Pima Bajo, Puritan Butternut, Qasim, Quaker Pie, Rampicante Trombocino, Really Big, Rebenque, Rhode Island Butternut, Rugo- sa Butternut, Sangol, Seminole pumpkin, Seoulmadi, Shakertown Field, Shishigatani, Sigol, Soler, St. Petersburg, Sucrine du Berry, Tahitian Melon Squash (Tahitian But- ternut), Tamala, Texas Indian, Thai Pumpkin, Toonas, Tripolitan, Trombone Squash (Lungo Trombocino, Tromboncino, Trombolino dAlbenga, Zucca dAlbenga, Zuc- chetta Trombolina, Zucchino Rampicante), Ultra Butternut, Upper Ground Sweet Po- tato, Violina, Virginia Mammoth, Waltham Butternut, Waltham Delite, White Rind Sugar, Wisconsin Canner, Yoeme Segualca, Yokohama, Zenith

Kabocha Group (interspecific F C. maxima x C. moschata) 1 Aiguri, Dandy Boy, Delica (Ebisu), Golden Debut, Hokkaido, Home Delite, Honey Delite, Iron Cap, Kikusui, Naguri Squash, Shintosa, Supreme Delight, Tetsukabuto

C. moschata possibly introgressed with C. argyrosperma Rio Fuerte Mayo Arrote

*Synonyms are shown in parentheses, but there are likely more synonyms than are indicated

109 Table 2. Infraspecific groupings of Cucurbita moschata

Author Year Classification scheme

Naudin 1856 3 groups Alefeld 1866 8 Latin trinomials Castetter 1925 3 groups Zhiteneva 1930 5 botanical varieties Whitaker and Davis 1962 3 groups Filov 1966 8 subspecies, each with up to 6 botanical varieties for a total of 27 botanical varieties Grebenscikov 1969 4 convarieties Fursa and Filov 1982 6 subspecies, each with 36 botanical varieties for a total of 29 botanical varieties Jeffrey 2001 6 cultivar-groups

The early European and American classifications were limited in scope since most of the diversity in the species was unknown. The Russian plant geographers of the 1920s, led by the eminent Nikolai I. Vavilov, were the first systematically to collect and document landraces of C. moschata from Mexico to Colombia and from Asia. Zhitenevas (1930a) infraspecific classification is based on these collections and is organized by locality. The var. colombiana, which has unique dark brown-colored seeds, was not only raised to the rank of subspecies by Filov (1966), but was considered possibly to be a new species (Bukasov, 1930). But there are intermediates with seeds of lighter shades of brown and some landraces that have dark brown seeds with lighter-colored seed margins. Zhiteneva (1930b) noted that var. indica, that is, those from India, is similar to the C. moschata grown in Cuba. The same could be said for some of the other groups. For example, var. japonica is distinguished by having flattened to disc-shaped fruits. Identical-appearing fruits can be found in the Yucatan Peninsu- la of Mexico. In Mexico alone, there is a wide range of fruit sizes, shapes, rind texture, color, and flesh color. Filov (1966) and Fursa and Filov (1982) are the only ones who have designated subspecies within C. moschata. The most recent infraspecific classification listed in Table 2 is by Jeffrey (2001) with six groups, five of which are based on geography. As Jeffrey stated, there is no wholly satisfactory classification of the cultivars. His approach was intended only as an interim grouping.

Discussion

Taxonomic surveys within C. moschata have been limited both geographically and in the characters examined. Temperate-adapted cultivars tend to be only moderate in size with non-lignified rinds that are smooth to moderately furrowed and uniformly buff-colored or reticulated dark green at maturity with orange flesh, seeds that are uniformly light tan in color, and are grown primarily for the culinary use of their mature fruits. Elsewhere, the fruit shapes remarkably resemble most of the fruit shapes of the

110 cultivar-groups of C. pepo as defined by Paris (1986). Fruit color ranges from nearly white, buff (pale orange), sometimes yellow or orange, to dark or even black green, and variously mottled or reticulated. The surface is often ridged and may be warty, especially in South America. The fruit flesh ranges from pale yellow to deep orange, or even inky blackish-green. Fruit sizes range from fist size to over 100 kg in Tripo- litan, a cultivar from the Middle East. As in C. pepo, a number of landraces and cultivars, often long-fruited, are grown partly or primarily for culinary use of the young fruit (Paris, 1989); these include some landraces from tropical America, the European cultivars Rampicante Trombocino and Longue de Nice, and the Korean cultivars Aehobag, Sangol, Seoulmadi and Sigol. In certain regions, such as in southern Mexico, the fruits are preferred for their edible seeds than for their flesh. The flowers, leaves, and young stems may also be eaten as potherbs or added to soups and stews. These, along with the seeds, are used in traditional medicines, such as in Suriname. Given that C. moschata has a high number of named cultivars and is highly polymorphic, an infraspecific classification scheme would be beneficial in organizing this diversity. A useful, defensible infraspecific classification has been presented for both, the extremely polymorphic C. pepo (Paris, 1986, 2001) and for the less polymorphic C. argyrosperma (Merrick and Bates, 1989; Jeffrey, 2001). However, for C. moschata, none of the proposed infraspecific groupings appear to be as clearly defined and none are adequate because the extent of genetic variation in the center of diversity of the species as well as worldwide is not well known. A comprehensive, systematic survey of the species is needed to determine whether there are unambiguous subspecies and/ or cultivar-groups. This should include both phenotypic as well as molecular analyses. As Wilson (1989) pointed out, characters, such as those based on the seed, fruit, and pedicel, that traditionally are proven useful in distinguishing related species, may not exhibit adequate variation for the discrimination at the infraspecific level. Other characters may need to be examined, such as the shape of the anther, trichomes on the leaf blade and stem, and shape of the hypanthium and sepal lobes. At vegetable stands and markets, various fruit types often carry familiar designations. Obviously, these designations of market types are useful for communicating and classifying the diversity of regional cultivars. In the United States, for example, Butternut is a familiar market type and Winter Crookneck and Cheese Pump- kin are popular heirloom types. A new market type, Calabaza Pumpkin (also called Caribbean Pumpkin, West Indian Pumpkin, or Cuban Pumpkin), is becoming popular and receiving the attention and efforts of American plant breeders.

References

Alefeld, F. 1866. Landwirthschaftliche Flora. Wiegandt and Hempel, Berlin. Bukasov, S.M. 1930. The cultivated plants of Mexico, Guatemala and Colombia. Trudy Prikl. Bot., Priloz. (Suppl.), 47: 301-310. Castetter, E.F. 1925. Horticultural groups of cucurbits. Proc. Amer. Soc. Hort. Sci., 22: 338-340. Filov, A.I. 1966. Ekologiya i klassifikatsiya tykvy. Byull. Glavn. Bot. Sada, 63: 33-41. Fursa, T.B. and Filov, A.I. 1982. Cucurbitaceae. In: Korovina, O.N. and Fursa, T.B. (Eds.), Flora of Cultivated Plants. Kolos, Moscow.

111 Grebenscikov, I. 1969. Notulae cucurbitologicae VII. Unterteilung von Cucurbita moschata Duch. ex Poir. (English summary: Subdivision of Cucurbita moschata Duch. ex Poir.). Kulturpflan- ze, 17: 109-120. Jeffrey, C. 2001. Cucurbita. In: Hanelt, P. and Institute of Plant Genetics and Crop Plant Research (Ed.), Mansfelds Encyclopedia of Agricultural and Horticultural Crops (Except Ornamentals). Springer, Berlin, pp. 1541-1552. Merrick, L.C. and Bates, D.M. 1989. Classification and nomenclature of Cucurbita argyrosperma. Baileya, 23: 94-102. Naudin, C. 1856. Nouvelles recherches sur les caractères spécifiques et les variétés des plantes du genre Cucurbita. Ann. Sci. Nat., Bot., ser. 4, 6: 5-73. Pangalo, K.I. 1930. A new species of cultivated pumpkins. Bull. Appl. Bot. Genet. Plant Breed., 23: 253-265. Paris, H.S. 1986. A proposed subspecific classification for Cucurbita pepo. Phytologia, 61: 133- 138. Paris, H.S. 1989. Historical records, origins, and development of the edible cultivar groups of Cucurbita pepo (Cucurbitaceae). Econ. Bot., 43: 423-443. Paris, H.S. 2000. First two publications by Duchesne of Cucurbita moschata (Cucurbitaceae). Taxon, 49: 305-319. Paris, H.S. 2001. History of the cultivar-groups of Cucurbita pepo. In: Janick, J. (Ed.), Horticul- tural Reviews, Vol. 25. Wiley, New York, pp. 71-170, 4 pl. Wessel-Beaver, L. 2000. Cucurbita argyrosperma sets fruit in fields where C. moschata is the only pollen source. Cucurbit Genet. Coop. Rep., 23: 62-63. Whealy, K. 1999. Garden Seed Inventory: An Inventory of Seed Catalogs Listing All Non-hybrid Vegetable Seeds Available in the United States and Canada, 5th ed. Seed Savers Exchange, Decorah, Iowa. Whitaker, T.W. and Davis, G.N. 1962. Cucurbits: Botany, Cultivation, and Utilization. Interscience, New York. Wilson, H.D. 1989. Discordant patterns of allozyme and morphological variation in Mexican Cucurbita. Syst. Bot., 14: 612-623. Wilson, H.D. 1990. Gene flow in squash species: domesticated Cucurbita species may not represent closed genetic systems. BioScience, 40: 449-455. Zhiteneva, N.E. 1930a. The worlds assortment of pumpkins. Bull. Appl. Bot. Genet. Plant Bre- ed., 23: 157-207. Zhiteneva, N.E. 1930b. Cucurbits of the northern part of tropical America (according to the samples collected by S. Bukasov). Supplementary article In: Bukasov, S.M. The cultivated plants of Mexico, Guatemala and Colombia. Trudy Prikl. Bot., Priloz. (Suppl.), 47: 311-331.

112 Diversity in tropical pumpkin (Cucurbita moschata): cultivar origin and history

T.C. Andres The Cucurbit Network, 5440 Netherland Ave., D24, Bronx, New York, USA; e-mail: [email protected]

Summary

Of the three main cultivated species of squash and pumpkin, Cucurbita moschata Duchesne is the least studied. This is, in part, due to its having few important cultivars outside of the tropics and subtropics, with the notable exception of the butternut squash and cultivars used for commercial canning. It is, however, the most widely cultivated Cucurbita in the tropics and has high nutritional value. But worldwide production statistics do not reflect the importance of C. moschata as a crop since it is primarily grown on a small-scale basis for local consumption where production data are not tabulated. There is, however, a growing recognition of the crops importance in the tropics for the export market, especially as improved cultivars are being developed for large-scale production. The greatest diversity lies in the Neotropics where the vines are grown under a wide range of ecological conditions, including under hotter conditions than are tolerated by the other cultivated Cucurbita species. Therefore, the English name for the species, tropical pumpkin, is an appropriate one. Wild forms of the species have not been described, but primitive-appearing landraces are known from Central America to northern Peru. This, coupled with the archaeological evidence, suggests that the center of origin is in northwestern South America.

Keywords: squash, pumpkin, domestication, landraces

Introduction

Cucurbita moschata Duchesne is one of the three main cultivated species of Cucurbita, C. pepo L. and C. maxima Duchesne being the other two. Unlike the other two species, C. moschata has not yet been recorded as growing wild. Although generally considered to be less polymorphic than the other two species, it has been less well studied and there are few descriptions of its diversity over the southern half of its traditional distribution, which includes the northern half of South America. At the time of European contact, it was being cultivated over a wider range of the Americas than any other species in the genus, even wider than that of C. ficifolia Bouché, which also extended into both North and South America. The purpose of this paper is to review the archaeological record, history, and economic importance of C. moschata, especially concerning its center of domestication. Tropical pumpkin is a most appropriate English-language name for C. moschata, given that the center of diversity of the species lies in the tropics rather than in the temperate zone as in the other two important pumpkin species. All three species are commonly referred to simply as pumpkin or squash, so those names do not help distinguish between them. Some C. moschata are called winter squash, mea- ning that the fruit is consumed when mature, but immature fruits of some C. moschata are eaten like summer squash, and the other two species also contain winter squash.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 113 The Algonkian word cushaw is still used today in parts of southern United States for both C. moschata and C. argyrosperma C. Huber. In Australia, C. moschata fruits are called gramma. Further south in the traditional range of C. moschata, other names used are ayote in Guatemala to Costa Rica, auyama or ahuyama in Panama, Colombia, Venezuela, and Dominican Republic, jiroumon in Haiti, abóbora and geremú or jerimú or jerimum in Brazil, lacayote in Peru, joko in Bolivia, and anco in Argentina. In Mexico and much of the Caribbean and elsewhere, the more generic Spanish name calabaza is used to refer to all squash species. Likewi- se, in Colombia, Ecuador, and Peru the generic word zapallo or sapayo is used, as is calabacín in Argentina. In Bolivia, zapallo refers specifically to C. maxima. Where Náhuatl is spoken in Mexico, tamalayota is used to mean C. moschata.

Pre-Columbian

Unfortunately, the hot, humid climate of much of the range where C. moschata is typically grown is not conducive to preservation of archaeological macro remains and thus records are incomplete. Seeds of C. moschata dating to 4000 B.C.E. have been found in southern Mexico (Flannery, 1973). While the archaeological record in Me- xico predates that of the dry coast of Ecuador and Peru, there is greater abundance of early archaeological seeds, rinds, and pedicels in the latter. The arrival of C. moschata in Peru preceded that of maize (Zea mays L.). In Ecuador, C. moschata may have displaced an incipient domesticate, C. ecuadorensis H. C. Cutler & Whitaker, in pre- historic times (Andres and Robinson, 2002). Parodi (1966) stated that C. moschata must have been cultivated in ancient northwest Argentina, but this has been little studied. By 200700 C.E., Moche pottery from northern Peru include casted C. moschata fruits, an indication that these were highly valued. In relatively recent pre-Columbian times, C. moschata was grown in eastern and southwestern United States.

European contact

On December 3, 1492, Columbus saw near the east end of Cuba fields planted with a rich assortment of crop plants, including calabazas, that he described as a glori- ous sight (que era gloria vella). The calabazas must have been C. moschata since that is the only species of Cucurbita grown in the region. Other early reports of Eu- ropean explorers observing what was likely C. moschata in the New World include Oviedo in 1526 observing on Hispaniola melones, pepinos, and calabaças that Indians commonly cultivated in their gardens. Cabeça de Vaca encountered pumpkins in July 1528 near Tampa Bay, Florida. The earliest known illustration of such an encounter is shown in a manuscript on Francis Drakes voyages to the Caribbean between 1577 and 1587 by an unknown accompanying artist/seaman. There is a watercolor of fruits on a vine that while not readily identifiable to species, given their location, they must be C. moschata fruits. Given these encounters, C. moschata must have been brought to Europe at least as early as C. pepo. Yet, it did not appear until later and never as abundantly. For exam-

114 ple, in the herbal by Fuchs (1542) and his subsequent unpublished manuscript (Me- yer, 1999), a number of detailed and accurate drawings of fruits show different cultivars of C. pepo but no C. moschata. Other herbals have illustrations that are too cru- de to be identifiable to species. The first herbal with a readily identifiable illustration of C. moschata is that of van Rheede tot Draakestein (1688), but this is over 100 years later and the drawing was from India and not Europe. Therefore, it was first assumed that C. moschata came from India. Later, it was thought to have come to Europe from the Americas indirectly via India (Whitaker, 1947). But the Italian botanist Matthioli (1560) noted that various cucurbits came to Italy from the West Indies with fruits lasting well into winter and having a sweetish taste. A plausible explanation for the comparatively late recording of C. moschata in Europe, even later than that of the South American species C. maxima, is that the first introductions came from the Caribbean region; these were short-day, late-maturing plants that failed to produce fruit in Euro- pe. Less ambiguous than the illustrations in the herbals are the highly detailed still- life Renaissance paintings of Dutch and Flemish artists. The first paintings showing C. pepo were drawn soon after contact with the New World, but nearly 50 years passed before Lucas Van Valkenborch, Jan Anton van der Baren, and other artists depic- ted an occasional C. moschata fruit among a mixture of other cucurbit species. The cultivar that first appeared in these paintings resembled the Cheese pumpkin, a popular heirloom first reported in the eastern United States. This cultivar would grow better further north in Europe than would cultigens from the Caribbean. Cucurbita moschata was named and first described in detail by Duchesne in 1786 (Paris, 2000a). Out of 364 beautifully realistic watercolor plates by Duchesne in France, only three show mature fruits of C. moschata, most of the rest being C. pepo. One painting resembles the Cheese pumpkin, while the other two resemble Caribbean- like calabazas (Paris, 2000b).

Today

By the end of the 19th century, C. moschata had spread worldwide. It is the predominant Cucurbita species in lowland tropical areas, and is grown to a lesser extent in temperate regions. The altitudinal range for the species is not limited, however, to the lowlands, but reach up to 2631 m in Colombia (Zhiteneva, 1930). It is more tolerant of heat, insolation, and humidity than any other domesticated species of Cucur- bita. Once plants are established, they are able to withstand not just wet conditions, but dry conditions as well (Ibrahim et al., 1996). The fruits of C. moschata are often covered with a waxy bloom like that of the wax gourd, Benincasa hispida (Thunb.) Cogn., which imparts protection from the intense tropical sun and good keeping quality. There are both, short-season (34-month-long) and long-season (67-month-long) cultigens (Lira Saade, 1995). Tropical pumpkins are consumed primarily near to where they are grown on small family farms under low-input agricultural systems, not exceeding a few hectares in size. These are usually landraces, i.e. locally adapted, traditional cultivars, rather than commercial cultivars. This primary use of the species is not included in national pro-

115 duction statistics and therefore its economic value is under estimated. Production statistics do not often distinguish between this species and the other species of squash and pumpkin. Cucurbita moschata is reported to be one of the most important vegetables in most of tropical Africa (Gwanama and Nichterlein, 1995) and Saudi Arabia (Alsa- don et al., 1998). In Puerto Rico, it is the most important non-root vegetable in amount consumed (Alamo, 1990). In Jamaica and Haiti, tropical pumpkin is the principal ingredient of pumpkin soup, a national dish of both countries. Nutritionally, tropical pumpkin plays an important role in local diets. The fruits, though variable, are usually high in carotenoids, particularly in the deeper orange- fleshed cultivars, and therefore provide an important source of vitamin A. Even the immature fruits tend to be better sources of vitamin A than C. pepo summer squash (Holmes et al., 1945). As tropical pumpkin plants are naturally pest-resistant and tolerant of poor, tropical soils, they are an environmentally friendly crop. Due to their success under marginal conditions, tropical pumpkins are being tested as a rootstock for melons (Tra- ka-Mavrona et al., 2000). There is enormous genetic diversity that is still largely untapped by scientific breeders. Since C. moschata is usually grown in fields with low-input agriculture in the poorer regions of developing countries, the fruits generally have a low value in public markets. One exception to this is a group of landraces in northern Peru called Loche. These fruits with typically warty rinds have a deep orange flesh that is highly estee- med locally. The fruits are relatively expensive and are used in small amounts as a flavoring for stews. There is a growing export market for C. moschata in Mexico, several Central and South American countries, as well as from the West Indies and southern Florida. Modern cultivars are being bred with improved productivity, flesh characteristics, and shipping quality. These are being grown under more intensive agriculture in larger field plots. In temperate regions, there are relatively few well-known cultivars of C. moschata. The most familiar are the butternut-type winter squash that are grown primarily for consumption of their high quality flesh. Cucurbita moschata is also the main species used in the pumpkin canning industry.

Origin Whitaker and Bemis (1964) proposed that C. moschata was first domesticated in Mexico. Later, they (Whitaker and Bemis, 1975) believed that the center of origin was in southern Mexico with subsequent domestications both northward throughout Mexico and secondarily into South America. Two independent domestication events in Mexico and Colombia were also proposed (Mangelsdorf et al., 1964). Pickersgill and Heiser (1977) suggested that there was a southern Mexico/Guatemala origin, with an early spread to South America. As we have gained a better understanding of the tropical pumpkin over the last 40 years, the proposed center of its origin of domestication has moved southward from Mexico to northern South America. Based on undocumented reports of a wild gourd

116 in northern Colombia where there is suitable habitat for a wild ancestor of C. moschata to exist, Nee (1990) hypothesized that this is where to look. Primitive-appearing fruits that are small with lignified rinds and poor flesh quality occur in Colom- bia (Wessel-Beaver, 2000). Colombia offers the most likely location for where tropical pumpkin was originally domesticated, but proof that the center of origin is in Colombia awaits the finding of the wild progenitor. The great diversity of landraces in Colombia has only begun to be described (Wessel- Beaver, 2000). Dark brown-seeded forms occur in Colombia and its neighboring countries, but not elsewhere. These are short-day plants and therefore cannot be grown at higher latitudes, such as at Geneva, New York (Andres and Robinson, unpublished). In Me- xico, only forms having light tan-colored seeds are grown. But it is not known how common the light-colored seeds are in Colombia. South of Colombia, the tan-colored seeds appear to be more common but this also has not been adequately studied. If there were two independent domestication events in Mexico and Colombia, one might expect the brown-seeded form to be more common in Peru and Bolivia. There are no distinct gaps in the morphology of the landraces, suggesting that there was only one major ancient domestication event rather than two. Secondary centers of domestication have occurred relatively recently in tropical Asia, Africa and the Mediterra- nean Basin, and the Caribbean and southern parts of North America. Molecular-marker technology may be helpful in defining infraspecific relationships, including cultivar-groups. Using random amplified polymorphic DNA (RAPD) markers, Gwanama et al. (2000) detected significant genetic variation among C. moschata landraces from south-central Africa, which constitute just a small sample from outside the center of diversity of the species.

References

Alamo, C.I. 1990. Hortalizas. In: Antoni, M., Cortes, M., Gonzales, G.M. and Velez, S. (Eds.), Situacion y Perspectivas: Empresas Agricolas de Puerto Rico en 1987-88. Estacion Experi- mental Agricola, Univ. Puerto Rico, Mayaguez, pp. 4-13. Alsadon, A.A., Hegazi, H.H. and Almousa, I.A. 1998. Evaluation of local pumpkin genotypes in the central region of Saudi Arabia. In: McCreight, J.D. (Ed.), Cucurbitaceae 98: Evaluation and Enhancement of Cucurbit Germplasm. A.S.H.S. Press, Alexandria, Virginia, pp. 43-50. Andres, T.C. and Robinson, R.W. 2002. Cucurbita ecuadorensis, an ancient semi-domesticate with multiple disease resistance and tolerance to some adverse growing conditions. In: Maynard, D.N. (Ed.), Cucurbitaceae 2002. A.S.H.S. Press, Alexandria, Virginia, pp. 95-99. Flannery, K.V. 1973. The origins of agriculture. Ann. Rev. Anthropol. 2: 271-310. Fuchs, L. 1542. De Historia Stirpium. Basle. Gwanama, C. and Nichterlein, K. 1995. Importance of cucurbits to small-scale farmers in Za- mbia. Zamb. J. Agric. Sci., 5: 5-9. Gwanama, C., Labuschagne, M.T. and Botha, A.M. 2000. Analysis of genetic variation in Cucurbi- ta moschata by random amplified polymorphic DNA (RAPD) markers. Euphytica, 113: 19-24. Holmes, A.D., Spelman, A.F. and Jones, C.P. 1945. Ascorbic acid, carotene, chlorophyll, ribof- lavin, and water content of summer squashes. Food Res., 10: 489. Ibrahim, A.M., Al-Suliman, A.I. and Al-Zeir, K.A. 1996. Hamdan and Qasim desert-adapted winter squashes. HortScience, 31: 889-890. Lira Saade, R. 1995. Estudios Taxonómicos y Ecogeográficos de las Cucurbitaceae Latinoameri- canas de Importancia Económica. Instituto de Biología, UNAM, Mexico City; IPGRI, Rome. Mangelsdorf, P.C., MacNeish, R.S. and Willey, G.R. 1964. Origins of Agriculture in Middle America.

117 In: West, R.C. (Ed.), Handbook of Middle American Indians, vol. 1, Natural Environment and Early Cultures. Univ. Texas Press, Austin, pp. 427-445. Matthioli, P.A. 1560. Commentarii Secundo Aucti, in Libros Sex Pedacii Dioscoridis Anazarbei de Medica Materia, Venice, Italy. Meyer, F.G. 1999. The Great Herbal of Leonhart Fuchs: De Historia Stirpium Commentarii Insignes, 1542 (Notable Commentaries on the History of Plants). Stanford University Press, Stanford, California Nee, M. 1990. The domestication of Cucurbita (Cucurbitaceae). Econ. Bot., 44: 56-68. Paris, H.S. 2000a. First two publications by Duchesne of Cucurbita moschata (Cucurbitaceae). Taxon, 49: 305-319. Paris, H.S. 2000b. The cucurbit legacy of Antoine Nicolas Duchesne (17471827). In: Katzir, N. and Paris, H.S. (Eds.), Proceedings of Cucurbitaceae 2000. Acta Hort., 510: 89-94. Parodi, L.R. 1966. La Agricultura Aborigen Argentina. Univ. Buenos Aires, Buenos Aires. Pickersgill, B. and Heiser, C.B. 1977. Origins and distribution of plants domesticated in the New World tropics. In: Reed, C.A. (Ed.), Origins of Agriculture. Mouton, The Hague, pp. 803-835. Traka-Mavrona, E., Koutsika-Sotiriou, M. and Pritsa, T. 2000. Response of squash (Cucurbita spp.) as rootstock for melon (Cucumis melo L.). Sci. Hort., 83: 353-362. Van Rheede tot Draakestein, H. A. 1688. Horti Malabarici. Amsterdam. Wessel-Beaver, L. 2000. Evidence for the center of diversity of Cucurbita moschata in Colombia. Cucurbit Genet. Coop. Rep., 23: 54-55. Whitaker, T.W. 1947. American origin of the cultivated cucurbits. I. Evidence from the herbals. II. Survey of old and recent botanical evidence. Ann. Missouri Bot. Gard., 34: 101-111. Whitaker, T.W. and Bemis, W.P. 1964. Evolution in the genus Cucurbita. Evolution, 18: 553-559. Whitaker, T.W. and Bemis, W.P. 1975. Origin and evolution of the cultivated Cucurbita. Bull. Torrey Bot. Club, 102: 362-368. Zhiteneva, N.E. 1930. Cucurbits of the northern part of tropical America. (According to the samples collected by S. Bukasov.) Supplementary article in: The cultivated plants of Mexico, Gua- temala and Colombia, by S.M. Bukasov. Trudy Prikl. Bot., Priloz (Suppl.), 47: 311-331.

118 Morphological variation of cultivated Cucurbita species

E. Køístková1, A. Køístková2 and V. Vinter3 1Research Institute of Crop Production, Division of Genetics and Plant Breeding, Department of Gene Bank, Workplace Olomouc, lechtitelù 11, 783 71 OlomoucHolice, Czech Republic; e-mail: [email protected] 2Secondary School, Tomkova 45, 779 00 OlomoucHejèín, Czech Republic 3Palacký University in Olomouc, Faculty of Science, Department of Botany, lechti- telù 11, 783 71 OlomoucHolice, Czech Republic; e-mail: [email protected]

Summary

Plant morphology was studied in five cultivated Cucurbita species: C. argyrosperma, C. ficifolia, C. maxima, C. moschata and C. pepo. A total of 51 characteristics of leaves, flowers, fruits and seeds were described and documented by photographs, scanning and herbarization. The most important characteristics for distinguishing species are located on male flowers (shape of the calyx, shape and indumentation on column), female flowers (colour of stigma), seeds (seed shape and colour, seed margin shape and colour, shape at funicular part), peduncles (shape) and leaf lamina (shape of basal sinus). Other features such as the leaf shape in outline, the silver mottling on the leaves and the fruit shape vary considerably even within particular species. Such characteristics are valuable for the determination of the extent of infraspecific morphological variation. The assessment of C. pepo and C. maxima accessions in the collection of genetic resources highlighted the discrepancies between some unusual morphological features and species classification of some accessions (such as C. maxima PI 175698). Assignment of accessions to species should be done after assessment of several plant characteristics at different developmental stages. The results gained in this work will be useful for the creation of the national and international descriptor lists for the genetic resources of the genus Cucurbita.

Keywords: Cucurbitaceae, Cucurbita argyrosperma, Cucurbita ficifolia, Cucurbita maxima, Cucurbita moschata, Cucurbita pepo, plant morphology, genetic resources, descriptors

Introduction

The genus Cucurbita (Cucurbitaceae) is native to the Americas (Whitaker and Davis, 1962) and consists of 15 or fewer species (Merrick, 1995; Nee, 1990). Five of the species are cultivated, with worldwide or regional economic importance. These are C. pepo L., C. maxima Duchesne, C. moschata Duchesne, C. argyrosperma Huber, and C. ficifolia Bouché. Cultivated Cucurbita species are monoecious, have long trailing vines and a pro- strate growth. Some C. pepo and C. maxima have short internodes and a bushy growth habit. Their rapidly established root system is horizontally extensive, although relatively shallow. Flowers are bright yellow to yellow-orange, usually borne singly in leaf axils, and open for one day. Some Cucurbita are day neutral, others have photoperiod sensitivity. The fruits are botanically fleshy berries and vary in size, shape and colour. The fruit flesh varies in texture, colour and nutritional composition. Seeds are ellip- tically flattened, seed coat colours vary from white to beige, tan, orange, brown and black (Whitaker and Davis, 1962). Some C. pepo cultivars produce seeds with a very

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 119 thin, nearly transparent seed coat. This feature detected as a spontaneous mutation facilitates seed consumption and oil extraction (Frimmel, 1957). All species are diploid and have the same chromosome number 2n = 40 (Singh, 1990). Despite the existence of much genetic differentiation within Cucurbita, none of the cultivated species in the genus is completely reproductively isolated from all of the others in terms of barriers to artificial hybridization. C. moschata is considered to be the extant species with the most ancestral-like genome because of its wide cross- compatibility (Merrick, 1995). Introgression can result from natural as well as artificial crossing (Decker-Walters et al., 1990; Køístková, 1991; Merrick, 1990; Rubatzky and Yamaguchi, 1995) and can cause complications in distinguishing species and determining taxonomic range. Similarly, the interpretation of scientific results, such as plant-pathogen interactions should be based on exact determination and morphological description of plant material. Genetic resources of the genus Cucurbita in the Czech Republic are maintained by the Research Institute of Crop Production (RICP) in PrahaRuzynì, Gene Bank at Olomouc. Cucurbita is represented in the collection by nearly 600 accessions of cultivated and wild species (Køístková, 2002). Passport data on accessions are available on the web site http://www.vurv.cz (part databases, EVIGEZ). International cooperation of European gene banks is promoted by the IPGRI (In- ternational Plant Genetic Resources Institute) within the framework of the European Cooperation Program on Plant Genetic Resources (ECP/GR) and its Working Groups. The most important tasks of the Cucurbitaceae Working Group were formulated at its informal meeting in Adana (Turkey) in 2002 (Díez et al., 2002). The creation of descriptors, that is systems of description data, has fundamental importance. It improves information on genotypes and enables its utilization. International descriptors for the Cucur- bita genetic resources have not been developed yet. The Czech national descriptor list will be finalized in 2004. The descriptor list should provide a tool for species discrimination and for the description of infraspecific variation. The purpose of this study was to observe morphological characteristics of the five cultivated Cucurbita species, to compare our observations with information in the available scientific literature and to prepare a set of the most important features for the creation of the national and international descriptor lists for genetic resources of cultivated Cucurbita species.

Material and methods

A basic set of 11 genotypes representing the five cultivated Cucurbita species (Table 1) was morphologically characterized during the growing season of the 2002. The plants were grown in the fields of the Gene Bank RICP at OlomoucHolice. Seeds were sown in the beginning of May. The distance between rows was 5 m; the distance within rows was 0.5 m for genotypes with a bushy growth habit and 1 m for those with viney growth habit. Each genotype was represented by ten plants in one replication. Plants were grown using standard cultivation practices, no chemical protection was used.

120 Table 1. List of Cucurbita spp. genotypes

Taxon Name Donor, seed company State of origin EVIGEZ number**

C. maxima Goliá Seva Flora, Valtice Czech Republic 09 H42 00137 C. ficifolia - Petr Kohout, Smrice Germany 09 H42 00616 C. argyrosperma Chicayote Adav PGR Unit Griffin, Mexico 09 H42 00790 (Fima) Georgia, USA C. moschata Butternut SEMO, Smrice unknown - C. pepo VM* Ghada F1 Royal Sluis The Netherlands - C. pepo ZU* Black Beauty Tézier France - C. pepo SC* Patina SEMO, Smrice Czech Republic - C. pepo CN* Early Yellow Seneca Seeds USA - Crookneck C. pepo SN* Early Prolific Seneca Seeds USA - Straightneck C. pepo PU* Adam Eva Køístková, Czech Republic selection from the accession Gene Bank RICP 09 H42 - 00126 C. pepo OG* - SEMO, Smrice Czech Republic -

* C. pepo morphotypes according to Paris (1989): VM=vegetable marrow, ZU= zucchini, SC=scallop, CN=crookneck, SN=straightneck, PU=pumpkin, OG=ornamental gourd ** accession number in the Czech National Germplasm Database EVIGEZ http://www.vurv.cz (part databases, EVIGEZ)

During the vegetative period, plants of the basic set were characterized morphologically, photo-documented, scanned and herbarized. The expression of a character was evaluated visually and/or with a light microscope. The list of 51 characters evaluated is given in Table 2. The terminology of botanical morphology was based on Futák (1966), Dostál (1989), Slavíková (1997) and Sugden (1984). Obtained data were compared to available information in the scientific literature (Esquinas-Alcazar and Gulick, 1983; Lira Saade, 1995). In 2003 a set of 53 C. pepo and C. maxima accessions of genetic resources was regenerated and morphologically assessed for the most distinct features.

121 Table 2. List of morphological characters described for Cucurbita plants

Plant part Characteristics

Plant habit (1)* evaluated at time of the opening of the first female flowers and by the end of the vegetative growth Stem (3) shape on the cross-section, longitudinal ribs, indumentum Leaf (10) shape in outline, division of lamina, number of lobes, shape of apex of the terminal lobe, shape of lamina base, ratio of length of the base (distance between horizontal line and * in Fig. 1) and length of lamina (distance between leaf base * and apex in Fig. 1), shape of basal sinus (below the horizontal line in Fig. 1), lamina * Fig. 1 margin, mottling on lamina, venation Male flower (14) shape of the calyx, shape of calyx leaves (sepals), shape of corolla, division of corolla, shape of the apex of corolla leaves (petals), colour of corolla, height and colour of anthers, height of filaments, fusion of filaments, shape of column, indumentum of column, shape of disk, size of pollen grains Female flower (9) shape of calyx, shape of calyx leaves (sepals), shape of corolla, shape of the apex of corolla leaves (petals), colour of corolla, colour of stigma, division and shape of style, shape of staminodia Fruit (7) shape of peduncle, shape and size of fruit, surface of fruit, colour of fruit, flesh colour, flesh texture, flesh thickness Seed (7) seed shape, colour and size, seed length-to-width ratio, weight, shape and colour of the margin, shape of the funicular attachment

* number of characters evaluated

Results and discussion

The list of characters which enable distinguishing the five Cucurbita species is presented in the Table 3. While the majority of characteristics are valid at the species level, some of them, such as the seed coat and seed margin colours, are valid only for the studied genotypes. The longitudinal stem ribs enable to distinguish certain species. They are sharp on C. pepo, but obtuse-angular on C. maxima. Based on the shape of leaf lobes, it is possible to clearly recognize C. ficifolia. Leaf characteristics of the other four species express a high infraspecific variation. The most important leaf characteristics, not mentioned by Lira Saade (1995) are loca- lised at the base of the leaf lamina, i.e. the length of the base and the shape of the basal sinus. Our observations of these characteristics correspond to drawings published by Whitaker and Davis (1962). Several characteristics essential for distinguishing the species are located on the male flowers. The calyx of C. maxima is, in contrast to the other four species, obco- nical to acetabuliforme (dish-shaped), not compressed below the calyx lobes. While

122 the shape of C. maxima calyx lobes are generally filiform and not very well developed, it varies in the other four cultivated Cucurbita species from linear lanceolate, triangular, to spatulate or forked-shape on the apex. C. maxima also has a unique shape of the column; it is cylindrical and enlarged at the base, while in other four Cucurbita species it is conical. Indumentum on the column was typical for C. ficifolia and C. maxima in our observations, but it is not reported for C. maxima by Lira Saade (1995). The intense orange colour of the stigma is a unique feature of C. moschata; in other species the stigma is yellow. The relation of presence and/or absence of staminodia and pistilodia and their shapes are further studied. Our observations confirm that there is much variation for fruit shape and colour, as well as the texture and colour of the flesh, in C. pepo, C. maxima and to some extent in C. moschata (Lira Saade, 1995). On contrast, these features are relatively uniform in C. ficifolia. Its fruits are elongate to round with the green-and-white exte- rior colour and the flesh is white. The characteristics of the peduncles are generally considered as very suitable for species determination (Fig. 2a). The peduncle of C. argyrosperma is rigid, angular, very often enlarged by a cork and abruptly contracted at its attachment to the fruit. The C. ficifolia peduncle is rigid and angular with obtuse ribs, slightly getting wider at its attachment to the fruit; the ribs do not tend to extend towards the fruits. The peduncle of C. maxima is soft, non-angular, round to oval on the cross-section, enlarged by irregular cork stripes, generally not enlarged, or moderately enlarged at the attachment to the fruit. The peduncle of C. moschata is rigid, angular, with obtuse ribs, enlarged at the attachment to the fruit and then abruptly contracts; the ribs do not tend to extend towards the fruit. The peduncle of C. pepo is rigid, angular with acute ribs, trumpet-like enlarged at the attachment to the fruit; the ribs tend to extend towards the fruit. Morphological characters of seeds provide reliable information for species determination. Until now, seeds without a lignified seed coat are known only in the species C. pepo. Black and/or dark brown colour of the seed coat is typical for C. ficifolia; however the seed coat colour of some genotypes can be light brown and/or cream (Lira Saade, 1995). The seed coat colour of C. pepo, C. argyrosperma, C. maxima and C. moschata varies from white, greyish, to light brown or bronze (Lira Saade, 1995). For the purpose of this study the selected genotypes of these four species have white or light seed coat colour. This selection enables a better comparison of seed characters. The seed coat and seed margin colours mentioned in Table 3 are only for the genotypes included to this study. Differences in seed coat colour among C. pepo, C. argyrosperma, C. maxima and C. moschata were observed, but it is more important to distinguish the colour of the central part of the seed from the colour of the margin. The colour of the seed margin of C. argyrosperma and C. moschata is darker than in the central part of the seed and varies from light brown to brown, respectively. Seeds of C. maxima and C. pepo possess the same colour of the central parts as on the margins. Seed shape varies from lanceolate, to elliptic, ovoid and very broadly elliptic. This last feature was recorded in C. ficifolia seeds. The shape of funicular attachment is essential for the distinguishing of the two most often cultivated species, C. pepo and C. maxima. It is transver-

123 Figure 2a. Fruits and details of peduncles of five Cucurbita species.

124 (expression (expression of the feature typical for the species Cucurbita Basic morphological characters distinguishing cultivated species is written in italics) Table 3. Table * feature not observed in our study, added to the Table characteristics from Lira Saade (1995) to complete the list of the most important

125 sely tapered to the longitudinal seed axis in C. maxima, and obtusely truncate in C. pepo, including longitudinal seed axis angle of 90°. The line of the funicular part of seed can be also slightly deflected in C. argyrosperma and C. ficifolia. The combination of the features characterizing the texture of the seed margin enables the distinguishing of four species having a light-coloured seed coat. While the seed margin of C. maxima and C. pepo is obtuse, smooth and vertically not wavy, C. argyrosperma and C. moschata have an acute, fibrous and vertically wavy seed margin. In 2003, a set of 26 accessions of C. pepo and 27 accessions of C. maxima were regenerated and morphologically assessed for their most distinct features (data not shown). Among them the stem ribs and peduncle of C. maxima accession PI 175698 (Fig. 2b) did not correspond to the species description. The peduncle is dark green even at a stage of botanical maturity, not enlarged on the base, sharply angular and covered by numerous trichomes. As some other morphological characters of this accession (position of corolla leaves and seed features) corresponded to the description of C. maxima, its taxonomic ranking should be verified. Actually this accession is listed as C. maxima in the donor GRIN database (Dr. J. Wiersema, USDA/ARS, Belt- sville, E-mail from 7 January 2004).

Figure 2b. Peduncles of two C. maxima accessions, PI 169466 (with species-typical peduncle) and PI 175698.

The majority of results from recent observations correspond to reports in the literature. Other characters, like the shape of the leaf blade and indumentum of the column, should provide additional information on the variation among and within Cucurbita species. As stressed by Paris (Dr. H.S. Paris, reviewer of this paper, pers. communication), one of the most fundamental differentiating factors among species, the type of hairiness, or indumentation of the foliage, was pointed in 1786 by Duchesne. The spiculate hairiness is typical for C. pepo, stiffly hairs for C. maxima and softly hairs for C. moschata. This characteristics completed by exact terms of botanical morphology will be included to the descriptor list. The classification of accessions should be done after assessment of several plant characteristics at different developmental stages. In 2003, the set of Cucurbita genotypes was supplemented by one genotype of C. pepo subsp. fraterna, one genotype of C. pepo subsp. texana, 20 morphotypes of C.

126 pepo, three genotypes of C. moschata and 5 genotypes of C. argyrosperma. Data to be obtained from these genotypes will be used for the elaboration of descriptor lists.

Acknowledgements

The authors are obliged to Prof. Ale Lebeda for his friendly intellectual support and to Veronika Køístková for the translation of selected chapters from the monograph of Lira Saade from Spanish. We thank Dr. H.S. Paris for valuable comments and suggestions to this manuscript. We thank Mr. Losík and Mrs. Vyhnánková, Calábko- vá, Golová, Sklenáøová a Kocmánková for their technical assistance. The work on morphological assessment of Cucurbita genetic resources was supported by the Mi- nistry of Agriculture (Czech Republic) through Grant E-97/01-3160-0200 National programme of conservation and utilization of plant genetic resources and by the Ministry of Education (the Czech Republic) through the Grant 38/2004 Visual aids to practical exercises of the selected botanical subjects; anatomical atlas of vascular plants (Foundation of the development of universities). Morphological assessment of plants and elaboration of data have been performed within the framework of student Se- condary school research activity.

References

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127 Paris, H.S. 1989. Historical records, origins, and development of the edible cultivar groups of Cucurbita pepo (Cucurbitaceae). Econ. Bot., 43: 423-443. Rubatzky, V.E. and Yamaguchi, M. 1996. Cucumber, melons, watermelons, squash, and other cucurbits. In: World Vegetables. Principles, Production and Nutritive Values, Second Edition, Chapman & Hall, International Thompson Publishing, New York, pp. 577-639. Singh, A.K. 1990. Cytogenetics and evolution of the Cucurbitaceae. In: Bates, D.M., Robinson, R.W. and Jeffrey, C. (Eds.), Biology and Utilization of the Cucurbitaceae. Cornell University Press, Ithaca, New York, pp. 3-7. Slavíková, Z. 1997. Terminologický slovník (Terminological dictionary). In: Hejný, S. and Sla- vík, B. (Eds.), Kvìtena Èeské republiky I (druhé vydání), Academia Praha, pp. 130-153. (in Czech) Sugden, A. 1984. Longman illustrated dictionary of botany, the elements of plant science illustrated and defined. Longman, York Press, Beirut. Wiersema, J. 2004. Personal communication from the USDA/ARS, Service Systematic Botany and Mycology, Beltsville, USA, E-mail from 7 January 2004 ([email protected]) Whitaker, T.W. and Davis, G.N. 1962. Cucurbits. In. Polunin, N. (Ed.), World Crops Books. Leonard Hill, London, 249 pp.

128 Management, conservation and valorization on genetic resources of Cucumis melo and wild relatives

M.L. Gomez-Guillamón1, E. Moriones1, M.S. Luis-Arteaga2, V. Carnide3, A. Börner4, N. Sari5, K. Abak5 and J.M. Alvarez2 1Estación Experimental La Mayora, CSIC, 29750 Algarrobo, Málaga, Spain 2SIA-DGA, Apdo. 727, 50080-Zaragoza, Spain 3UTAD, Department of Genetics and Biotechnology, Apdo. 202, 5000-911 Vila Real, Portugal 4Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersle- ben, Germany 5Department of Horticulture, Faculty of Agronomy, Çukurova University, 01330 Ada- na, Turkey

Summary

A list of passport, description and second characterization data has been established after three years of running an EU project on genetic resources in Cucumis melo aimed to homogenize and harmonize melon collections in Europe. A total of 392 melon accessions and wild relatives have been characterized attending a previously established list which included fruit quality related characters and susceptibility/resistance responses to several pathogens. A preliminary core collection has been established. Several areas of high risk of genetic erosion has been explored in Turkey.

Keywords: melon, passport, description, second characterization, evaluation, resistance, susceptibility, core collection, prospection, genetic erosion

Introduction

There is not sufficient knowledge about the intraspecific variability in Cucumis melo for many characters related to fruit quality and disease resistance. Melon collections in Europe contain mainly landraces and cultivars of C. melo and some related wild species. There are important germplasm collections in different countries where seeds are stored in optimal conditions in order to maintain their viability. There is, however, very little information about the horticultural features of the accessions of those collections. Germplasm collections are then undervalued and useless since their evaluation costs much time, effort and money. A list including passport data and descriptors for primary and further characterization and evaluation was published by the IPGRI (Esquinas-Alcázar and Gullick, 1983). Although different research groups have tried to evaluate their material (Gómez-Guillamón et al., 1985; Hammer et al., 1986; Nuez et al., 1986) following that list, and some of the accessions have been used for different research programs (Gómez-Guillamón and Tores, 1989; Nuez at al. 1992; Gómez-Guillamón et al., 1994, 1998), description of the accessions in most of the collections is insufficient, even for characters involved in the primary characterization. Moreover, the available passport and characterization data used by each group

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 129 require to be compared among them to make the melon collections comprehensive and available. Collecting melon landraces and varieties in Spain, Portugal and Turkey have been an activity developed by several research groups through national programs, and the results of some of those collections have been published in different reports (Gómez- Guillamón et al., 1985; Nuez et al., 1986, 1988; Gómez-Guillamón et al., 1994, 1998). But there are still areas in Turkey where old melon cultivars and melon landraces have the risk to be lost soon. With that art status five European groups have received financial support from the EU for a three years project. The aim was to uniform and homogenize passport and characterization data from germplasm characterization collections through the collaboration of five germplasm holdings and research institutions in the EU: Experimen- tal Station La Mayora (EELM-CSIC), Servicio de Investigación Agraria (SIA-DGA) from Spain, Universidade Trás-os Montes e Alto Duoro (UTAD) from Portugal, Insti- tute of Plant Genetics and Crop Plant Research (IPK) from Germany and Çukurova University from Turkey. With this proposal a better knowledge of the existing melon variability was intended. Important characters related to fruit quality and fruit resistance have been evaluated and seed stocks have been regenerated making the European gene banks more useful to melon breeders. The main objectives in the project were: the establishment of a minimum list of descriptors to identify easily the accessions in any germplasm bank, the establishment of a minimum number of characteristics related with fruit quality and with the susceptibility/resistance to different diseases, identification of duplicates and gaps among the five collections and the establishment of a temptative core collection of melon.

Material and methods

A previous list of passport and primary descriptors (first characterization) and second characterization traits listed in the IPGRI recommendations was used. To confirm the suitability of the vegetative and fruit characters chosen as descriptors five melon accessions were selected to be evaluated every year: PMR-45 (American cantaloupe type), Casca de Carvalho, (Portuguese cultivar, fruits of elliptical shape, intermediate netting, green flesh), Doublon (cantaloupe Charentais type), Kirkagac (Turkish cultivar, with ovated fruits of wrinkled, yellow and spotted skin, white flesh), and Negro (Spanish tendral type). A total of 392 melon accessions and relatives were evaluated according to the chosen characters. At least 10 plants/accession were observed. A minimum of 10 fruits per accession was evaluated. The list of tested accessions together with their origins could be obtained from the web site www.eelm.csic.es. The regeneration and description of all the melon accessions were carried out under different environments depending on the research group and the most traditional way to grow melons in each area and country. In Spain, the characterization was carried out under polyethylene plastic houses, with gravelled soil and drip irrigation. In Portugal, half of the material was sown at Vairão (North littoral) and half at Mirandela (North

130 inner). Trials were carried out in open field. In Germany, melon plants were sown in a greenhouse and then transferred to beds covered with glass. In Turkey, trials were carried out in the open field, but a replication of the accessions was grown under plastic- house to accurate the regeneration of the accessions. The following pathogens were used in the evaluation carried out looking for resistance: Fusarium oxysporum f.sp. melonis (Fom); races 0, 1 and 2, Sphaerotheca fuliginea; races 1 and 2, Cucumber Mosaic Virus (CMV); Watermelon Mosaic Virus (WMV); Zuchini Yellow Mosaic Virus, (ZYMV); Papaya Ring Spot Virus (PRSV-W); Melon Necrotic Spot Virus (MNSV) and Cucumber Yellow Stunting Disorder (CYSDV). Methodology and specific isolates of the pathogens used for the artificial inoculations could be obtained from the web site www.eelm.csic.es. Several areas with high risk of melon genetic erosion were prospected in Turkey by the Çukurova University team.

Results and discussion

The melon collection lists from the five research groups together with their passport data were adapted to an Excel file format and a preliminary inventory of European melon collections was established. The inventory was composed by 1762 melon accessions most of them belonging to the species Cucumis melo. Several accessions of C. africanus, C. anguria, C. dipsaceus, C. ficifolius, C. metuliferus, C. figarei, C. meeusii, C. myriocarpus, C. prophetarum, C. sagitatus, C. zeihery, and Lagenaria siceraria were also included. The identification of the duplicates and gaps among those collections was also done. A total of 149 duplicates have been found among the different melon collections. Several gaps have also been detected, and the research groups informed the EU about the localization of the melon collections where those gaps could be found. The following descriptors for passport data and primary characterization together with the scale to record them have been consolidated.

Passport data

Accession data: Accession number, Donor name, Donor identification name, Scienti- fic name (Family, Species, Taxonomic variety/convariety), Cultivar local name, Ac- quisition year, Date of the latest regeneration, Accession size, Number of times accession regenerated. Collection data: Collectors number, Collecting Institute, Date of collection, Country of collection (or cultivar where variety bred), Province/State, Location of collection site.

Descriptors

Vegetative data Sex type: Monoecious, Gynomonoecious, Andromonoecious, Hermaphroditic, And- roecious, Gynoecious, Dioecious. Ovary pubescence: recorded when short or absent.

131 Fruit data Fruit shape: Flattened, Globular, Ovate, Elliptical, Other. Fruit ribs: Absent, Present. Predominant fruit skin colour at maturity: White, Green, Blue, Cream, Yellow, Orange, Red, Pink, Brown, Grey, Black, Other. Design produced by secondary skin colour: No secondary, Speckled, Spotted, Strip- ped, Streaked, Other (specify). Fruit skin texture: Smooth, Grainy, Finely wrinkled, Shallowly wavy, Netted, Warts, Spines. Fruit abscision: No abscision, Abscises. Blossom scar: Obscure, Intermediate, Conspicuous. Fruit length (cm) Fruit width (cm) Fruit weight (kg) Flesh colour: White, Green, Yellow, Orange. Flesh thickness (mm) at fruit diameter Soluble solids content (°Brix) Seed coat colour: White, Yellow, Brown, Other. Seed size: 100 seed weight.

Evaluation

Vegetative and fruit data Leaf pubescence: recorded when no hairs. Ease of peduncle separation from fruit at mature stage: Easy, Intermediate, Difficult. Fruit splitting: recorded when splitting. External aroma: Absent, Present. Writting: Absent, Slight, Intermediate, Strong. Netting: Absent, Sparse, Intermediate, Strong. Fruit skin thickness (mm at maximum fruit diameter) Cavity diameter (mm at maximum fruit diameter) Seed shape: Pineseed shape, Normal shape, Other.

Disease and pest susceptibility Natural conditions Every research group tested the accessions against any disease or pest that occurred during the trial under natural conditions of infection. The following scoring scale was used: 1 = No infection, 3 = Low susceptibility, 5 = Medium susceptibility, 7 = High susceptibility.

Artificial conditions Scoring was done using the following scale: r: resistant, s: susceptible, v: variability in the response. Regarding F. oxyosporum f.sp. melonis races 0, 1 and 2 some variability in the response of the accessions have been observed. Only the accession of C. melo, CUM- 334 coming from Tadshikistan has shown resistance to the races 0, 1 and 2. Resistan-

132 ce to the three races has also been found in the wild species C. metuliferus, C. africanus, C. zeyheri, C. anguria var. longipes. The Spanish accessions Maduro Amarillo, Amarillo Cáscara Pinta, Banda de Godoy, Amarillo manchado, the Russian Korça, the Italian Cucumarazzo and the Turkish accessions, ref. 4, 16 and 51, showed a resistant response when inoculated with races 0 and 2. One of the tested accessions of C. melo var. conomon showed resistance to races 0 and 1 but susceptibility to race 2 and the Japanese Shiroubi Okayama showed resistance to races 0 and 1 but had a heterogeneous response to race 2. C. myriocarpus, Melone Gialle from Italy, SE-2811-1C from Spain, Muchane- svi from Russia and CUM-355 from Irak were resistant to race 0 but susceptible or with heterogeneous response to races 1 and 2. The Turkish accession ref. 30 and the Spanish Hidalgo were resistant to race 1 and susceptible or with heterogeneous response to races 0 and 2. The Turkish accession ref. 60 and CUM-85 from Greece showed resistance to race 2, heterogeneous response to race 0 and susceptibility to race 1. The Spanish accessions Amarillo Alargado, Maduro Negro ANC-57 and Kreta from Greece, have shown heterogeneity in their responses whatever the race of the fungus was used. Seven more melon accessions showed heterogeneity when inoculated with races 0, 1 or 2. Variability for resistance to F. oxysporum f.sp. melonis races 0, 1, and 2 was found within the tested accessions, showing, in most of the cases, lack of uniformity of the material that may be due to a lack of proper isolation of the accessions during its multiplication. A total of 149 accessions were tested against S. fuliginea races 1 and 2. When plants of the Spanish accessions ANC-29, ANC-57 and Negro de Ardales were inoculated with races 1 and 2 of S. fuliginea, a resistant response to both races was observed. The accessions, Negro de Zaragoza and ANC-42 were resistant to race 1 but susceptible to race 2. None of the evaluated accessions have shown any resistance to CMV, MNSV nor CYSDV. The resistance to these viruses seems to be very infrequent, at least within the materials that have been evaluated. The accessions Togo and Golden Champlain showed variability for resistance to ZYMV and WMV respectively that could be of interest in breeding. The Spanish accessions Amarillo manchado and Japonés showed variability when inoculated with PRSV. Summing up, when inoculated with the different viruses, none resistant response was observed in any new C. melo accessions. Variability to some of the tested viruses was found in several accessions which means that there is the possibility to find resistance in those accessions when selected to be used in breeding programs. A preliminary core collection was established. The list of the included accessions could be reached from the web site www.eelm.csic.es. Seeds of the core collection will be maintained by each research group and they will be in long term storage as safety duplicate at the IPK-Gatersleben. The areas prospected in Turkey during 2001, were Manisa, Balikesir, Cannakale, Adana, Sanliurfa, Diyarbakir, Mardin, Batman, Bitlis and Van. There, a total of 136 melon accessions were collected. In 2002, the areas prospected were Ankara, Edirne, Tekirdag and Balikesir, where 24 new accessions were accumulated. Besides of that during the year 2000, 23 genotypes were collected from Eastern and Central Anato-

133 lian Regions (provided by the University of Van), and 17 genotypes were provided from AARI (Aegean Agricultural Research Institute). With the prospections carried out by the Turkish team, 176 new melon accessions have been added to the collections. It is assumed that an important decreasing of the risk of genetic erosion in an important melon area, as Turkey is, have been done

Note: A complete information about the project, including all the results obtained could be reached from de web site www.eelm.csic.es.

Acknowledgements

Authors thank C. Soria, R. Camero, F. Sánchez, M. Crespillo, C. Cotilla, R. Gil (EELM-CSIC), R. González-Torres, C. Mallor (SIA-DGA), F. Miranda, E. Varandas (DRAEDM, Vairâo), M.R. Barroso (DRATM, Mirandella), B. Schmidt, K. Weisse, S. Eikmeier (IPK), and E. Ekbic (Çukurova University) for their worthly collaboration to make possible this work. This research was supported by the project GENRES CT98- 108.

References

Esquinas-Alcázar, J.T. and Gulick, P.J. 1983. Genetic resources of Cucurbitaceae. A.G.P.G.R., I.B.P.G.R., 83/84: 20, Rome, 101 pp. Gómez-Guillamón, M.L., Abadía, J., Cuartero, J., Cortés, C., and Nuez, F. 1985. Characterization of melon cultivars. Cucurbit Genet. Coop. Rep., 8: 39-40. Gómez-Guillamón, M.L., Moriones, E., Luis-Arteaga, M.S., Alvarez, J.M., Torés, J.A., López- Sesé, A.I., Cánovas, I., Sánchez, F. and Camero, R. 1998. Morphological and disease resistance evaluation in Cucumis melo and its wild relatives. Proceedings of Cucurbitaceae98, Eva- luation and Enhancement of Cucurbit Germplasm: Pacific Grove, California, U.S.A., pp. 53- 61. Gómez-Guillamón, M.L. and Torés, J.A. 1989. Resistance to Sphaerotheca fuliginea in Spanish muskmelon cultivars. Cucurbit Genet. Coop. Rep., 12: 39-40. Gómez-Guillamón, M.L., Torés, J.A., Soria, C. and López-Sesé, A.I. 1994. Screening for resistances to S. fuliginea and two yellowing diseases in C. melo and related Cucumis species. Proceedings of Cucurbitaceae94, Evaluation and Enhancement of Cucurbit Germplasm, South Padre Is- land, Texas, U.S.A., pp. 205-208. Hammer, K.P., Hanelt, P. and Perrino, P. 1986. Carosello and the taxonomie of Cucumis melo especially of its vegetable races. Kulturpflanze, 34: 249-259. Nuez, F., Anastasio, G., Cortés, C., Cuartero, J., Gómez-Guillamón, M.L., and Costa, J. 1986. Germplasm resources of Cucumis melo, L. from Spain. Cucurbit Genet. Coop. Rep., 9: 60-63. Nuez, F., Esteva, J., Soria, C. and Gómez-Guillamón, M.L. 1992. Search for sources of resistance to a whitefly-transmitted yellowing disease in melon. Cucurbit Genet. Coop. Rep., 14: 59-60. Nuez, F., Ferrando, C., Diez, M.J., Costa, J., Catalá, M.S., Cuartero, J. and Gómez-Guillamón, M.L. 1988. Collecting Cucumis melo in Spain. Cucurbit Genet. Coop. Rep., 11: 54-56.

134 Evaluation of Portuguese melon landraces conserved on farm by morphological traits and RAPDs

V. Carnide1, S. Martins1, F.J. Vences2, L.E. Sáenz de Miera2 and M.R. Barroso3 1Departamento de Genética e Biotecnologia, CGB/ICETA-UTAD, Universidade de Trás- os-Montes e Alto Douro, 5000-911 Vila Real, Portugal; e-mail: [email protected] 2Área de Genética, Depto. Ecología, Genética y Microbiología, Universidad de León, E-24071 León, Spain 3Centro Experimental da Terra Quente, Direcção Regional de Agricultura de Trás- os-Montes, 5370-087 Carvalhais MDL, Portugal

Summary

Morphological traits and RAPD markers were used to evaluate the genetic diversity among ten Portuguese landraces of melon. Morphological data were collected for 20 traits. Ten numerical agronomical traits were analysed by ANOVA and revealed statistically significant differences (P<0.05) between landraces. The total of these traits was used for principal component analysis and cluster analysis. A high concordance between these two analyses was observed with a clear separation of the landraces by geographic regions. The 30 primers used generated 388 amplified fragments with an average of 37.9 polymorphic fragments. The landraces from the Trás-os-Mon- tes region were shown to be less polymorphic than the ones from the Minho region. The cluster analysis from RAPD data did not reveal a clear relationship between genetic diversity and geographic origin of the landraces. In summary a great genetic diversity was registered among these landraces which can be used in the future in breeding programmes.

Keywords: Cucumis melo, genetic diversity, landraces, morphological traits, Portugal, RAPDs

Introduction Attempts to conserve and utilize plant diversity are as old as humankind itself (Maxted et al., 1997). The importance of in situ conservation was recognised in the 1980s and the Convention on Biological Diversity held in Rio de Janeiro in 1992 stresses the complementarity of in situ and ex situ conservation. In situ conservation is characterized by the possibility of combining it with the goals of nature conservation and agricultural production (Brockhaus and Oetmann, 1996). On-farm conservation is one form of in situ conservation, which involves the traditional crop varieties and the cropping systems by farmers. Traditionally, farmers do not buy seeds but keep a proportion of harvested seeds for resowing (Maxted et al., 1997). The local varieties/landraces, which are an example of the dynamic process of in situ conservation, are highly adapted to the local environment, to varying degrees of mass selection by farmers and to the uses of local populations. The new techniques employed in plant breeding programmes result in new variations but isozymes and molecular data indicate that wild relatives and landraces remain the main sources of genetic diversity of crop gene pools (Miller and Tanksley, 1990). The conservation of these sources is crucial because the replacement of local

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 135 varieties by improved or exotic cultivars or species, overexploitation, deforestation and land clearance, environmental effects, introduction of new pests and diseases, population pressure, urbanisation, economic policies and legislation are causing an erosion of genetic resources (FAO, 1998). The characterization of plant genetic resources is a necessity for plant breeders. Morphological, biochemical or molecular markers permit this characterization. Mor- phological characterization is the oldest method and it involves a description of variation, particularly agromorphological characteristics, of direct interest to users. However, this analysis has some limitations such as highly heritable traits, which often show little variation although traits expression is subject to environmental variation and may be difficult to measure (Karp et al., 1997). Biochemical techniques such as the electrophoresis of isozymes and protein (Hunter and Markert, 1957) and molecular techniques for the analysis of DNA polymorphism are complementary to morphological characterization (Karp et al., 1997). RAPD-PCR is a molecular technique that has been used to evaluate genetic diversity in several species of the Cucurbitaceae genera, namely in Cucumis melo (Baudracco- Arnas and Pitrat, 1996; Katzir et al., 1996; Garcia et al., 1998; Silberstein et al., 1999; Garcia-Mas et al., 2000). However, it has two main limitations: RAPDs are dominant genetic markers and have reproducibility problems (IPGRI, 1996). Genetic diversity reduction in traditional agrosystems, caused in part by moderni- zation of agricultural systems, in particular the introduction of new cultivars, is occurring in different parts of Portugal. In the North of the country there are several landraces of melon which have suffered great erosion mainly due to replacement by new cultivars. Given this the knowledge of genetic variability maintained in landraces could prove interesting. Therefore, ten landraces from North Portugal (Minho and Trás-os- Montes regions) were selected for on-farm conservation and for diversity evaluation.

Material and methods Ten melon landraces (Cucumis melo L.) from North Portugal plus a Turkish control (Kirkagaç) were analysed in this study (Table 1). Two groups of landraces were formed: one of six landraces from the Minho region and other of four landraces from the Trás-os-Montes region. A landrace was defined as a traditional variety unimpro- ved by modern methods of plant breeding.

Morphological characterization Each landrace and the control were represented by 15 plants and from each plant two fruits were characterized. Characterization was made according to IBPGR descriptors (Esquinas-Alcázar and Gulick, 1983) for twenty traits.

RAPD markers Total DNA from six plants of each landrace/control was isolated from fresh leaves according to Sandra et al. (unpubl.). Forty oligonucleotides primers from kits E and R (Operon Technologies) were purchased for RAPD-PCR amplifications, PCRs were conducted with a Biometra UNO II Ther-

136 mocycler using the following cycle profile: an initial denaturing step of 5min at 94°C; 9 cycles of 94°C/15sec, 33°C/45sec, 72°C/75sec; 35 cycles of 94°C/15sec, 37°C/45sec, 72°C/75sec with a final step of 7min at 72°C. The amplified fragments were scored as 1 for the presence and 0 for the absence of the fragment.

Statistical analysis For the purpose of statistical analysis, data relative to morphological traits analysis of variance was deduced. Principal component analysis and hierarchical clustering was done using the GENESTAT program. Similarity tree was produced by clustering the similarity data with the unweighted pair group method using the arithmetic average (UPGMA). For analysis of RAPD markers data genetic diversity statistics (HT, HS, DST, GST) (Nei, 1973) and its counterparts Kx values (Nei and Kumar, 2000) were used to estimate the genetic variability and the dendrogram was constructed by UPG- MA. All statistical procedures were carried out by the TULKAS software program developed by Sáenz de Miera (unpubl.).

Results

Morphological traits The ten landraces of melon came from two different regions of North Portugal - Minho and Trás-os-Montes - which have different climatic conditions. Minho has an Atlantic climate while the area of the Trás-os-Montes landraces (Vilariça valley) has a Mediterranean climate. The mean values for the eight fruit traits and for the duration of the vegetative period are presented in Table 1. For all traits, ANOVA shows significant differences between landraces at 0.001 level. For the fruit traits, the six landraces from Minho, in comparison with the four from Trás-os-Montes, showed the highest average for fruit width and weight, fruit skin and flesh thickness, while the Trás-os-Montes landraces had the highest soluble solids, 100 seeds weight and fruit length. For the other fruit traits and for duration of the vegetative period the average was similar between the two groups of landraces. The landrace M325 from Minho showed the highest average fruit width and weight and cavity diameter being statistically different (P<0.05) from all others landraces and the control Kirkagaç, and also had one of the higher average fruit length and flesh thickness. For this trait, it was not statistically different (P>0.05) only from the landrace M277, also from Minho, which had the highest flesh thickness. The lowest flesh thickness, and consequently the lower volume of flesh, was registered in the landrace M1, from the Trás-os-Montes region, which had also one of the lowest soluble solids content. For this trait the landrace from Trás-os-Montes M7 had the highest content, being statistically different (P<0.05) from all others landraces and from the control. The average duration of the vegetative period ranged between 131 days in the landrace M318, and 152 days in the landrace M368, both from the Minho region.

137 Mean of nine traits for ten melon landraces, one control, and assemble of landraces from the Minho * Table 1. Table and the Trás-os-Montes regions

138 To study the inter-relationships between all the accessions indicated by the 20 traits a principal component analysis was performed. The three principal components explain 89,98% of the whole variation. Duration of the vegetative period is the main trait correlated with the first principal component (0.9732). All the other traits have a low correlation with this component. The second component is mainly correlated with netting and writting (0.6089 and 0.5751, respectively). The most correlated trait to the third component is the fruit length (0.8387). Fig. 1 represents the projection of each landrace and the control onto the plan defined by components 1 and 2. The most obvious result is that all landraces from the Trás-os-Montes region and the control are grouped on the negative side of axis 2 and all landraces from the Minho region are grouped on the positive side of this axis. Hierarchical classification leads to the dendrogram presented in Fig. 2. Three main clusters can be identified. The first cluster includes the four landraces from the Trás- os-Montes region (M4, M17, M1 and M7) and the control Kirkagaç. In the second cluster we find two landraces from Minho and in the third cluster the remaining four landraces from the Minho region. In this group the landrace M251 is the least similar. The dendrogram and ordination of the landraces/control in the plan defined by the two principal components are consistent.

Figure 1. Projection of the 10 landraces Figure 2. Dendrogram of the 10 land- and the control onto the plan defined by races and the control on the basis of the principal components 1 and 2. the mean of the Euclidean distance for the 3 principal components.

RAPDs The landraces analysed by RAPD markers and morphological traits were the same. In order to discriminate the ten landraces of melon and the control, 40 random primers were screened and thirty were selected using the criterion of Lynch and Milli- gan (1994).

139 The number of landraces that each primer can identify was variable. The primers OPE 7, OPE 8, OPE 9, OPE 16, OPE 20, OPR 1, OPR 2, OPR 14, OPR 15 and OPR 16 can identify only one landrace by accession specific amplified fragments, but the primer OPE 1 can identify six landraces. On the other hand, six primers of the kit OPE and thirteen other primers from kit OPR cannot identify any landrace by specific amplified fragments. These primers generated 388 amplified fragments with an average of 9.7 amplified fragments/primer. This value was higher than the one observed by Garcia et al. (1998) of 5.4 amplified fragments. The average of amplified fragments in the set of the landraces/control was 153, the average number of amplified fragments for the landraces from Minho 154 being and for the Trás-os-Montes landraces 149 (Table 2). The average number of polymorphic amplified fragments was 37.91 (24.75%). For the Minho landraces this number was 38.83 while for the Trás-os-Montes landraces it was only 28.25. The control Kirkagaç had the highest number of polymorphic amplified fragments with 71 (18.3%) while the landrace M25 from the Minho region had the lowest number at 10 (2,6%).

Table 2. Total of amplified fragments, polymorphic and specific amplified fragments

Material Region Amplified Polymorphic Specific Nei-Kumar fragments amplified amplified diversity fragments fragments

M 4Trás-os-Montes 153.00 48.00 2.00 0.0773 M 17 Trás-os-Montes 170.00 16.00 13.00 0.0182 M 1 Trás-os-Montes 123.00 15.00 1.00 0.0245 M 7 Trás-os-Montes 150.00 34.00 1.00 0.0540 M 387 Minho 157.00 57.00 2.00 0.0823 M 368 Minho 160.00 45.00 3.00 0.0668 M 251 Minho 150.00 10.00 5.00 0.0161 M 277 Minho 153.00 54.00 1.00 0.0909 M 318 Minho 164.00 26.00 7.00 0.0318 M 325 Minho 141.00 41.00 2.00 0.0645 Kirkagaç Turkey 164.00 71.00 6.00 0.1159 Average 153.18 37.91 3.91 0.0584 Minho 154.16 38.83 3.340.0587 Trás-os-Montes 149.00 28.25 4.25 0.0435

The total genetic variability (T) was higher for the set landraces/control then for each one of the sets of landraces. The set of landraces from Trás-os-Montes showed the lowest intrapopulational variability (S) while the set of landraces from Minho presented the lowest interpopulational variability (ST). The average value at an intra- inter- populational level (GST) was higher for the set of Trás-os-Montes landraces but infe- rior to 0.90. Furthermore, the genetic variability shown by the control Kirkagaç was higher than the estimated in any of the ten Portuguese landraces (Table 3). Total genetic variability was similar in the two regions and the two components of inter- and intra-population level had a similar distribution. The inter-population component was

140 about five times higher than the intra-population component. This could be because the seeds analyzed belong to local varieties and traditionally every year the farmers keep a small sample of harvested seeds for resowing the following year.

Table 3. Comparison between H and Kx parameters at inter- and intra-landraces/ control level

Landraces/control Minho Trás-os-Montes

HT 0.235 0.199 0.208 HS 0.037 0.037 0.029 DST 0.198 0.162 0.179 GST 0.841 0.812 0.859 DST/HS 5.275 4.334 6.159 Kx T 0.389 0.325 0.337 Kx S 0.058 0.059 0.043 Kx ST 0.331 0.267 0.294 Kx ST/T 0.885 0.820 0.871 Kx ST/S 5.661 4.545 6.749

The cluster analysis divided the landraces/control into four main clusters (Fig. 3). The first cluster contains the landraces M1 and M7 from the Trás-os-Montes region and the landrace M251 from the Minho region. The three landraces included in the second cluster (M277, M325 and M387) are all from the Minho region. The third cluster contains the landrace M17 from the Trás-os-Montes region and the landraces M368 and M318 from the Minho region. The control Kirkagaç and the landrace M4 from Trás-os-Montes are in the fourth cluster. This analysis did not show a clear relationship between the geographic origin of the landraces and its distribution by the clusters. Additionally a Mantels test (Mantel, 1967) was carried out between the genetic distance (1-Qxy) matrix and the Euclidean distance matrix obtained previously from agronomical traits, and no correlation was found (r = 0.021, P = 0.342).

Figure 3. Dendrogram from Qxy similarity index.

141 From the above data we can conclude that the morphological and agronomical traits studied have been selected in an independent way for each one of the landraces analyzed. Additional studies with a high number of control accessions from different countries are desirable in order to compare the genetic variability maintained in the traditional Portuguese melon landraces.

Acknowledgements

This work was supported by project Agro I&D 149.

References

Baudracco-Arnas, S. and Pitrat, M. 1996. A map genetic with melon (Cucumis melo L.) with RFLP, RAPD, isozyme, disease resistance and morphological markers. Theor. Appl. Genet., 93: 57-64. Brockhaus, R. and Oetmann, A. 1996. Aspects of the documentation of in situ conservation measures of genetic resources. Plant Genet. Res. Newslet., 108: 1-16. Esquinas-Alcázar, J.T. and Gulick, P.J. 1983. Genetic resources of Cucurbitaceae. A.G.P.G.R., I.B.P.G.R., 83/84: 20, Rome, 101 pp. FAO, 1998. The state of the worlds plant genetic resources for food and agriculture. FAO, Rome. Garcia, E., Jamilena, M., Alvarez, J.I., Arnedo, T., Oliver, J.L. and Lozano, R. 1998. Genetic relationshpis among melon breeding lines revealed by RAPD markers and agronomic traits. Theor. Appl. Genet., 96: 878-885. Garcia-Mas, J., Oliver, M., Gómez-Paniagua, H. and de Vicente, M.C. 2000. Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon. Theor. Appl. Genet., 101: 860-864. Hunter, R.L. and Markert, C.L. 1957. Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels. Science, 125: 1294-1295. IPGRI, 1996. Measuring genetic variation using molecular markers unit 10-1-4. International Plant Genetic Resource Institute, Rome, Italy. Karp, A., Kresovich, S., Bhat, K.V., Ayad, W.G. and Hodgkin, T. 1997. Molecular tools in plant genetic resources conservation: a guide to the technologies. IPGRI Technical Bulletin no. 2. International Plant Genetic Resources Institute, Rome, Italy. Katzir, N., Danin-Poleg, T., Tzuri, G., Karchi, Z., Lavi, U. and Cregan, P.B. 1996. Length polymorphism and homologies of microsatellites in several Cucurbitaceae species. Theor. Appl. Genet., 93: 1282-1290. Lynch, M. and Milligan, B.G. 1994. The analysis of population genetic structure with RAPD markers. Mol. Ecol., 3: 91-99. Mantel, N. 1967. The detection of disease clustering and a generalized regression approach. Cancer Res., 27: 209-220. Maxted, N., Ford-Lloyd, B.V. and Hawkes, J.G. 1997. Complementary conservation strategies. In: Maxted, N., Ford-Lloyd, B.V. and Hawkes, J.G. (Eds.), Plant Genetic Conservation. Chap- man & Hall, London, pp. 15-39. Miller, J.C. and Tanksley, S.D. 1990. RFLP analysis of phylogenetic relationships and genetic variation in the genus Lycopersicon. Theor. Appl. Genet., 80: 437-448. Nei, M. 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci. USA, 70: 3321-3323. Nei, M. and Kumar, S. 2000. Molecular Evolution and Phylogenetics. Oxford University Press, Oxford, New York. Silberstein, L., Kovalski, I., Huang, R., Anagnostou, K., Jahn, M. and Perl-Treves, R. 1999. Molecular variation in melon (Cucumis melo L.) as revealed by RFLP and RAPD markers. Scientia Hort., 79: 101-111.

142 Comparative analysis of melon landraces from South Portugal using RAPD markers

M.R. Barroso1, S. Martins2, F.J. Vences3, L.E. Sáenz de Miera3 and V. Carnide2 1Centro Experimental da Terra Quente, Direcção Regional de Agricultura de Trás- os-Montes, 5370-087 Carvalhais MDL, Portugal; e-mail: mariarosario@min-agricultura.dratm.pt 2Departamento de Genética e Biotecnologia, CGB/ICETA-UTAD, Universidade de Trás- os-Montes e Alto Douro, 5000-911 Vila Real, Portugal 3Área de Genética, Depto. Ecología, Genética y Microbiología, Universidad de León, E-24071 León, Spain

Summary

Genetic diversity among nine melon landraces from South Portugal was evaluated by RAPD markers. A total of 371 bands were generated by the 30 primers used, amplifying each primer an average of 12.4 bands. These 371 bands revealed that eight of the nine landraces and the cultivar Doublon can be identified by the amplification of a single marker band. The Nei-Kumars total diversity was 0.351. When all plants were treated as a whole, the inter-populational variability (0.300) was the main component of the total variability. The dendrogram obtained by UPGMA method showed that Tendral landrace is the most genetically distant from all the others landraces and the control Doublon. Although a clear relationship between the geographic origin of the landraces and its distribution by clusters was not observed.

Keywords: Cucumis melo, germplasm, landraces, RAPDs

Introduction Melon is an economically important crop in Portugal, mainly in the Center and South of the country. Here, there are several local landraces which differ from modern cultivars, especially in their morphological and organoleptical characteristics. Fre- quently this type of under-utilized germplasm behaves as a reservoir of genetic variation, unlike modern crop varieties, where limited genetic variation makes them more susceptible to disease epidemics. Thus these landraces are important to ensure the retention of the existing genetic diversity and for identifying populations and/or accessions which contain important genes for the improvement of quantitative traits (Tanksley and Nelson, 1996). Methods of analysing variation in DNA sequences allow those involved in conservation and use of plant genetic resources to analyse the extent and distribution of genetic diversity at the DNA level and to use this information to make more effective conservation choices (Ayad et al., 1997). Several molecular markers, such as isozymes (Esquinas-Alcazar, 1977; Pearl-Treves et al., 1985; Staub et al., 1987, 1997; Meglic et al., 1994), RFLPs (Shattuck-Eidens et al., 1990; Neuhausen, 1992; Silberstein et al., 1999), SSR (Katzir et al., 1996; Staub et al., 2000; Danin-Poleg et al., 2001; López- Sesé et al., 2002 ), AFLPs (Garcia-Mas et al., 2000) have been used to characterize

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 143 relationships among melon accessions or cultivars. Also, there are many examples of the use of inherited RAPD loci in the assessment of genetic diversity in melon germplasm (Staub et al., 1997; Mo-Suk et al., 1998; Silberstein et al., 1999; Stepansky et al., 1999; Mliki et al., 2001; López-Sesé, et al., 2002, 2003). Because the assessment of genetic variability and its partitioning is an important matter in plant breeding, RAPD markers were used to estimate genetic diversity in a set of nine melon landraces from South Portugal.

Material and methods

Plant material and sample preparation Nine melon accessions from South Portugal were used to evaluate genetic diversity between and within these landraces. The French cultivar Doublon was used as control (Table 1). Plants were grown under identical greenhouse conditions.

Table 1. Melon (Cucumis melo L.) accessions assessed for diversity analysis

Accession Geographical Local Name RAPD banding morphotype1 No. origin P % A % V %

ACC03351 Faro (Castro Melão do Algarve 122.0 32.9 72.0 49.4 77.0 20.3 Marim) ACC03370 Faro (Castro Melão Casca 143.0 38.5 204.0 55.0 24.0 6.5 Marim) de Carvalho ACC05726 Faro (Olhão) Melão 131.0 35.3 209.0 56.431.0 8.4 ACC02494 Évora Melão Amarelo 165.0 44.5 176.0 47.4 30.0 8.1 (Redondo) ACC02495 Évora Melão Branco 148.0 39.9 181.0 48.8 42.0 11.3 (Redondo) ACC02501 Évora Melão de Casca 144.0 38.8 179.0 48.2 48.0 12.9 (Redondo) Preta ACC02496 Évora Melão Casca 167.0 45.0 177.0 47.7 27.0 7.3 (Redondo) de Carvalho ACC02435 Beja (Salvada) Melão 151.0 37.9 182.0 49.1 38.0 10.2 Campo Maior Tendral 107.0 28.8 225.0 60.3 39.0 10.5 France Doublon (control) 117.0 31.5 182.0 49.1 72.0 19.4 Average 139.5 37.6 188.7 46.4 42.8 11.5

1 values for the 371 RAPD bands analysed. (P) Present presence of marker band in all individuals of an accession; (A) Absent absence of marker band in all individuals of an accession; (V) Variable Presence of marker band in some individuals of an accession.

Six individuals of each Portuguese accession and Doublon cultivar were evaluated for intra- and inter-accession variation. Genomic DNA was extracted from young

144 leaves as follows: two leaves of 2-week-old seedlings were ground in a mortar with liquid nitrogen. Then, 1.5 ml of extraction buffer (Tris-HCL 100mM, pH=7; EDTA 100mM, pH=7; NaCl 3M) and SDS at a final concentration of 1% were added. Sam- ples were shaken and 1.5 ml equilibrated phenol was added. The phases were separated by centrifugation at 13 000 rpm for 30 min. For DNA precipitation, 2 vol of 100% ethanol and 50 µl of 3M sodium acetate were added to 500 ml µl of supernatant. The pellets were washed with 70% ethanol and re-suspended in 200 µl of TE. Finally samples were treated with RNAse and the DNA purity was estimated by measuring the OD (optical density) at 260/280 nm.

Polymerase chain reaction (PCR) reactions and primer selection Amplification reaction was performed in a Biometra UNO II thermocycler. The primers used were obtained from Operon Technologies (Alameda, Kits E and R). The optimized reaction contained 700 ng DNA, 25 ng primer, 2.5 mM dNTPs, 25 mM MgCl , 2.5 2 µl of 10x Taq DNA polymerase buffer and 2.5 U of Taq polymerase MBI Fermentas in a 25µl final volume. After 5 min of heating at 94°C, amplifications were performed under the following regime: 9 cycles of 15 sec at 94°C, 45 sec at 33°C, and 75 sec at 72°C; followed by 35 cycles of 15 sec at 94°C, 45 sec at 37°C, 75 sec at 72°C, and a final extension reaction of 7 min at 72°C. PCR products were separated by electrophoresis in 1.5% agarose gels and stained with ethidium bromide. Gels were then photographed under UV light with Polaroid 667 film. Under these conditions, 40 primers were screened using two individuals of each accession present in this analysis. Those primers showing no amplification or no consistent banding pattern were rejected for subsequent analysis. After this first screening 30 primers were selected and tried with the rest of the individuals of all accessions in two reactions set. Only the strong reproducible bands produced by these primers were scored.

Data analysis Each polymorphic band was scored as either present (1) or absent (0) for all genotypes, resulting in a binary data matrix. This matrix was used to calculate Neis (1973) genetic diversity statistics (H , H , D , G ) and its counterparts Kx values (Nei and T S ST ST Kumar, 2000). Both were used to estimate the genetic variability. Nei-Kumars (2000) genetic diversity (index Q ) between accessions was calculated. From these values a XY dendrogram was constructed by unweighted pair-group method using arithmetic average (UPGMA) (Sneath and Sokal, 1973). All statistical procedures were carried out using computer programme TULKAS developed by Sáenz de Miera (unpublished).

Results and discussion

The 30 primers used provided 371 reproducible bands for examination. Bands were scored, ranging in size between 300 and 2700 bp approximately, amplifying each primer an average of 12.4 bands. This average is close to that recorded by López Sesé et al. (2002) (11.1 bands per primer). The number of variable bands within any accession (present and absent bands) was, on average, 42.8 (Table1). The percentage of polymorphic loci per accession was

145 higher for the control cultivar (19.4%) and for the accessions ACC03351 (20.8%) and ACC02501 (12.9%). On average, 37.6% of the markers examined were monomorphic (band present in all individual in an accession) ranging from 29 to 45% within accessions. The lowest frequency of monomorphic markers was detected in Tendral, Doublon and ACC03351. The remaining accessions exhibited a number of monomorphic markers higher than the average (Table 1). On average, 50.9% of the markers examined were absent in every individuals in any one accession. The accessibility of RAPD methodology associate with its widely distribution throughout melon genome (Baudracco-Arnas and Pitrat, 1996), support the utility of this marker for melon genome analysis and germplasm evaluation. The variation detected by this marker system has proved to be informative not only for the genetic analysis of cultivated melon, but also in melon germplasm management and breeding programme purposes (Garcia et al., 1998; Staub et al., 1997, 2000; López-Sesé et al., 2002, 2003).

Table 2. Specific amplified marker bands for the nine Portuguese melon accessions and Doublon Primer ACC03351 ACC03370 ACC05726 ACC02494 ACC02495 ACC02501 ACC02496 ACC02435 Tendral Doublon OPR7 1 ------OPR8 ------1 - OPR9 ------1 - OPR12 2 - - - - - 1 - - - OPR14- - - 1 ------OPR17 ------1 - - - OPE1 - 1 1 - - 1 1 - - 1 OPE5 ------1 OPE6 ------1 - - OPE7 - - 1 - - 1 - - - - OPE8 - - - - - 1 - 1 - - OPE9 ------1 - - - OPE14- - 1 - - - - 1 - - OPE15 - - 1 - - 1 - 1 - - OPE16 - - - - - 1 - - - - OPE18 1 ------OPE19 - - 1 - - - - 1 - - OPE20 - - - - - 1 - - - -

Total 41 41 0 6 45 2 2

146 The RAPD primer array used in this study detected 28.3 % polymorphism which was higher than that observed by Baudracco-Arnas and Pitrat (1996) (18%). The average number of polymorphic marker bands per primer was 3.5. This degree of polymorphism is lower then that recorded by Garcia et al. (1998) (49%), in a set of elite melon breeding lines, but higher than the polymorphism level detected by López-Sesé et al. (2002, 2003) (25.6 % polymorphism in an average of 2.8 polymorphic marker bands per primer). A fingerprinting study with the 371 RAPD loci analysed showed that all accessions, except for ACC02495, can be identified by the amplification of one single specific marker band (Table 2). Two accessions (ACC03370 and ACC02494) show only one fixed and specific marker band, which do not exist in any other accession. Tendral and the cultivar used as control, Doublon, show another two marker bands, and the remaining accessions more then two specific marker fragments. Heterozygosity index ranged from 0.021 in the accessions ACC03351 to 0.063 in the accession ACC3351. Nei-Kumars total diversity parameter (Kx T) was 0.351, and the average diversity (Kx S) per accession was 0.051 (ranging from 0.028 to 0.096). The lowest diversity value is associated to accession ACC02496 and the highest with the accession ACC03351. Values found for inter (DST; ST) and intra-populational variability (S), indicate that inter-populational variability is the main component of the total variability (T) found when all plants are treated as a whole (Table 3).

Figure 1. Dendrogram obtained from Qxy similarity index.

147 Cluster analysis (UPGMA) resulted in a dendrogram with two main branches; one containing only one accession (Tendral) and the other containing all the others. Thus, Tendral, is genetically distant from all other accessions (Fig. 1). The first branch contains three groups all formed by accessions from different, but close geographic origin. The second one has only one group. The accessions ACC02496 and ACC03351 and the cultivar Doublon are grouped together (Table 3; Fig. 1). The results obtained by Mliki et al. (2001) indicate that African accessions are different from a reference array accessions, and that African accessions are different according its origin. In this study, no association was detected with landraces geographic origin and local name with its cluster distribution. This fact is probably due to its close geographic origin. Nevertheless, Portuguese landraces conserve an important genetic diversity, for what it is desirable to carry out additional studies with a higher number of samples on the structuring of the genetic variability for different markers.

Table 3. Statistics of genetic diversity for 9 Portuguese accessions and Doublon as measured by RAPD loci

Accession No. HS1 Kx2 Clustering Group3

ACC03351 0.063 0.096 1 ACC03370 0.021 0.033 2 ACC05726 0.023 0.039 3 ACC02494 0.0240.032 2 ACC02495 0.0340.050 2 ACC02501 0.041 0.059 3 ACC02496 0.021 0.028 1 ACC02435 0.027 0.039 3 Tendral 0.022 0.045 4 Doublon 0.055 0.0941 Total (PT, HT, KxT) 0.220 0.351 Average (PS, HS, Kx S) 0.033 0.051 DST (T S) 0.186 GST(DST/T) 0.849 DST/S 5.599 Kx ST 0.300 Kx ST/T 0.855 Kx ST/S 5.882

1 Genetic heterozygosity index according to Neis method (1973). 2 Genetic variability by the Kx parameter of Nei-Kumars method (2000). 3 Group number given in Fig. 1.

Acknowledgements

The authors express gratitude to A. Barata da Silva from Banco Português de Ger- moplasma Vegetal (Braga), M. E. Ferreira from Estação Agronómica Nacional (Oeiras)

148 and M.L. Gomez-Guillamon from Estacion Experimental La Mayora (Malaga, Spain) for providing seed samples for this study. This work was supported by project AGRO IED 149.

References

Ayad, W.G., Hodgkin, T. Jaradat, A. and Rao, V.R. (Eds.) 1997. Molecular genetic techniques for plant genetic resources. Report of an IPGRI workshop, 9-11 October 1995, Roma, Italy. Inter- national Plant Genetic Resources Institute, Rome, Italy. Baudracco-Arnas, S. and Pitrat, M. 1996. A genetic map of melon (Cucumis melo L.) with RFLP, RAPD, isozyme, disease resistance and morphological markers. Theor. Appl. Genet., 93: 57-64. Danin-Poleg, Y., Reis, N., Tzuri, G. and Katzir, N. 2001. Development and characterization of microsatellite in Cucumis. Theor. Appl. Genet., 102: 61-72. Esquinas-Alcazar, J.T. 1977. Allozyme variation and relationships in the genus Cucumis. Ph.D. Diss., University California, Davis, 170 pp. Garcia, E., Jamilena, M., Alvarez, J.I., Arnedo, T., Oliver, J.L. and Lozano, R. 1998. Genetic relationships among breeding lines revealed by RAPD markers and agronomic traits. Theor. Appl. Genet., 96: 878-885. Garcia-Mas, J., Oliver, M., Gómez-Paniagua, H. and de Vicente, M.C. 2000. Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon. Theor. Appl. Genet., 101: 860-864. Katzir, N., Danin-Poleg, T., Tzuri, G., Karchi, Z., Lavi, U. and Cregan, P.B. 1996. Length polymorphism and homologies of microsatellites in several Cucurbitacea species. Theor. Appl. Ge- net., 93: 1282-1290. López-Sesé, A.I., Staub, J.E., Katzir, N. and Gómez-Guillamón, M.L. 2002. Estimation of between and within accession variation in selected Spanish melon germplasm using RAPD and SSR markers to assess strategies for large collection evaluation. Euphytica, 127: 41-51. López-Sesé, A.I., Staub, J.E. and Gómez-Guillamón, M.L. 2003. Genetic analysis of Spanish melon (Cucumis melo L.) germplasm using a standardized molecular-marker array and geographically diverse reference accessions. Theor. Appl. Genet., 108: 41-52. Meglic, V.V., Horejsi, T.F., McCreight, J.D. and Staub, J.E. 1994. Genetic diversity and inheritance and linkage of isozyme loci in melon (Cucumis melo L.). Hort Sci., 29: 449. Mliki, A., Staub, J.E., Zhangyong, S. and Ghorbel, A. 2001. Genetic diversity in melon (Cucumis melo L.): An evaluation of African germplasm. Genet. Res. Crop Evol., 48: 587-597. Mo-Suk, Y., Im-Sung, H., Go-Gawn, D., Ann-Chong, M. and Kim-Doo, H. 1998. RAPD analysis of genetic diversity of melon species. Korean J. Hort. Sci. Tech., 16: 21-24. Nei, M. 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci. USA 70: 3321-3323. Nei, M. and Kumar, S. 2000. Molecular Evolution and Phylogenetics. Oxford University Press, Oxford, New York. Neuhausen, S.L. 1992. Evaluation of restriction fragment length polymorphism in Cucumis melo. Theor. Appl. Genet., 83: 379-384. Perl-Treves, R., Zamir, D., Navot, N. and Galun, E. 1985. Phylogeny of Cucumis based on isozyme variability and its comparison with plastome phylogeny. Theor. Appl. Genet., 71: 430-436. Shattuck-Eidens, D.M., Bell, R.N., Neuhausen, S.L. and Hellentjaris, T. 1990. DNA sequence variation within maize and melon: observations from polymerase chain reaction amplification and direct sequencing. Genetics, 126: 207-217. Silberstein, L., Kovalski, I., Huang, R., Anagnostou, K., Jahn, J.M. and Perl-Treves, R. 1999. Molecular variation in melon (Cucumis melo L.) as revealed by RFLP and RAPD markers. Sci. Hort., 79: 101-111. Sneath, P.H.A. and Sokal, R. 1973. Numerical Taxonomy. Freeman, San Francisco. Staub, J.E., Box, J., Meglic, V., Horejsi, T.F. and McCreight, J.D. 1997. Comparison of isozyme and random amplified polymorphic DNA data for determining intraspecific variation in Cucu- mis. Genet. Res. Crop Evol., 44: 257-269.

149 Staub, J.E., Danin-Poleg, Y., Fazio, G., Horejsi, T., Reis, N. and Katzir, N. 2000. Comparative analysis of cultivated melon groups (Cucumis melo L.) using random amplified polymorphic DNA and simple sequence repeat. Euphytica, 115: 225-241. Staub, J.E., Frederick, L. and Marty, T.L. 1987. Electrophoretic variation in cross-compatible wild diploid species of Cucumis. Can. J. Bot., 65: 792-798. Stepansky, A., Kovalski, I., Perl-Treves, R. and Naudin, C.V. 1999. Intraspecific classification of melons (Cucumis melo L.) in view of their phenotypic and molecular variation. Plant Syst. Evol., 217: 313-332. Tanksley, S.D. and. Nelson, J.C. 1996. Advanced backcross QTL analysis: a method for the simultaneous discovery and transfer of valuable QTLs from unadapted germplasm into elite breeding lines. Theor. Appl. Genet., 92: 191-203.

150 Screening of melon (Cucumis melo) germplasm for consistently high sucrose content and for high ascorbic acid content

Y. Burger1, Y. Yeselson2, U. Saar1, H.S. Paris1, N. Katzir1, Y. Tadmor1 and A.A. Schaffer2 1Department of Vegetable Crops, Institute of Field and Garden Crops, Agricultural Research Organization, Newe Yaar Research Center, P. O. Box 1021, Ramat Yishay 30-095,Israel; e-mail: [email protected] 2Department of Vegetable Crops, Institute of Field and Garden Crops, Agricultural Research Organization, Volcani Center, P. O. Box 6, Bet Dagan 50-250, Israel

Summary

Melon fruit quality is determined by a number of components that affect taste and nutritional value. Sucrose content is a primary determinant of taste and ascorbic acid (vitamin C) is an important nutritional benefit. Over the past three years, approximately 350 melon accessions grown in the field were screened for sucrose and ascorbic acid contents of their fruit flesh. A few accessions were identified that had consistently high soluble solids and sucrose contents and/or high ascorbic acid content.

Keywords: melon, germplasm, total soluble solids, sucrose, ascorbic acid, genetic variability

Introduction

The improvement of fruit quality is a most important goal of cucurbit breeding programs conducted in Israel and throughout the world. The value of horticultural produce is determined by its quality, which in turn is governed by a number of components. The accumulation of sugars, particularly sucrose, is perhaps the primary determinant of fruit quality in melons and other cucurbits (Burger et al., 2000). Both environmental and genetic factors affect sugar content of C. melo fruit. Genetic variability for total sugar concentration of the fruit flesh of C. melo is accounted for mainly by differences in the level of sucrose (Stepanski et al., 1999; Burger et al., 2000). Germplasm having high sucrose content that is relatively uniform over a wide range of conditions would of great value for breeding new high-quality melon varieties. It is widely accepted that fruits and vegetables have many healthful properties, including provision of essential nutrients and lowering the incidence or severity of various diseases or adverse health conditions. Studies of nutrient contents of different cultivars have been conducted on various fruits and vegetables, including cucurbits. For example, pumpkin and squash cultivars differ more than 15-fold among themselves in carotenoid content (Paris, 1994). Red-fleshed watermelon cultivars vary markedly in lycopene content (Perkins-Veazie et al., 2001), and melon cultivars vary in ascorbic acid and carotenoid contents (Swamy and Dutta, 1985; Lester, 1997). Over the past 40 years, several hundred melon accessions have been collected at the Newe Yaar Research Center in northern Israel. The Directorate of the Agricultural Research Organization, the research arm of Israels Ministry of Agriculture, has recently fun-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 151 ded a multidisciplinary project, Center for the Genetic Enhancement of Cucurbit Fruit Quality. Within this framework, we have screened and characterized this collection for important fruit quality traits. The aim of this report is to illustrate and describe some of our findings.

Materials and methods

Our seed collection of melons includes accessions obtained from public and private breeders and plant introduction centers from various countries, such that the accessions are derived from countries throughout the temperate, sub-tropical, and tropical regions of the world. Some of the accessions contain obvious phenotypic variability. Approximately 350 of the accessions in our collection were grown and compared in the field during the spring-summer seasons of 2001-2003 at Newe Yaar. Seeds were sown in trays consisting of 128 inverse pyrimidal cells (35 mm × 35 mm at the top, 60 mm deep) on 15 March. Eight seedlings of each accession were transplanted to the field in April. During the first week of flowering of each accession, female flowers were tagged on the day of anthesis. Most plants were self-pollinated. At fruit maturity (abscission or rind color change), melons were harvested and measured for total soluble solids (TSS) by refractometer, and sampled for sucrose and ascorbic acid contents with HPLC. Seeds obtained by self-pollination of plants having the highest levels of TSS, sucrose, and/or ascorbic acid were saved and used for planting in the succeeding year.

Results and discussion Germplasm consistently high for sugar was found in widely differing C. melo germplasm. PI 321005, a finely netted, orange-fleshed muskmelon (Reticulatus Group) from Taiwan, was outstanding for consistently high soluble solids content over the three years (Table 1). Its sucrose content was among the highest measured during 2001. Another muskmelon, AR5, also had consistently good soluble solids over the three years. AR5 and a third muskmelon, Ananas Yoqneam, had high sucrose contents in both years that this variable was measured, 2001 and 2003. The casaba (Inodorous Group) Arka Jeet had the highest sucrose content of all in both years and Sakatas Sweet (Makuwa Group) also was very high in sucrose content. Additionally, the data suggest that the process of self-pollination and selection for high sugar content within Far East 5, Gold King, Grand Gold, and PI 234607 may have resulted in inbreds that are genetically superior in fruit quality to their respective original accessions. In this survey, a 50-fold range in ascorbic acid content was observed (Table 2). Values ranged from 0.7 mg/100 g fresh weight in Hogolyo Storable to 35.3 mg/100 g fresh weight in PI 200819 (Table 2). Sakatas Sweet and several other accessions and inbreds also had relatively high ascorbic acid contents. Accessions AR5, Arka Jeet, Far East 5, Grand Gold, PI 321005, and Saka- tas Sweet had both, high sucrose content and high ascorbic acid content. These accessions are potentially valuable germplasm for the improvement of melon fruit quality. Combining various components of fruit quality can lead to new tastes, such as sweet-and-sour melons (Burger et al., 2003).

152 Table 1. Total soluble solids (TSS) and sucrose contents in 24 representative accessions of the melon germplasm collection at Newe Yaar

2001 2001 2002 2003 2003 Sucrose Sucrose Accession Group* TSS (mg/g TSS TSS (mg/g fresh fresh weight) weight)

AR5 Reticulatus 11.5 55.3 10.1 11.8 65.6 Ananas Yoqneam Reticulatus 9.3 48.5 9.1 53.1 Arka Jeet Inodorus 14.5 75.4 13.7 78.3 Branco De Rebatejo Inodorus 9.0 33.0 Doublon Cantalupensis 8.0 37.8 Dulce Reticulatus 11.2 48.5 Early Silver Line Makuwa 8.2 30.2 Far East 5 Inodorus 11.5 37.6 14.0 11.5 59.9 Gold King Inodorus 9.3 38.4 13.6 63.6 Golden Beauty Inodorus 9.457.9 Grand Gold Reticulatus 9.3 30.9 14.0 12.2 62.4 Hogolyo Storable Inodorus 5.3 6.5 Honduras Wild Melon (subsp. melo) 2.1 6.9 3.3 Imperial 45 Reticulatus 5.6 9.5 PI 125863 Dudaim 5.1 12.9 PI 149169 Flexuosus 4.9 2.8 PI 157070 Conomon 7.1 8.0 10.2 42.5 PI 157080 Conomon 9.3 32.5 PI 200819 (subsp. agrestis) 8.8 5.7 2.9 1.6 PI 234607 Reticulatus 9.3 40.5 11.1 10.9 61.1 PI 321005 Reticulatus 14.8 58.4 13.2 12.5 Revigal Reticulatus 7.426.4 6.5 9.3 Rochet Inodorus 9.440.3 Sakatas Sweet Makuwa 12.5 76.5

*Groups designated in accordance with infraspecific classification of Pitrat et al. (2000).

The ability to accumulate sucrose in the mesocarp of melon fruit is conferred by one recessive gene; sucrose accumulators are defined as having at least 14 mg/g fresh weight sucrose content in the fruit flesh (Burger et al., 2002). Nonetheless, there is much genetic variation for the amount of sucrose accumulated (Table 1). Sucrose accumulation is also strongly affected by growing conditions. In melons as well as in other dessert fruits, high sugar content is essential and this characteristic must be expressed under a wide range of growing conditions. The germplasm collected and conserved at Newe Yaar is a valuable reservoir of genes for improved fruit quality.

153 Table 2. Ascorbic acid content in 20 representative accessions of the melon germplasm collection at Newe Yaar

Accession Group* 2001 2002 2003

Ananas Yoqneam Reticulatus 11.4 AR5 Reticulatus 33.3 24.0 Arka Jeet Inodorus 22.3 Branco De Rebatejo Inodorus 2.3 Charmy Cantalupensis 27.5 Dulce Reticulatus 23.8 Early Silver Line Makuwa 15.1 Far East 5 Inodorus 25.410.4 Gold King Inodorus 5.6 Grand Gold Reticulatus 24.6 24.7 Hogolyo Storable Inodorus 0.7 Honduras Wild Melon (subsp. melo) 21.0 PI 125863 Dudaim 21.4 PI 149169 Flexuosus 2.8 PI 157070 Conomon 16.2 PI 157080 Conomon 28.3 PI 200819 (subsp agrestis) 35.3 15.6 PI 234607 Reticulatus 14.4 18.1 PI 321005 Reticulatus 34.2 Sakatas Sweet Makuwa 32.0

*Groups designated in accordance with infraspecific classification of Pitrat et al. (2000)

References

Burger, Y., Saar, U., Katzir, N., Paris, H.S., Yeselson, Y., Levin, I. and Schaffer, A.A. 2002. A single recessive gene for sucrose accumulation in Cucumis melo fruit. J. Amer. Soc. Hort. Sci., 127: 938-943. Burger, Y., Saar, U., Distelfeld, A., Katzir, N., Yeselson, Y., Shen, S. and Schaffer, A.A. 2003. Development of sweet melon (Cucumis melo) genotypes combining high sucrose and organic acid content. J. Amer. Soc. Hort. Sci., 128: 537-540. Burger, Y., Shen, S., Petreikov, M. and Schaffer, A.A. 2000. The contribution of sucrose to total sugar content in melons (Cucumis melo). In: Katzir, N. and Paris, H.S. (Eds.), Proceedings of Cucurbitaceae 2000. Acta Hort., 510: 479-485. Lester, G. 1997. Melon (Cucumis melo L.) fruit nutritional quality and health functionality. Hort. Tech., 7: 222-227. Paris, H.S. 1994. Genetic analysis and breeding of pumpkins and squash for high carotene content. In: Linskens, H.-F. and Jackson, J.F. (Eds.), Modern Methods of Plant Analysis, vol. 16. Veg. and Veg. Prod., pp. 93-115. Perkins-Veazie, P., Collins, J.K., Pair, S.D. and Roberts, W. 2001. Lycopene content differs among red-fleshed watermelon cultivars. J. Food Agric. Sci., 81: 10-15 . Pitrat, M., Hanelt, P. and Hammer, K. 2000. Some comments on infraspecific classification on cultivars of melon. In: Katzir, N. and Paris, H.S. (Eds.), Proceedings of Cucurbitaceae 2000. Acta Hort., 510: 29-36.

154 Stepanski, A., Kovalski, I., Schaffer, A.A. and Perl-Treves, R. 1999. Variation in sugar levels and invertase activity in mature fruit representing a broad spectrum of Cucumis melo genotypes. Genet. Res. Crop Evol., 45: 53-62. Swamy, K.R.M. and Dutta, O.P. 1985. Inheritance of ascorbic acid content in muskmelon (Cucu- mis melo L.). Sabrao J., 17: 157-163.

155 156 Pre-breeding in the watermelon germplasm bank of the Northeast of Brazil

M.A. Queiroz1, M.L. Silva2, L.M. Silveira3, R.C.S. Dias4, M.A.J.F. Ferreira4, S.R.R. Ramos4, R.L. Romão5, J.G.A. Assis6, F.F. Souza4 and M.C.C.L. Moura7 1UNEB, P.O. Box 171, 48905-680, Juazeiro-BA, Brazil; e-mail: [email protected] 2UFPE, Recife-PE, Brazil; 3ESAM, Mossoró-RN, Brazil 4Embrapa Petrolina-PE, Brasília-DF, Teresina-PI, Porto Velho-RO, Brazil 5UEFS, Feira de Santana-BA, Brazil; 6UFBA, Salvador-BA, Brazil 7UEMA, São Luís-MA, Brazil

Summary

The use of germplasm from gene banks is limited and there is great concern in the scientific community about this problem. Ounce in the management of a germplasm bank the accessions can be studied in any of its different phases, this approach has been followed at the watermelon germplasm bank as a strategy to increase the use of the accessions in breeding programmes to improve the watermelon crop. Therefore, during the seed multiplication and characterization in field trials, both plant and fruit characters were recorded. It was found genetic variability for several descriptors used. In a multivariate analysis it was found nine different groups, although one group comprised around 68% of the accessions. It is expected that molecular markers e.g. RAPD or AFLP, help in the establishment of a core collection for watermelon. In another experiment, some accessions were evaluated against PRSV-w, WMV-2 and ZYMV, using ELISA test. It was found potential resistant plants for the three viruses which were transplanted to get selfed and crossed seeds. The resulting progenies and segregating populations could be analyzed using molecular tools. Past experiments revealed agronomic traits (powdery mildew resistance, prolificacy and small fruits) which were introgressed into commercial backgrounds to obtain lines and hybrid combinations of different ploidy levels and fruit patterns. With this approach around 84% of the accessions of the watermelon gene bank were used.

Keywords: Citrullus lanatus, genetic resources, gene bank management

Introduction

The watermelon crop in Brazil covers an area of 70,000 hectares in different parts of the country and produces around 1,000,000 tons of fruits annually (Queiroz et al., 2000). As also mentioned by the authors, the cultivars available for the farmers are few and were not developed for the Brazilian conditions, with very few exceptions. As a result, the productivity is low and the crop needs sprays against pests and diseases. The watermelon has a great genetic variability in the traditional agriculture in the Northeast of Brazil. Several expeditions were performed and the samples collected gave rise to a watermelon germplasm bank (Queiroz et al., 1999) which comprises more than 500 accessions. The use of accessions from a gene bank in breeding programmes has been of great concern in the scientific community (Brown et al., 1989), and Marshall (1989) analy- zes the question in depth. Marshall (1989) discusses an appropriate measure for the

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 157 use of an accession of a gene bank. Peeters and Williams (1984) concluded that the number of independent requests for accessions is probably the simplest and the most appropriate measure. However, apart from the measure of usage there are other factors which have great influence in the germplasm use, e.g., lack of information in germplasm collections, lack of tuning between curator and breeders, legal constraints, low number of plant breeders and lack of pre-breeding among others (Marshall, 1989). On the other hand, the management of a germplasm bank involves collecting, the multiplication, the characterization, the evaluation and the conservation, all focused in the use of the germplasm (Hawkes, 1982). Therefore, some of these phases were used to study the accessions of the watermelon germplasm bank, taking into account the points raised by Marshall (1989) regarding the use of accessions as it is described as follows.

Material and methods

The accessions were collected in the traditional agriculture in the Northeast of Brazil as seeds or fruits from the farmers fields or from open fairs. The seeds were stored in a cold chamber at 10°C and 40% of relative humidity (Queiroz et al., 1999). The multiplication of the accessions was done under field conditions. During the multiplication trials plant and fruit characteristics were recorded. In a particular experiment, carried out in the year 2002, 48 accessions from Vitoria da Conquista, State of Bahia were planted in rows of fifteen plants for each accession. It was used self pollination for the majority of the plants, but some open pollinated plants were also harvested (half-sib progenies). The same experiment was also used in order to record disease resistance (grading scale: 1 = in the harvest, healthy plants with green leaves from the cotiledonary ones; 2 = defoliated plants with typical symptoms of powdery mildew and/or alternaria leaf blight; and 3 = other symptoms, including nutritional problems), fruit length and diameter, total soluble solids, flesh color and external color (Silveira and Queiroz, 2003). In another experiment carried out in the year 2003, a set of 43 accessions from three different regions of the State of Bahia (Vitória da Conquista, Chapada Diaman- tina and Irecê) was characterized using morphological descriptors (stem length, number of stems, yield per plant, fruit weight, length and diameter, flesh color in a scale from 1 (red) to 5 (white) and total soluble solids). The accessions were replicated in a randomized block. Univariate and multivariate analyses were performed (Cruz and Regazzi, 1994). Finally, a set of nine accessions plus a check (cv. Crimson Sweet) were evaluated against papaya ring spot virus, strain watermelon (PRSV-w) and watermelon mosaic virus 2 (WMV-2). Another set of five treatments plus the same check were evaluated against zucchini yellow mosaic virus (ZYMV). A sample of each treatment was planted under green house conditions, using a subsample of eight plants to be inoculated with each virus. After ten days a second inoculation was done in the symptomless plants. A sample of 0.1g of leaf tissue of each plant was collected and analyzed using ELISA test (Almeida and Lima, 2000). Non inoculated plants (control) from the accessions were used. The plants which gave absorbancy values below twice the mean

158 absorbancy of non inoculated plants and not presented virus symptoms were regar- ded as resistants and were transplanted under field conditions in order to get seeds.

Results and discussion

Almost 50% of the evaluated accessions presented resistant plants to powdery mildew (Sphaerotheca fuliginea) and alternaria leaf blight (Alternaria sp.), but the percentage of resistant plants in each accession, ranged from 7 to 37%. Doing the morphological characterization of the fruits simultaneously to the seed multiplication, allowed to identify a reasonable genetic variability for fruit weight (0.6 to 7.6 kg), total soluble solids (2.0 to 10.2 ° Brix), fruit length (14.1 to 41.5 cm) and diameter (7.2 to 18.9 cm), external color (light to dark green, solid and strips of different widths) and flesh color (pink to red). The morphological characterization in a replicated trial has also shown that the accessions collected in the State of Bahia presented genetic variation for plant and fruit characters, although the precision of the trial has been low, particularly for characters that are more influenced by the environment as yield and fruit weight (Table 1).

Table 1. Summary of the analysis of variance, average and amplitudes for some characters evaluated in 43 watermelon accessions collected in the State of Bahia, Petro- lina-PE, 2003

Mean Squares Amplitude Characters1 C.V. Blocks Accessions Error (%) Mean Lower Upper (2d.f.)1 (42d.f.) (84d.f.) Value Value

Stem length 4.047 3.265** 0.685 15.57 5.32 2.59 8.12 Number of stems 12.770 4.676* 2.842 19.05 8.843.67 13.67 Yield per plant 0.163 4.597* 2.383 28.64 5.38 1.53 10.27 Fruit weight 0.291 4.497** 0.915 28.30 3.38 1.20 11.00 Fruit length 0.454 50.040** 10.440 14.49 22.29 13.27 34.33 Fruit width 1.288 8.750** 1.782 8.88 15.02 10.70 23.63 Flesh color 0.195 1.055** 0.209 11.53 3.96 1.00 5.00 Total soluble solids 1.908 2.438** 0.387 9.69 6.41 3.73 11.13

1d.f. Degree of freedom; **,* Significant at 1% and 5% by F test, respectively.

A multivariate analysis showed two groups. One was made up by the check (Crim- son Sweet) and the second was composed by all the other accessions. When the check was excluded from the analysis, nine groups were formed (Table 2). When analyzing the nine groups, the first cluster comprises accessions from the three regions, but the other clusters were formed by accessions of the same regions which, in turn, indicates a reasonable discrimination of the accessions. Molecular markers (Levi et al., 2000) could be used to analyze the same set of accessions in order to examine if the power

159 of discrimination increases, since they are not affected by the environment. They can also be used to analyze a representative sample of the accessions from the bank to establish a core collection (Brown, 1989) for long term conservation. The absorbancy of the plants evaluated for PRSV-w, WMV-2 and ZYMV varied among the progenies in the accessions evaluated (Table 3). The non inoculated plants (control) also varied in the absorbancy values for the three viruses analyzed (Table 3).

Table 2. Similarity groups among 40 watermelon accessions according the Tochers method based on the Mahalanobis distances. Petrolina-PE, 2003

Groups Accessions

I 03 05 07 08 09 11 12 13 1415 16 19 20 25 29 30 31 32 33 34 35 36 37 38 40 41 42 II 01 0406 III 18 22 23 27 IV 24 V21 VI 17 VII 2 VIII 28 IX 10

Table 3. Absorbancy values of the watermelon treatments evaluated against the virus PRSV-w, WMV-2 and ZYMV. UFC. Fortaleza-CE, 2003

Absorbancy Treatments Absorbancy Amplitude Treatments Amplitude

Virus PRSV-w Virus WMV-2 Virus ZYMV

11 0.409 3.063 1 0.360 3.075 2 0.123 2.136 0.189 2.0842 (check) 0.243 3.166 3 0.122 0.6240.178 0.765 3 0.239 3.163 40.127 0.155 40.205 3.515 5 0.116 0.329 5 0.247 2.365 6 0.120 0.166 6 0.192 2.212 7 0.114 2.997 8 0.102 0.115 0.152 0.172 9 0.104 0.116 0.156 0.248 10 (check) 0.106 0.177 0.142 0.197 Control 0.114 0.174 0.156 0.196 0.106 0.263

1Not evaluated

160 The mean absorbancies for the control for the three viruses were 0.146 (PRSV-w), 0.175 (WMV-2) and 0.166 (ZYMV). It was found some plants with absorbancy values below the double mean for each virus (resistant plants), although some of them were discarded despite the low amount of virus, because they presented virus symptoms. It was selected 26 potential plants to be source of resistance to PRSV-w and 30 to WMV-2. For the virus ZYMV seven plants were selected. It will be necessary to evaluate the progeny of the selected plants in order to identify homozygous sources for the three viruses, which, in turn, allows to obtain segregating populations for the study using molecular markers associated with resistance to those viruses (Danin-Poleg et al., 2000). Considering past and recent experiments in the watermelon gene bank, several traits as powdery mildew resistance (Borges, 1996), gummy stem blight (Dias, 1993), virus (Oliveira et al., 2000), prolificacy and small fruits (Ferreira, 1996) were identified. In fact, as stated by Romão (1995) the majority of the genes described in the current watermelon literature have been identified in the traditional agriculture of the Nor- theast of Brazil. Also, tetraploid lines from the accessions have been developed (Souza et al., 1999). Some traits have been transferred to commercial backgrounds and, then, inbred lines and hybrid combinations of different ploidy levels and fruit patterns, resistant to powdery mildew were developed (Queiroz et al., 2003).

Conclusions With the approach described, the use of the accessions in the watermelon breeding programme has been around 84% which is superior to the use of several other crops according to the current literature. Therefore, this strategy can be applied to increase the use of accessions in gene banks of annual crops.

References

Almeida, A.M.R. and Lima, J.A.A. 2001. Princípios e técnicas de diagnose aplicadas em fitoviro- logia. Londrina, Embrapa Soja/Brasília, Sociedade Brasileira de Fitopatologia, 186 pp. Borges, R.M.E. 1996. Estudo da herança da resistência ao oídio Sphaerotheca fuliginea (Schlecht. ex Fr.) Poll em melancia Citrullus lanatus Thunb. Mansf. UFPE, Recife, 46 pp. (Dissertação de Mestrado) Brown, A.H.D. 1989. The case for core collections. In: Brown, A.D.H., Marshall, D.R., Frankel, O. and Williams, J.T. (Eds.), The Use of Plant Genetic Resources. Cambridge University Press, Cambridge, pp. 136-156. Brown, A.H.D., Marshall, D.R., Frankel, O.H. and Williams, J.T. (Eds.) 1989. The Use of Plant Genetic Resources. Cambridge University Press, Cambridge, 382 pp. Cruz, C.D. and Regazzi, A.J. 1994. Modelos biométricos aplicados ao melhoramento genético. UFV, Viçosa, 390 pp. Danin-Poleg, Y., Tzuri, G., Reis, N., Karchi, Z. and Katzir, N. 2000. Search for molecular markers associated with resistance to viruses in melon. Acta Hort., 510: 399-404. Dias, R.C.S. 1993. Características Fisiológicas de Didymella bryoniae (Auersw) Rehm e Fontes de Resistência em Melancia (Citrullus lanatus) (Thunb.) Mansf. UFRPE, Recife, 140 pp. (Disser- tação de Mestrado)

161 Ferreira, M.A.J.F. 1996. Análise dialélica em melancia Citrullus lanatus (Thunb.) Mansf. UNESP- FCAV, Jaboticabal, 83 pp. (Dissertação de Mestrado) Hawkes, J.G. 1982. Germplasm collection, preservation, and use. In: Frey, K.J. (Ed.), Plant Bre- eding II. Kalyani, Ludhiana, pp. 57-83. Levi, A., Thomas, C.E., Keinath, A.P. and Wehner, T.C. 2000. Estimation of genetic diversity among Citrullus accessions using RAPD. Acta Hort., 510: 385-390. Marshall, D.R. 1989. Limitations to the use of germplasm collections. In: Brown, A.D.H., Mar- shall, D.R., Frankel, O. and Wiliams, J.T. (Eds.), The Use of Plant Genetic Resources. Cam- bridge University Press, Cambridge, pp. 105-120. Oliveira, V.B., Queiroz, M.A. and Lima, J.A.A. 2002. Fontes de resistência aos principais poty- vírus isolados de cucurbitáceas no Nordeste brasileiro. Hort. Bras. (Brasília), 20: 589-592. Peeters, J.P. and Williams, J.T. 1984. Towards better use of genebanks with special reference to information. Plant Gen. Res. Newslett., 60: 20-32. Queiróz, M.A., Costa, N.D. and Dias, R.C.S. 2003. Avaliação de combinações híbridas de melancia no Submédio São Francisco. Hort. Bras. (Brasília), 21(Suplemento 1): 361-362. Queiróz, M.A., Dias, R.C.S., Souza, F.F., Ferreira, M.A.J.F. and Borges, R.M.E. 2000. Waterme- lon breeding in Brazil. Acta Hort., 510: 105-112. Queiróz, M.A., Ramos, S.R.R., Moura, M.C.C.L., Costa, M.S.V. and Silva, M.A.S. 1999. Situação atual e prioridades do Banco Ativo de Germoplasma (BAG) de cucurbitáceas do Nordeste brasileiro. Hort. Bras. (Brasília), 17: 25-29. Romão, R.L. 1995. Dinâmica evolutiva e variabilidade de populações de melancia Citrullus lanatus (Thunb.) Matsum. & Nakai em três regiões do Nordeste brasileiro. USP-ESALQ, Piracica- ba, 75 pp. (Dissertação de Mestrado) Silveira, L.M. and Queiróz, M.A. 2003. Variabilidade genética de acessos de melancia coletados na região de Vitória da Conquista-BA. In: Congresso Brasileiro de Melhoramento de Plantas, 2, 2003, Porto Seguro. SBMP/Embrapa Mandioca e Fruticultura, Anais. Porto Seguro. (CD- ROM) Souza, F.F., Queiróz, M.A. and Dias, R.C.S. 1999. Melancia sem sementes: desenvolvimento e avaliação de híbridos triplóides experimentais de melancia. Biotecnol. Ciên. & Desenvolv., 9: 90-95.

162 Use of genetic resources in a dual approach toward selecting improved scion/rootstock grafting combinations of melon (Cucumis melo) on Cucurbita spp.

M. Koutsika-Sotiriou1, E. Traka-Mavrona2, A.L. Tsivelikas1, G. Mpardas3, A. Mpeis3 and E. Klonari3 1Aristotelian University of Thessaloniki, Department of Agriculture, Laboratory of Genetics and Plant Breeding, 541 24 Thessaloniki, Greece 2National Agricultural Research Foundation (N.AG.RE.F.), Agricultural Research Center of Macedonia-Thrace, 570 01 Thermi, Thessaloniki, Greece 3Aristotelian University of Thessaloniki, Department of Agriculture, Laboratory of Plant Pathology, 541 24 Thessaloniki, Greece

Summary

A dual approach toward improved grafting of melons (Cucumis melo) was applied through breeding and selection within local cultivars for use as rootstocks and scions. Specifically described are the breeding and selection of Cucurbita L. rootstocks from local variable germplasm of Cucurbita moschata and Cucurbita maxima and the breeding and selection of the scion, from local variable germplasm of melons of the Inodorous Group. Homozygosity of rootstocks Kalkabaki (C. moschata) and Kolokitha (C. maxima) was increased by selfing, and the resulting lines were examined and compared for their compatibility with the melons Thrakiotiko and Lefko Amynteou. Kalkabaki had low compatibility with both melon cultivars (7-34%), while Kolokitha showed high compatibility (79-96%), equal to that of commercial rootstocks. Both Cucurbita rootstocks were completely resistant to the soil-borne fungi Fusarium oxysporum f.sp. melonis and F. oxysporum f.sp. radici-cucumerinum, as tested in planta. Three cycles of pedigree breeding and selfing were applied to the melon cultivars, with the goal of increasing their yield and quality. The improved scion cultivars were added to the European List of Vegetable Cultivars.

Keywords: Cucumis melo, Cucurbita, grafting, compatibility, soil-borne pathogens

Introduction

Cucurbit grafting has a 50-year history. Grafting is often advantageous because it can reduce crop susceptibility to soil-borne diseases such as Fusarium wilt, increase plant tolerance to sub-optimal temperatures, enhance water and nutrient uptake, and allow for a more sustainable production of fruit-bearing vegetables. However, grafting requires time, space, materials, and expertise. In addition, rootstocks must be carefully chosen, because they may have undesirable effects on the scion (Lee, 1994; Oda, 1995; Robinson and Decker-Walters, 1997). A glance at the present status of vegetable grafting shows that in the U.S.A. and other countries where land use is not intensive, allowing adequate crop rotation to be practiced, grafting is seldom used for vegetables. In Asia and Europe, where land use is often intensive and farming area restricted, grafting is popular. In Japan and Korea, the proportion of the total production area using grafted plants is almost 92% and

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 163 90% for watermelon, 55% and 42% for cucumber, 0% and 85% for melon, 8% and 0% for tomato, and 43% and 0% for eggplant, respectively, under field and tunnel cultivation (Lee and Oda, 2003). In Greece, grafting is popular too, especially in southern areas, where early cropping of watermelons and melons under low tunnels is practiced. The proportion of the production area using grafted plants in the southern part of Greece is almost 100% for watermelons and 80% for melons. Vegetable grafting is becoming increasingly popular in other Mediterranean countries as well (Traka-Ma- vrona and Koutsika-Sotiriou, 2002). The present study describes a dual approach toward grafting of melons on Cucur- bita. This dual approach, breeding and selecting both rootstock and scion, is based on local plant genetic resources, as these could be expected to offer adaptation to local environmental conditions. Preliminary results suggested that survival of the local melon cultivars on the local Cucurbita rootstocks was not as high as on the commercial ones, but could be increased through breeding both the melons and Cucurbita for improved compatibility (Traka-Mavrona et al., 2000; Koutsika-Sotiriou and Tra- ka-Mavrona, 2002). This approach has been continued with the goal of further increasing compatibility and has been expanded to include selection for resistance to soil- borne pathogens and improved fruit yield and quality. Some of the results are presented herein.

Materialp and methods

For breeding and selection of the rootstock, two variable local cultivars were employed: Cucurbita moschata Duchesne Kalkabaki and Cucurbita maxima Duchesne Kolo- kitha. In 2001, four S -lines of Kalkabaki and the initial Kolokitha were tested in 1 comparison with the commercial rootstock TZ-148, for compatibility with the melon cultivars used as scions. The experiments were conducted in the open field with a plant spacing of 150 × 200 cm. Seedlings were grafted by the tongue-approach method. A randomized complete block design was adopted with four replications. Intact plants of each cultivar acted as controls. Observed and recorded were the number of plants that survived until transplanting, the number of fruits per plant, and fruit yield per plant. The data obtained were subject to analysis of variance. Fruit descriptive and qualitative characteristics were also observed. The two Cucurbita spp. cultivars and inbreds derived from them were tested for resistance to two isolates of F. oxysporum f.sp. melonis and F. oxysporum f.sp. radici- cucumerinum. Plants were grown for one week in seed beds composed of peat moss and perlite at a ratio 10:1. Plants were transplanted to pots when they had one true leaf. Prior to transplanting, roots were dipped in a conidial suspension of 106 conidia/ml. Then the inoculated plants were irrigated with a spore suspension of the same density three times, once a day for the following three days after the transplanting. The inoculated plants were kept in a growth chamber at 23oC, RH 70% and 16/8 hours of light and darkness. Ten days later, the response of each plant was examined and scored on a disease scale from 0 to 3 (0: no symptoms, 3: dead plants). Selection for yield and quality of the melon cultivars was based on the performance of the progeny of individual plants, 21 plants per breeding cycle. The following

164 data for each cycle were gathered for both cultivars: (a) fruit number per plant, (b) marketable yield (kg/plant), and (c) fruit size (kg/fruit). Additionally, fruit descriptive and qualitative characteristics were observed (data not shown).

Results and discussion

The survival of melon transplants grafted onto TZ 148 was 80-87%. When grafted onto Kolokitha, survival was 79% for Thrakiotiko and 96% for Lefko Amyn- teou (Table 1).

Table 1. Survival (%) and yield components of grafted and non-grafted melon plants.

Fruit yield

Marketable Total Treatment Survival (%) Fruits/plant kg/plant kg/plant

Thrakiotiko/Kalkabaki S 20 1.9 a1 2.87 bc 4.16 ab 1.1 Thrakiotiko/Kalkabaki S 342.9 a 4.51 a 5.06 a 1.2 Thrakiotiko/Kalkabaki S 241.9 a 2.50 bc 3.39 bcde 1.3 Thrakiotiko/Kalkabaki S 10 2.5 a 3.16 b 3.56 bcd 1.4 Thrakiotiko/Kolokitha 79 1.9 a 2.76 bc 3.40 bcde Thrakiotiko/TZ-148 80 1.3 a 1.62 c 2.15 e Thrakiotiko (control) 100 1.7 a 1.99 bc 2.58 de L. Amynteou/Kalkabaki S 20 2.1 a 3.00 bc 3.34 bcde 1.1 L. Amynteou/Kalkabaki S 7 1.7 a 2.28 bc 2.93 bcde 1.2 L. Amynteou/Kalkabaki S 7 2.2 a 3.18 b 3.98 abc 1.3 L. Amynteou/Kalkabaki S 21 2.6 a 2.78 bc 3.47 bcd 1.4 L. Amynteou/Kolokitha 96 1.6 a 1.89 bc 2.41 de L. Amynteou/TZ-148 87 1.8 a 2.36 bc 2.70 de L. Amynteou (control) 100 2.3 a 3.05 b 3.36 bcde

1Means within a column followed by the same letter are not significantly different at P=0.05 as determined using Duncans multiple range test.

The S lines of Kalkabaki had 10-34% survival with Thrakiotiko and 7-21% with 1 Lefko Amynteou. Fruit yield of most stock/scion combinations was not significantly affected by grafting except for the combinations S /Thrakiotiko and S /Thrakiotiko. Fruit 1.1 1.2 descriptive and qualitative characteristics were also not significantly affected by grafting (data not presented). The two Cucurbita spp. cultivars were completely resistant to F. oxysporum f.sp. melonis and F. oxysporum f.sp. radici-cucumerinum (Table 2).

165 Table 2. Response of the genetic materials to Fusarium oxysporum

Genetic material F. oxysporum f.sp. melonis F. oxysporum f.sp. radici- cucumerinum

Scale of disease1 (%) Scale of disease1 (%) 0123 0123 Thrakiotiko 100 100 L. Amynteou 10 90 20 80 C. maxima (C ) 100 100 0 C. maxima (C ) 100 100 1 C. maxima (S ) 100 100 1 C. moschata (C ) 100 100 0 C. moschata (C ) 100 100 1 C. moschata (S ) 100 100 1 C. moschata (S ) 100 100 2.1 C. moschata (S ) 100 100 2.2

1Scale of disease is 0, no symptoms; 1, Slight yellowing of bottom leaves; 2, Leaves with dead areas, leaf senescence, yellow spots on the upper leaves, slight wilting; 3, plant dead.

Table 3. Means of fruit yield and fruit size and coefficient of variability (CV%) of source material and of each cycle of selection for both melon cultivars

Genetic material Fruit yield Fruit size

No./plant kg/plant kg/fruit

Mean CV% Mean CV% Mean CV%

Thrakiotiko Source material 3.17 52 5.86 63 1.82 35 1st cycle 2.61 44 2.47 43 1.99 25 2nd cycle 2.25 44 4.26 48 1.92 26 3rd cycle 2.64 43 3.74 43 1.70 25 L. Amynteou Source material 1.55 52 1.5457 1.56 23 1st cycle 1.87 47 2.06 55 1.27 30 2nd cycle 1.73 542.13 67 1.58 27 3rd cycle 1.66 52 2.07 541.4529

166 As for the scion, three cycles of selection resulted in improved quality, uniformity, and yield of melons (Table 3; data not presented). As expected, selfing lowered plant- to-plant variability. The improved Thrakiotiko and Lefko Amynteou have now been registered and included in the European List of Vegetable Cultivars. Inbreeding and selection of single plants of genetic resources resulted in the development of improved germplasm that meets current market standards. Improvement of old, local cultivars can allow them to retain their competitiveness in the market against newer, inter- nationally grown cultivars, thereby contributing to the retention and maintenance of crop diversity.

References

Koutsika-Sotiriou, M. and Traka-Mavrona, E. 2002. The cultivation of grafted melons in Greece. Current status and prospects. In: Paroussi, G., Voyiatzis, D. and Paroussis, E. (Eds.), 2nd Balkan Symposium on Vegetables and Potatoes. Acta Hort., 579: 325-330. Lee, J.M. 1994. Cultivation of grafted vegetables. I. Current status, grafting methods, and benefits. HortScience, 29: 235-239. Lee, J.M. and Oda, M. 2003. Grafting of herbaceous vegetable and ornamental crops. In: Janick, J. (Ed.), Horticultural Reviews, 28: 61-124. Fasoulas, A.C. 1988. The honeycomb methodology of plant breeding. A.C. Fasoulas, Thessaloni- ki, Greece. Oda, M. 1995. New grafting methods for fruit-bearing vegetables in Japan. Jarq, 29: 187-194. Robinson, R.W. and Decker-Walters, D.S. 1997. Cucurbits. CAB International, Wallingford, U.K. 226 pp. Traka-Mavrona, E. and Koutsika-Sotiriou, M. 2000. Compatibility between scions of two Greek melon cultivars (Cucumis melo L.) and two rootstocks (hybrids of Cucurbita spp.). Scientific Annals of the Faculty of Agriculture of the School of Agricultural Sciences, Aristotelian Uni- versity of Thessaloniki, 32: 19-34 (in Greek with English summary). Traka-Mavrona, E. and Koutsika-Sotiriou, M. 2002. The application of grafting on fruit bearing vegetables. Geotechnical Scientific Issues: 2 (4): 47-59 (in Greek with English summary). Traka-Mavrona, E., Koutsika-Sotiriou, M. and Pritsa, T. 2000. Response of squash (Cucurbita spp.) as rootstock for melon (Cucumis melo L.). Scientia Hort., 83: 353-362.

167 168 Effects of seed maturation and temperature in germination of squash accessions: implications for gene flow

A. López-Sesé1 and J.E. Staub2 1Estación Experimental La Mayora, Consejo Superior de Investigaciones Científicas, E-29750 Algarrobo-Costa, Málaga, Spain 2USDA/ARS, Vegetable Crops Unit, Department of Horticulture, 1575 Linden Dr., University of Wisconsin, Madison, WI 53706, USA; e-mail: [email protected]

Summary

Seed dormancy and germination characteristics are fitness-related traits that effect gene flow from transgenic to either cross-compatible horticultural varieties or wild, free-living populations. Such factors are directly related to the formation of persistent soil-seed-banks and are intrinsic to the dynamics associated with such biological systems. Germination of three Cucurbita pepo accessions were examined and include the ornamental gourd Orange Ball, the transgenic cultivar Destiny III, and the wild free-living species form, subsp. ovifera var. ozarkana. Germination rate and percentage was recorded for radical emergence of seeds extracted from open-field-grown fruit at different times (Storage Period) and temperatures (15, 20, and 25°C). Significant differences were recorded in germination rate and percentages among the accessions examined when seed were germinated at different temperature, and different seed extraction dates (Storage Peri- od). The rate and percentage of seed germination of Orange Ball and Destiny III was significantly greater than subsp. ovifera var. ozarkana at any temperature, and storage period had no effect on Orange Ball germination. Time- and temperature-related seed dormancy was observed in subsp. ovifera var. ozarkana, but competitive advantage (fitness) could not be implied from these experiments. Nevertheless, hybrids produced by intermatings between wild free-living populations and transgenic plants could result in escaped hybrid populations that possess a wide array of seed germination/dormancy characteristics that possess increased fitness. The determination of the inheritance of seed dormancy in subsp. ovifera var. ozarkana and the fitness of cross progeny derived from matings with transgenic cultivars in such populations would provide supportive information for the estimation of socio-ecological risk.

Keywords: Cucurbita pepo, var. ozarkana, dormancy, landrace, socio-ecological risk, transgenic

Introduction

The recent commercialization of numerous transgenic crops has provided many benefits for agriculture. Because of this commercialization, awareness of this innova- tions social and economic importance has necessitated appraisal of socio-ecological risk. Assessment of risk requires scientific appraisal of the introduction of transgenes into free-living (i.e., self-sustaining) relatives of agricultural crops, since recent studies have concluded that genes will escape from cultivation (Hancock et al., 1996). In the U.S., squash (Cucurbita pepo L.), which is native to North America, is the only example of a vegetable species in which commercial transgenic cultivars are grown in close proximity to cross-compatible native wild free-living populations of C. pepo subsp. ovifera var. ozarkana. Previous studies have clearly shown the occurrence of gene flow via pollen transfer between free-living and cultivated taxa of C. pepo

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 169 (Kirkpatrick and Wilson, 1988). Research that assesses socio-ecological risk of the flow of transgenes from cultivated to wild C. pepo populations requires an appraisal of the relative competitive and reproductive success of commercial, free-living, and transgenic genotypes and their hybrids, as well as factors related to survivorship of escapes from cultivation. Factors, such as seed germination and dormancy are important for risk assessment because of their fitness values. For instance, the fitness (e.g., dormancy) of plants (i.e., segregating progeny of horticultural varieties and/or wild populations) possessing newly acquired transgenes after initial introgression may differ and thus vary in their contribution to the structure and persistency of soil-seed-banks. Preliminary studies in our laboratory indicated that there might be differences in germination of C. pepo germplasm. Therefore, a study was designed to evaluate the seed germination of a commercial transgenic squash variety (Destiny III), a landrace cultivar Orange Ball, and a free-living C. pepo subsp. ovifera var. ozarkana accession seed under different temperatures and storage times. Differences in germination rate and/or percentage, if they exist, would allow for the design of more in depth evaluations of segregating progeny as a first step in characterizing potential fitness differences among progeny, and hence their contribution to soil-seed banks.

Material and methods Three squash accessions were examined for germination: the ornamental gourd landrace cultivar C. pepo subsp. pepo var. pepo Orange Ball (Stokes seeds, USA), the cultivar Destiny III that possesses transgene-mediated viral resistance to zucchini yellow mosaic virus (ZYMV) and watermelon mosaic virus 2 (WMV-2) (Seminis Seeds, initially sold as a proprietary product of Asgrow Vegetable Seeds subsidiary of Seminis Seeds, USA), and the wild free-living C. pepo subsp. ovifera var. ozarkana accession # 892 [The Cucurbit Network (TCN) number, USA]. Plants of each accession were grown on separated plots on 0.3 x 0.9 m centers in each of two blocks per accession consisting of 80 experimental units (plants) on the University of Wisconsin Agricul- tural Experiment Station at Hancock, Wisc. Seeds of the similar maturity were obtained from controlled self-pollination of random plants in each replication. The standard seed germination protocols of Nienhuis et al. (1983) were followed to examine responses to temperature and the time of seed extraction from fruits and storage after harvest. Fruits were obtained from similar pollination dates and grouped in lots according to storage period. Seeds were extracted from fruits approximately 40 days after pollination and designated Lot 1 for germination tests (Storage Period 1). Every 30 days thereafter, seeds were extracted from fruits and given a Lot designation (e.g., Lot 2 for second extraction date) and a new germination test (e.g., Stora- ge Period 2) was initiated with the most recently extracted seeds and remaining seeds from the previous lots (e.g., remnant of Lot 1 test with Lot 2). Germination tests were carried out under constant temperatures of 15, 20, and 25°C at > 90% RH in growth chambers, and under fluctuating temperatures (~20 to 30°C) in a greenhouse (Madison, Wisc.), in which each treatment contained 15 to 20 seeds and was arranged in a completely randomized design. For controlled experiments (constant temperatures), seeds were placed in plastic Petri dishes containing filter paper

170 discs moistened with distilled water and germinated in darkness. Water was added when necessary, and the positions of each Petri dish were re-randomized after each data collection interval in order to reduce environmental effects due to position. In the greenhouse, all seeds were initially sowed in vermiculite. In some germination tests, the seed coat (i.e., integument) of seeds of C. pepo subsp. ovifera var. ozarkana were removed in order to examine the relationship between seed coat removal and germination for this accession. Seeds of each treatment were examined daily for 30 days or until germination in a treatment had ceased for at least five days. Seeds were considered germinated when the radicle protruded > 2mm, and germinated seeds were removed. Temperature effects were examined by comparing total germination percentage across entries, temperature and storage (seed extraction) treatments (periods and lots). Treatment means for percentage germination were calculated for each entry. Differences in treatment means provides for an assessment of percentage are rate changes over time within treatments.

Results

Dates of the first and last seeds to germinate in an experimental unit (a fruit) were recorded for each entry in each treatment. For simplicity of presentation, means are given as pooled data by entry over replicates (Fig. 1 and Table 1). Fig. 1 presents percentage of germination data for seed of each entry (3) germinated in either greenhouse and control temperature over three extraction periods (Storage Periods 1 to 3). Table 1 presents percentage of germination over time for seeds challenged in the third time period (Storage Period 3) for the three seed lots of all accessions.

Figure 1. Percentage of germinated seeds of three squash (Cucurbita pepo L.) accessions (landrace Orange Ball (Ob), commercial transgenic Destiny III (T), and wild free-living C. pepo subsp. ovifera var. ozarkana (Oz)) at various germination temperatures (greenhouse (GH; fluctuating temperature) and controlled 15, 20, and 25°C), according to seed extraction date (Lots 1 (white), 2 (gray), and 3 (black)) and germination test (Store periods 1, 2, and 3).

171 Germination was relatively high in all treatments for Orange Ball and the transgenic Destiny III, where the percentage of germination increased with increasing temperature regardless the time of seed extraction (Storage Period; Fig. 1). Generally, the lower the temperature, more time is required after seed extraction to approach 100% germination. Regardless of seed extraction interval and germination temperature, the longer the time after extraction the fewer the days that were required for the germination percentage reach a plateau (Table 1).

Table 1. Germination percentage of Cucurbita pepo L. accessions (cultivated and wild) at different constant temperatures (15, 20, and 25°C) at the third germination interval (Storage Period 3) after harvest from an open-field nursery (Hancock, Wisc.)

15°C 20°C 25°C 313 43134 3134 Genotype1/ # seeds days week weeks weeks days week weeks weeks days week weeks weeks extraction lot2 Orange Ball / 1st 20 0.0 60.0 65.0 70.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 Orange Ball / 2nd 40 2.5 15.0 20.0 30.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 Orange Ball / 3rd 80 21.3 47.5 55.0 56.3 86.3 86.3 86.3 86.3 96.3 96.3 98.8 98.8 Transgenic / 1st 35 0.0 88.6 91.491.491.497.1 100.0 100.0 82.9 100.0 100.0 100.0 Transgenic / 3rd 40 0.0 7.5 22.5 25.0 42.5 95.0 100.0 100.0 80.0 100.0 100.0 100.0 Ozarkana / 1st 15 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6.7 6.7 13.3 13.3 Ozarkana / 2nd 15 0.0 0.0 0.0 0.0 0.0 6.7 13.3 13.3 20.0 40.0 46.7 46.7 Ozarkana / 3rd 80 0.0 0.0 0.0 0.0 0.0 1.3 7.5 8.8 12.5 22.5 37.5 38.8

1Orange Ball = C. pepo var. pepo cv. Orange Ball, Transgenic = C. pepo var. pepo cv. Destiny III (transgenic cultivar), and Ozarkana = C. pepo subsp. ovifera var. ozarkana (wild free-living accession). 2Extraction lot where 1st = seeds extracted at time of harvest; 2nd = seeds extracted 30 days after harvest, and 3rd = seeds extracted 60 days after harvest.

The percentage of germination of seed from individual fruits of Orange Ball from was more variable than the other entries, especially at low temperatures (15°C) and when newly-extracted seeds (e.g., Lot 1 at Storage Period 1, Lot 2 at Storage Period 2) were evaluated (Fig. 1). The increase of germination percentage was more rapid in seed lots extracted first (i.e., Lot 1) (Table 1), rapidly reaching 100% germination at 20 and 25°C in tests at Storage Period 3 (Table 1). Effect of time was only observed at 15°C (Fig. 1). The percentage of germination of transgenic Destiny III approached 100% at 20 and 25°C, and was similar to Orange Ball. However, the germination variability of Destiny III was lower in all storage treatments than Orange Ball. Germination after three weeks was similar for the landrace Orange Ball and the transgenic Destiny III (Fig. 1), but for Orange Ball the number of germinated seeds increased gradually over the time interval (Table 1). The number of germinated seeds increased rapidly in the transgenic accession in almost all treatments (Table 1). Germination did not occur in subsp. ovifera var. ozarkana at 15°C, and was comparatively low at 20°C. The highest germination percentages (25-50%) were recorded

172 under greenhouse conditions and at constant 25°C mainly for seeds sampled from Storage Period 2 and 3. These observations include seeds that were kept in storage for extended periods since extraction from fruits (Lot 1) and seeds more recently extracted but kept for extend periods in fruits before extraction (Lots 2 and 3 in Storage Period 3) (Fig. 1). These data therefore indicate a clearly defined storage period (i.e., seeds held in fruit) effect on germination percentage where a gradual but noticeable increase was detected in subsp. ovifera var. ozarkana (Fig. 1 and Table 1) over time (extraction interval) and germination temperature. Likewise, seed coat removal remarkably increased germination percentage at 20 and 25°C (data not presented). Greenhouse temperatures and light conditions (intensity and duration) fluctuated during the test period. Temperatures were relatively high (>28°C), even during dark periods (night). Germination rate and percentage of Orange Ball and transgenic Destiny III were similar to values obtained when seeds were germinated at constant 25°C under controlled conditions (dark, relatively high RH). In contrast, germination of subsp. ovifera var. ozarkana seed was higher and more variable than those obtained 25°C (Fig. 1).

Discussion

Some free-living plant populations in Southern states in the U.S. occur as aggres- sive weeds in areas near or adjacent to non-cucurbit crops (Oliver et al., 1983; Smith et al., 1992). It has been suggested that some weedy-habitat populations (Decker and Wilson, 1987; Wilson, 1990; Cowan and Smith, 1993; Decker-Walters et al., 1993) may have evolved as feral cultivar escapes. Such populations would likely experience a wide range of reciprocal gene introgression between annually planted cucurbit species under cultivation and nearby cross-compatible weedy wild C. pepo native populations if present. Commercial cucurbit seeds possess little or no dormancy. Orange Ball and De- stiny III germinated at close or equal to 100% at temperatures slightly below the optimum as defined by Oliver et al. (1983; 25 and 30°C) without apparent dormancy. Germination rate and variability among fruits was, however, considerable where pro- longed germination was observed only for Orange Ball. Germination patterns such as this in Orange Ball and its hybrid progeny could limit losses from a seed-bank because only a few seeds germinate under favorable conditions, and thus facilitate survivorship as escapes of cultivation in weedy habitats. Seed dormancy of feral forms and landraces cultivars (e.g., Orange Ball) can be similar to wild, free-living forms, and this appears be the case with the subsp. ovifera var. ozarkana accession examined herein. Cucurbitaceae includes species having physical and/or physiological dormancy (Baskin et al., 2000). The seed dormancy of subsp. ovifera var. ozarkana defined herein has not been previously reported (Decker and Wilson, 1987; Decker-Walters et al., 1993). Dormancy facilitates differential germination of dispersed seeds that have been incorporated into soil seed-banks (Baskin et al., 2000). Physical dormancy mechanisms operate in this subsp. ovifera var. ozarkana accession since seed coat removal appreciably increased germination. Thus, it is likely that environmental conditions in the seed-bank play a definitive role in its germination-associated fitness.

173 Slightly higher germination rates were observed for seeds of subsp. ovifera var. ozarkana that remain longer in the fruit after harvest than seed extracted immediately from fruit at the same maturity. This implies that after-ripening mechanisms also could be present in this accession. After-ripening factors involve the breakdown of germination inhibitors and alteration in respiration or gene expression (Adkins et al., 2002), and are affected by environmental conditions during fruit maturation (Baskin and Baskin, 2000). For instance, seeds remaining in Ozark Gourd fruit for ~20 days after harvest have higher germination rates than freshly extracted seed (Oliver et al., 1983). The persistence of seed banks and the timing of seed dispersal, along with germination/dormancy characteristics could be viewed as survival strategies (Baskin and Baskin, 2000). The assessment of the effects of gene flow and introgression between cultivated and wild, weedy populations is critical to an understanding the socio-ecological risk. A major such consideration in such assessments is the fitness of cross- progeny and their fitness in subsequent generations. The characterization of the inheritance of dormancy in subsp. ovifera var. ozarkana would further define this important fitness factor.

References

Adkins, S.W., Bellairs, S.M. and Loch, D.S. 2002. Seed dormancy mechanisms in warm season grass species. Euphytica, 126: 13-20. Baskin, C.C. and Baskin, J.M. 2000. Ecology and evolution of specialized seed dispersal, dormancy and germination strategies. Plant Species Biol., 15: 95-96. Baskin, J.M., Baskin, C.C. and Li, X. 2000. Taxonomy, anatomy and evolution of physical dormancy in seeds. Plant Species Biol., 15: 139-152. Cowan, C.W. and Smith, B.D. 1993. New perspectives on a wild gourd in eastern North America. J. Ethnobiol., 13: 17-54. Decker, D.S. and Wilson, H.D. 1987. Allozyme variation in the Cucurbita pepo complex: C. pepo var. ovifera vs. C. texana. Syst. Bot., 12: 263-273. Decker-Walters, D.S., Walters, T.W., Cowan, C.W. and Smith, B.D. 1993. Isozymic characterization of wild populations of Cucurbita pepo. J. Ethnobiol., 13: 55-72. Hancock, J.F., Grumet, R. and Hokanson, S.C. 1996. The opportunity for escape of engineered genes from transgenic crops. Hort. Sci., 31: 1080-1085. Kirkpatrick, K.J. and Wilson, H.D. 1988. Interspecific gene flow in Cucurbita: C. texana vs. C. pepo. Amer. J. Bot., 75: 519-527. Nienhuis, J., Lower, R.L. and Staub, J.E. 1983. Selection for improved low temperature germination in cucumber (Cucumis sativus L.). J. Amer. Soc. Hort. Sci., 108: 1040-1043. Oliver, L., Harrison, S. and McClelland, M. 1983. Germination of Texas gourd (Cucurbita texana) and its control in soybeans (Glycine max). Weed Sci., 31: 700-706. Smith, B.D., Cowan, C.W. and Hoffman, M.P. 1992. Is it an indigene or a foreigner? In: Smith, B.D. (Ed.), Rivers of Change: Essays on the Origins of Agriculture in Eastern North America. Smithsonian Institution Press, Washington, D.C., pp. 67-100. Wilson, H.D. 1990. Gene flow in squash species. Bioscience, 40: 449-455.

174 Characteristics and inheritance of a high hermaphroditic flower- bearing accession of watermelon (Citrullus lanatus)

M. Sugiyama1, K. Sugiyama1, T. Ohara1, M. Morishita2 and Y. Sakata1 1National Institute of Vegetable and Tea Science, Ano, Mie 514-2392, Japan 2National Agricultural Research Center for Hokkaido Region, Hitsujigaoka, Sapporo, Hokkaido, 062-8555, Japan

Summary

Obtaining high levels of fruit set in watermelon is a major challenge in Japan, because sex expression in this species is easily influenced by environmental conditions and by its production of fewer pistillate flowers than other cucurbitaceous vegetables such as melon or cucumber. We have developed a line of watermelon (HHF) that bears a high proportion of hermaphroditic flowers. HHF was developed by crossing the accession Red Seeded 3b, which bears a high proportion of pistillate flowers, with an accession Bei Jing C. HHF is andromonoecious and sets flowers in a ratio of about 1 staminate : 1 hermaphroditic. The ability of HHF to produce high levels of hermaphroditic flowers is controlled by incompletely dominant polygenes, and its andromonoecy is controlled by a single recessive gene. HHF should be useful in the development of high-pistillate-flower-bearing cultivars.

Keywords: Citrullus lanatus, female, flower, fruits set, hermaphroditic, inheritance, watermelon

Introduction

Watermelon (Citrullus lanatus Thunb. Matsum. et Nakai) plants produce fewer pistillate flowers than other cucurbitaceous vegetables such as melon or cucumber. Sex expression in watermelon is easily influenced by environmental conditions such as temperature and day length. Stable fruit set is required to obtain high yields and efficient cultivation. As most Japanese watermelon cultivars set flowers in a ratio of 5 staminate : 1 pistillate, increasing the production of pistillate flowers would increase the opportunities for pollination and fruit set. An investigation of multiple watermelon varieties to determine their ability to bear female flowers revealed definite differences among accessions (Su- giyama, 2001), with Red Seeded 3b and Africa 22860 bearing a high proportion of pistillate flowers. We have developed a watermelon accession, HHF, which bears more hermaphroditic flowers than common watermelon cultivars, using Red Seeded 3b as the breeding source for the high-hermaphroditic-flower-bearing trait. In this paper, we report the morphological characteristics of HHF and its mode of inheritance.

Materials and methods

Morphological characteristics of HHF The pedigree of HHF is shown in Fig. 1. HHF is a breeding line that originated from the cross Red Seeded 3b x Bei Jing C. Bei Jing C originates from China.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 175 Fujihikari TR and Miyako 3 are Japanese cultivars (Table 1). HHF and Fujihika- ri TR were sown on 3 March 2003 and transplanted into the greenhouse on 17 April. Lateral branches were pruned upon appearance. The main stems of the plants were trained vertically and one fruit per plant was allowed to set. The numbers of pistillate and hermaphroditic flowers from the 10th through the 30th nodes were counted.

Miyako 3 Fujihikari TR F F F · · · · F 1 2 3 8 (HHF) Bei Jing C F F F · · · · F 2 3 6 F 1 Red Seeded 3b

Figure 1. Pedigree of HHF.

Table 1. Morphological characteristics of HHF and the other watermelon cultivars used

Cultivars and Origin Pistillate or hermaphroditic Sex type Flesh color breeding line flower-bearing ability

Red Seeded 3b Unknown Very high Monoecious White Bei Jing C China High Monoecious Red Miyako 3 Japan High Andromonoecious Red Fujihikari TR Japan Middle Monoecious Red HHF - Extremely high Andromonoecious Red

Mode of inheritance of the high-hermaphroditic-flower-bearing trait Kleckley Sweet (P ) which sets few pistillate flowers, HHF (P ), F , F , and back- 1 2 1 2 cross progenies were used. Plants were sown on 10 May 2001 and transplanted into the greenhouse on 4 June. Lateral branches were pruned upon appearance. The main stems of the plants were trained vertically. The numbers of pistillate and hermaphroditic flowers from the 10th to the 40th nodes were counted and the sex expression of each flower was observed.

Results and discussion

Morphological characteristics of HHF The Japanese F cultivar Fujihikari TR sets flowers in the ratio of ca. 5 stamina- 1 te: 1 pistillate (Fig. 2). In contrast, HHF is andoromonoecious and sets flowers in the ratio of ca. 1 staminate : 1 hermaphroditic. A continuous hermaphroditic flower set is notable in HHF. The cultivar produces a high fruit set (Fig. 3). HHF fruits have green skin, black stripes and red flesh of about 12 Brix degrees (Table 1).

176 : Staminate flower

: Pistillate flower

: Hermaphroditic flower

Red Seeded 3b Fujihikari TR HHF

Figure 2. Patterns of sex expression along the main stem from the 10th to the 30th nodes.

Figure 3. Fruit set and mature fruits of HHF.

177 Mode of inheritance of the high-hermaphroditic-flower-bearing trait The trait of high production of hermaphroditic flowers is polygenic in HHF, as determined by tests with the F , F , and backcross progenies (Fig. 4). The segregation of sex expres- 1 2 sion in the F , F , and backcross progenies fit the expected ratio, assuming that andromono- 1 2 ecy is controlled by a single recessive gene (Table 2). Andromonoecy is recessive to mono- ecy in watermelon, as in cucumber and melon. Sex expression in the Cucurbitaceae can be altered by exogenous treatment with ethylene. The ethylene-releasing compound ethephon is particularly effective in promoting gynoecy in the monoecious melon (Cucumis melo L.) (Karchi, 1970) and cucumber (Cucumis sativus L.) (McMurray and Miller, 1968; Iwahori et al., 1969). Ethylene evolution is highly correlated with sex expression in cucumber (Rudich et al., 1972). However, in watermelon, ethephon inhibits rather than promotes pistillate flowering. The ethylene inhibitors aminoethoxyvinyl glycine (AVG) and AgNO reduce the number of 3 staminate flowers and promote hermaphroditic flowering (Christopher and Loy, 1982). We investigated the ethylene evolution of HHF and Kleckley Sweet and found no difference (data not shown). We obtained six andromonoecious plants from the BCP line in spite of the 1 expected BCP ratio of 1 monoecious : 0 andromonoecious. We have not yet determined whether 1 this result is controlled by genetic or environmental factors. In general, the formation of pistillate flowers and the fruit set in watermelon are highly sensitive to environmental conditions. However, the fruit set and high production of hermaphroditic flowers by HHF are very stable. In addition, andromonoecy is recessive to mono- ecy in watermelon. Therefore, it should be possible to develop high-pistillate-flower-bearing cultivars by crossing HHF with common parental accessions.

20 35 HHF 30 F2 Kleckley Sweet 15 25 F‚P 20 10 15 10 5 Number of plants 5 Number of plants 0 0 1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15 Total number of pistillate or hermaphroditic Total number of pistillate or hermaphroditic flowers from 10th to 40th node flowers from 10th to 40th node

40 40 35 35 BC1(P1) BC1(P2) 30 30 25 25 20 20 15 15 10

10 Number of plants Number of plants 5 5 0 0 1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15 Total number of pistillate or hermaphroditic Total number of pistillate or hermaphroditic flowers from 10th to 40th node flowers from 10th to 40th node

Figure 4. Segregation of the total number of pistillate and hermaphroditic flowers from the 10th to the 40th node in F , F , and BC progenies from the cross between 1 2 1 Kleckley Sweet (P ) and HHF (P ). 1 2

178 Table 2. Segregation of sex expression in the F , F , and BC progenies of the cross 1 2 1 between Kleckley Sweet and HHF.

Total number Sex expression c2 P

Line of plants Monoecious Andromonoecious Expected ratioZ

Kleckley Sweet (P )20 20 0 1 HHF (P )20020 2 F (P x P )20200 1 1 2 F (P x P ) 100 73 27 3 : 1 0.21 0.64 2 1 2 BCP 100 946 1 : 0 1 BCP 100 41 59 1 : 1 3.24 0.07 2

ZOn the assumption that monoecious trait is controlled by a single dominant gene.

References

Christopher, D.A. and Loy, J.B. 1982. Influence of foliarly applied growth regulators on sex expression in watermelon. J. Amer. Soc. Hort. Sci., 107: 401-404. Iwahori, S., Lyons, J.M. and Sims, W.L. 1969. Induced femaleness in cucumber by 2-chloroethanephosphonic acid. Nature, 222: 171-172. Karchi, Z. 1970. Effects of 2-chloroethanephosphonic acid on flower types and flowering sequences in muskmelon. J. Amer. Soc. Hort. Sci., 95: 515-518. McMurray, A.L. and Miller, C.H. 1968. Cucumber sex expression modified by 2-chloroethanephosphonic acid. Science, 162: 1397-1398. Rudich, J., Baker, L.R., Scott, J.W. and Sell, H.M. 1972. Ethylene evolution from cucumber plants as related to sex expression. Plant Physiol., 49: 998-999. Sugiyama, K. 2001. Studies on breeding of watermelon for female flower-bearing ability and cracking resistance. Bull. Natl. Res. Inst. Veg., Ornam. Plants & Tea Japan, 16: 265-310.

179 180 Fruit coloration in watermelon: lessons from the tomato

Y. Tadmor1, N. Katzir1, S. King2, A. Levi3, A. Davis4 and J. Hirschberg5 1Department of Vegetable Crops, Agricultural Research Organization, Newe Yaar Research Center, P. O. Box 1021, Ramat Yishay 30-095, Israel; e-mail: [email protected] 2Department of Horticultural Sciences, Texas A&M University, 2119 TAMU, College Station, TX 77843-2119, USA 3U.S. Vegetable Laboratory, U.S.D.A., A.R.S., 2875 Savannah Highway, Charleston SC 29414, USA 4South Central Agricultural Research Laboratory, U.S.D.A., A.R.S., P. O. Box 159, Lane, OK 74555, USA 5Department of Genetics, The Hebrew University of Jerusalem, Givat Ram, Jerusa- lem 91-904, Israel

Summary

The characteristic red pigmentation of watermelon and tomato fruits is determined by accumulation of the carotenoid pigment lycopene. As carotenoids are known to have health-promoting activities and watermelon can be a significant source of lycopene and other carotenoids, it is important to understand the genetic basis of fruit-specific carotenoid biosynthesis. In contrast to tomato, very little is known about the regulation of carotenoid biosynthesis during fruit development in watermelon. We analyzed carotenoids in watermelon fruits of various flesh colors and compared their carotenoid patterns to known tomato fruit-color variants. We detected genes in watermelon that appear to be equivalent to the r, t, og and B tomato genes. By comparing the fruit carotenoid biosynthetic pathways of these two unrelated species, we can contribute to the understanding of the evolution of fruit-development processes and carotenogenesis.

Keywords: Citrullus lanatus, C. colocynthis, Lycopersicon esculentum, gene orthology, carotenoids

Introduction

Carotenoids influence fruit color and health benefits (Galili et al., 2002). Tomato, Lycopersicon esculentum Mill., and the cultivated edible watermelon, Citrullus lanatus (Thunb.) Matsum. & Nakai subsp. vulgaris (Schrad. ex Eckl. & Zeyh.) Fursa, accumulate lycopene as their major fruit carotenoid (Rick, 1995; Perkins-Veazie et al., 2001). The closely related wild taxa C. lanatus subsp. lanatus and C. colocynthis (L.) Schrad. have white-fleshed fruits. Thus the genetic changes that led to the development of colored fruit probably occurred after watermelon was domesticated. Although wild taxa closely related to tomato have green fruits, the presumed ancestor of the cultivated tomato had colored fruit (Rick, 1995). Furthermore, tomato and watermelon differ in the way lycopene accumulates during fruit development. The immature tomato fruit is green and accumulates b-carotene and xanthophylls similarly to green leaf tissues (Hirschberg, 2001), while the young watermelon fruit endocarp (fruit flesh) is usually colorless and contains only trace amounts of carotenoids (our unpublished data). Moreover, carotenoid biosynthesis in tomato is ethylene-enhanced (climacteric fruit) while watermelon fruit is not climacteric. Hence, we hypothesize that the regulation of lyco-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 181 pene accumulation is different in watermelon and tomato, and that color variants of these species do not necessarily share the same carotenoid composition, genotype, and gene-expression patterns. The genetic basis of fruit-color variation in tomato and its association with carotenoid composition is well-established (Hirschberg, 2001). Variant fruit colors were assigned to specific carotenogenesis genes; the yellow fruit r results from a non-functional mutated Psy-1 (Fray and Grierson, 1993), the orange fruit tangerine (t) is due to a mutated carotenoid isomerase (CRTISO; Isaacson et al., 2002), the high d-carotene fruit of the mutant delta is caused by an over-expressing Lcy-e allele (Ronen et al., 1999), the high b-carotene fruit of the mutant Beta (B) is conferred by a dominant allele of Cyc-b, and the crimson phenotype (og, ogc) is a null allele of Cyc-B (Ronen et al., 2000). In contrast to tomato, very little is known about carotenoid biosynthesis in watermelon fruits. Yet, watermelon exhibits a wide range of fruit-flesh color variants. We have analyzed fruit carotenoids in various watermelon accessions and compared their profiles with characterized tomato mutants, in order to infer related- ness. We present here results concerning carotenoid composition of yellow-, orange-, and red-flesh watermelon accessions and compare them to known tomato fruit-color mutants.

Material and methods

Plant material The watermelon accessions analyzed were 32 open-pollinated cultivars having red, orange, yellow, or white fruit-flesh color. Four tomato fruit-color mutations, r (yellow fruit), t (orange fruit), og (intense red) and B (orange-red) as well as a wild-type tomato were also included in this study as references (Fig. 1). Plants were grown in the field at the Newe Yaar Research Center and at the Akko Experiment Station, both in northern Israel, during the summer of 2003 using standard cultural practices. Samples for carotenoid extraction were taken from at least three fruits of each accession.

Carotenoid analysis Watermelon and tomato carotenoids were extracted and fractionated according to Tadmor et al. (2000) with slight modifications. Carotenoids were extracted by grinding 0.5 g fresh fruit in hexane:acetone:ethanol (50:25:25), followed by 5 minutes saponification in 8% (w/v) KOH. The saponified material was extracted twice with hexane and then dried. The solid pellet was resuspended in 400 ml of acetonitri- le:methanol:dichloromethane (45:5:50), passed through a 0.2 micron nylon filter, and kept at room temperature in darkness for no more than 24 h before analysis. Forty ml were injected into a 2996 Waters HPLC equipped with Waters PDA detector 996, C18 Nova-Pak column (250 × 4.6 mm i.d.; 60 A°; 4 mm), and a Nova-Pak Sentry Guard cartridge (Waters, Milford, MA U.S.A.). Compounds were identified by comparison of retention times, co-injection spiking, and by comparing their UV-visible spectra with authentic standards. Quantification was performed by integrating the peak areas of the HPLC results using Millennium chromatography software (Waters).

182 Figure 1. Representative color variation of watermelon and tomato fruits.

Table 1. List of some of the accessions, their endocarp color, their major carotenoid, and the tomato mutant color equivalent

Accession Color Major carotenoid(s) Tomato equivalent

Calsweet Red Lycopene wild type Moon and Stars Red Lycopene og Malali Orange-Red Lycopene & b-carotene B NY162003 Yellow-Orange b-carotene B Orange Flesh Tendersweet Orange Pro-lycopene t Yellow Crimson Salmon yellow Pro-lycopene t Early Moonbeam Canary yellow Lutein (traces) r

Results and discussion

Representative samples of all carotenoid analyses of watermelon fruits and known tomato fruit-color variants indicated similarities between watermelon and tomato fruit- flesh colors and carotenoid content. Major carotenoids of some of the watermelon accessions and the equivalent tomato fruit-color mutants are presented in Table 1. A

183 typical red watermelon, Calsweet, contains mainly lycopene (Fig. 2, I) while the canary- yellow watermelon, Early Moonbeam, contains only trace amounts of the chloroplastic carotenoids lutein and b-carotene (Fig. 2, IV). White-fleshed watermelon did not have any detectable amount of carotenoids (data not shown). These phenotypes are analo- gous to the tomato r mutation in the Psy-1 gene (Fray and Grierson, 1993). The difference between the white and yellow phenotypes is most likely at the plastid level; the yellow fruit has carotenoid-producing plastids (chloroplasts or chromoplasts) while the white fruit lacks them. Malali showed increased levels of b-carotene at the ex- pense of lycopene (Fig. 2, III), a carotenoid profile similar to the tomato B mutant where increased expression of the chromoplast-specific lycopene b-cyclase (CYC-B) occurs during fruit maturation (Ronen et al., 2000). The yellow-orange type profile, represented by accession NY162003, has mainly b-carotene (Fig. 2, V). This phenotype looks like an extreme case of the tomato B mutation where all lycopene had been converted to b-carotene. The red Moon and Stars seems to contain lycopene with only trace amounts of b-carotene (Fig. 2, VI), unlike Calsweet that contains both lycopene (90% of total carotenoids) and b-carotene (Fig. 1, I) and thus the former may carry a mutation similar to the tomato og which is a null allele of the B gene (Ronen et al., 2000). A tangerine-type was represented by Orange Flesh Tendersweet (Figure 2, II), which has intense orange fruit flesh with poly-cis-lycopene (pro-lycopene) as its major pigment. In general, the carotenoid profile of Orange Flesh Ten- dersweet is similar to the tomato t mutant that has a mutated CRTISO (Isaacson et al., 2002). A salmon-yellow fleshed watermelon, Yellow Crimson, also had pro-lycopene as its major carotenoid, but in low amounts (Table 1).

Figure 2. HPLC chromatograms of carotenoids extracted from red (I, Calsweet), orange (II, Orange Flesh Tendersweet), orange-red (III, Malali), canary-yellow (IV, Early Moonbeam), yellow-orange (V, NY162003) and red (VI, Moon and Stars) watermelons. Carotenoids are a = lycopene; a = cis-lycopene; b = b-carotene; c = z-carotene; d = di-cis-lycopene; e = phytoene; f = phytofluene; g = neurosporene; h = lutein.

184 Henderson et al. (1998) described five fruit color phenotypes: Canary yellow, Salmon yellow, Orange, Red, and White, and defined the genetic relationships among these colors based on segregating populations. Our designation of the watermelon flesh- color variants is in accordance with the results presented therein. It seems that the tomato fruit color mutations r, t, og and B have gene orthologues in the watermelon genome. Comparison of fruit carotenoid biosynthetic pathways of species from different plant families has potential for understanding the genetics and evolution of fruit development and carotenogenesis.

Acknowledgements

We thank Ayala Meir, Yaniv Azoulai and Boris Wasserman for technical assistance. Special thanks to Erica Clair Renaud from Seeds of Change (Santa Fe, New Mexi- co, U.S.A.) for providing the watermelon seeds.

References

Fray, R.G. and Grierson, D. 1993. Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression. Plant Mol. Biol., 22: 589-602. Galili, G., Galili, S., Lewinsohn, E. and Tadmor, Y. 2002. Utilization of genetic, molecular and genomic approaches to improve the value of plant food and feeds. Critical Rev. Plant Sci., 21: 167-204. Henderson, W.R., Scott, G.H. and Wehner, T.C. 1998. Interaction of flesh color genes in watermelon. J. Hered., 89: 50-53. Hirschberg, J. 2001. Carotenoid biosynthesis in flowering plants. Curr. Opin. Plant Biol., 4: 210- 218. Isaacson, T., Ronen, G., Zamir, D. and Hirschberg, J. 2002. Cloning of tangerine from tomato reveals a carotenoid isomerase essential for production of ß-carotene and xanthophylls in plants. Plant Cell, 14: 333-342. Perkins-Veazie, P., Collins, J.K,, Pair, S.D. and Roberts, W. 2001. Lycopene content differs among red-fleshed watermelon cultivars. J. Sci. Food Agric., 81: 983-987. Rick, C.M. 1995. Tomato. In: Smartt, J. and Simmonds, N.W. (Eds.), Evolution of Crop Plants. Longman Scientific and Technical, Essex, England, U.K., pp. 452-457. Ronen, G., Cohen, M., Zamir, D. and Hirschberg, J. 1999. Regulation of carotenoid biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon cyclase is down- regulated during ripening and is elevated in the mutant Delta. Plant J., 17: 341-351. Ronen, G., Carmel-Goren, L., Zamir, D. and Hirschberg, J. 2000. An alternative pathway to ß- carotene formation in plant chromoplasts discovered by map-based cloning of Beta (B) and old-gold (og) color mutations in tomato. Proc. Natl. Acad. Sci. U.S.A., 97: 11102-11107. Tadmor, Y., Larkov, O., Meir, A., Minkoff, M., Lastochkin, E., Edelstein, M., Levin, S., Wong, J., Rocheford, T. and Lewinsohn, E. 2000. Reversed-phase high performance liquid chromatographic determination of vitamin E components in maize kernels. Phytochem. Analysis, 11: 370-374.

185 186 Scientific contributions

III. Diseases and pests, disease resistance

187 188 Some disease resistance tests in Cucumis hystrix and its progenies from interspecific hybridization with cucumber

J.F. Chen1, G. Moriarty2 and M. Jahn2 1The State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China 2Department of Plant Breeding, Cornell University, Ithaca NY, 14853, USA

Summary

Several disease screens were carried out to characerize the response of C. hystrix and its progenies derived from the interspecific hybridization to common cucurbit diseases. The results indicated the resistances to gummy stem blight, downy mildew, and three viruses, e.g. CMV-C, ZYMV, PRV existed in C. hystrix. The resistance to gummy stem blight was partially transmitted to cultivated cucumber through interspecific hybridization and backcrossing with cucumber.

Keywords: Cucumis sativus, C. hytivus, disease resistance, downy mildew, gummy stem blight, viruses, interspecific hybridization

Introduction

Cucumis hystrix Chakr. was the first wild Cucumis species found in Asia with chromosome 2n = 24 (Chen et al., 1995). Moreover, it was the first wild Cucumis relative that is sexually compatible with cultivated Cucumis species, e.g. C. sativus, 2n = 14 (Chen et al., 1997). Although the original interspecific hybrid was sterile, fertility was res- tored by doubling the chromosome number from 2n = 19 to 2n = 38 (Chen et al., 1998). This amphidiploid has been successfully backcrossed with cucumber to produce allotriploid (Chen et al., 2003a), which was further crossed with cucumber to produce Monosomic Alien Addition Lines (Chen et al., 2003b). This synthetic amphidiploid species was designated C. hytivus Chen et Kirkbride (Chen and Kirkbride, 2000) and has been characterized cytogenetically (Chen et al., 2003c). Utilization of this interspecific bridge may create unique germplasm with desirable characteristics which has great potential for cucumber improvement. Little was known, however, concerning characteristics of the wild parental species, C. hystrix, and the bridge genotype, C. hytivus. In the present paper, several disease screens were undertaken to characterize the response of C. hystrix and its progenies derived from the interspecific hybridization to common cucurbit diseases.

Materials and methods Plant materials C. hytivus (HH) used in this study were from the original collection of J.F. Chen in 1990 (Chen et al., 1994). Several cucumber (Cucumis sativus) cultivars (CC) were used

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 189 for comparison in different experiment, in which Beijing jietou and Jinlu from Nan- jing Agricultural University, China, and Marketmore 97 from Cornell University, USA. The amphidiploid species C. hytivus (HHCC) was generated by self-pollinating the primary amphidiploid CC1-33. A variety of progenies from reciprocal backcrossing between the C. hytivus and C. sativus were also used, including C1-56 representing the allotriploid (HCC, 2n = 26), HH1-9-7 representing the BC3 which set long fruits and HH1-8-57 also representing the BC3 but characterized by short fruits (CC, 2n = 14).

Resistance tests Gummy Stem Blight. Resistance evaluations were made in a field at Cornell Uni- versity using the highly virulent Didymella bryoniae isolate NY1 (Keinath et al., 1995). C. hystrix, CC3, HH1-8-57, and HH1-9-7 were evaluated in this test. Spore suspensions were applied to stem and leaf surfaces of 3 to 4 week old seedlings at 5 x 105 spore/ml using a backpack sprayer. A 72 hour inoculation period at 25°C in a mist chamber followed inoculation to encourage disease development. Disease severity was measured 21 days post inoculation using a 1 to 5 scale based on stem damage ratings (Zuniga et al., 1999) where 5 = no damage; 4 = a single lesion 1-10 mm long or coa- lesced lesions 1-20 mm long with no girdling of the stem; 3 = lesions 21 to 80 mm and/ or girdling of the stem; 2 = withered stem; 1 = dead seedling. The number of individuals falling into resistant (stem damage rating 3-5) and susceptible (stem damage rating 1-2) categories was determined. Powdery mildew. C. hystrix, CC3, C. hytivus, C1-56, HH1-9-7, Jinlu were used in this test in Nanjing Agricultural University. 24 hours before inoculation the existing spores on the cucumber leaves were dusted off to let new spores developed. The leaves were harvested from the field for inoculation. Six replicates for each treatment. The inoculated leaves were maintained in the sealed Petri dish for two day prior to the second inoculation. Scores were taken in 12 days. Downy mildew. C. hystrix, CC3, C. hytivus, C1-56, HH1-9-7, Jinlu were used in this test in Nanjing Agricultural University. Two days before inoculation, the infected leaves were harvested and cultured fro 2448 hours under moisture. The spores were used to make inoculum (10000/ml). The health leaves were harvested and co- cultured with the inoculum in a 9×9 cm Petri dish. Scores were taken in 8 days. Viruses. C. hystrix was evaluated for resistance to four viruses: CMV-C, WMV-2, PRV, and ZYMV in the field at Cornell University. 12 plants were mechanically inoculated with each virus separately. The inoculated plants were transplanted to the field for evaluation throughout the growing season. Nematodes. Nematode resistance tests were carried out both in Nanjing Agricultu- ral University of China and at the N.Y. Agricultural Expt. Station, Cornell University, USA. In the test in China, C. hystrix, C. hytivus, CC3, and Jinlu were used to test resistance to Meloidogyne incognita race 3, M. hapla, and M. javanica. In the test in USA, C. hystrix, HH1-8-57, and Marketmore 97 were tested for resistance to M. incognita and M. hapla. Nematode inoculum was obtained by collecting eggs with 0.5% NaOCl as described by Hussey and Barker (1973). Seeds were germinated in vermiculite in a greenhouse, and seedlings at the two- leaf-stage were transplanted into 4-inch pots filled with sand. Plantlets from in vitro

190 culture were also transplanted into that medium at the same time. Plants were fertilized weekly with a commercial nutrient formulation (N: P: K = 20: 20: 20), and kept in a greenhouse at 28°C. Four days after planting, two holes 2-3 cm in depth and 0.6 cm in diameter. were made with a bamboo stick around the plant roots. One ml of inoculum containing ~2500 eggs was pipetted into each hole (~5000 eggs per plant). Plants were placed on a table in a completely randomized design. Seven weeks after inoculation, the root systems were carefully washed free of sand, and evaluated for number of galls under a stereoscopic microscope at 10x magnification. The number of galls for each root system was counted, and a gall index was calculated using a 0-5 scale, with 0 = no galls; 1=1 - 2; 2 = 3-10; 3 = 11-30; 4 = 31-100; and 5 > 100 galls.

abc Figure 1. Gummy Stem Blight resistance shown in C. hystrix (a) and HH1-8-57 (c) in the field. Segregation could be seen in the HH1-8-57 (b).

Table 1. Single plant ratings for Gummy Stem Blight resistance

Plants C. hystrix CC3 HH1-8-57 HH1-9-7 Stem Leave R/S Stem Leave R/S Stem Leave R/S Stem Leave R/S

1L43R11R21S11S 1R43R11R11S11S 2L43R11R11S11S 2R43R11R11S11S 3L43R11R11S11S 3R43R11R11S11S 4L4 3R---4 3R11S 4R4 3R---11S11S 5L 43 R - - - 1 1 S 1 1 S 5R 43 R - - - 1 1 S 3 2 R/S 6L 43 R - - - 1 1 S 1 1 S 6R 43 R - - - 3 2 R/S 1 1 S

Results Gummy Stem Blight. Resistance was found in C. hystrix plants in the field (Fig. 1a). Few symptoms were found on stem, and slightly more on the leaves. There was no segregation observed among the plants. The plants derived from the interspecific

191 hybridization set a large number of fruits, especially in the HH1-8-57 (Fig. 1c), however, segregation for resistance was observed. In 12 HH1-8-57 plants tested, three were found to show strong stem resistance and one also showed good leaf ratings (Fig. 1b). In the 12 HH1-9-7 plants tested, only one plant was found resistant. All the CC3 plants tested were susceptible and died without setting any fruits. The ratings for the plants tested shown in Table 1. Powdery mildew. Similar to the cultivar Jinlu, C. hystrix is also susceptible to powdery mildew. The CC3 is resistant. C. hytivus, the interspecific hybrid between C. hystrix and CC3 showed similar resistance to CC3, however, only moderate resistance was apparent in the backcross progenies, C1-56 and HH1-9-7. Data are summarized in Table 1.

Table 2. Results of powdery mildew resistance in C. hystrix, and some other cultigens

Genotypes Replicates Disease 1 2 3 4 5 6 Index

C. hystrix II III IV III II II 70.8 CC3 I I I 0 0 0 12.5 C. hytivus 0 I II 0 0 I 16.5 C1-56 IV III IV IV IV III 87.5 HH1-9-7 I I II II I 0 29.1 Jinlu III III III II IV II 66.7

Grade was made according to the ratio of infected area/leaf area. 0: infected area 0; I: <1/4; II: 1/4<1/2; III: 1/2<3/4; IV: 3/4<1.

Downy mildew. The disease index in C. hystrix was 5.3, indicating it is highly resistant to downy mildew. This resistance was partially transmitted to the C. hytivus, and the progenies from backcross. Compared to the susceptible cucumber cultivar Jinlu, all the materials derived from this interspecific hybridization possess at least moderate resistance (Table 2).

Figure 2. C. hystrix plants showing resistance to PRV, CMV-C, and ZYMV (a) and susceptibility to WMV-2 (b).

192 Table 3. Results of downy mildew resistance test

Genotypes Replicates Disease 1 2 3 4 5 6 Index

C. hystrix 0 0 0 I 0 I 5.3 CC3 0 0 I I I 0 16.7 C. hytivus I II I I I I 38.9 C1-56 0 I I II I I 33.3 HH1-9-7 0 II 0 0 II I 27.8 Jinlu I I III III II I 61.0

0: infected area 0; I: 0<20%; II: 20<50%; III: >50%.

Viruses: C. hystrix plants generally suffered from the WMV-2 inoculation (Fig. 2b), but showed stronger resistance to PRV and moderate resistance to CMV-C and ZYMV (Fig. 2a).

Nematodes. Slight resistance was found to M. incognita, M. hapla, and M. javanica in C. hystrix in the test in China according to the gall index (Table 3). The average galls per root was 58 for M. incognita, 60 for M. hapla, and 43 for M. javanica, respectively; fewer than the others where the count were 150 or above (data not shown). No significant transmission of resistance was observed in C. hytivus. But in the test with M. javanica, the average galls per root in the C. hytivus was 97, indicating a possible transmission from C. hystrix to C. hytivus.

Table 4. Root gall ratings after inoculated with nematodes in NAU, China

Nematode Genotypes Number of Gall index inoculation 0 1 2 3 4 5

M. incognita C. hystrix 6 000060 CC36 000006 C. hytivus 6 000006 Jinlu 6 0 0 0 0 0 6 M. hapla C. hystrix 6 000060 CC36 000006 C. hytivus 6 000006 Jinlu 6 0 0 0 0 0 6 M. javanica C. hystrix 10000190 CC310000019 C. hytivus 10000046 Jinlu 10 0 0 0 0 0 10

Gall index: 0 = no gall; 1 = 1-2 galls/root; 2 = 3-10 galls/root; 3 = 11-30 galls/root; 4 = 31-100 galls/root; 5 = more than 100 galls/root.

193 The results from Cornell largely agree with the results obtained in China. But based on the results of gall index and the egg count, the partial transmission of resistance to M. hapla was observed in HH1-8-57, the progenies from selfing the BC3 (Table 4).

Table 5. Average egg counts after inoculation with M. hapla in Cornell, USA

Genotypes Rgs Root wt Egg count Total Eggs/g (g) 5/100ml eggs root

C. hystrix 3.1 1.60 0.14 20 1.8 HH1-8-57 3.5 1.90 12.70 2300 232.0 Marketmore 5.3 1.35 28.00 5060 336.0

Discussion

Although the results obtained in this paper were preliminary, the information provided will be useful to guide further genetic research and breeding. Gummy stem blight is the second most important disease of cucumber in North Carolina (Wehner and Shetty, 2000). In Europe, gummy stem blight is a serious disease of greenhouse cucumber. Previous studies indicated that the estimates of genetic gain for resistance ranged from low to moderate (Amand and Wehner, 2001). Some other related Cucumis species, e.g. C. myriocarpus Naud., C. zeyheri Sond., C. anguria L., were also found to be resistant (Wehner and Amand, 1993) but those species are cross-incompatible with either cucumber or melon. The resistance we found in C. hystrix is likely to be different from that identified in cucumber or melon. The resistance was partially transmitted to cucumber through the interspecific hybridization, and the resistance in one of the HH1- 8-57 plants was quite high. We dont know how this resistance inherited yet, but the gummy stem blight resistance in cucumber would likely be improved by combining this resistance genes with the existing most resistance cucumbers. Downy mildew is an important disease in most cucumber production areas worldwide, and it can be devastating to European growers (Van Vliet and Meysing, 1974). Reportedly, the different local isolates may represent different races of the pathogen (Shetty et al., 2002). Meanwhile, due to differences in rating, the downy mildew resistance in C. hystrix described in this paper could not be directly compared to the existing resistance cucumbers. Although resistant genes (dm-1, dm-2, and dm-3) were identified, they do not provide adequate control of disease damage (Horejsi et al., 2000), and higher levels of resistance are needed to avoid yield losses (Wehner and Shetty, 1997). An extensive evaluation indicated that no plant introduction accessions were found to be more resistant than the most resistant cultivars or breeding lines (Wehner et al., 1997). Therefore, the resistance found in C. hystrix is potentially significant and could be used as a new source of resistance to serve breeding programs. All the viruses tested in this paper are transmitted by aphids and have had great economic impact on fresh cucumber market, as well as pickling industry. CMV-C and ZYMV not only transmitted through mechanical, but also seeds (Provvidenti, 1993), which makes them even harder to control. Resistance to CMV-C is conferred by three

194 partial dominant factors (Risser et al., 1977). While resistance to PRV and ZYMV are monogenically recessive (Pitrat, 1990), the resistance to WMV was monogenically dominant (Robinson et al., 1976). Additional resources for resistance to viruses in cucumber could strengthen the durability of the monogenic resistances already known.

Acknowledgements

George Abawi for assistance with the nematode screens and the Tang Family Foundation for support of J.F. Chens fellowship at Cornell University. This research was also partially supported by National Hi-Tech R & D Program Nos. 2001AA241123, 2002AA241251, and 2002AA244021.

References

Amand, P.C.St. and Wehner, T.C. 2001. Generation means analysis of leaf and stem resistance to gummy stem blight in cucumber. J. Amer. Soc. Hort. Sci., 126: 95-99. Chen, J.F., Adelberg, J.W., Staub, J.E., Skorupska, H.T. and Rhodes, B.B. 1998. A new synthetic amphidiploid in Cucumis from C. sativus × C. hystrix F1 interspecific hybrid. In: McCreight, J.D. (Ed.), Cucubitaceae ´98. ASHS Press, Alexandria, VA, USA, pp. 336-339. Chen, J.F., Isshiki, S., Tashiro, Y. and Miyazaki, S. 1995. Studies on a wild cucumber from China (Cucumis hystrix Chakr.). I. Genetic distance between C. hystrix and two cultivated Cucumis species (C. sativus L. and C. melo L.) based on isozyme analysis. J. Jpn. Soc. Hort. Sci., 64 (suppl. 2): 264-265. Chen, J.F. and Kirkbride, J.H. Jr. 2000. A new synthetic species Cucumis (Cucurbitaceae) from interspecific hybridization and chromosome doubling. Brittonia, 52: 315-319. Chen, J.F., Luo, X.D., Qian, Ch.T., Jahn, M.M., Staub, J.E., Zhuang, F.Y., Lou, Q.F. and Ren, G. 2003b. Cucumis monosomic alien addition lines: morphological, cytological and genotypic analysis. Theor. Appl. Genet. (in press). Chen, J.F., Luo, X.D., Staub, J.E., Jahn, M.M., Qian, Ch.T., Zhuang, F.Y. and Ren, G. 2003a. An allotriploid derived from a amphidiploid × diploid mating in Cucumis I: production, micro- propagation and verification. Euphytica, 131: 235-241. Chen, J.F., Staub, J.E., Qian, Ch.T., Jiang, J.M., Luo, X.D. and Zhuang, F.Y. 2003c. Reproduc- tion and cytogenetic characterization of interspecific hybrids derived from Cucumis hystrix Chakr. × C. sativus L. Theor. Appl. Genet., 106: 688-695. Chen, J.F., Staub, J.E., Tashiro, Y. and Miyazaki, S. 1997. Successful interspecific hybridization between Cucumis sativus L. and C. hystrix Chakr. Euphytica, 96: 413-419. Chen, J.F., Zhang, S.L. and Zhang, X.G. 1994. The xishuangbanna gourd (Cucumis sativus var. xishuangbannesis Qi et Yuan), a traditionally cultivated plant of the Hanai people, xishuangbanna, Yunnan, China. Cucurbit Genet. Coop. Rep., 17: 18-20. Horejsi, T., Staub, J.E. and Thomas, C. 2000. Linkage of random amplified polymorphic DNA markers to downy mildew resistance in cucumber (Cucumis sativus L.). Euphytica, 115: 105-113. Hussey, R.S. and Barker, K.R. 1973. A comparison of methods of collecting inocula of Meloido- gyne spp. including a new technique. Plant Dis. Rep., 57: 1025-1028. Keinath, A.P., Farnham, M.W. and Zitter, T.A. 1995. Morphological, pathological, and genetic differentiation of Didymella bryoniae and Phoma spp. isolated from cucurbits. Phytopatholo- gy, 85: 364-369. Pitrat, M. 1990. Gene list for Cucumis melo L. Cucurbit Genet. Coop. Rep., 13: 58-70. Provvidenti, R. 1993. Resistance to viral diseases of cucurbits. In: Kyle, M.M. (Ed.), Resistance to Viral Diseases of Vegetables. Timber Press, Inc., Portland, OR 97225, USA. Risser, G., Pitrat, M. and Rode, J.C. 1977. Etude de la resistance du melon (Cucumis melo L.) au virus de la mosaique du concombre. Annal. Amel. Plantes, 27: 509-522.

195 Robinson, R.W., Munger, H.M., Whitaker, T.W. and Bohn, G.W. 1976. Genes of the Cucurbitace- ae. HortScience, 11: 554-568. Shetty, N.V., Wehner, T.C., Thomas, C.E., Doruchowski, R.W. and Shetty, K.P.V. 2002. Evidence for downy mildew races in cucumber tested in Asia, Europe, and North America. Sci. Hort., 94: 231-239. Van Vliet, G.J.A. and Meysing W.D. 1974. Inheritance of resistance to Pseudoperonospora cubensis Rost and in cucumber (Cucumis sativus L.). Euphytica, 23: 251-255. Wehner, T.C. and Amand, P.C.St. 1993. Field tests for cucumber resistance to gummy stem blight in North Carolina. HortScience, 28: 327-329. Wehner, T.C. and Shetty, N.V. 2000. Screen the cucumber germplasm collection for resistance to gummy stem blight in North Carolina field tests. HortScience, 35: 1132-1140. Zuniga, T.L., Jantz, J.P., Zitter, T.A. and Jahn, M.K. 1999. Monogenic dominant resistance to gummy stem blight in two melon (Cucumis melo) accessions. Plant Dis., 83: 1105-1107.

196 Searching for resistance to cucumber vein yellowing virus in Cucumis melo

T. Montoro1, S. Sánchez-Campos2, R. Camero1, C.F. Marco1, P. Corella2 and M.L. Gómez-Guillamón1 1Experimental Station La Mayora, CSIC, 29750-Algarrobo, Málaga, Spain 2Rijk Zwaan Ibérica, Paraje El Mamí, Ctra. Viator, 04120-La Cañada, Almería, Spain

Summary

A total of 235 melon accessions originating from different geographic areas have been evaluated against CVYV, during 2002 and 2003, using mechanical inoculation. In early autumn 2003, a total of 93 additional melon accessions have been evaluated in the greenhouse using Bemisia tabaci as CVYV vector. Autumn conditions were more favourable for the plants to show virus symptoms of infection. Neither resistance nor tolerance to CVYV has been found in the Cucumis melo accessions tested.

Keywords: melon, Bemisia tabaci, CVYV, artificial inoculations, tolerance

Introduction

The Cucumber vein yellowing virus was described by Cohen and Nitzany (1960) in Israel in 1960. Later on it was also observed in Jordan (Al Musa et al., 1985), Turkey (Yimaz et al., 1989), in Sudan (Desbiez et al., 2001), and recently it has been described affecting protected melon, watermelon, and cucumber crops in Almería (Cuadrado et al., 2001). Since fall 2001, the virus has been spread drastically being the causal agent of serious economic losses observed in protected cucurbits crops in Southeast Spain. The virus belongs to the Ipomovirus genus of the Potyviridae family (Lecoq et al., 2000) attending to their biological, cytological, and molecular features (Jones, 2003). It is transmitted by the sweet potato whitefly Bemisia tabaci (Gennadius), in a semiper- sistant manner (Mansour and Musa, 1993). In laboratory conditions it is also mechanically transmitted. Its host range seems to be restricted to cucurbits (Mansour and Musa, 1993), although Janssen et al. (2002) reported that the virus also hosts other species belonging to Convolvulaceae (Convolvulus arvensis L.), Malvaceae (Malva parviflora L.) and Asteraceae (Sonchus oleraceus, S. asper and S. tenerrimus) families. The symptomatology in melons is characterised by a yellowing of the leaf vein area, becoming systemic, with chlorosis of the youngest leaves. In extreme infections, plant stunting and fruit damages have been described. Affected fruits show chlorotic spots on their skin and/or internal necrosis. The high virus transmission efficiency of B. tabaci and its difficult control make necessary searching for genetic resistance/tolerance to the virus. There is not either total or useful resistance to CVYV described in melon so far and the behaviour of any wild Cucumis species against the virus is unknown. Once the mechanical inoculation techniques have been optimised and an accurate molecular diagnostic method has been

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 197 established in our laboratory, we started an evaluation against CVYV of the melon collection maintained at the Experimental Station La Mayora, CSIC, Spain.

Material and methods

Two-hundred and thirty five melon accessions originating from different geographical areas have been evaluated for CVYV resistance/tolerance during 2002 and 2003 (Table 1). The melon commercial cultivar Rochet has been used as susceptible control in all the experiments.

Table 1. Accessions of C. melo evaluated against CVYV

Origin Number of accessions Mechanical inoculation Mass inoculation B. tabaci

Europe Central 15 9 Mediterranean 138 54 East 8 - Middle East 13 - Asia 18 24 North America 30 1 Africa 13 5

Total 235 93

The virus isolate CVYV-A1LM was obtained by the authors from a commercial cucurbit crop in Almería (Spain ) and was maintained on melon plants of Rochet cultivar.

Mechanical inoculations Five to 10 plants per accession have been mechanically inoculated with the virus and two plants per accession without any contact to the virus were used as control. The plants at the fully expanded cotyledons growth stage were inoculated mechanically by rubbing active carbon and carborundum-dusted on the cotyledons. The inoculum was made with extracts from infected foliar tissue homogenized in 0.01 M K HPO 2 4 buffer (pH 7). Mock-inoculated and non inoculated controls were included routinely. To ensure efficient virus infection, plants were reinoculated five days after the first inoculation. Presence or absence of virus symptoms was recorded for each plant ten days after the second inoculation. Both plants without symptoms and plants with unclear symptoms were tested for the virus presence by molecular hybridization using a RNA digoxigenin probe containing a cDNA insert of 1,5 Kb (Marco et al., 2003). Plants were grown in an insect-proof glass-house and the mechanical inoculations of the accessions were made in different batches due to space availability reasons in

198 the glasshouse. In each batch, 25 melon accessions and 8-10 plants/accession were tested. When the percentage of infected/inoculated plant in one experimental test was less than 50%, that accession was included in the next experiment. Temperatures ranged from 15 to 38°C in all experiments during spring (from February to May) and autumn (from September to December).

B. tabaci-mediated inoculations Accessions with non-infected plants after inoculations in two separated tests were also inoculated using B. tabaci as a virus vector. The virus transmission was carried out by using leaf cages containing 20 and 60 whiteflies that were allowed to feed on the inoculum source for 24 hours and then transferred to healthy test plants for 24 hours (Mansour and Musa, 1993). Whiteflies were then sprayed and plants were protected with insect-proof muslin net and remained in the greenhouse waiting for symptoms. Presence or absence of virus symptoms was recorded for each plant once a week during three weeks after inoculation. In early autumn 2003, a total of 93 additional melon accessions were evaluated in the greenhouse using B. tabaci as CVYV vector (Table 1). Two separated experiments were carried out in Almería and Málaga. In Almería, 20 healthy plants of 41 accessions at the two-true- leaf stage were inoculated using mass inoculation inside an insect-proof screened box. In Málaga, 11 plants of 52 accessions at the two-true- leaf stage were planted in a greenhouse where infected plants and high populations of B. tabaci had been maintained. Plants were observed for presence or absence of virus symptoms during eight weeks.

Results and discussion Mechanical inoculations All inoculated plants of the susceptible control accession Rochet showed systemic infection twelve days after their mechanical inoculation with CVYV. Plants showed strong interveinal chlorosis in the second true leaf, affecting drastically the plant tips. (Fig. 1).

Figure 1. Melon plant with severe symptoms of CVYV.

199 All plants of the 235 melon accessions mechanically inoculated showed systemic response after 12-15 days after inoculation. Two melon accessions, C-29 (Casaba Golden Beauty) and C-867 (Ambrus-Fele) showed a heterogeneus response to the virus in previous experiments (Marco et al., 2003). These accessions showed a clear susceptibility to CVYV when inoculated in a second experiment. Three melon accessions, C-30 (Honey Dew Green Flesh), C-32 (Tam Dew Impro- ved), and C-41 (Freemans cucumber) showed a low percentage of infection (10-40%) in two different experiments.

B. tabaci-mediated inoculations Those accessions tended to escape of the infection in two experiments were tested using B. tabaci as virus vector. Plants started to show symptoms of infection seven days after inoculation, but their response was generalized twenty-one days after inoculations (Table 2). Twenty and sixty viruliferus whiteflies were used in two separated experiments and, according to Mansour and Musa (1993), when using 20 whiteflies, plants became infected in a percentage higher than 55%. Although no significant differences have been observed, inoculations using 60 whiteflies per plant seem to be more appropriate. However, the inoculation method should be revised because, in any case, the 100% of the plants were infected even using 60 whiteflies. Similar results were also observed by Harpaz and Cohen (1965) and Mansour and Musa (1993) attributed to the low virus concentration in the infected plants.

Table 2. Evaluation against CVYV of three melon accessions using B. tabaci as a vector

Accession N.whiteflies/plant Infect/Inocul* Infect/Inocul* Infect/Inocul* 7dpi 14dpi 21dpi

C-30 20 9/10 9/10 9/10 60 8/10 8/10 8/10 C-32 20 5/9 6/9 6/9 60 6/10 8/10 8/10 C-41 20 2/10 6/10 6/10 60 6/10 9/10 9/10 Rochet 20 2/10 6/10 6/10 60 7/10 8/10 8/10

* Infected plants/inoculated plants at 7, 14 and 21 days post-inoculation (dpi).

When CVYV inoculations were carried out in a greenhouse using mass inoculation of B. tabaci, all inoculated plants showed clear symptoms of virus infection 48-55 days after inoculations. Plants started showing symptoms a week after inoculation and some of those accessions showed a delay in the symptom expression. Most of them (90-95%) became infected between 30 and 35 days after viruliferus whiteflies were allowed to infect them (data not showed). Mechanical inoculation is an efficient method to test melons against CVYV, although the environmental conditions should be revised to obtain uniformity in the

200 experiments. The experiments were carried out in glasshouse in spring and autumn seasons. Although any relation between susceptibility/resistant response and temperature maintained in each separate experiment has been observed, the uniformity in the response appeared to be higher when inoculations were done in early autumn. B. tabaci showed to be a very efficient CVYV vector in our experiments, as previously described by Cohen and Nitzany (1960) and Mansour and Musa (1993). In our experiments, the systemic response in the inoculated plants, using leaf cages, takes the same period of time as when mechanical inoculation is used but requires more work and facilities to be carried out. However, it seems to be an accurate method when variability in the response of the plants is observed. Although the plants without symptoms likely escaped the infection, the behaviour against CVYV of the accessions C-30, C- 32, and C-41 should be considered and compared with the control. Thus, some plants of Rochet escaped also the infection when using B. tabaci as vector, but whereas all the plants of Rochet showed symptoms of infection after mechanical inoculations, the three melon accessions, C-30, C-32 and C-41 showed a low rate of infection (10- 40%) in two different experiments using mechanical inoculation. It should be interesting to examine the existence of variability for resistance to CVYV in those three accessions. When using mass inoculation, either in insect-proof cages or in greenhouses, a delay in the symptom expression have been observed, since the observation period should be extended to 30-35 days until most of the plants became infected. However, this test method could be very useful when high populations of B. tabaci and high level of CVYV in the plot is expected. Until now no useful resistance to CVYV has been described in C. melo. Marco et al. (2003) found resistance in C. prophetarum, one accession of C. africanus, and two of C. dipsaceus. However, strong sexual barriers between those wild species and melons make very difficult the use of these found resistances.

Acknowledgements

This work was partially supported by the Research Projects GENRES-108 and AGL2002- 04554-CO2-02 .

References

Al Musa, A.M., Qusus, S.J. and Mansour, A.N. 1985. Cucumber vein yellowing virus on cucumber in Jordan. Plant Dis., 69: 361. Cohen, S. and Nitzany, F.E. 1960. A whitefly-transmitted virus of cucurbits in Israel. Phytopa- thol. Mediter., 1: 44-46 (abstract). Cuadrado, I.M., Janssen, D., Velasco, L., Ruiz, L. and Segundo, E. 2001. First report of cucumber vein yellowing virus in Spain. Plant Dis., 85: 336. Desbiez, C., Delecolle, B., Wipf-Scheibel, C. and Lecoq, H. 2001. Le Cucumber vein yellowing virus, virus transmis par l´aleurode Bemisia tabaci, est un member des Ipomovirus, Potyviri- dae. 8th Rencontres de Virologie Végétale´, Aussois, France. Harpaz, I. and Cohen, S. 1965. Semipersistent relationship between Cucumber vein yellowing virus (CVYV) and its vector the tobacco whitefly Bemisia tabaci. Phytopathol. Z., 54: 240-248

201 Janssen, D., Ruiz, L., Velasco, L. and Cuadrado, I.M. 2002. Non-cucurbitaceous weed species shown to be natural hosts of cucumber vein yellowing virus in south eastern Spain. New Di- sease Reports, Vol. 5, January-July 2002. Jones, D.R. 2003. Plant viruses transmitted by whiteflies. Europ. J. Plant Pathol., 109: 195-219. Lecoq, H., Desbiez, C., Delecolle, B., Cohen, S. and Mansour, A. 2000. Cytological and molecular evidence that the whitefly-transmitted Cucumber vein yellowing virus is a tentative member of the family Potyviridae. J. Gen. Virol., 81: 2289-2293. Mansour, A. and Musa, A.A. 1993. Cucumber vein yellowing virus: host range and virus vector relationships. J. Phytopathology, 137: 73-78. Marco, C.F., Aranda, M.A., Montoro, T. and Gómez-Guillamón, M.L. 2003. Evaluation of several accessions and wild relatives of Cucumis melo against CVYV. Cucurbit Genet. Coop. Rep., 26: (in press) Yimaz, M.A., Ozaslan, M. and Ozaslan, D. 1989. Cucumber vein yellowing virus in Cucurbita- ceae in Turkey. Plant Dis., 73: 610.

202 Behaviour of Cucumis melo Cantaloup Haogen against melon necrotic spot virus (MNSV)

C. Mallor, J.M. Álvarez and M. Luis-Arteaga Centro de Investigación y Tecnología Agroalimentaria de Aragón, Apartado 727, 50080 Zaragoza, Spain; e-mail: [email protected]

Summary

The behaviour of Cucumis melo Cantaloup Haogen when inoculated with melon necrotic spot virus (MNSV) was studied. After cotyledon mechanical inoculation C. Haogen plants remained symptomless when maintained at 25°C, however at 25°C day / 18°C night they began to show some local necrotic symptoms in a few plants that became more frequent when the temperature decreased to 20°C. Nevertheless, when this variety was inoculated on the cotyledons and first leaf, all plants developed necrotic local lesions at any temperature. The genetic study of the absence of local lesions when plants were inoculated at 25°C showed that this may be a polygenic character. This hypothesis may be supported by the fact that a huge variability in number of local lesions per plant was observed. When C. Haogen was inoculated using the vector Olpidium bornovanus no symptoms at all were observed on the foliage of the plants. This results may support the presence in C. Haogen of some kind of resistance different to those described up to now.

Keywords: melon, virus resistance, temperature effects, Olpidium bornovanus, virus transmission, genetic control

Introduction

Melon necrotic spot virus (MNSV) has been found causing important economic losses on melon in several parts of the world (Hibi and Furuki, 1985; Tomlinson and Thomas, 1986; Avgelis, 1990; Matsuo et al., 1991), including the Mediterranean Spanish areas (Martínez de Salinas et al., 1987; Cuadrado et al., 1993). The only genetic resistance to MNSV described until 2003 is controlled by a single recessive gene nsv; plants that carry the nsv gene in homozygous condition will remain symptomless when mechanically inoculated with MNSV (Coudriet et al., 1981). This resistance is effective against all the virus strains tested (González Garza et al., 1979; Coudriet et al., 1981; Pitrat and Lecoq, 1984; Maestro, 1992), except to a recently described one, which is able to overcome it (Díaz et al., 2002). Mallor et al. (2003) found that after mechanical inoculation of the virus on the cotyledons, C. melo cv. Doublon developed local necrotic lesions but failed to develop any systemic symptoms. This behaviour was kept under different environmental conditions and the virus could not be detected by ELISA analysis except in the local lesion tissue. This resistant behaviour of Doublon appeared to be controlled by two dominant genes Mnr1 and Mnr2 (Mallor et al., in press). When Doublon plants were inoculated with MNSV by using the natural vector Olpidium bornovanus, the virus was detected on the roots and on the base of the hypocotyls but not on the aerial part of the plants (Mallor et al., 2002a).

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 203 During the screening of some C. melo accessions for MNSV resistance, using two virus isolates differing in their ability to infect melon plants carrying the nsv gene, we found that Cantaloup Haogen was the only genotype that remained symptomless after cotyledon mechanical inoculation (Mallor et al., 2002b). Nevertheless, in subsequent inoculations on the cotyledons and the first leaf, all plants developed necrotic local lesions on the first leaf. The aim of this work was the study of this peculiar behaviour of C. Haogen against MNSV.

Material and methods

Plant material The accessions provided by the Banco de Germoplasma de Hortícolas (Vegetable Germplasm Bank) of Zaragoza (Spain) Cantaloup Haogen, PMR-5 that carries the resistance nsv gene, and ANC-42 a Spanish autochthonous line susceptible to MNSV were used. Seeds were pregerminated at 30°C for three days. Then, they were transplanted into 8 x 8 x 8 cm plastic pots filled with a standard melon substrat. Plants were placed in a growth chamber at 27°C, 60% RH and 16 h photoperiod (300 mmol.m-2.s-1) until they reached the optimal mechanical inoculation stage.

Viral material and mechanical inoculation procedure The MNSV isolate used was M-8-85, a common strain collected from melon crops in Almería area (South-eastern Spain) during 1985. For mechanical inoculation 1 g of inoculated melon cotyledons exhibiting necrotic local lesions was harvested 4 or 5 days after inoculation and ground in 4 ml of 0.03 M Na HPO solution containing 2 4 0.2% sodium-diethyldithiocarbamate (DIECA). The extracted juice was mixed with 400-mesh carborundum (0.375 g/ml) and activated charcoal (0.375 g/ml) and then rubbed on the cotyledons and first leaf of melon seedlings.

Temperature conditions The experiment was performed in three growth chambers at 25°C, 25°C day/18°C night, and 20°C temperature, 60% RH and 16 h photoperiod (300 mmol.m-2.s-1). Twenty plants of each of the above accessions were mechanically inoculated and placed into the growth chambers at the mentioned temperatures. The inoculation was done at cotyledon and first leaf stages on the cotyledons and first leaf.

Genetic studies F , F and backcross populations were derived from C. melo cv. C. Haogen and 1 2 the melon lines ANC-42 and PMR-5. Seeds were sown in pots and placed in a growth chamber at 25°C, 16 h photoperiod (300 mmol.m-2.s-1) and 60% RH. 100-140 plants from each segregating population and 14 from the F progenies were mechanically 1 inoculated at cotyledonar stage. Every experiment included 14 plants of each parental genotype. Plants were observed for 30 days, and presence or absence of local and systemic symptoms were recorded.

204 Olpidium inoculation Six plants per accession were grown on sterilized sand and placed in a growth chamber at 25°C. Then they were inoculated by irrigating them with 5 ml/plant of a mixture of a 3x106 zoospores/ml suspension of O. bornovanus and an extract of MNSV infected plants made by grinding 0.015 g of symptomatic plant tissue into 1 ml of 0.03 M Na HPO solution containing 0.2% sodium-diethyldithiocarbamate (1 plant extract : 2 4 9 spore suspension) (Tomlinson and Thomas, 1986; Campbell and Lecoq, 1996). The O. bornovanus isolate used was M-202 originated from Israel and provided by Dr. Julio Gómez from CIFA (Almería, Spain). Plants were observed during thirty days and presence or absence of symptoms were recorded. Thirty days after inoculation the roots of each plant were microscopically observed for O. bornovanus presence and ELISA analysed for MNSV presence. ELISA test was performed with polyclonal MNSV antisera from Loewe Phytodiagnostica (Otterfing, Germany) according to manufacturers recommendation. Samples were considered as positive when their absorbance values exceeded three times the values observed in healthy plants used as negative controls.

Results and discussion

Temperature effects The development of local lesions on cotyledons and first leave was different in the three varieties used (Table 1). PMR-5 did not show any local symptom at any of the tested temperatures. At 25°C only the susceptible accession ANC-42 developed local necrotic lesions that appeared four days after inoculation on the inoculated cotyledons of all the plants. However at 25°C / 18°C (day / night) C. Haogen began to show some local necrotic symptoms in a few plants that became more frequent when the temperature was 20°C. Lecoq and Pitrat (1982) reported that in natural conditions symptoms of MNSV infection are influenced by environmental conditions, appearance being favoured by low temperature and short photoperiod. We have observed that in artificial conditions low temperatures favoured the appearance of systemic symptoms in MNSV mechanically inoculated melon plants (Mallor et al., 2003). Results shown here may indicated that temperatures also affect the development of the local necrotic lesions. Coudriet et al. (1981) and Pitrat and Lecoq (1984) reported that when melon plants were mechanically inoculated with MNSV on the cotyledons and no necrotic local lesions reaction was observed, they were resistant to MNSV and homozygous for the nsv gene. According to our results this does not hold true for every genotype and temperature, and it is necessary to determined the optimal experimental environmental conditions for testing melon accessions when looking for presence or absence of the nsv gene.

Genetic studies The inoculation of both F generations (C. Haogen x PMR-5 and C. Haogen x ANC- 1 42), always produced many necrotic local lesions on the inoculated cotyledons (Table 2). According to this results it appears that the failure in developing necrotic local lesions after MNSV inoculation of the cotyledons of C. Haogen seems to be a recessive character. From the study of the F s and BCs generations it appeared that the 2

205 segregations found did not fit with a simple hypothesis (regulation by 1-3 genes). So we may be facing a polygenic character. This hypothesis may be supported by the fact that a huge variability in number of local lesions per plant was observed, from plants without any lesion to plants that showed a great amount of lesions (80-100 lesions), passing by plants that only developed 1, 2 or a few local lesions.

Olpidium inoculation All the ANC-42 plants showed necrotic streaks along the hypocotyls, and 4 of them died. The fungus was microscopically observed on the roots, and the virus was detected in the streaks and roots by mechanical backinoculation on cucumber. C. Haogen and PMR-5 behaved alike, no symptoms at all were observed on the foliage of the plants, but the fungus was microscopically observed and the virus was detected by ELISA on the roots of all plants. In addition to the resistance controlled by nsv (Gonzalez-Garza et al., 1979; Coudriet et al., 1981), another resistance characterized by the presence of local necrotic lesions on inoculated cotyledons but failure in developing systemic symptoms (Mallor et al., 2003) controlled by two dominant genes (Mallor et al., in press) has been described in Doublon. It seems that C. Haogen has some kind of resistance to MNSV that is different to that controlled by nsv (C. Haogen develops local lesions when inoculated on first leaf), and also to that described by Mallor et al. (2003) (C. Haogen does not develop local lesions when inoculated on cotyledons). Until now we have studied the development of local symptoms (temperature influence and genetic control) of C. Haogen against MNSV mechanically inoculated as well as its behaviour when inoculated with virus and Olpidium bornovanus. Fur- ther investigations on the resistance to systemic symptoms development should be carried out and, in that case, the genetic control determination of the resistance.

Table 1. Number of plants showing local and systemic symptoms after inoculation on the cotyledons and first leaf of 20 plants of different varieties of melon with the MNSV isolate M-8-85 and incubation at different temperatures

20°C 25°C / 18°C 25°C

local symptoms systemic local symptoms systemic local symptoms systemic

nl nl symptoms nl nl symptoms nl nl symptoms Genotype cotyle- first cotyle- first cotyle- first dons leaf dons leaf dons leaf

C. Haogen 14(1) 20 0 3 (1) 20 0 0 20 0 ANC-42 20(1) 20 18 2 0 (1) 20 1 20 20 0 PMR-5 0(1) 00 0(1) 00 00 0

nl = necrotic lesions (1) Very few local lesions (13 lesions per cotyledon)

206 Table 2. Response of Cantaloup Haogen, PMR-5, ANC-42 and the progenies derived from their crosses to inoculation with MNSV (isolate M-8-85) at the cotyledon stage

Parent or progeny Number of plants Parent or progeny Number of plants no few (1) no few (1) lesions lesions lesions lesions lesions lesions Cantaloup Haogen 140 0 Cantaloup Haogen 140 0 PMR-5 140 0 ANC-42 0 0 14 F 0014F 0014 1 1 C. Haogen x PMR-5 (C. Haogen x ANC-42) F 0014F 0014 1 1 PMR-5 x C. Haogen (ANC-42 X C. Haogen) F 58 31 32 F 10 26 89 2 2 (C. Haogen x PMR-5) (C. Haogen x ANC-42) BC 1429 60 BC 20 46 43 1 1 (C. Haogen x PMR-5) ((C. Haogen x ANC-42) x C. Haogen x C. Haogen) BC 85 3 54 BC 3 2 129 2 2 (C. Haogen x PMR-5) ((C. Haogen x ANC-42) x PMR-5 x ANC-42)

(1) : 1-3 lesions / cotyledon

References

Avgelis, A.D. 1990. Melon necrotic spot virus in plastic houses on the island of Crete. Acta Hort., 287: 349-354. Campbell, R.N. and Lecoq, H. 1996. Melon necrotic spot virus: transmission by Olpidium bornovanus and vector assisted seed transmission in melon. Proc. VI Eucarpia Meeting on Cucurbit Genetics and Breeding. Málaga, 28-30 Mayo, pp. 313-321. Coudriet, D.L., Kishaba, A.N. and Bohn, G.W. 1981. Inheritance of resistance to muskmelon necrotic spot virus in a melon aphid-resistant breeding line of muskmelon. J. Amer. Soc. Hort. Sci., 106: 789-791. Cuadrado, I.M., Gomez, J. and Moreno, P. 1993. El virus de las manchas necróticas del melón (MNSV) en Almería. I. Importancia del MNSV como causa de la muerte súbita del melón. Bol. San. Veg. Plagas, 19: 93-106. Díaz, J.A., Nieto, C., Moriones, E. and Aranda, M.A. 2002. Spanish Melon necrotic spot virus isolate overcomes the resistance conferred by the recessive nsv gene of melon. Plant Dis., 86: 694. Gonzalez-Garza, R., Gumpf, D.J., Kishaba, A.N. and Bohn, G.W. 1979. Identification, seed transmission, and host range pathogenicity of a California isolate of melon necrotic spot virus. Phytopathology, 69: 340-345. Hibi, T. and Furuki, I. 1985. Melon necrotic spot virus. AAB Descriptions of Plant Viruses. No. 302, 4pp. Lecoq, H. and Pitrat, M. 1982. Note sur les virus de cucurbitacées presents en France. I.N.R.A.- C.R.A. dAvignon-Monfavet, 29 pp. Maestro, C. 1992. Résistance du melon aux virus. Interaction avec les pucerons vecteurs. Analyse génétique sur des lignées haplodiploides. Thèse Docteur Sciences Université dAix-Marseille, 134 pp.

207 Mallor, C., Álvarez, J.M. and Luis-Arteaga, M. 2002a. Transmisión artificial de MNSV a melón mediante inoculación mecánica y con el hongo vector Olpidium bornovanus. XI Congreso So- ciedad Española de Fitopatología. Almería,14-18 Octubre, p. 170. Mallor, C., Álvarez, J.M. and Luis-Arteaga, M. 2002b. Nueva resistencia en Cucumis melo L. al virus de las manchas necróticas del melón (MNSV). Actas de Horticultura, 34: 207-214 Mallor, C., Álvarez, J.M. and Luis-Arteaga, M. 2003. A resistance to systemic symptom expression of melon necrotic spot virus in melon. J. Amer. Soc. Hort. Sci., 128: 541-547. Mallor, C., Álvarez, J.M. and Luis-Arteaga, M. 2003. Inheritance of resistance to systemic symptom expression of melon necrotic spot virus (MNSV) in Cucumis melo L. Doublon. Euphy- tica (in press) Martínez de Salinas, J., Fraile, A., Solís, I. and García-Arenal, F. 1987. Characterization of a Spanish isolate of melon necrotic spot virus. Proc. 7th Congress Mediterr. Phytopath. Union. Granada, 142. Matsuo, K., Kameya Iwaki, M. and Ota, T. 1991. Two new strains of melon necrotic spot virus. Ann. Phytopath. Soc. Jap., 57: 558-567. Pitrat, M. and Lecoq, H. 1984. A rapid method to test muskmelons for several virus resistances. EUCARPIA. Cucumis and melons 84 IIIrd Meeting 2-5 July, 1984, Plovdiv, Bulgaria, pp. 104-107. Tomlinson, J.A. and Thomas, B.J. 1986. Studies on melon necrotic spot virus disease of cucumber and on the control of the fungus vector (Olpidium radicale). Ann. appl. Biol., 108: 71-80.

208 A physical map covering the Nsv locus in melon

J. Garcia-Mas1, M. Morales1,3, H. van Leeuwen1, A. Monfort1, P. Puigdomenech1, P. Arús1, C. Nieto2,3, M.A. Aranda2, C. Dogimont3, G. Orjeda4, M. Caboche4 and A. Ben- dahmane4 1Laboratori CSIC-IRTA de Genetica Molecular Vegetal, Departament de Genetica Vegetal IRTA, Carretera de Cabrils s/n, 08348 Cabrils (Barcelona), and Departament de Genetica Molecular, IBMB-CSIC, Jordi Girona 18-36, 08034 Barcelona, Spain; e-mail: [email protected] 2Centro de Edafología y Biología Aplicada del Segura (CEBAS)-CSIC, Apdo. corre- os 164, 30100 Espinardo, Murcia,Spain 3Unité de Génétique et dAmélioration des Fruits et Légumes, Domaine St Maurice, BP 94, 84 143 Montfavet Cedex, France 4INRA-URGV, 2 Rue Gaston Crémieux CP 5708, 91057 Evry Cedex, France

Summary

The recessive allele of the Nsv gene confers resistance to Melon necrotic spot virus (MNSV). A physical map spanning the nsv locus has been developed based on a high-resolution genetic map of the corresponding region located in melon linkage group 11. The BAC contig contains 15 clones covering a genetic distance of 1.2 cM and a physical distance of approximately 0.4 Mb. Here we show that BAC 1-21-10 contains the Nsv gene and further experiments have been started in order to identify the gene responsible of the Nsv genotype.

Keywords: Cucumis melo, Melon necrotic spot virus (MNSV), resistance, Nsv, positional cloning

Introduction

Melon necrotic spot virus (MNSV; family Tombusviridae, genus Carmovirus) is a single-stranded RNA virus that infects cucurbits grown under glasshouse. MNSV spread can be controlled using the genetic resistance conferred by the recessive allele of the Nsv gene, present in the Korean accession PI 161375 (Coudriet et al., 1981). This gene confers resistance against all known strains of the virus except the recently described strain MNSV-264 that is able to overcome the resistance (Díaz et al., 2003). Chimeric mutants between the MNSV-264 isolate, which overcomes the Nsv resistance, and the MNSV-Ma5 isolate which does not, allowed to establish that the virulence determinant of MNSV-264 on Nsv genotypes is located in the 3UTR of the virus sequence (Aranda et al., unpublished). The Nsv gene has been mapped to linkage group 11 of the melon genetic map described by Oliver et al. (2001) and also in the melon genetic map reported by Périn et al. (2002). More recently, several AFLP and RAPD markers closely linked to the resistance gene have been reported (Morales et al., 2002). Here we describe the recent progress towards the map-based cloning of the Nsv gene with the construction of a high-resolution map of the region and the identification of a BAC clone that contains the Nsv gene.

Plant material A melon (Cucumis melo L.) F segregating population was used for the high-reso- 2 lution mapping. The Piel de Sapo line T111 (PS) and the resistant Korean accession PI 161375 (PI) were crossed to obtain 408 F individuals that were used for the gene- 2 tic mapping. The F recombinant plants identified between two flanking markers were 2 selfed in order to obtain F seed for the MNSV resistance progeny test. 3

MNSV resistance assays Isolate M-8-85 of MNSV was propagated on the susceptible melon line PS by mechanical inoculation of the cotyledons and the infected plants were kept as a source of virus. One gram of tissue discs from cotyledons containing fresh MNSV lesions was ground in 4 ml of 0.03 M Na HPO , pH 8,5 that contained 0.2 % DIECA and ac- 2 4 tivated charcoal (75 mg·ml-1). This extract was spread over the cotyledons of 2 week- old melon plants. The cotyledons were dusted previously with carborundum or in some cases it was added to the extract before inoculation. Finally the inoculated cotyledons were washed with water to remove excess of carborundum and charcoal. Plants were visually scored as susceptible 3-5 days after the mechanical inoculation if they showed MNSV necrotic spots on the cotyledons. If no symptoms were detected 10 days after the inoculation plants were scored as resistant to MNSV (Pitrat and Lecoq, 1984). The MNSV test was performed on the cotyledons of 20 F seedlings of each of 3 46 F recombinants. 2

BAC isolation BAC clones positive for the genetic markers (Morales et al., 2002) were obtained after PCR amplification of our melon BAC library (van Leeuwen et al., 2003). The library contains 23040 clones that are arrayed in sixty 384-well plates. A pool of DNA is available for each plate and six DNA pools of 10 plates are also available (superpools). A first PCR reaction in the 6 superpools identifies a group of plates with positive BACs, and a second PCR reaction in the individual pools identifies the positive plate. PCR analysis in each row and column of the plate yields the individual positive BAC clone. BACs were end-sequenced using the ABI Prism BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) in a ABI Prism 377 genetic analyser. The sequences obtained were used to design specific primers that amplified the corresponding BAC end region.

BAC end mapping Markers based on BAC ends were amplified in the parental lines PI and PS. The PCR products were sequenced and the sequences were aligned using the GCG package (Genetic Computer Group, Madison, WI) and SNPs and insertion-deletions (indels) were detected (Morales et al., 2004). The newly discovered SNPs were mapped in the recombinant individuals of the Nsv region using either CAPS markers (Konieczny and Ausubel, 1993) or SNaPshot (Applied Biosystems, Foster City, CA).

210 Results and discussion

In order to increase the resolution of the map reported in Morales et al. (2002), 408 F plants from the cross PS x PI were analysed with two flanking markers, the 2 AFLP M29 and the RAPD D08, that were previously transformed into CAPS markers for an efficient screening. Ninety-one individuals with a recombination event between the flanking markers were identified (Fig.1). AFLP marker M132 was positioned in this interval at 26 recombinants from M29 and 65 recombinants from D08. The Nsv gene was mapped in this interval using only 42 of the 91 recombinants, as we did not get F seed from all F recombinant plants. Nsv was mapped at 7 recombinants from 3 2 M29 and 5 from M132. M29 and M132 markers were used to screen a melon BAC library obtained from a resistant double haploid line derived from the PI x PS cross (van Leeuwen et al., 2003). BAC 38B12, positive for M132, was used to obtain new markers from its end sequences. BAC end 38B12u was mapped closer to the Nsv gene than M132 and used to characterise new BAC clones in this region. The new BAC clone ends were sequenced, PCR markers were developed from these sequences, and the new markers were positioned in the BAC contig. Again, the closest BAC end to Nsv was positioned in the genetic map and a new walking step was performed. After several walking rounds, BAC end 52K20sp6 from clone 52K20 was shown to cosegregate with Nsv and BAC end 1L3 from clone 1-21-10 was separated from Nsv by two recombination events in the M29 side. BAC clone 1-21-10 was isolated from a BAC library obtained from the susceptible line WMR29 (Bendahmane et al., unpublished). Here we show that BAC

Figure 1. High-resolution genetic map and BAC contig obtained for the Nsv region in melon linkage group 11. BAC clones shown in the contig are not in scale. Smaller boxes represent other BAC clones physically mapped in the contig. Genetic distances are expressed as recombinant individuals. In red, recombination events found in the interval M29/M132/D08. In blue, recombination events found in the M29/M132 interval with MNSV tested individuals.

211 1-21-10 physically contains the nsv gene as both BAC ends are genetically separated from the gene in the high-resolution map. Further work involves sequencing the 100 kb 1-21-10 BAC clone and identifying the candidate gene responsible for the Nsv resistance. At the same time two other segregating populations derived from the cross PI 161375 (R) x Védrantais (S), 200 recombinant inbred lines (Périn et al., 2002) and 2700 BC individuals are under analysis with the same markers to confirm the genetic and physical data with a higher genetic resolution. Besides, in order to verify that 1-21-10 BAC clone contains the Nsv gene we will use a complementation strategy based on a transient expression system based on Agrobacterium-mediated infiltration (Bendahmane et al., 2000) of both the candidate gene and the MNSV strains MNSV-264 and MNSV-Ma5.

Acknowledgements

This work has been supported in part with funds from the project 2FD97-0286- C02 of the Spanish Ministry of Science and Technology.

References

Bendahmane, A., Querci, M., Kanyuka, K. and Baulcombe, D. 2000. Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato. Plant J., 21: 73-81. Coudriet, D.L., Kishaba, A.N. and Bohn, G.W. 1981. Inheritance of resistance to muskmelon necrotic spot virus in a melon aphid-resistant breeding line of muskmelon. J. Amer. Soc. Hort. Sci., 106: 789-791. Díaz, J.A., Bernal, J.J, Moriones, E. and Aranda, M.A. 2003. Nucleotide sequence and infectious transcripts from a full-length cDNA clone of the carmovirus Melon necrotic spot virus. Arch. Virol., 148: 599-607. Konieczny, A. and Ausubel, F. 1993. A procedure for mapping Arabidopsis mutations using co- dominant ecotype-specific PCR-based markers. Plant J., 4: 403-410. Morales, M., Luís-Arteaga, M., Álvarez, J.M., Dolcet-Sanjuan, R., Monfort, A., Arús, P. and Garcia- Mas, J. 2002. Marker saturation of the region flanking the gene Nsv conferring resistance to the melon necrotic spot Carmovirus (MNSV) in melon. J. Amer. Soc. Hort. Sci., 127: 540-544. Morales, M., Roig, E., Monforte, A.J., Arús, P. and Garcia-Mas, J. 2004. Single nucleotide polymorphism discovery from ESTs in melon. Genome (in press). Oliver, M., Garcia-Mas, J., Cardús, M., Pueyo, N., López-Sesé, A.I., Arroyo, M., Gómez-Pania- gua, H., Arús, P. and Vicente M.C. 2001. Construction of a reference linkage map for melon. Genome, 44: 836-845. Périn, C., Hagen, L., de Conto, V., Katzir, N., Danin-Poleg, Y., Portnoy, V., Baudracco-Arnas, S., Chadoeuf, J., Dogimont, C. and Pitrat, M. 2002. A reference map of Cucumis melo based on two recombinant inbred line populations. Theor. Appl. Genet., 104: 1017-1034. Pitrat, M. and Lecoq. H. 1984. A rapid method to test muskmelon for several virus resistances. IIIrd Eucarpia Meeting on Cucumber and Melons, Plovdiv (Bulgaria), 2-5 July, pp. 104- 107. Van Leeuwen, H., Monfort, A., Zhang, H-B. and Puigdomenech, P. 2003. Identification and characterisation of a melon genomic region containing a resistance gene cluster from a constructed BAC library. Microcolinearity between Cucumis melo and Arabidopsis thaliana. Plant Mol. Biol., 51: 703-718.

212 Detection of resistant melons to the Indonesian isolate of KGMMV

B.S. Daryono1,2, S. Somowiyarjo3 and K.T. Natsuaki1 1Laboratory of Tropical Plant Protection, Graduate School of Agriculture, Tokyo University of Agriculture, Setagaya-Ku, Tokyo 156-8502, Japan 2Laboratory of Genetics, Faculty of Biology, Gadjah Mada University, Yogyakarta 55281, Indonesia; e-mail: [email protected] 3Laboratory of Plant Pathology, Faculty of Agriculture, Gadjah Mada University, Yogyakarta 55281, Indonesia

Summary

Source of genetic resistance to an Indonesian isolate of melon infecting Kyuri green mottle mosaic virus (KGMMV-YM) in thirty melon cultivars was screened by artificial inoculation of the virus. The level of resistance to KGMMV-YM was examined by a combination of symptoms observation and enzyme-linked immunosorbent assay (ELISA). The results revealed that cultivars Mawatauri and Kohimeuri which were Japanese origin were resistance to KGMMV-YM, while other cultivars tested were clearly susceptible.

Keywords: Cucumis melo, Kyuri green mottle mosaic virus, inoculation, symptoms, ELISA

Introduction

Several viruses have been reported infecting cucurbit plants in Indonesia such as: Cucumber mosaic virus (CMV), Papaya ring spot virus (PRSV-W), Zucchini yellow mosaic virus (ZYMV), and Watermelon mosaic virus (WMV) (Somowiyarjo et al., 1993). Presently, a new virus infecting melon in Indonesia was identified as Kyuri green mottle mosaic virus (Daryono et al., 2003a). KGMMV belongs to the genus Tobamovirus and it was first reported in Japan described as cucumber strain of Cucumber green mottle mosaic virus (Inouye et al., 1967; Tan et al., 2000). KGMMV is a serious disease agent of cucurbit crops and causing significant economical loses in Japan and Korea (Tan et al., 2000; Yoon et al., 2001). The use of genetic resistance against this virus has the possibility to be an effective control strategy (Khertapal et al., 1998). In this study, several related local melon cultivars were screened and evaluated for source of genetic resistance to KGMMV-YM by artificial inoculation. Furthermore, the levels of resistance to virus accumulation in melon leaf tissues were evaluated using a combination of symptom observation and ELISA.

Material and methods Melon genotypes, virus maintenance and inoculation procedures Thirty Asian melon cultivars were used in this study. Seeds of each cultivar were then planted in plastic pots in growth chambers under continuous illumination (8000

Serological detection Upper leaves were harvested from each plant and weighted. Three to five plants were sampled for each cultivar. Analysis of samples for the presence of KGMMV-YM involved ELISA using standard sandwich method as described by Clark and Adams (1977). Antiserum and alkaline phosphatase enzyme conjugate of KGMMV were obtained from Agdia Inc., USA. To directly compare absorbance values among plates, a positive sample of KGMMV-YM and a healthy melon were included in ELISA analysis. The average of absorbance values of three wells for each sample was used to evaluate virus infection. A sample was considered positive for KGMMV infection when the ELISA absorbance value was two times greater than the average absorbance value of healthy control tissue.

A B Figure 1. Symptoms comparison of KGMMV-YM on melon cultivar Vakharman (A) and Mawatauri (B).

214 Table 1. Melon cultivars used in this study and their reactions against KGMMV-YM

Cultivars Country origin Symptom1) ELISA2) Reactions3)

Blewah Bhisma Indonesia M, D 1.929±0.037 S Timunsuri-Jakarta Indonesia M, D 1.865±0.007 S Timunsuri-Samarinda Indonesia M, D 1.854±0.011 S Blewah Sragen Indonesia M, CL 1.818±0.006 S Tembikai Susu Malaysia M 1.699±0.016 S Sunnet 858 Thailand M, D 1.782±0.025 S Dua Gang Tay Vietnam M, CL 1.668±0.018 S Dua Hoang Kim Vietnam M, D 1.663±0.033 S Dua An Tiem 95 Vietnam M, CL 1.956±0.003 S Yamatouri Japan M, CL 1.847±0.047 S Mawatauri Japan CL 0.146±0.002 R Miyamauri Japan M, D 1.702±0.062 S Kohimeuri Japan 0 0.173±0.014R Mi Tang Ting China M 2.030±0.096 S Shinjong China M, D 1.748±0.044 S Vakharman Turkmenistan M, D 1.813±0.018 S PAK 0010035 Pakistan M, D 2.178±0.012 S PAK 0010538 Pakistan M 2.278±0.052 S OU 627 a Laos M, CL 0.261±0.049 S Bali 16 a Indonesia M 1.709±0.052 S PI 200817 Myanmar M, CL 0.287±0.012 S PI 116738 India M 2.120±0.038 S PI 210077 India M 2.250±0.016 S Ames 20947 a India M 2.128±0.056 S PI 210542 India M 2.394±0.041 S OU 641 a Nepal M 2.328±0.084S PI 125976 Afghanistan M, D 2.293±0.073 S PI 230185 Iran M, D 2.255±0.094S PI 435290 Iraq M, D 2.333±0.057 S PI 169379 Turkey M 2.287±0.054S Healthy (Bhisma) Indonesia 0.147±0.001 Positive control Indonesia 2.132±0.032

1) Observed in upper leaves: 0=No symptoms, M=Mosaic, D= Leaf deformation, CL=Chlorotic; 2) Mean ELISA values of upper leaves ±SD based on O.D. at 405 nm 14 days after inoculation; 3) R: Resistant, S: Susceptible; a accession number in the Laboratory of Plant Breeding, Faculty of Agriculture, Oka- yama University, Japan, and provided by Dr. K.Kato.

215 Results and discussion

Symptoms observation Twenty six of 30 melon cultivars inoculated with KGMMV-YM were homogenous in their response to KGMMV-YM inoculation and showed systemic symptoms that included systemic chlorosis, mottling or speckling of leaves and deformed leaves (Fig. 1). Symptoms appeared by 7 to 20 days after inoculation in all the cultivars except OU 627 and PI 200817, in which the mosaic symptoms appeared 24 days after inoculation (data not shown). On the other hand, chlorotic local lesions appeared on Mawatauri by 26 days after inoculation, whereas Kohimeuri did not show any symptoms by 30 days after inoculation (Table 1).

Serological detection KGMMV-YM was detected with variable titers in upper leaves with high titers (>1.6) of twenty six cultivars tested, indicating susceptible to KGMMV-YM. In contrast, the virus could not be detected in upper leaves of cultivars Mawatauri and Kohimeuri. However, KGMMV-YM was only detected in the upper leaves of OU 627 and PI 200817 with low titers (<0.3). This result revealed that cultivars Mawatauri and Kohimeuri are resistant to KGMMV-YM (Table 1), while OU 627 which originated from Nepal and PI 200817 which originated from Myanmar are semi-tolerant. Mawatauri and Kohimeuri are Oriental melons (C. melo L. var. makuwa) and these cultivars have been reported resistant to CMV (Hirai and Amemiya, 1989; Daryono et al., 2003b). In addition, two RAPD markers linked to CMV-B2 resistant melons showed in resistant Mawatauri (Daryono and Natsuaki, 2002). As Mawatauri and Kohimeuri are resistant to KGMMV-YM, these cultivars will be used as a donor of resistance in melon breeding and in the study of the inheritance of resistance to KGMMV.

Acknowledgement

The authors thank Dr. Kenji Kato (Faculty of Agriculture, Okayama University, Japan) for supporting and providing some melon seeds.

References

Clark, M.F. and Adams, A.N. 1977. Characteristics of the micro plate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J.Gen.Virol., 34: 475-483. Daryono, B.S. and Natsuaki, K.T. 2002. Application of random amplified polymorphic DNA markers for detection of resistant cultivars of melon (Cucumis melo L.) against cucurbit viruses. Acta Hort., 588: 321-329. Daryono, B.S., Somowiyarjo, S. and Natsuaki, K.T. 2003a. Characterization of Kyuri green mottle mosaic virus infecting melon in Indonesia. Jap. J. Phytopathol., 69: 334. (Abstract) Daryono, B.S., Somowiyarjo, S. and Natsuaki, K.T. 2003b. New source of resistance to Cucum- ber mosaic virus in melon. SABRAO J. Breed. Genet., 35: 19-26. Hirai, S. and Amemiya, Y. 1989. Studies on the resistance of melon cultivars to Cucumber mosaic virus (I) virus multiplication in leaves or mesophyll protoplasts from a susceptible and a resistant cultivars. Ann. Phytopath. Soc. Jap., 55: 458-465.

216 Inouye, T., Inouye, N., Asatani, M. and Mitsuhata, K. 1967. Studies on Cucumber green mottle mosaic virus in Japan. Nogaku Kenkyu, 51: 175-186. (in Japanese) Khertapal, R.K., Maisonneuve, B., Maury, Y., Calhoun, B., Dinant, S., Lecoq, H. and Varma, A. 1998. Breeding for resistance to plant viruses. In: Hadidi, A., Khertapal, R.K. and Koganeza- wa, H. (Eds.), Plant Virus Disease Control. APS Press, St. Paul, MN, pp.14-32. Somowiyarjo, S., Sako, N. and Tomaru, K. 1993. The use of dot immunobinding assay for detecting cucurbit viruses in Yogyakarta. In: Triharso, and Maeda, E. (Ed.), Production of Virus- free Tropical Crops. NODAI Center for International Programs, Tokyo University of Agricul- ture, Tokyo, pp. 3-11. Tan, S.H., Nishiguchi, M., Murata, M. and Motoyoshi, F. 2000. The genome structure of kyuri green mottle mosaic tobamovirus and its comparison with that of cucumber green mottle mosaic tobamovirus. Arch Virol., 45: 1067-1079. Yoon, J.Y., Min, B. E., Choi, J.K. and Ryu, K.H. 2001. Completion of nucleotide sequence and generation of highly infectious transcript to cucurbits from full-length cDNA clone of Kyuri green mottle mosaic virus. Arch. Virol., 146: 2085-2096.

217 218 Progress in breeding melon for resistance to lettuce infectious yellows virus

J.D. McCreight U.S. Department of Agriculture, Agricultural Research Service, 1636 E. Alisal St., Salinas, California 93905, USA

Summary

Resistance to lettuce infectious yellows closterovirus (LIYV) is being transferred from PI 313970 to western U.S. shipping type orange flesh cantaloupe. Although resistance is inherited as a single dominant gene, uncertainty of symptom expression requires use of ELISA assays and serial transfers to indicator plants in order to reduce false negative and false positive segregants in each backcross generation. Straight backcrossing of selected putative resistant individuals has been carried through the fourth backcross: Top Mark (BC ), PMR 5 (BC ), and breeding line AR 5 (BC and BC ). Selection has 1 2 3 4 also been made for resistance to powdery mildew race 2U.S. found in PMR 5, AR 5 and PI 313970.

Keywords: Cucumis melo, source of resistance, LIYV, inheritance, backcrossing, selection, powdery mildew resistance

Introduction Lettuce infectious yellows closterovirus (LIYV), transmitted by the A biotype of sweetpotato whitefly, Bemisia tabaci Genn. Sweetpotato whitefly biotype A seriously affected melons (Cucumis melo L.) grown for fall harvest (October to December) in the lower elevation desert areas of Arizona and California in the U.S.A. from about 1981 through 1990 ( Duffus and Flock, 1982; Duffus et al., 1986; Duffus, 1995; Wis- ler et al., 1998). LIYV was first observed in 1981 in Imperial Valley, California on lettuce (Duffus and Flock, 1982; Duffus et al., 1986). Melon was also adversely affected by LIYV although the yellowing symptoms were not obvious until late in plant development after fruit-set. Symptoms on lettuce and melon are characterized by interveinal yellowing and eventual brittleness of the older leaves (Fig. 1), and are similar to those produced by beet pseudo-yellows virus (Duffus, 1965) and cucurbit yellow stunting disorder virus (Celix et al., 1996). Symptoms appear first in the basal (crown) leaves and progress acropetally. Fall melons (generally planted in July-Au- gust) were a major source of LIYV for the winter lettuce crop (planted September- November) in the southwest United States. Resistance to LIYV in melon would reduce losses in the fall melon crop as well as in the larger and more valuable winter lettuce crop planted in surrounding fields.

Genetics of resistance

Resistance to LIYV in melon PI 313970 is conditioned by a single dominant gene (McCreight, 2000a). Despite the simple inheritance of this resistance, selection pro-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 219 gress has been slowed by several factors. First, the sudden and virtually complete displacement of sweetpotato whitefly biotype A by sweetpotato whitefly biotype B (synonymous with silverleaf whitefly, Bemisia argentifolii Bellows & Perring) in 1991 in the lower elevation deserts of Arizona and California brought an abrupt end to the LIYV epidemic (Brown and Costa, 1992). The B biotype is a very poor vector of LIYV (Duffus, 1995) and LIYV essentially disappeared from California and Arizona. This sudden change in whitefly biotype eliminated the use of uniformly and naturally infected field tests for selection of resistance to LIYV. Selection had to be done, therefore, using controlled inoculations in greenhouses. Second, relatively large numbers of biotype A sweetpotato whiteflies are needed per test plant (3 40) to approach 100% infection of susceptible segregants. Third, foliar symptoms of LIYV in melon appear 5 to 8 weeks post-inoculation at which time plants in greenhouses are large and may be crowded and infected with powdery mildew, and senescence of older leaves may confound symptom expression. Uncertainty of symptom expression in greenhouse tests necessitates use of ELISA assays of inoculated plants, and serial transfers using sweetpotato whiteflies from inoculated test plants to LIYV-indicator hosts for verification of infection in order to reduce false negative and false positive segregants (McCreight, 1998).

Melon breeding for LIYV resistance Despite the apparent disappearance of LIYV from the lower desert areas, breeding for resistance was initiated because of the adverse effect that LIYV had on melon and lettuce production in the affected areas. Although LIYV disappeared from Imperial Valley and Arizona it may re-occur to again cause losses to melon and lettuce production. Moreover, the USDA-ARS station in Salinas was uniquely positioned to carry out this work. LIYV was discovered and characterized by virologists at the station, and the virus, its vector, antisera, and indicator hosts were maintained by them and familiar to this melon breeding project. Transfer of resistance to LIYV began with crosses of progeny 90625, which had been derived from PI 313970 (McCreight, 2000b), with three susceptible melons adapted to the desert southwest U.S.: Top Mark and breeding lines AR 5 (McCreight et al., 1984) and PMR Honeydew (McCreight et al., 1987). The F progenies were challen- 1 ged with LIYV-inoculation to verify the dominant nature of resistance in PI 313970 (McCreight, 2000a); all plants were asymptomatic and virus-free as determined by ELISA (McCreight, 1998) prior to backcrossing to their respective susceptible parents to produce the BC generation, which was then evaluated in a greenhouse test for 1 resistance to LIYV. The BC generation segregated after correction in a good fit to 1 the expected 1 resistant: 1 susceptible ratio. The BC data were corrected for esca- 1 pes of the susceptible control plants (usually Top Mark) by proportionally adjusting (usually downward) the number of putative resistant plants in the backcross family. For example, if three of 10 inoculated susceptible control plants were asymptomatic and virus-free as determined by ELISA and serial transfers to an indicator host, the number of putative resistant segregants in a backcross family was reduced by 30% (3/ 10) for chi-square analysis. It should be noted that all of the putative resistant plants were backcrossed to produce the next backcross generation; none were discarded.

220 Beginning with the BC generation, the AR 5 cross was emphasized, i.e., the Top 2 Mark and PMR Honeydew crosses were not continued past the BC generation, due 2 to the resources required each generation and combine powdery mildew resistances from AR 5 and PI 313970. The particular line being carried forward originated from a Top Mark cross; PMR 5 was used as the first recurrent susceptible parent to produce the BC ; AR 5 was used to produce the BC and BC generations (Fig. 2). Pow- 2 3 4 dery mildew resistance in AR 5 has not been fully characterized; it may have Pm-1 and Pm-2 from PMR 5 (Bohn and Whitaker, 1963) and one of several genes from PI 414723 (McCreight et al., 1987). PI 313970 has one gene for resistance to race 1 and two genes for resistance to race 2 (McCreight, 2003).

Figure 1. LIYV-symptomatic leaf in a greenhouse test.

F Top Mark x PI 313970 (90625) 1

BC Top Mark 1

BC PMR 5 2

BC AR 5 3

BC AR 5 4

Figure 2. Progress in the development of LIYV-resistant melon using PI 313970 as the source of resistance.

221 References

Bohn, G.W. and Whitaker, T.W. 1963. Genetics of resistance to powdery mildew race two in muskmelon. Phytopathology, 54: 587-591. Brown, J.K. and Costa, H.S. 1992. First report of whitefly-associated squash silverleaf disorder of Cucurbita in Arizona and of white streaking disorder of Brassica species in Arizona and California. Plant Dis., 76: 426. Celix, A., Lopez-Sese, A., Almarza, N., Gomez-Guillamon, M.L. and Rodriguez-Cerezo, E. 1996. Characterization of cucurbit yellow stunting disorder virus, a Bemisia tabaci transmitted closterovirus. Phytopathology, 86: 1370-1376. Duffus, J.E. 1965. Beet pseudo-yellows virus, transmitted by the greenhouse whitefly (Trialeuro- des vaporariorum). Phytopathology, 55: 450-453. Duffus, J.E. 1995. Whitefly transmitted yellowing viruses of the Cucurbitaceae. In: Lester, G.E. and Dunlap, J.R. (Eds.), Cucurbitaceae 94: Evaluation and Enhancement of Cucurbit Germ- plasm, November 1-4, 1994, South Padre Island, Texas. Gateway Printing and Office Supply, Edinburg, Texas, pp. 12-16. Duffus, J.E. and Flock, R.A. 1982. Whitefly-transmitted disease complex of the desert southwest. Calif. Agr., 36: 4-6. Duffus, J.E., Larsen, R.C. and Liu, H.Y. 1986. Lettuce infectious yellows virusa new type of whitefly-transmitted virus. Phytopathology, 76: 97-100. McCreight, J.D. 1998. Breeding melons for resistance to lettuce infectious yellows virus. In: McCreight, J.D. (Ed.), Cucurbitaceae 98: Evaluation and enhancement of cucurbit germplasm. ASHS Press, Alexandria, Va, pp. 241-247. McCreight, J.D. 2000a. Inheritance of resistance to lettuce infectious yellows virus in melon. HortScience, 35: 1118-1120. McCreight, J.D. 2000b. Molecular and phenotypic variation in melon PI 313970. In: Katzir, N. and Paris, H. (Eds.), Proceedings of 7th EUCARPIA meeting on Cucurbit Genetics and Bree- ding, March 19-23, 2000. Maale Ha Hamisha, Israel. Acta Hort., 510: 235-239. McCreight, J.D. 2003. Genes for resistance to powdery mildew races 1 and 2U.S. in melon PI 313970. HortScience, 38: 591-594. McCreight, J.D., Bohn, G.W. and Whitaker, T.W. 1987. PMR honeydew muskmelon. HortScience, 22: 177. McCreight, J.D., Kishaba, A.N. and Bohn, G.W. 1984. AR Hales Best Jumbo, AR 5, and AR Topmark, melon aphid-resistant muskmelon breeding lines. HortScience, 19: 309-310. McCreight, J.D., Pitrat, M., Thomas, C.E., Kishaba, A.N. and Bohn, G.W. 1987. Powdery mildew resistance genes in muskmelon. J. Amer. Soc. Hort. Sci., 112: 156-160. Wisler, G.C., Duffus, J.E., Liu, H.Y. and Li, R.H. 1998. Ecology and epidemiology of whitefly transmitted closteroviruses. Plant Dis., 82: 270-280.

222 Resistance to a severe strain of zucchini yellow mosaic virus in watermelon

N. Guner and T.C. Wehner Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695-7609, USA; e-mail: [email protected]

Summary

The watermelon germplasm collection of 1653 plant introduction (PI) accessions, breeding lines and cultivars were screened for resistance to ZYMV in watermelon. High resistance was identified in PI 595203, PI 386019, PI 490377, PI 596662, PI 485580, and moderate resistance was identified in other accessions. The F , F , and BC generations derived from the cross Cal- 1 2 1 houn Gray x PI 595203 and New Hampshire Midget x PI 595203 were used to study the inheritance of resistance to ZYMV. A single recessive gene was found to control resistance to ZYMV. Additional work is needed to determine whether the gene is allelic to the previously published gene, zym-FL, for resistance to the Florida strain of ZYMV.

Keywords: Citrullus lanatus, inheritance, genetics, resistance, ZYMV

Introduction Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is a major cucurbit crop that accounts for 6.8% of the world area devoted to vegetable crops (FAO, 2002). In the United States, watermelon is used fresh as a dessert, or in salads. U.S. production is concentrated in Florida, California, Texas, and Georgia (USDA, 2002), increasing from 1.2 M tons in 1980 to 3.9 M tons in 2002, with a farm value of $329 million (USDA, 2002). Plant diseases caused by viruses are a major limiting factor in commercial watermelon production worldwide. Around the world, over 10 viruses are known to be a problem in watermelon production (Provvidenti, 1986). The major viruses affecting watermelon in the United States are zucchini yellow mosaic virus (ZYMV), papaya ringspot virus-watermelon strain (PRSV-W, formerly watermelon mosaic virus-1), and watermelon mosaic virus (WMV, formerly watermelon mosaic virus-2) (Adlerz and Crall, 1967). Zucchini yellow mosaic virus is one of the most destructive viruses in watermelon production (Nameth et al., 1985). ZYMV infects all the agriculturally important species of the Cucurbitaceae (Provvidenti, 1991). ZYMV was first described in 1981 in squash from northern Italy (Lisa and Dellavalle, 1981), and spread within a decade to the major cucurbit producing regions worldwide. ZYMV is spread in a non-persistent manner by a number of aphid species, and is easily transmitted mechanically. In areas where cucurbits are not grown continuously, the virus overwinters on wild species. Natural infection appears to be limited to members of the Cucurbitaceae, but members of 11 families of dicotyledons are considered diagnostic hosts. At least 25 strains of ZYMV have been identified (Desbiez and Lecoq, 1997). Provvidenti et al. (1984) reported the occurrence of Connecticut

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 223 (CT) and Florida (FL) strains of ZYMV, with the FL strain occurring more widely in the United States. Symptoms of severe ZYMV infection in cucurbits include yellow mosaic, stunting, blistering, and laminar reduction on leaves, and knobby, malfor- med, stunted, and mottled fruit (Provvidenti, 1996). ZYMV causes yield reductions in watermelon, squash, melon, cucumber, and other cultivated cucurbits (Nameth et al., 1985). Researchers have screened other cucurbit species for resistance to ZYMV and the inheritance of the resistance has been determined. Sources of resistance to ZYMV have been found in cucumber (Cucumis sativus), melon (Cucumis melo), and squash (Cucurbita spp.). ZYMV resistance was controlled by a single recessive gene in cucumber (Ka- belka et al., 1997), a single dominant gene in melon (Pitrat and Lecoq, 1984), and a single dominant gene in squash (Munger and Provvidenti, 1987; Paris et al., 1988; Gilbert-Albertini et al., 1993; Brown, 2001). Watermelon germplasm was screened for resistance to ZYMV by Provvidenti (1991), who evaluated 68 plant introduction (PI) accessions, breeding lines and commercial cultivars. Provvidenti (1991) reported ZYMV resistance in four landraces (PI 482322, PI 482299, PI 482261, and PI 482308). The resistance was specific to the Florida strain (ZYMV-FL). Resistance was conferred by a single recessive gene, zym (Provvidenti, 1991). A second screening of watermelon germplasm was made by Boyhan et al. (1992), who evaluated 153 PI accessions, breeding lines, and commercial cultivars. They identified five accessions (Egun, PI 482261, PI 494528, PI 396026, and PI 386025) resistant to ZYMV-FL. The watermelon germplasm collection at the Plant Genetic Resources Unit in Griffin, Georgia has increased in size, and more accessions have been made available for researchers to evaluate since part of the collection was first screened for ZYMV in 1991. To date, 221 PI accessions, breeding lines and commercial cultivars have been screened for ZYMV resistance by Provvidenti (1991) and Boyhan et al. (1992), or 14% of the collection. There may be additional sources of ZYMV resistance, or higher levels of resistance in the remaining 86% of the watermelon germplasm collection. The objectives of this study were: 1) to screen the U.S.D.A. watermelon germplasm collection along with watermelon cultivars to identify additional sources of ZYMV resistance; 2) to verify resistance of PI accessions that were identified in previous studies; and 3) to study the inheritance of any ZYMV resistance identified.

Material and methods

Germplasm screening The experiments were run in the North Carolina State University plant pathology and horticultural science greenhouses. Greenhouse temperatures ranged 23 to 43°C (day) and 12 to 24°C (night). The virus isolate was obtained from Dr. Ernest Hiebert, University of Florida, Gainesville. Three Florida isolates of ZYMV were tested to determine virulence (data not shown). The ZYMV isolate used for screening was a subculture of isolate 2088, a severe isolate of ZYMV described by Wisler et al. (1995). The virus isolate was maintained on Gray Zucchini squash (Cucurbita pepo L.) from Seminis Vegetable Seeds (Woodland, CA). All Citrullus PI accessions were obtained from the

224 Southern Regional Plant Introduction Station at Griffin, Georgia. PI accessions originated in 68 different countries, with 46 countries having fewer than 10 accessions each. Countries (number shown in parentheses) with the most accessions in the collection were Turkey (310), Yugoslavia (185), Zimbabwe (156), India (151), Spain (77), China (73), Zambia (68), South Africa (58), Nigeria (49), Iran (41), United States (33), and Syria (31). The inoculation procedure used for increasing the ZYMV isolate on squash, and for the screening experiment was the leaf rub method (Guner et al., 2002). Inoculum was produced by grinding infected Gray Zucchini squash leaves using mortar and pestle in 0.02 M phosphate buffer, pH 7.0. Leaf to buffer ratio was 1:5 (1 g infected leaf to 5 ml buffer). Inoculation consisted of dusting one leaf on each three-week-old plant with 800-mesh carborundum, then applying the inoculum to the leaf with a pestle which was rotated in a circular motion eight to ten times as if painting the leaf with inoculum. After inoculation, carborundum was rinsed off the leaves to improve light interception, and the plants were maintained in aphid-proof cages. All Gray Zucchi- ni squash plants were seeded in metromix 200 (Scotts-Sierra Horticultural Products Company, Marysville, Ohio) in 160 mm diameter (1550 ml volume) clay pots. Plants were fertilized weekly with 150 mg.kg-1 Peters Professional 20-20-20 N-P-K (Scotts- Sierra Horticultural Products Company, Marysville, OH). The germplasm screening was a randomized complete block with four replications of 1613 PI accessions and 41 watermelon cultivars. Each plot was a 100 x 100 mm square pot (600 ml volume) planted with two seeds and thinned to one plant before inoculation. By using single-plant plots, we were able to fit each complete block into each greenhouse run. In addition to the accessions tested, there were 10 check plants per replication of Charleston Gray that were inoculated with the virus, and 10 check plants of Charleston Gray that were not inoculated. The inoculated checks served as verification of viral infection and the uninoculated checks served as an indicator of other disease in the greenhouse that might confound symptom expression. Plants were inoculated at the first true leaf stage, and rated weekly for three weeks on a scale of 1 to 9 on the basis of severity of viral symptoms, where 1=none, 2=tendrils absent, 3=tendrils absent, slightly stunted growth, 4=mosaic patches and/or necrotic spots on leaves, 5=leaves near apical meristem deformed, meristem yellow and reduced in size, 6=apical meristem withered and brown, 7=apical meristem dead with more basal leaves dying, 8=most leaves dead, main stem green/yellow, 9=plant dead (Guner et al., 2002). Data were summarized as the average, the maximum, and the best of the three ratings. The best rating was the one with the greatest range over the 1654 cultigens. The best rating was considered useful for identifying resistance because early ratings often have little disease damage on any of the cultigens, and later ratings have much disease on most of the cultigens. Data were analyzed using the MEANS, ANOVA, CORRELATION, and GLM procedures of the SAS statistical package (SAS Institute, Cary, NC). Data were based on ratings from single-plant plots, and each rating date was analyzed separately.

Inheritance of resistance The inbred parental inbreds, as well as the F , F , and BC generations from the 1 2 1 crosses Calhoun Gray x PI 595203 and New Hampshire Midget x PI 595203 were used to study the inheritance of resistance to ZYMV. Calhoun Gray (CG) and New

225 Hampshire Midget (NHM) were highly susceptible to ZYMV, and PI 595203 (Egun) was highly resistant. Two families were developed by crossing Egun with Calhoun Gray and New Hampshire Midget. All crosses were made using controlled hand pollination in the greenhouse. Six generations were developed for inheritance study of resistance: susceptible parent, resistant parent, F , F , and BC to the susceptible and resistant 1 2 1 parents. For each cross, 5 Ps, 5 Pr, 30 BC s, 30 BC r, 10 F , and 100 F plants tested 1 1 1 2 for a total of 360 plants. All experiments were run in the Plant Pathology greenhouse at North Carolina State University in Raleigh, NC. Greenhouse temperatures ranged 23 to 43°C (day) and 12 to 24°C (night). Plants at the first true leaf stage were inoculated using the rub method described by Guner et al. (2002), and rated three times per week on a 1 to 9 scale starting two weeks after inoculation. The chi-square tests for goodness-of-fit (Ramsey and Schafer, 1997) and homogeneity were used to examine segregation ratios in populations with the SAS statistical package (SAS Institute, Cary, NC) and the SASGene 1.2 program (Liu et al., 1997).

Results and discussion

Germplasm screening Data were obtained for 1643 cultigens because 11 PIs did not germinate in any of the four replications. These PI accesssions were Grif 1420, PI 271468, PI 271767, PI 274034, PI 381745, PI 386014, PI 532670, PI 542113, PI 542118, PI 596679, and PI 596691. The complete dataset was submitted to the Germplasm Resources Information Network (http:/ /www.ars-grin.gov/) for those interested in ratings for particular cultigens. The most resistant and most susceptible cultigens are presented here along with checks (Table 1). The ANOVA indicated that there were highly significant differences (P=0.01) among accessions for all rating dates (data not shown). Since the best and average ratings were highly correlated (r=0.80), and the maximum rating had a smaller F ratio than the other ratings, only the best rating was given in Table 1 to save space. We observed significant differences for virus resistance in our study, as did Boyhan et al. (1992) working with ZYMV, Gillaspie and Wright (1993) working with WMV, and Strange et al. (2002) working with PRSV. PI 595203 had high resistance to ZYMV in our study, and also was resistant to PRSV-W and WMV (unpublished data). PI 482299 (best rating of 4.8) also had some resistant to ZYMV. However, PI 482261 (best rating of 6.0) and PI 255137 (best rating of 6.8) were not resistant to the isolate of ZYMV used in our study. PI 244018 (best rating of 2.8) and PI 244019 (best rating of 3.3) were reported to be resistant to WMV by Gillaspie and Wright (1993) and by Strange et al. (2002), and also had some resistance to ZYMV. Other accessions that were reported to be resistant to WMV (PI 189316, PI 189317, and PI 248178) did not have resistance to ZYMV. The PI accessions with the most resistance (best rating less than 3.0 and less) and having complete data (missing in no more than one replication) were: PI 595203, PI 386019, PI 490377, PI 596662, PI 485580, PI 560016, PI 494528, PI 386016, PI 482276, PI 386025, PI 595201, PI 494530, PI 494529, PI 482265, PI 596696, PI 485583, PI 244018, PI 482293, PI 386015, PI 482286, PI 559992, and PI 485581 (Table 1). The PI accessions having resistance to other watermelon viruses in addition to ZYMV were PI 244018, PI 595203, and PI 485583.

226 Table 1. Disease rating for 1643 watermelon accessions inoculated with ZYMV in the screening study+

Accession Country Best Rank or cultivar of origin rating

Resistant 1 PI 595203 United States 0.3 2 PI 386019 Iran 0.7 3 PI 490377 Mali 1.0 4PI 596662 South Africa 1.0 5 PI 485580 Botswana 1.0 6 PI 560016 Nigeria 1.3 7 PI 494528 Nigeria 1.3 8 PI 386016 Iran 1.5 9 PI 482276 Zimbabwe 1.8 10 PI 386025 Iran 2.0 11 PI 595201 United States 2.0 12 PI 494530 Nigeria 2.0 13 PI 494529 Nigeria 2.0 14PI 482265 Zimbabwe 2.0 15 PI 596696 South Africa 2.3 16 PI 485583 Botswana 2.5 17 PI 244018 South Africa 2.8 18 PI 482293 Zimbabwe 2.8 19 PI 386015 Iran 3.0 20 PI 482286 Zimbabwe 3.0 21 PI 559992 Nigeria 3.0 22 PI 485581 Botswana 3.0 Checks 1 Calhoun Gray United States 7.3 2 Crimson Sweet United States 8.3 Susceptible 1 PI 542117 Botswana 9.0 2 PI 596682 South Africa 9.0 3 Tendersweet OF United States 9.0 4PI 182935 India 9.0 5 PI 278058 Turkey 9.0 6 PI 270143 India 9.0 7 PI 269679 Belize 9.0 8 PI 273480 Ethiopia 9.0

+ Rank indicates the ranking of the cultigen for resistance to ZYMV, based on best rating (as well as average and maximum ratings; data not shown).

227 The susceptible checks in this study were Charleston Gray and Crimson Sweet, widely available cultivars. However, we identified accessions having more susceptibility to ZYMV than the checks. Some PI accessions would make excellent susceptible checks because they have high germination rates, and best ratings of 9.0 (compared to Charleston Gray and Crimson Sweet, which had a best rating of 7.3 and 8.3, respectively). Some of those were: PI 542117, PI 596682, Tendersweet Orange Flesh, PI 182935, PI 278058, PI 270143, PI 269679, and PI 273480 (data in GRIN).

Inheritance of resistance The inbred parental inbreds, as well as the F , F , and BC generations from the 1 2 1 crosses Calhoun Gray x PI 595203 and New Hampshire Midget x PI 595203 behaved as expected for a single gene for resistance (Table 2). In the F generation, all 1 plants developed severe systemic symptoms of ZYMV, indicating that resistance in PI 595203 was recessive. This hypothesis was confirmed by the reaction of F plants, 2 which segregated in a ratio 3 susceptible to 1 resistant. A segregation of 1 susceptible to 1 resistant was observed in the BC to the resistant parent, whereas plants of 1 the backcross to susceptible parents were all susceptible. Data pooled over family confirm the same results (Table 2).

Table 2. Single locus goodness-of-fit-test for resistance to ZYMV-FL in watermelon for two crosses and the pooled data Population No. resistant No. susceptible Expected a c2 P value Calhoun Gray (CG) x PI 595203 (Egun) Calhoun Gray (CG) 0 5 - - - PI 595203 (Egun) 5 0 - - - (CG x Egun) F 010--- 1 (CG x Egun) x CG 0 30 - - - (CG x Egun) x 0.133 0.72Egun 1416 1:1 (CG x Egun) F 26 70 1:3 0.222 0.64 2 New Hampshire Midget (NHM) x PI 595203 (Egun) NH Midget (NMH) 0 5 - - - PI 595203 (Egun) 5 0 - - - (NMH x Egun) F 010--- 1 (NMH x Egun) x NMH 0 30 - - - (NMH x Egun) x Egun 15 15 1:1 0 1 (NMH x Egun) F 2476 1:3 0.053 0.82 2 a Expected was the hypothesized segregation ratio for single gene inheritance for each segregating generation. Table 2. continued b Pooled over crosses Susceptible parents 0 10 - - - Resistant parent 10 0 - - - F 020--- 1 BC s060--- 1 BC r 29 31 1:1 0.066 0.80 1 F 50 146 1:3 0.027 0.87 2 b Data combined from two families: Calhoun Gray x PI 595203 and New Hampshire Midget x PI 595203.

228 The high level of resistance to ZYMV-FL in PI 595203 was controlled by a single recessive gene. These results were similar to those of Provvidenti (1991) who reported a single recessive gene for resistance to the Florida strain of ZYMV in PI 482261. Guner et al. (2003) tested PI 482261 for resistance to ZYMV-FL and reported no resistance, possibly because the isolate of Guner et al. (2003) was more virulent. ZYMV isolates have been reported to differ in pathogenicity, aphid transmissibility, and serological or molecular properties (Lecoq and Purcifull, 1992; Desbiez et al., 2002). Thus, additional research is needed to determine whether the two ZYMV strains are the same, and whether the two resistant genes are allelic.

References

Adlerz, W.C. and Crall, J.M. 1967. Epidemiology of control of watermelon mosaic virus. Florida Agr. Exp. Sta. Ann. Rep., 403. Boyhan, G., Norton, J.D., Jacobsen, B.J. and Abrahams, B.R. 1992. Evaluation of watermelon and related germplasm for resistance to zucchini yellow mosaic virus. Plant Dis., 76: 251-252. Brown, R.N. 2001. Traditional and molecular approaches to zucchini yellow mosaic virus resistance in Cucurbita. PhD Thesis. Dept. of Horticulture, Oregon State University, Corvallis, Oregon. Desbiez, C., Wipf-Scheibel, C. and Lecoq, H. 2002. Biological and serological variability evolution and molecular epidemiology of zucchini yellow mosaic virus with special reference to Caribbean island. Virus Res., 85: 5-16. Desbiez, C. and Lecoq, H. 1997. Zucchini yellow mosaic virus. Plant Pathol., 46: 809-829. FAO. 2002. Agricultural statistics for 2002. Food and Agriculture Organization of the United Nations, Rome; http://apps.fao.org/page/collections?subset=agriculture. Gilbert-Albertini, F., Lecoq, H., Pitrat, M. and Nicolet, J.L. 1993. Resistance of Cucurbita moschata to watermelon mosaic virus type 2 and its genetic relation to resistance to zucchini yellow mosaic virus. Euphytica, 69: 231-237. Gillaspie, A.G. and Wright, J.M. 1993. Evaluation of Citrullus sp. germplasm for resistance to watermelon mosaic virus 2. Plant Dis., 77: 352-354. Guner, N., Strange, E.B., Wehner, T.C. and Pesic-VanEsbroeck, Z. 2002. Methods for screening watermelon for resistance to papaya ringspot virus type-W. Sci. Hort., 94: 297-307. Guner, N., Wehner, T.C. and Pesic-VanEsbroeck, Z. 2003. Screening for resistance to zucchini yellow mosaic virus in 1654 plant introduction accessions and watermelon cultivars. Crop Sci. (in preparation). Kabelka, E., Ullah, Z. and Grumet, R. 1997. Multiple alleles for zucchini yellow mosaic virus resistance at zym locus in cucumber. Theor. Appl. Genet., 95: 997-1004. Lecoq, H. and Purcifull, D.E. 1992. Biological variability of potyviruses, an example: zucchini yellow mosaic virus. Arch. Virol. Suppl., 5: 229-234. Lisa, V. and Dellavalle, G. 1981. Characterization of two potyviruses from zucchini squash. Phy- topathology, 100: 279-286. Liu, J.C., Wehner, T.C. and Donaghy, S.B. 1997. SASGENE: A SAS computer program for genetic analysis of gene segregation and linkage. J. Hered., 88: 253-254. Munger, H.M. and Provvidenti, R. 1987. Inheritance of resistance to zucchini yellow mosaic virus in Cucurbita moschata. Cucurbit. Genet. Coop. Rep., 10: 80-81. Nameth, S.T., Dodds, J.A., Paulus, A.O. and Kishaba, A.K. 1985. Zucchini yellow mosaic virus associated with severe diseases of melon and watermelon in Southern California desert valleys. Plant Dis., 69: 785-788. Paris, H.S., Cohen, S., Burger, Y. and Yoseph, R. 1988. Single-gene resistance to zucchini yellow mosaic virus in Cucurbita moschata. Euphytica, 37: 27-29. Pitrat, M. and Lecoq, H. 1984. Inheritance of zucchini yellow mosaic virus resistance in Cucumis melo L. Euphytica, 33: 57-61. Provvidenti, R. 1986. Reactions of accessions of Citrullus colocynthis to zucchini yellow mosaic virus and other viruses. Cucurbit. Genet. Coop. Rep., 9: 82-83. Provvidenti, R. 1991. Inheritance of resistance to the Florida strain of zucchini yellow mosaic virus in watermelon. HortScience, 26: 407-408.

229 Provvidenti, R. 1996. Zucchini yellow mosaic virus. In: Zitter, T.A., Hopkins, D.L. and Thomas, C.E. (Eds.), Compendium of Cucurbit Diseases. APS Press, St. Paul, Minnesota, p. 44. Provvidenti, R., Gonsalves, D. and Humaydan, H.J. 1984. Occurrence of zucchini yellow mosaic virus in cucurbits from Connecticut, New York, Florida, and California. Plant Dis., 68: 443-446. Ramsey, F.L. and Schafer, D.W. 1997. The statistical sleuth. Thomson Publ. Co., Belmont, Calif. Strange, E.B., Guner, N., Pesic-VanEsbroeck, Z. and Wehner, T.C. 2002. Screening the watermelon germplasm collection for resistance to papaya ringspot virus type-W. Crop Sci., 42: 1324-1330. U.S. Department of Agriculture. 2002. Agricultural Statistics. US Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.; http://www.usda.gov/nass/pubs/agr02/acro02.htm. Wisler, G.C., Purcifull, D.E. and Hiebert, E. 1995. Characterization of the P1 protein and coding region of the zucchini yellow mosaic virus. J. Gen. Virol., 76: 37-45.

230 Preliminary evaluation of squash cultivars for resistance to a Czech isolate of zucchini yellow mosaic virus

J. Svoboda and J. Polák Research Institute of Crop Production, Division of Plant Medicine, Drnovská 507, 161 06 Prague 6-Ruzyne, Czech Republic

Summary

The cultivars of squash commonly grown in the Czech Republic are highly susceptible to zucchini yellow mosaic virus (ZYMV). The resistance of three summer squash (Cucurbita pepo) cultivars described as ZYMV-resistant, Jaguar, Cougar, and Hurakan, was examined by ar- tificially inoculating them with a highly virulent Czech ZYMV isolate, ZYMV-H. The degree of resistance of these cultivars was assessed by visual inspection of virus symptoms and by determining with ELISA the relative concentration of virus protein in leaves. Zelená, a Czech cultivar susceptible to ZYMV-H, served as the control. Of the three summer squash, Jaguar possessed the highest degree of resistance to ZYMV-H. Nonetheless, virus concentration measured in the leaves of Cougar and Hurakan was 20 times lower than that in the susceptible Zelená Cucur- bita moschata cv. Menina 15 was also tested, and observed to be immune to ZYMV-H.

Keywords: Cucurbita pepo, C. moschata, ZYMV, resistance, cultivars

Introduction

Recently, in comparison with the beginning of 1990s (Lebeda et al., 1996), zucchini yellow mosaic virus (ZYMV) has caused considerable losses of squash (Cucurbita L. species) grown in South Moravia, and is spreading gradually to Central Moravia (M. indelková, 2002, person. commun.). Protection against the virus is difficult, with the virus overwintering in biennial and perennial weeds (Svoboda and Polák, 2002). Growing cultivars resistant to ZYMV might solve the problem. The Cucurbita moschata Duchesne landrace from Portugal, Menina, carries a single dominant gene for ZYMV resistance (Gilbert-Albertini et al., 1993). The summer squash, C. pepo L., Dividend, Hurakan, Jaguar, Cougar, Puma, Revenue and Tigress, from the U.S.A., were bred for ZYMV resistance, the source of which was another ZYMV-resistant C. moschata landrace referred to as Nigerian Local (Provvidenti, 1997). Other C. pepo hybrids were reported by Ansanelli et al. (1997) to have tolerance to ZYMV. Lebeda et al. (1999) tested 50 varieties of squash grown in the Czech Republic and observed that all of them were susceptible to the ZYMV. Similar conclusion made Køístková and Lebeda (1999) who tested more than 400 accessions of the Cucurbitaceae, however none of them was resistant to the ZYMV. Re- cently, resistance to the ZYMV was identified in cantaloupes (Diaz et al., 2003). Considering this, we have obtained foreign sources of resistance, the objective being to observe the degree of their resistance to the highly pathogenic Czech ZYMV isolate.

Plant material The seeds of the American summer squash (Cucurbita pepo) Jaguar, Cougar, and Hurakan and of Menina 15 (Cucurbita moschata) were obtained from the Harris Moran Company, California, and the Institute for Agrobiotechnology, Tulln, Austria, respectively. The susceptible Czech summer squash Zelená served as the control. Eight plants of each cultivar were grown in a greenhouse. Once reaching the cotyledon stage, the plants were mechanically inoculated with ZYMV. The resistance of the plants was evaluated three weeks after inoculation by determining the relative concentration of the virus, using the ELISA method. The determination of the virus by ELISA was repeated after another three weeks. In a second sowing of these cultivars, the plants were inoculated with ZYMV by aphids. Four weeks after inoculation, ELI- SA was carried out and the symptoms evaluated. Virus symptoms were observed on all plants for another two months.

ZYMV isolates In 2001, six ZYMV isolates were obtained from various South Moravian locations; from inoculating them on cucurbit plants, three distinct pathogenicity reactions were observed: K = mild, L = moderate, and H = severe (Svoboda and Polák, 2002). The severely pathogenic ZYMV-H isolate was then maintained in squash plants and was used to test the resistance of the cultivars. Leaves from systemically infected plants were used for inoculation of the plants to be tested.

Mechanical transmission To prepare the mechanical transmission, 1 g of leaves from plants infected with the ZYMV-H isolate was homogenized with 3 ml of 0.03 M Na HPO containing 0.2% 2 4 DIECA, pH 9.3 (Mahgoub et al., 1997). Active charcoal and carborundum (200 mesh) were added to the homogenate and the cotyledons of the plants were then inoculated mechanically.

Aphid transmission For the purpose of non-persistent transmission of ZYMV by Myzus persicae, the aphids sucked leaves of summer squash plants infected by the ZYMV-H isolate for 2 to 3 minutes after fasting for two hours. After this acquisition time, the aphids were transferred to the first true leaves of the tested plants for a four-hour inoculating su- ction, 25 aphids per plant. At the end of the inoculation period, the aphids were killed using Pirimor insecticide. The infectious properties of the aphids were controlled by a parallel transmission of the same number of aphids undergoing the acquisition time to two indicator plants of Zelená. Transmission success rate was evaluated on the basis of symptoms and applying ELISA.

ELISA The DAS-ELISA-specific polyclonal antiserum and the method developed by the Loewe Company were used. Sample leaves were homogenized with an extraction buffer at a rate of 1 : 20 according to the instructions. A diluting series was prepared with

232 the same extraction buffer. The highest dilution resulting in a positive reaction in ELI- SA was considered as the reciprocal value of relative concentration of ZYMV protein.

Results and discussion

The results of evaluation of the squash cultivars for ZYMV resistance, based on symptoms, are listed in Table 1. The results also show significant differences in the relative concentrations of the virus between the susceptible control Zelená and the resistant cultivars (Fig. 1). Furthermore, there were differences among individual plants of a single cultivar in relative virus concentration, suggesting the possibility that further increase of resistance would be possible by selection. Assessed as immune, C. moschata Menina 15 was clearly superior to all of the others, irrespective of whether it was inoculated mechanically or by aphids; it can be recommended to breeders as a source of resistance against ZYMV. Jaguar and Cougar showed viral infection symptoms on leaves as local lesions or mild mosaic and their fruits were only slightly deformed. In comparison, the plants of the susceptible control Zelená were stunted and had severe mosaic and deformed leaves; the fruits, if any, were severely deformed. In our tests, Jaguar was evaluated as resistant, Cougar as moderately resistant, and Hurakan as moderately susceptible to ZYMV-H.

Table 1. Evaluation of selected squash cultivars for resistance to the Czech isolate of zucchini yellow mosaic virus (ZYMV-H)

Symptoms in Relative Species/Cultivar concentration Evaluation Leaves Fruits of ZYMV

C. moschata no symptoms no symptoms 0 immune Menina 15

C. pepo Jaguar mild mosaic mild deformations 0.4 × 103 resistant

C. pepo Cougar mosaic mild deformations 1.0 × 103 medium resistant

C. pepo Hurakan mosaic, deformations 2.7 × 103 medium deformations susceptible

severe mosaic, C. pepo Zelená severe severe 12.2 × 103 very susceptible deformations deformations

233 ZYMV concentration was higher in Zelená than in Jaguar, Cougar and Hura- kan (Fig. 1). Virus concentration in Jaguar was particularly low and therefore this cultivar could be the best adapted for areas severely affected by ZYMV in the Czech Republic. To obtain a final evaluation of cultivar resistance to ZYMV, it would be necessary to repeat the greenhouse test and to conduct field evaluations in an area seriously infected with ZYMV.

Figure 1. Relative concentration of ZYMV-H in leaves of squash plants.

Acknowledgements The authors thank Dr. Martin Pachner of the Institute for Agrobiotechnology, Tulln, Austria and the Harris Moran Company for furnishing the squash seeds. This work was supported by Project MZE 0002700603 of the Ministry of Agriculture, Czech Republic.

References

Ansanelli, C., Giovannantonio, C., Tomassoli, L. and Di-Giovannantonio, C. 1997. Tolerant varieties and reflective soil coverings to control viruses of courgette. Informatore Agrario. 53: 43-45. Diaz, J.A., Mallor, C., Soria, C., Camero, R., Garzo, E., Fereres, A., Alvarez, J.M., Gomez-Guillamon, M.L., Luis-Arteaga, M. and Moriones, E. 2003. Potential sources of resistance for melon to nonpersistently aphid-borne viruses. Plant Dis., 87: 960-964. Gilbert-Albertini, F., Lecoq, H., Pitrat, M. and Nicolet, J.L. 1993. Resistance of Cucurbita moschata to watermelon mosaic virus type 2 and its genetic relation to resistance to zucchini yellow mosaic virus. Euphytica, 69: 231-237. Køístková, E. and Lebeda, A. 1999. Disease resistance of Cucurbita pepo and C. maxima genetic resources. Cucurbit Genet. Coop. Rep., 22: 53-54.

234 Lebeda, A., Kozelská, S., Køístková, E. and Novotný, R. 1996. The occurrence of viruses on Cucurbita spp. in the Czech Republic and resistance of squash cultivars to CMV and WMV-2. J. Plant Dis. Protec., 103: 455-463. Lebeda, A., Køístková, E., Kozelská, S., Joke, M. and Rodová, J. 1999. Response of Cucurbita pepo and Cucurbita maxima genotypes to Czech isolate of zucchini yellow mosaic virus. Pe- tria, 9: 335-336. Mahgoub, H.A., Desbiez, C., Wipf-Scheibel, C., Dafalla, G. and Lecoq, H. 1997. Characterizati- on and occurrence of zucchini yellow mosaic virus in Sudan. Plant Pathol., 46: 800-805. Provvidenti, R. 1997. New american summer squash cultivars possessing a high level of resistance to a strain of zucchini yellow mosaic virus from China. Cucurbit Genet. Coop. Rep., 20: 57-58. Svoboda, J. and Polák, J. 2002. Distribution, variability and overvintering of Zucchini yellow mosaic virus in the Czech Republic. Plant Protect. Sci., 38: 125-130.

235 236 Different genes for resistance to zucchini yellow mosaic virus (ZYMV) in Cucurbita moschata

M. Pachner1 and T. Lelley2 1University of Natural Resources and Applied Life Sciences, Vienna, Austria 2Department for Agrobiotechnology, Division Biotechnology in Plant Production, IFA- Tulln, Konrad Lorenz Str. 20, A-3034 Tulln, Austria; e-mail: [email protected]

Summary

Five genotypes of Cucurbita moschata from widely dispersed geographic regions were used to study inheritance of resistance to ZYMV. Four of the five genotypes exhibited resistance to an Austrian isolate of ZYMV. Eight F s, 7 F s and 4 three-way crosses were produced and investiga- 1 2 ted for mode of inheritance of resistance after artificial inoculation. The results indicate that these C. moschata genotypes contain as many as five loci for resistance to ZYMV. The resistances of the landraces Nigerian Local and Menina are conferred by genes at separate loci.

Keywords: Cucurbita pepo, C. maxima, Mendelian segregation, virus resistance

Introduction

Zucchini yellow mosaic virus (ZYMV), first described by Lisa et al. (1981) in Ita- ly and Lecoq et al. (1981) in France, is a serious threat to all cucurbits. As a conse- quence of a devastating epidemic of ZYMV in Austria in 1997, a breeding program was initiated to introduce resistance to Austrian oil pumpkin (Cucurbita pepo L.). Initially, the American zucchini hybrids Tigress, Jaguar and Puma (Harris Moran Seed Company), as well as Dividend and Revenue (Rogers Seed Company) were used as sources of resistance. The resistance of all these can be traced to the C. moschata Duchesne landrace Nigerian Local (Provvidenti, 1997) (NL resistance). Later and independently, other ZYMV-resistant C. pepo germplasm was developed. A ZYMV- resistant near-isogenic line of the zucchini True French was described (Paris and Cohen, 2000) and the zucchini hybrid Dundoo (Enza Zaden) was released. The original source of this resistance was the Portuguese C. moschata landrace Menina (M- resistance) (Paris and Cohen, 2000). We have transferred both resistances into the same oil-pumpkin genotype, a breeding line of Saatzucht Gleisdorf, Austria, by a backcross program. Valuable breeding material was selected in the BC F -generation. Selection for resistance was based on 3 3 artificial inoculation using an Austrian isolate of ZYMV (Riedle-Bauer, 1998; Pfos- ser and Baum, 2002). In infection experiments with oil pumpkin, the two resistances showed different symptom development and a different level of protection against the virus. The NL-resistance was monogenically inherited and the resistance was recessive. After inoculation, symptom development was delayed. First symptoms started to appear on the second true leaf, later symptom expression increased continuously until the end of the experiment.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 237 The M-resistance, on the other hand, was controlled by a single dominant gene. Symptom development started right after inoculation already on the first true leaf and the plants were stunted at around the third-leaf stage. One week later the plants recovered and became almost symptom-free, producing healthy fruit. The different kinds of resistance, both in expression and in efficiency, originating from different landraces of C. moschata coming from different continents, prompted us to start an investigation into the inheritance of ZYMV resistance in C. moschata.

Material and methods

Plant material Seed samples of Menina 15, a selection from the Portuguese landrace Meni- na, and Nigerian Local, a landrace from Nigeria, were obtained from M. Pitrat, INRA, France. Seeds of Soler were provided to us by Linda Wessel-Beaver, Agronomy and Soils Dept. Univ. Puerto Rico; seeds of Nicklow´s Delight were obtained from Clark W. Nicklow, U.S.A. Seeds of the susceptible Waltham Butternut, from the U.S.A., were obtained from Harry Paris, A.R.O., Newe Ya´ar Research Center, Israel. All resistant genotypes were crossed with the susceptible Waltham Butternut. Moreover, the resistant genotypes were crossed in all possible combinations, but no reciprocal crosses were made. F s with Waltham Butternut were selfed to obtain segregating F generati- 1 2 ons. F s among the resistant genotypes were crossed with Waltham Butternut. All crosses 1 were made under controlled conditions in the greenhouse during the winter season.

Artificial inoculation Inoculation experiments were performed in a growth chamber. Seeds were sown in 8 x 8 x 9 cm pots containing a 10:1 mixture of ready-to-use garden mould (2.5g salt/l) and sand. Plants were grown at 23°-25°C day and 20°-22°C night temperatures at 50-70% RH. Natural illumination was supplemented with a combination of mercury and sodium va- por lamps maintaining a day length of 14 hours during the whole experiment. An Austrian isolate of ZYMV was used for inoculation. The inoculum was prepared from infected susceptible plants of oil pumpkin (C. pepo). Young leaves with clear symptoms of infection were collected and stored at 20°C in plastic bags, not longer than 6 months. The infectious homogenate was prepared from 5 g of infected leaves, ground in a mortar on ice in 50 ml inoculation buffer containing 1% K HPO . Finally, 2 4 1 g Celite® 545 was added. Seedlings were inoculated twice: first, when the first true leaf just appeared, the two cotyledons were inoculated, and three days later the first true leaf was inoculated. Inoculation was carried out by gently rubbing the leaf surface with a finger in rubber gloves.

Symptom evaluation Plants were observed 10, 17 and 24 days after the first inoculation. The first evaluation was conducted only to ensure the success of inoculation by observing typical reactions to the inoculum. Classification of the plants as resistant or susceptible was done throughout by the first author by using the scale 0 to 5 (Fig. 1). Segregation data were subjected to c2 analysis.

238 Figure 1. Leaves of cucurbit plants inoculated with the Austrian isolate of ZYMV. Leaves without symptoms after inoculation are considered as non-infected (0). Lea- ves of resistant plants classified as 1 or 2 show chlorotic spots or dots or are lightly marbled. Susceptibility is expressed by blistered and/or distorted leaves and stunted growth of the plants (3, 4 and 5, respectively).

Results and discussion

Prior to this study, we analyzed the five C. moschata genotypes for homogeneity of their reaction to artificial infection with the Austrian isolate of ZYMV. Moreover, all following experiments included as control the respective parental genotypes. In all cases the parents showed a uniform reaction to infection, showing the homogeneity of their genetic constitution regarding resistance or susceptibility. Inoculation of parental material resulted in characteristic symptom development on the different genotypes. Menina 15 showed small chlorotic dots right on the in-

Figure 2. Symptom development after inoculation with ZYMV on the Cucurbita moschata germplasm used in this study. From left: (a) Menina 15, (b) Nigerian Local, (c) So- ler, (d) Nicklow´s Delight, (e) Waltham Butternut.

239 fected first true leaf which disappeared already on the third leaf (Fig. 2a). The reaction of Nigerian Local was very similar to that of Menina 15 with the difference that, instead of chlorotic dots, short chlorotic streaks along the veins appeared (Fig. 2b). Soler developed chlorotic spots approximately 1 cm in diameter that persisted throughout the whole observation period (Fig. 2c). Nicklow´s Delight behaved similar to So- ler, but instead of round spots irregularly shaped chlorotic areas of the same size appeared (Fig. 2d). Waltham Butternut developed typical symptoms of susceptibility, including mosaic and leaf distortion (Fig. 2e).

Table 1. Segregation data for resistance (R) and susceptibility (S) in C. moschata progenies in crosses among the genotypes Menina 15 (Men), Soler (Sol), Nigerian Local (NL), Nicklow´s Delight (Nic) and Waltham Butternut (WB) in artificial inoculation experiments using the Austrian isolate of ZYMV.

Generation Description No. of plants Expected c2 p total observed expected ratio

R S R S R:S F WB x Men 18 18 0 1 F WB x Sol 18 0 18 1 F WB x NL 18 18 0 1 F Sol x Men 18 18 0 1 F NL x Sol 12 12 0 1 F NL x Men 12 12 0 1 F Nic x WB 17 17 0 1 F Nic x NL 12 12 0 1

F WB x Men 21416253.5 52 160.5 3:10 0.06 0.81 2 F Sol x Men 211 170 41 171 40 13:30 0.06 0.80 2 F NL x Men 179 177 2 176 3 63:10 0.23 0.63 2 F WB x Sol 113 39 7428 850 1:35.45 0.02 2 F WB x Sol alternative R : S 42 71 6:10 0.43 0.51 2 F NL x Sol 189 185 4180 9 61:30 2.80 0.09 2 F NL x Sol alternative R : S 182 7 246:10 1.61 0.20 2 F Nic x WB 216 16452 162 54 3:10 0.10 0.75 2 F Nic x NL 33 33 0 33 0 1:00 2 F WB x NL 0 2

TWC* WB x (NL x Men) 106 91 15 93 13 7:10 0.26 0.61

TWC (Sol x Men) x WB 108 57 51 5454 1:1 0 0.33 0.56

TWC WBx (NLx Sol) 1413 1 11 43:1 0 2.38 0.12

TWC WBx (Nic x Sol) 57 26 31 28.5 28.5 1:10 0.44 0.51

* three-way cross

240 Data collected from F -, F - and three-way crosses are presented in Table 1. Hypo- 1 2 thetical genetic constitution of each of the five genotypes with respect to resistance or susceptibility, derived from the segregation data, is presented in Table 2. In this experiment, we did not make reciprocal crosses systematically because under our growing conditions very often crossing was possible only in one direction. Mo- reover, such crosses between Menina and Waltham Butternut were made by Paris et al. (1988) who stated that no difference in reaction was observed among progenies of reciprocal crosses. Paris et al. (1988), in crossing Waltham Butternut with an inbred line of Menina that had been selected for resistance to ZYMV, found a single dominant gene to be responsible for the high resistance of Menina. In our experiment, with the slight difference of using a different selection from Menina, we arrived at the same conclusion. Menina 15 has a single dominant gene (A) conferring strong resistance to ZYMV (Table 1). As two, and only two, susceptible individuals occurred in an F population of 179 2 plants of the cross combination Menina x Nigerian Local, a two dominant-gene ratio of 15:1 is inadequate; this result can be accounted for by a three-gene hypothesis, with segregation pattern of 63:1. It follows that NL possesses duplicate genes (BB and CC) for resistance. It is interesting to mention that all American summer squash varieties obtained their resistance from NL (Provvidenti, 1997), and our first resistant oil-pumpkin genotype mentioned above also derives its resistance from NL, via Tigress. Munger and Provvidenti (1987) described the behavior of the progeny of an NL x WB cross, showing a single incompletely dominant gene for ZYMV resistance. The difference from our results may be due to heterogeneity of NL and that it may harbor two genes for resistance to ZYMV. So far we were not able to obtain viable F seeds 2 from our cross NL x WB grown in the greenhouse. However, the segregation of the three-way cross WB x (NL x Men) did not fit the two-gene 3:1 testcross ratio but instead the three-gene 7:1 testcross ratio, consistent with the idea that NL contains duplicate resistance genes. A similar result could be expected, however, if the genes from NL and Men are linked; resolution of this problem could be expected by studying the F of the cross of NL and WB, or the backcross to WB. 2 From correspondence with Clark W. Nicklow, we know that Nicklow´s Delight obtained its resistance from a Nigerian line provided by Provvidenti at Cornell University. The cross Nic x NL in our study showed no segregation in the F (Table 1), confirming the 2 presence of a common resistance gene in Nic and NL. However, Nicklows Delight would contain only one of the resistance genes carried by NL, as indicated by the nice fit to the 3:1 segregation in the F of the cross Nic x WB (Table 1). 2 The resistance of Soler from Puerto Rico is different from that of Men and NL. The F of WB x Sol is uniformly more susceptible to the Austrian isolate of ZYMV 1 than WB itself. This suggests a recessive resistance in Soler. The segregation of 113 F plants, however, did not fit into a clear 1:3 pattern (Table 1). A 6:10 (3:5) segrega- 2 tion with a probability of 0.51 can be explained if we assume a further weak resistance gene (ee) in WB with the dominant allele (EE) in Soler. This gene interacts with (dd) in Soler if it is in heterozygous state (Dd). The recessive monogenic resistance of Soler is apparent in the cross Sol x Men which segregates 13:3, P = 0.8 (Table 1). The 61:3 segregation of the cross NL x Sol

241 had a low P value. A segregation pattern of 246:10 (P = 0.2) could be explained with the presence of the gene (ee) in NL (Table 2). The three-way cross (Sol x Men) x WB showed a clear 1:1 segregation (Table 1), confirming the presence of a single recessive gene for resistance in Soler. A hypothetical genetic constitution of loci and alleles conditioning resistance or susceptibility in the five C. moschata genotypes investigated in this study is presented in Table 2.

Table 2. Hypothetical genetic constitution of loci and alleles conferring resistance (underlined in italics) or susceptibility in the investigated five C. moschata genotypes

Menina 15 AA bb cc DD Nigerian Local aa BB CC DD (ee) Soler aa bb cc dd (EE) Nicklow´s Delight aa BB cc DD Waltham Butternut aa bb cc DD (ee)

Conclusions

The results (Table 1) can be most simply explained by the hypothesis that C. moschata contains as many as five genes that confer resistance to ZYMV (Table 2). Thus, C. moschata offers a most valuable source of resistance to this virus. Provvidenti and Alconero (1985) tested 418 accessions of C. maxima Duchesne from 35 countries on six continents with two isolates of ZYMV; none of the accessions were resistant to either isolate. To our knowledge, no resistance to ZYMV originating in C. pepo has been found. Thus, it appears that, of the economically important species of pumpkins, only C. moschata contains germplasm that is resistant to ZYMV. Three independent teams of investigators (Munger and Provvidenti, 1987; Paris et al., 1988; Gilbert- Albertini et al., 1993) have identified genes for ZYMV resistance in C. moschata. The single resistance gene found in Menina by Paris et al. (1988) and by Gilbert-Alberti- ni et al. (1993), referred to as Zym or as Zym-1 (Paris and Cohen, 2000), probably corresponds with the gene designated A in Table 2 and is at a locus separate from that of the gene(s) conferring resistance in Nigerian Local. The results of the three-way crosses (Table 1) revealed that C. moschata harbors genes at various loci that confer resistance to ZYMV, which has obvious implications for breeding ZYMV-resistant pumpkins.

Acknowledgements

This study was financially supported by a grant (Project 1228) of the Austrian Federal Ministry of Agriculture, the federal states of Styria and Burgenland as well as the Breeding Company Saatzucht Gleisdorf. We gratefully acknowledge the seeds received from Michel Pitrat, Harry Paris, Clark W. Nicklow and Linda Wessel-Beaver.

242 References

Gilbert-Albertini, F., Lecoq, H., Pitrat, M. and Nicolet, J.L. 1993. Resistance of Cucurbita moschata to watermelon mosaic virus type 2 and its genetic relation to resistance to zucchini yellow mosaic virus. Euphytica, 69: 231-237. Lecoq, H., Pitrat, M. and Clement, M. 1981. Identification et caracterisation dún potyvirus pro- voquant la maladie du rabougrissement jaune du melon. Agronomie, 1: 827-834. Lisa, V., Boccardo, G., D´Ágostino, G., Dellavalle, G. and D´Aquilio, M. 1981. Characterization of a potyvirus that causes zucchini yellow mosaic. Phytopathology, 71: 667-672. Munger, H.M. and Provvidenti, R. 1987. Inheritance of resistance to zucchini yellow mosaic virus in Cucurbita moschata. Cucurbit Genet. Coop. Rep., 10: 80-81. Paris, H.S. and Cohen, S. 2000. Oligogenic inheritance for resistance to zucchini yellow mosaic virus in Cucurbita pepo. Ann. Appl. Biol., 136: 209-214. Paris, H.S., Cohen, S., Burger, Y. and Yoseph, R. 1988. Single-gene resistance to zucchini yellow mosaic virus in Cucurbita moschata. Euphytica, 37: 27-29. Pfosser, M. and Baum, H. 2002. Phylogeny and geographical differentiation of zucchini yellow mosaic virus isolates (Potyviridae) based on molecular analysis of the coat protein and part of the cytoplasmic inclusion protein genes. Archiv. Virol., 147: 1599-1609. Provvidenti, R. 1997. New American summer squash cultivars possessing a high level of resistance to a strain of zucchini yellow mosaic virus from China. Cucurbit Genet. Coop. Rep., 20: 57-58. Provvidenti, R. and Alconero, R. 1985. Lack of resistance to zucchini yellow mosaic virus in accessions of Cucurbita maxima. Cucurbit Genet. Coop. Rep., 8: 76-77. Riedle-Bauer, M. 1998. Ölkürbis & Co.: Was tun gegen das Zucchinigelbmosaikvirus? Der Pflan- zenarzt, 4: 1-4.

243 244 Cucumber screening for resistance to angular leaf spot

H. Olczak-Woltman1, M. B¹kowska1, M. Schollenberger2 and K. Niemirowicz-Szczytt1 1Department of Plant Genetics, Breeding and Biotechnology; Warsaw Agricultural University, Nowoursynowska 166, 02-787 Warsaw, Poland; e-mail: [email protected] 2Department of Plant Pathology, Warsaw Agricultural University, Nowoursynowska 166, 02-787 Warsaw, Poland

Summary

Increased occurrence of angular leaf spot on cucumber crops in Poland have brought about significant losses in the fruit yield. Therefore, it has become necessary to estimate the resistance of the commonly cultivated cucumber hybrids. The screening was performed for 42 cultivars and lines in the growth chamber conditions. All forms were inoculated with the same repeatable, strongly pathogenic isolate. About 20% of the screened forms showed higher resistance as compared with Gy14 line (resistant standard).

Keywords: Pseudomonas syringae pv. lachrymans, Cucumis sativus, cucumber hybrid, resistance

Introduction

Cucumber angular leaf spot is a well-known disease caused by Pseudomonas syringae pv. lachrymans (Smith et Bryan) Young, Dye et Wilkie, which brings about serious yield losses in cucumber (Cucumis sativus L.) crops (Dessert et al., 1981). Although the inheritance of resistance to this pathogen has been subject to numerous investigations for many years (Chand and Walker, 1963; Dessert et al., 1981), it has not been possible to develop highly resistant forms so far. Also wild forms of the genus Cucu- mis turned out to be not completely resistant to this pathogen (Kùdela and Lebeda, 1997). Recently, strong bacterial infections have been observed in the Polish open field plantations just before harvesting, which has inspired further experiments aimed to evaluate cucumber resistance to angular leaf spot.

Materials and methods

Pathogen Twelve different isolates of Pseudomonas syringae pv. lachrymans (Psl) were selected for the experiment. Three of them were derived from the plant pathogens banks (from the Netherlands and Belgium) and the others were isolated from cucumber leaves collected in different parts of Poland. The own isolates were tested and classified according to LOPAT tests (Lelliot et al., 1966). Then all collected bacterial isolates were checked for their pathogenicity on Wisconsin 18 SMR, susceptible cucumber cultivar. The most repeatable and pathogenic isolate was chosen and used for next inoculations.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 245 Plant materials Cucumber seeds were sown into plastic pots filled with pit moss so as to grow a single plant per pot. The plants were growing in the growth chamber conditions (25°C during the day and 22°C at night, illumination for 16 hours a day at 50 W/m2). Two independent experiments were carried out. In each experiment 24 plants (4 replicates x 6 plants) of each cultivar were inoculated. The screening was performed on 40 different hybrids and lines in comparison to two standards: susceptible Borszczagowski cultivar and resistant Gy14 line.

Inoculum preparation To prepare the inoculum bacteria were grown on King medium B plates for 24 hours at 26°C. Bacterial cells were washed with sterile distillate water to a sterile flask to get concentration of about 1x107 CFU (OD = 0.050). Three drops of Tween 20 were 600 added to each dm3 of inoculum. The inoculum was sprayed on plants immediately after preparation using a low-pressure sprayer (Williams and Keen, 1967; Dessert et al., 1981; Kùdela, 1986).

Plant inoculation Young plants (at the stage of 2-3 leaves) were inoculated according to the protocols of Dessert et al. (1981) and Kùdela (1986). Bacterial suspension was sprayed onto the underside of cucumber leaves. Inoculated plants were placed in darkness for 24 hours at 22°C in the controlled relative humidity of 100%. For the following 6 days the chamber was illuminated and the temperature of 25°C and relative humidity of 95-100% were applied. After seven days from inoculation the plants were evaluated according to the disease rating scale. The disease index were expressed by a nine-degree rating scale (Jenkins and Weh- ner, 1983): 9 = highly resistant; few minute lesions; 8 = resistant; few lesions on 3- 8% of leaf area, without chlorosis; 7 = moderately resistant; necrotic lesions on 8- 15% of leaf area, on few leaves or stems, without chlorosis; 6 = intermediate; large necrotic lesions on 15-25% of leaf area, on few leaves or stems, with light chlorotic halo; 5 = intermediate; necrotic or water-soaked lesions on 25-50% of leaves area or stems with chlorotic halo; 4 = susceptible; typical angular, water-soaked lesions on 50-75% of leaves area, with bacterial exudes and yellow borders; 3 = susceptible; lots of water-soaked lesions on 75-87% of leaves area, with bacterial exudes and large yellow chlorosis; 2 = highly susceptible; damage of 87-95% of leaves area, bacterial exudes, large chlorosis, necrosis; 1 = highly susceptible; damage up to 100% of plant leaves. Results were analyzed in statistical multiple range test. Student-Neu- man-Keuls test was used at 95% of confidence. Two independent experiments were analysed separately. To each analysing data from four replicates were introduced.

Results and discussion

The isolates collection which was qualified as group Ia (LOPAT +---+) showed a significant variation with respect to pathogenicity. The most pathogenic and stable isolate, with typical angular leafspot symptoms was strain 814/98 from the Nether-

246 lands plant pathogens bank. Two other strains from the plant pathogens banks caused weak symptoms on cucumber leaves i.e. dry, light-colored, papery lesions. Similar symptoms to this two strains, with additional limited chlorosis gave isolates obtained from the leaves collected in Poland. The results of screening for the resistance to angular leafspot are presented in Table 1. On the susceptible standard Borszczagowski were observed repeatable, water-soaked, angular, necrotic spots with bacterial exudes. In both independent experiments this lesions appearance on susceptible standard was rated as 3.3 to 3.8 according to the scale. On the other hand, line Gy14 used as the resistant standard was evaluated as closed to moderately resistant (6.3 to 6.7 according to the scale). The results of both experiments did not differ much and it was possible to divide screened cultivars into three groups. The largest group (60% in both experiments) was created by the cultivars with intermediate disease index (4.7 to 5.9). They formed necrotic spots or water-soaked lesions with chlorotic boarders (halo) on 25-50% of leaves or on stems. All the other varieties can be classified into two groups (about 20% each): the most susceptible cultivars (with index of 3.0 to 4.6) and those more resistant than Gy14 line (5.0 to 7.1) - this groups includes Hardwicki 603 and Gy3 lines and hybrids Cyryl F , Bazyl F , Parys F , Basza F , Izyd F and Regal F . In both experiments the most 1 1 1 1 1 1 resistant was Hardwicki 603 line, which was scored 7.1.

Table 1. Evaluation of cucumber hybrids and lines for resistance to angular leaf spot in two experiments

Cultivar/Line Average of disease index 1st Experiment 2nd Experiment

Borszczagowski (suscept. control) 3.78 a* 3.25 a Sander F 3.78 a 3.30 ab 1 Fason F 4.03 ab 3.30 ab 1 Polonez F 4.08 abc 4.63 abcdef 1 Wojan F 4.10 abc 4.18 abcde 1 Gracjus F 4.40 abcd 4.28 abcde 1 Barbakan F 4.40 abcd 4.65 abcdef 1 Malta F 4.45 abcd 4.55 abcdef 1 2gg (line) 4.70 abcde 3.35 abc Monika 4.75 abcde 3.83 abcd Moro 4.83 abcde 4.90 abcdef Wisconsin 18 SMR 4.85 abcde 3.30 ab Wawel F 4.85 abcde 4.65 abcdef 1 Anulka F 4.85 abcde 4.80 abcdef 1 Fortuna F 4.88 abcde 5.40 defg 1 Dar 4.93 abcdef 5.53 defg Polan F 4.96 abcdef 4.65 abcdef 1 remski 5.02 abcdef 4.58 abcdef Soplica F 5.08 abcdefg 4.83 abcdef 1 S³awko F 5.33 abcdefgh 5.05 bcdefg 1

247 Lider F 5.45 bcdefgh 5.38 defg 1 Metro F 5.53 bcdefghi 4.95 abcdef 1 Calypso 5.53 bcdefghi 5.73 defg Aladyn F 5.65 cdefghi 5.93 defg 1 Major 5.80 defghi 5.86 defg Royal F 5.83 defghi 5.43 defg 1 Cezar F 5.83 defghi 5.85 defg 1 Izyd F 5.88 defghi 6.38 efg 1 remianin F 5.95 defghi 5.55 defg 1 Gomez F 5.98 defghi 4.88 abcdef 1 Frykas F 5.98 defghi 5.68 defg 1 Hermes F 6.23 efghi 6.38 fg 1 Regal F 6.33 efghi 5.82 defg 1 Prymus F 6.50 fghi 5.35 cdefg 1 Atlas F 6.63 ghi 5.76 defg 1 Gy14 (line) (resistant control) 6.65 hi 6.30 efg Bazyl F 6.68 hi 6.13 efg 1 Gy3 (line) 6.68 hi 6.63 fg Cyryl F 6.73 hi 6.23 efg 1 Parys F 6.80 hi 5.86 defg 1 Basza F 6.93 hi 5.80 defg 1 Hardwicki line 603 7.08 i 7.07 g

*a-i homogenous groups according to Student-Newman-Keuls test

Conclusions The majority of screened cucumber cultivars (60%) were intermediate in their reaction to Pseudomonas syringae pv. lachrymans. However it was possible to determine several hybrids with higher resistance than the resistant standard Gy14. The Hard- wicki 603 line was showing the highest scores. Among 14 bacterial isolates selected for cucumber screening for angular leaf spot the one most pathogenic and repeatable was strain 814/98 from the Netherlands collection.

References

Chand, J. and Walker, J. 1963. Inheritance of resistance to angular leafspot of cucumber. Phyto- pathology, 54: 51-53. Dessert, J.M., Baker, L.R. and Fobes, J.F. 1981. Inheritance of reaction to Pseudomonas lachrymans in pickling cucumber. Euphytica, 31: 847-855. Jenkins, S.F. and Wehner, T.C. 1983. A system for the measurement of foliar diseases of cucumber. Cucurbit Genet. Coop. Rep. 6: 10-12. Kùdela, V. 1986. Pseudomonas syringae pv. lachrymans. In: Lebeda, A. (Ed.), Methods of Testing Vegetable Crops for Resistance to Plant Pathogens. VHJ Sempra, VÚZ Olomouc, pp. 77-80. Kùdela, V. and Lebeda, A. 1997. Response of wild Cucumis species to inoculation with Pseudo- monas syringae pv. lachrymans. Genet. Res. Crop Evol., 44: 271-275.

248 Lelliot, R.A., Billing, E. and Hayward, A.C. 1966. A determinative scheme for the fluorescent plant pathogenic Pseudomonads. J. Appl. Bacteriol., 29: 470-489. Williams, P. and Keen, N. 1967. Relation of cell permeability alterations to water congestion in cucumber angular leaf spot. Phytopathology, 57: 1378-1385.

249 250 Characteristics of resistance to Acidovorax avenae subsp. citrulli in the Citrullus lanatus accessions PI 482279 and PI 494817

D.L. Hopkins University of Florida, Mid-Florida Research and Education Center, 2725 Binion Road, Apopka, Fl 32703-8504, USA

Summary

Characteristics of the resistance of PI 482279 and PI 494817 to bacterial fruit blotch (Acido- vorax avenae subsp. citrulli) were compared with the susceptible watermelon cultivar Crimson Sweet. Hypersensitivity did not appear to be involved in the resistance. With leaf infiltrations at higher inoculum concentrations (>104 bacteria/ml), there were no obvious differences in symptom development between the PIs and Crimson Sweet watermelon. However, at lower inoculum concentrations, the bacterium multiplied more rapidly and reached higher final populations in the Crimson Sweet plants than in the PIs. This reduced colonization of the PIs when compared to Crimson Sweet may have been the mechanism of resistance that was clearly observed in the PIs after the more natural inoculation of misting the plants with water suspensions of A. avenae subsp. citrulli. This resistance found in PI 482279 and PI 494817 should be effective against bacterial fruit blotch in the greenhouse and field where spread of the disease is through splashing water droplets or aerosols.

Introduction

Bacterial fruit blotch (BFB) of watermelon (Citrullus lanatus), caused by Acido- vorax avenae subsp. citrulli, was first observed in commercial production areas in Guam in 1988 (Wall and Santos, 1988). The disease has occurred in one or more watermelon-producing states in the eastern U.S. every year since 1989 (Maynard and Hopkins, 1999). BFB has the potential to cause disaster in any cucurbit-growing area of the world. For example, bacterial fruit blotch was a problem in melons in Costa Rica in 2002. With BFB, exclusion of the pathogen has been the most successful means of control to this time. The intensive efforts of the seed industry and the transplant industry to produce seeds and transplants free of A. avenae subsp. citrulli have reduced significantly the incidence of BFB. In spite of these efforts, the bacterium still appears in a few fields every year, and there are still significant losses occurring to this disease in some years. In addition to infested seeds and infected transplants, the bacterium can invade a field from contaminated volunteer watermelons, other cultivated cucurbits, and wild cucurbits (Latin and Hopkins, 1995). The only control option for fruit blotch once it infests a field is multiple applications of copper-containing fungicides (Hopkins, 1991). Resistance is a preferred method of control for plant diseases; therefore, watermelon germplasm was screened for resistance to BFB (Hopkins and Thompson, 2002).

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 251 Accessions were found that have genes for resistance that may be useful in a watermelon breeding program. While the major loss to BFB of watermelon is due to fruit symptoms, the resistance that was identified in the accessions was a foliar resistance. Foliar resistance could eliminate the foliage as a reservoir of bacteria to infect the fruit and render the germinating seedling resistant to seed transmission of the bacterium. The objective of this research was to define the characteristics and mechanisms of the resistance identified in two of the accessions.

Materials and methods

Inoculations by leaf infiltration Two Citrullus lanatus accessions (PI 482279 and PI 494817), and the BFB-susceptible watermelon cultivar Crimson Sweet were used. Seedlings in the 3 to 4 true leaf stage were inoculated. A Florida strain of A. avenae subsp. citrulli (WFB89-1) isolated in 1989 from a commercial watermelon cultivar was used throughout these tests. For inoculations, WFB89-1 was grown on nutrient agar for 48 hr and washed from the agar surface with phosphate buffer. Bacterial suspensions were adjusted to A = 0.25 (107 CFU/ml of phosphate buffer) with a spectrophotometer and diluted 600nm with sterile buffer to the desired concentrations. Using a 25 gauge needle, inoculum was injected into the intercellular spaces of plant leaves. The infiltrated, water-soaked area was marked with a marking pen for future evaluations of symptoms and for sampling. Experiments were repeated at least once. In the first experiment, two true leaves per plant were injected with bacterial dilutions. The concentrations of inoculum were 107, 105, 103, and 102 CFU/ml. Plants were maintained in the greenhouse, where temperatures ranged from 24oC to 33oC, and observed for symptom development in the infiltrated areas for 7 days. In the second experiment, inoculum containing 102 CFU/ml was used to infiltrate cotyledons and true leaves. Three leaf disks each were taken from infiltrated areas of the two accessions and Crimson Sweet. The 0.03 g of tissue samples were ground in 1 ml phosphate buffer. These ground samples and 10-fold dilutions of the samples were plated on TC media, on which A. avenae subsp. citrulli colonies can be identified. 50

Inoculation by misting to runoff Inoculum preparation and host plants were as described above. Four plants were inoculated per treatment by misting the plants thoroughly until runoff occurred on both upper and lower leaf surfaces. Bacterial concentrations in inoculum varied from 107 to 102 CFU/ml of inoculum. After inoculation, seedlings were placed in a moist chamber on the greenhouse bench for two 18-hr nights. After 48 hr in the moist chamber, inoculated seedlings were placed in a greenhouse, where temperatures ranged from 24oC to 33oC. Disease ratings were made 10 days after inoculation using a scale based on symptom appearance: 1 = no symptoms; 2 = few small, necrotic lesions on cotyledons; 3 = small, necrotic lesions on cotyledon, <20% necrotic cotyledon; 4 = small, necrotic lesions on >20% of cotyledon; 5 = necrotic lesions with chlorosis on cotyledon, 20-50% necrotic cotyledon; 6 = necrotic lesions on 20-50% of cotyledon with restricted lesions on true leaf; 7 = large spreading lesions, >50% of cotyledon necro-

252 tic with restricted lesions on true leaves; 8 = large spreading lesions, >50% of cotyledon necrotic with lesions and chlorosis on true leaves; and 9 = >90% necrosis of the cotyledon and large spreading lesions on the true leaves, or a dead plant. Each plant was given a rating and an average rating was calculated for each treatment

Results and discussion

Leaf infiltration experiments With the 107 CFU/ml inoculum, Crimson Sweet and the two PIs had necrosis in the infiltrated areas of the leaves after 24 hr. In plants inoculated with the 105 CFU/ ml, yellowing was observed after 24 hr and necrosis at 72 hr. With the lower inoculum levels of 103, and 102 CFU/ml, visible symptoms were first observed at 72 hr and limited necrosis occurred at 120 hr or later. There were no obvious differences between the PIs and Crimson Sweet in development of BFB symptoms after leaf infiltration of A. avenae subsp. citrulli into leaves. To further characterize any possible resistance to colonization of leaf tissue in the PIs, multiplication of A. avenae subsp. citrulli in leaf tissue was determined for 5 days after infiltration. When more than 104 CFU/gram of tissue were injected into the leaves, bacterial populations reached more than 107 CFU/g in all 3 host plants. When less than 104 CFU/gram of tissue were injected into the leaves, bacterial populations reached that same level in Crimson Sweet, but were at least 100-fold lower in the 2 PIs (Fig. 1). Thus, there appears to be resistance to colonization of the PIs at low bacterial infiltration levels, but high inoculum levels overcomes this resistance.

Figure 1. Multiplication of A. avenae subsp. citrulli in resistant PIs compared with Crimson Sweet watermelon.

253 Inoculation by misting with A. avenae subsp. citrulli suspensions PI 482279 and PI 494817 developed much milder symptoms after misting with a bacterial suspension than did the susceptible watermelon Crimson Sweet (Table 1). The resistance was evident at all concentrations of inoculum from 103 to 107 CFU of bacteria/ml. There were some severe BFB symptoms in the resistant PIs at the highest inoculum level of 107 CFU of bacteria/ml, but symptoms were much more severe in Crimson Sweet watermelon. At all other inoculum levels, only small restricted lesion occurred on the cotyledons or true leaves of the PIs. At the lowest inoculum level of 102 CFU of bacteria/ml, only an occasional restricted lesion could be found on any of the seedlings. Misting is an inoculation method that closely resembles natural inoculations that occur in greenhouses where the bacteria are spread by overhead watering or in the field where the bacteria are spread by rain. Therefore, this resistance should be effective in the greenhouse and field.

Table 1. Rating of BFB symptoms in Crimson Sweet watermelon and PI 482279 and PI 494817 after inoculation by misting with A. avenae subsp. citrulli1

Inoculum concentration (CFU/ml) Cultivar 107 105 103 102

Crimson Sweet 8.3 4.6 2.7 1.3 PI 482279 3.8 1.8 1.3 1.3 PI 494817 5.8 2.6 1.5 1.0

1Disease ratings were made 10 days after inoculation using a scale based on symptom appearance: 1 = no symptoms; 3 = small, necrotic lesions on cotyledon, <20% necrotic cotyledon; 5 = necrotic lesions with chlorosis on cotyledon, 20-50% necrotic cotyledon; 7 = large spreading lesions, >50% of cotyledon necrotic; and 9 = >90% necrosis of the cotyledon.

In conclusion, resistance of the PIs to BFB was overcome by infiltration of the tissue with very high concentrations of bacteria. Hypersensitivity did not appear to be involved in the resistance. The resistances of PI 482279 and PI 494817 were very good under natural type inoculation, where the inoculum was misted or splashed onto the seedlings and entered the plant through stomata. This resistance of the PIs to BFB probably resulted from reduced colonization of the plants by A. avenae subsp. citrulli, similar to that observed after injection of the tissue with low concentrations of inoculum in the leaf infiltration experiments.

References

Hopkins, D.L. 1991. Chemical control of bacterial fruit blotch of watermelon. Proc. Fla. State Hort. Soc., 104: 270-272. Hopkins, D.L. and Thompson, C.M. 2002. Evaluation of Citrullus sp. germplasm for resistance to Acidovorax avenae subsp. citrulli. Plant Dis., 86: 61-64.

254 Latin, R.X. and Hopkins, D.L. 1995. Bacterial fruit blotch of watermelon: The hypothetical exam question becomes reality. Plant Dis., 79: 761-765. Latin, R.X., Tikhonova, I. and Rane, K.K. 1995. Factors affecting the survival and spread of Acidovorax avenae subsp. citrulli in watermelon transplant production facilities. Phytopatho- logy, 85: 1413-1417. Maynard, D.N. and Hopkins, D.L. 1999. Watermelon fruit disorders. HortTechnol., 9: 155-161. Wall, G.C. and Santos, V.M. 1988. A new bacterial disease of watermelon in the Mariana Islands. Phytopathology, 78: 1605 (Abstr.).

255 256 Plant eR genes encoding for glyoxalate aminotransferase enzymes confer resistance against downy mildew in melon

D. Kenigsbuch2, D. Taler1, M. Galperin1, I. Benjamin1 and Y. Cohen1 1Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel 2Department of Postharvest Science, Volcani Center, Beit-Dagan 50250, Israel

Summary

Downy mildew caused by the oomycete pathogen Pseudoperonospora cubensis is a devastating foliar disease of cucurbits worldwide. We previously demonstrated that the melon (Cucumis melo L.) line PI 124111F (PI) is highly resistant to all pathotypes of P. cubensis. That resistance was genetically controlled by two partially-dominant complementary loci. Here we show that unlike other plant disease resistance (R) genes, which confer an ability to resist infection by pathogens expressing corresponding avirulence (avr) genes, the resistance of PI to P. cubensis is controlled by enhanced expression of enzymatic resistance genes (eR) At1 or At2. These constitutively-expressed genes encode for the photorespiratory peroxisomal enzyme proteins glyoxylate-aminotransferases. The low expression of At1 and At2 in susceptible melon lines is mainly regulated at the transcriptional level. This regulation is independent of infection with the pathogen. Transge- nic melon plants overexpressing either one of these eR genes displayed enhanced activity of glyoxylate-aminotransferases and remarkable resistance against P. cubensis. The cloned eR genes provide a new resource for developing downy mildew-resistant melon varieties.

Keywords: Cucumis melo, Pseudoperonospora cubensis, aminotransferase, peroxisomes, eR-genes for resistance

Introduction

The oomycete, algal-like organism, P. cubensis, causes a devastating disease, downy mildew, in leaves (only) of cucumber, melon, watermelon, squash and luffa. It has six pathotypes (rather than physiological races) each infecting specific genera (rather than cultivars of the same species) of the Cucurbitacae (Thomas et al., 1987b; Cohen et al., 2003). Lebeda and Widrlechner (2003) proposed a set of 12 differential taxa of Cucurbitaceae to identify pathotypes of P. cubensis. Previously, we identified a genotype (PI 124111F = PI) of melon from India, which exhibited multiple resistance to several diseases including downy mildew (Cohen and Eyal, 1987). Upon inoculation of susceptible cultivars with P. cubensis, large chlorotic leaf lesions developed with abundant sporulation of the pathogen, whereas, the resistant PI showed minute, water-soaked, chlorotic lesions with no sporulation. Host cells within and around such minute lesions respond in massive accumulation of callose-like materials in their walls and phenolics and lignin-like substances in their cytoplasm (Cohen et al., 1989). PI 124111F is resistant to all six known pathotypes of P. cubensis (Thomas et al., 1987b; Cohen et al., 2003). Our previous genetic results suggested that two partially-dominant complementary loci, Pc and Pc , are responsible for disease resistance (Thomas et al., 1987a; Kenigsbuch 1 2 and Cohen, 1992). Further studies revealed that resistance in PI and in its F deri- 10

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 257 vative from the cross PI x Hemed was associated with the presence of a specific protein P45 (Balass et al., 1992). P45 was negligible in the near-isogenic Hemed susceptible melon line (Balass et al., 1992). In the F plants moderate amount of P45 was detected 1 (Balass et al., 1992). Most interestingly, resistance diminished when the temperature under which inoculated plants were incubated was lowered from >21°C to <15°C, indicating that resistance depends on metabolic activity of the host (Balass et al., 1993). In this study we report on cloning of the genes encoding for the P45 protein. These genes, when overexpressed into a susceptible melon turned it highly resistant to downy mildew. A full report on these results was published recently (Taler et al., 2004).

Material and methods

Plant material and transformation Our inbred melon line PI124111F (PI) (Cohen and Eyal, 1987) was used as a source for downy mildew resistance. The commercial line Hemed and our susceptible inbred line BU21/3 (with high competence for regeneration in culture) were used as susceptible controls and an acceptor, respectively. Plant transformation was performed as described by Galperin et al. (2003).

Pathogen inoculation and assessment of disease development A local isolate of Pseudoperonospora cubensis (Berk. & Curt. et de Toni) Rost. was used for inoculation. Propagation of the pathogen was done as described elsewhere (Kenigsbuch and Cohen, 1989). Resistance of F plants to P. cubensis was examined at 2 7 days post-inoculation using the RT index described before (Thomas et al., 1987a; Kenigsbuch and Cohen, 1992). Briefly, RT value was visually assessed in leaf 1 and leaf 2, resulting in a double-digit score for each plant. RT values ranged between 11- 44, with RT 11 = highly susceptible and 44 highly resistant. Resistance in detached leaves of T plants was similarly examined except that leaves were placed inside plas- 0 tic trays (20 x 20 x 2 cm) on moist filter paper. In order to assess resistance of T plants 1 under natural conditions, plants were raised in a shade house and then inoculated at the 5-leaf stage by spraying sporangial suspension (1x103 sporangia/ml) of the pathogen on their leaf surfaces. Disease development was periodically recorded until fruit harvest (at 4 months). A mean RT index was calculated for all leaves in a plant.

Microscopy Fluorescence micrographs of lesions taken from infected leaves were done under UV light as previously described (Cohen et al., 1989). Briefly, ethanol-clarified leaf discs were first stained with 0.05% aniline blue in 70 mM K HPO pH 8.9 buffer for 2 4 24 h and then with 0.01% calcofluor (Sigma) solution in water for 5 min. Sporangio- phores and sporangia emerging from stomata fluoresced blue, whereas callose depo- sits in plant cells fluoresced yellow.

Northern gel blot analysis Total RNA was extracted from plants using TRI reagent (Sigma, St Louis, MI). Ten mg of total RNA was size-fractionated on formaldehyde agarose gel and transferred to

258 Zeta probe membrane (Bio-Rad, Hercules, CA). The blot was hybridized with a three random primed 32P labeled fragments: 490 bp probe of At1 (777-1267 in the coding region), 380bp probe of At2 (1027-1407) and 466 bp probe of 18S RNA (981-1447) synthesized on RT-PCR product template with downstream primer 5-GTGTTGGCT- TCGGGATCGG-3 and upstream primer 5-CGCTCCACCAACTAAGAACGG-3.

Enzymes assays Homogenates from 0.5 g leaf tissue in 70 mM HEPES pH 7 were prepared at 4°C and 0.2 mg total protein in 10 µl was taken for each assay. AGT activity was measured at 340 nm by coupling the reduction of glyoxylate to the oxidation of NADH, in the presence of excessive lactate dehydrogenase. The reaction mixture contained 10 µl homogenate, 20 mM alanine 70 mM HEPES pH 7, 0.17 mM NADH, 0.1 mM pyri- doxal-5-phosphate, 0.03 U/ml lactate dehydrogenase and 1 mM glyoxylate in a final volume of 1ml. SGT activity was measured at 340 nm by coupling the reduction of glyoxylate to the oxidation of NADH, in the presence of excessive hydroxypyruvate reductase. The reaction mixture was as above except that alanine was replaced with 20 mM serine and lactate dehydrogenase was replaced by 0.05 U/ml hydroxypyruvate reductase. The reaction was started by adding the amino acid. Water was added instead of an amino acid to the reference cuvette (Rehfeld and Tolbert, 1972). Protein concentration was determined using Bradford reagent (Bio-Rad). Specific enzymatic activity for each aminotransferase reaction is expressed in µmol min-1 mg- 1 and was calculated using the extinction coefficient for NADH of 6.2 cm-1 mM-1. Glycolate oxidase (GO) activity was measured according to Macheroux et al. (1991) by an enzyme-coupled assay using horseradish peroxidase and o-dianisidine which utilizes the hydrogen peroxide generated by GO during oxidation of glycolate. A typical assay mixture contained 10 µl horseradish peroxidase (1 mg/ml), 50 µl o-dianisidine solution (8 mM, 20% Triton X-100), 10 µl sodium glycolate (1M), and 930 µl of 0.1M potassium phosphate buffer pH 8.3. The reaction was started by adding 10 µl of leaf homogenate. Formation of o-dianisidine radical cation which reflects the catalytic activity of glycolate oxidase was measured spectrophotometrically at 440 nm at 25°C.

Results and discussion

Cloning genes At1 and At2 from the resistant PI A P45 denatured protein was isolated from leaves of healthy, downy mildew-resistant melon PI124111F (herein PI). This protein was negligible in melon lines dis- playing susceptibility to the disease (Balass et al., 1992). Partial sequencing of P45 revealed 6 peptides. To retrieve the P45-encoding genes from PI we performed RT- PCR with degenerated oligonucleotides designed from these peptides. Two specific DNA bands were obtained, cloned and sequenced. The two DNA fragments were subjected to RACE reactions to obtain the 5 and 3 ends. This enabled the synthesis of the full length of both cDNAs. Two genes were cloned from PI, At1 (GenBank accession AY066012) and At2 (GenBank accession AF461048). They each encode for a 401 amino-acid protein. The two proteins share 93% homology in deduced amino

259 acids and 86% homology in nucleic acid sequence. BLAST analysis revealed >80% homology of the N-terminus of the proteins encoded by either At1 or At2 with serine- glyoxylate aminotransferase (SGT) from Fritilaria agrestis (Accession AF039000) and alanine-glyoxylate aminotransferase (AGT1) from Arabidopsis thaliana (Accession AF063901). Those enzymes occur in plants peroxisomes and participate in producing glycine during photorespiration. AGT1 in Arabidopsis primarily catalyses SGT trans- amination (Liepman and Olsen, 2001). Homologes of AGT1 are also known in other plants, animals, and bacteria (see literature cited in Liepman and Olsen, 2001, 2003).

Transformation of At1 and At2 into susceptible melon plants To confirm the role of At1 and At2 in resistance against downy mildew, the susceptible BU21/3 line of melon was used for transformation (Galperin et al., 2003). Detached cotyledon pieces of BU21/3 were inoculated with the Agrobacterium tumefaciens EHA105 strain carrying At1 or At2 from PI124111F driven by CaMV 35S promoter. About 200 regenerated plants (T ) showing resistance to kanamycin were grown in a shade house (50 o mesh). BU21/3 plants transformed with the empty vector were used as positive control and PI as a negative control. Detached leaves were exposed to infection with the downy mildew pathogen P. cubensis in growth chambers. All positive control plants and about one half of the At1 and one half of At2-transformed T plants were susceptible to the di- 0 sease, showing large leaf lesions (10-15 mm) with profuse pathogens sporulation. The other plants differ in their level of resistance from high (RT 34, 44) to moderate (RT 22, 23, 24, 33). Each highly resistant individual was selfed, seeds (T ) were collected and 1 germinated on kanamycin in culture. Germlings segregated into 3 : 1 resistant : sensitive to kanamycin, suggesting single insertions. The kanamycin resistant germlings were transferred to pots and thereafter to the shade house and examined for resistance to the disease. About 90-100% (in various lines) of these T plants were highly resistant (RT 33-44) to the di- 1 sease all along the season. The highly resistant plants were characterized by the appearance of small (1-2 mm) chlorotic, water-soaked lesions with no sporulation (HR, hypersensitive response) (Fig. 1A). Microscopical examinations of such lesions (Fig. 1B) revealed enhanced accumulation of callose similar to, or even stronger than, PI (Cohen et al., 1989). RT-PCR conducted with resistant individuals of the T and T generations revea- 0 1 led, in all, overexpression of At1 or At2. It should be noted that BU21/3 plants as well as BU21/3 plants transformed with At1 or At2 were susceptible to powdery mildew caused by the fungus Sphaerotheca fuliginea, confirming the specific action of At1 and At2 against P. cubensis. PI was completely resistant because it carries the genes Pm3 and Pm6 for resistance against S. fuliginea (Kenigsbuch and Cohen, 1989).

SGT and AGT activities in transgenic melon plants SGT and AGT enzymatic activities were comparatively analyzed in nine transgenic T plants (7 resistant, one moderately resistant and one susceptible), PI (resistan- 1 ce source) and the susceptible lines Hemed and BU21/3 (vector only). Results are shown in Fig. 2. Both enzymes were several folds more active in the resistant T plants 1 than in susceptible control plants. Some resistant T plants showed >60% higher ac- 1 tivity than PI, probably due to the CaMV 35S-promoter driving At1 or At2. The transgenic plant with intermediate level of resistance (#128) showed lower enzyme activities and the susceptible transgenic plant (#148) lowest activities (Fig. 2A, B)

260 T #133 BU21/3 PI124111F 1

Figure 1. Macroscopic and microscopic appearance of downy mildew in melon leaves 7 days postinoculation. A. Note a compatible interaction (large lesions occupying most of the leaf area) in the susceptible BU21/3 as against a hypersensitive response (HR) (small chlorotic flecks) in the resistant PI and T #133. The latter is a 1 transgene overexpressing At2. B. Fluorescen- Figure 2. SGT (A) and AGT (B) activi- ce micrographs of lesions taken with UV light. ty in melon leaves. Resistant PI, susceptible Leaf discs were stained with 0.005% aniline Hemed and BU21/3, At1-transgenic T 1 blue in 70 mM K HPO pH 8.9 buffer for 24h #148 susceptible, At2 # 128 moderate- 2 4 and then with 0.01% calcofluor solution in water ly-resistant and resistant At1-transgenic for 5 min. Note sporulation in the BU21/3 as T #s 114, 184, 185, and resistant At2 1 against no sporulation and callose surrounding transgenic T #s 101, 111, 133, 134. 1 the lesions in PI and T . 1

Northern analysis of At1 and At2 Low enzymatic activity of At1- or At2-encoded AGT and SGT occurs in leaf extracts of the susceptible melons Hemed and BU21/3, relative to the resistant PI or T 1 (Fig. 2A, B). Northern blot analysis (Fig. 3) indicated that At1 and At2 were transcribed in the resistant melon (PI) much stronger compared to the susceptible melon (He- med). This suggests that the difference in AGT and SGT enzymic activity between the susceptible and resistant lines is mainly regulated at the transcriptional level.

Figure 3. Northern analysis of At1 and At2 from leaves of downy mildew- susceptible (S) and resistant (R) plants. Northern blot was hybridized with specific probes for At1 and At2. The 18S RNA was used as standard.

261 AGT activity in segregating F family 2 PI 124111F was crossed with Hemed and the F plants, having 2 fully expanded 2 leaves, were subjected to P. cubensis infection in growth chambers. The population segregated in the response to disease into 3 phenotypic classes (Thomas et al., 1987a): resistant, moderately resistant, and susceptible. Leaf 3 from 6-10 representative individuals, expressing either resistance, moderate resistance, or susceptibility was thereafter examined for AGT enzymatic activity. All resistant individuals exhibited high activity of AGT, similar to the resistant parent, whereas all susceptible individuals showed a significantly lower activity, similar to that of the susceptible parent. The intermediate phenotype plants were also intermediate in AGT activity (Fig. 4) confirming co-segregation of resistance and enzyme activity.

Figure 4. AGT activity in segregating F melon population from resistant (PI) x sus- 2 ceptible (Hemed). F plants with 2 expanded leaves were inoculated with 2 P. cubensis. Seven day post inoculation plants were evaluated for resistance and the third leaf from 6-10 plants from each subgroup were analyzed for AGT activity. Dif- ferent letters on columns indicate a significant difference at 5% probability level (Duncans multiple range test). RT of the resistant, moderately-resistant, and susceptible offsprings ranged between 34-44, 23-33, and 11-12, respectively.

Glycolate oxidase (GO) activity in resistant and susceptible melon plants GO catalyzes the oxidation of glycolic acid to glyoxylate and H O in the peroxi- 2 2 somes during photorespiration. GO activity was measured in the resistant and susceptible melons with the premise that enhanced glyoxylate aminotransferase activity may affect its activity. Data in Table 1 indeed confirm that GO activity in leaf homogenates of the resistant PI was ~10-20 times higher compared to similar homogenates made from the susceptible Hemed. In the susceptible Hemed, activity in young and old leaves was quite similar whereas in the resistant PI activity in older leaves was about twice higher than in young leaves.

262 Other experiments revealed similar low activity of GO in Hemed, AY and BU21/3 (all susceptible) but a 4.1 and 4.8 fold enhanced activity in T #4 and T #133, respe- 1 1 ctively. Infection (2 days) of such plants with P. cubensis had only a minor effect on GO activity (data not shown). These data suggest that the enhanced SGT/AGT activity in the resistant genotypes is associated with accelerated activity of the upstream, peroxisomal, photorespiratory enzyme glycolate oxidase.

Table 1. Activity of Glycolate oxidase in leaf extracts of susceptible (Hemed) and resistant (PI 124111F) melons

DOD mg protein-1 min-1

Hemed PI Plant Leaf 1 Leaf 2 Leaf 1 Leaf 2

1 0.036 0.0240.4230.162 2 0.022 0.021 0.627 0.192 3 0.020 0.021 0.496 0.283

Mean ±SE 0.026±0.07 0.022±0.0140.515±0.0840.212±0.050 Fold increase 19.8 9.6

The present study describes two aminotransferase-encoding genes, At1 and At2, each capable of mediating resistance of melon against P. cubensis. These genes are constitutively and highly expressed in leaves of the resistant melon PI and transgenic plants which carry them. Highly homologous genes are present in susceptible melons but they are not expressed. Mendelian genetics conducted with the Cucumis melo P. cubensis pathosystem revealed two complementary loci, Pc1 and Pc2, controlling resistance (Thomas et al., 1987a; Kenigsbuch and Cohen, 1992). Molecular cloning of the resistance genes unexpectedly discovered that they do not belong to any class of known R genes (for review, see Dangl and Jones, 2001). Rather, they encode for aminotransferase enzymatic activity and therefore designated At1 and At2. The results from the F population from the cross (PI x Hemed) indicate a strong 2 linkage between the plants response to the pathogen and its AGT enzyme activity (Fig. 4). Resistant, moderately resistant and susceptible individuals exhibited high, moderate and low AGT activity, respectively. It seems most likely, therefore, that At1 and At2 are Pc1 and Pc2. The proof for the pivotal role of At1 or At2 in resistance against P. cubensis was obtained from transgenic melons. Transformed melon plants, carrying either At1 or At2, expressed high activity of serine or alanine glyoxylate aminotransferase and exhibited a strong resistance to the pathogen. Whereas in PI and its derivatives the complementary action of Pc1 and Pc2 is required for full protection against disease, in transgenic plants a single insert of either gene driven by a strong promoter sufficed for resistance. These genes, therefore, may be utilized for producing new transgenic culti-

263 vars of resistant melon while saving the excessive labor required for recurrent crossing and selection. Upon inoculation of PI with P. cubensis, a hypersensitive reaction is observed in the leaves, characterized as small chlorotic, water-soaked lesions embedded with callose and lignin depositions (Cohen et al., 1989). Transgenic melon plants carrying either At1 or At2 similarly reacted to infection with the pathogen. This suggests that At1 and At2 are responsible for defense gene activation (e.g., cell-wall strengthening, callose deposition and lignification) which culminates in cell death and arrest of the pathogen. The literature supplies ample evidence showing that ROI production is often induced during early stages of a plant resistance response (see Jackson and Taylor, 1996; Heath, 2000). However, we know of no plant species (excluding transformed) in which constitutive production of H O is associated with resistance. 2 2 This paper provides a link between photorespiration and resistance to disease. Photorespiration, although being a wasteful process, was shown to provide protection against stress caused by salinity, drought and intensive light (Wingler et al., 2000). The present study indicates that it also provides resistance against disease.

Acknowledgements The technical assistance of D. Levy and D. Gotlieb is gratefully acknowledged.

References

Balass, M., Cohen, Y. and Bar-Joseph, M. 1992. Identification of a constitutive 45 kD soluble protein associated with resistance to downy mildew in muskmelon (Cucumis melo L.) line PI 124111F. Physiol. Mol. Plant Pathol., 41: 387-396. Balass, M., Cohen, Y. and Bar-Joseph, M. 1993. Temperature-dependent resistance to downy mildew in muskmelon: structural responses. Physiol. Mol. Plant Pathol., 43: 11-20. Cohen, Y. and Eyal, H. 1987. Downy mildew-, powdery mildew- and fusarium wilt-resistant muskmelon breeding line PI-124111F. Phytoparasitica, 15: 187-195. Cohen, Y., Eyal, H., Hanania, J. and Malik, Z. 1989. Ultrastructure of Pseudoperonospora cubensis in muskmelon genotype susceptible and resistant to downy mildew. Physiol. Mol. Plant Pathol., 34: 27-40. Cohen, Y., Meron, I., Mor, N. and Zuriel, S. 2003. A new pathotype of Pseudoperonospora cubensis causing downy mildew in cucurbits in Israel. Phytoparasitica, 31: 458-466. Dangl, J.L. and Jones, J.D. 2001. Plant pathogens and integrated defence responses to infection. Nature, 411: 826-833. Galperin, M., Patlis, L., Ovadia, A., Wolf, D., Zelcer, A. and Kenigsbuch, D. 2003. A melon genotype with superior competence for regeneration and transformation. Plant Breeding, 122: 66-69. Heath, M.C. 2000. Hypersensitive response-related death. Plant Mol. Biol., 44: 321-334. Jackson, A.O. and Taylor, C.B. 1996. Plant-Microbe interactions: Life and death at the interface. Plant Cell, 8: 1651-1668. Kenigsbuch, D. and Cohen, Y. 1989. Independent inheritance of resistance to race 1 and race 2 of Sphaerotheca fuliginea in muskmelon. Plant Dis., 76: 615-617. Kenigsbuch, D. and Cohen, Y. 1992. Inheritance of resistance to downy mildew in Cucumis melo PI 124112 and commonalty of resistance genes with C. melo PI 124111F. Plant Dis., 76: 615- 617.

264 Lebeda, A. and Widrlechner, M.P. 2003. A set of Cucurbitaceae taxa for differentiation of Pseu- doperonospora cubensis pathotypes. J. Plant Dis. Prot., 110: 337-349. Liepman, A.H. and Olsen, L.J. 2001. Peroxisomal alanine: glyoxylate aminotransferase (AGT1) is a photorespiratory enzyme with multiple substrates in Arabidopsis thaliana. Plant J., 25: 487-498. Liepman, A.H. and Olsen, L.J. 2003. Alanine aminotransferase homologs catalyze the glutamate: glyoxylate aminotransferase reaction in peroxisomes of Arabidopsis. Plant Physiol., 131: 215- 227. Macheroux, P., Massey, V., Thiele, D.J. and Volokita, M. 1991. Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization. Biochemistry, 30: 4612- 4619. Rehfeld, D.W. and Tolbert, N.E. 1972. Aminotransferases in peroxisomes from spinach leaves. J. Biol. Chem., 247: 4803-4811. Taler, D. Galperin, M. Benjamin, I. Cohen, Y. and Kenigsbuch, D. 2004. Plant eR genes that encode photorespiratory enzymes confer resistance against disease. Plant Cell, 16: 172-184. Thomas, C.E., Cohen, Y., McCreight, Y.D., Jourdion, E.L. and Cohen, S. 1987a. Inheritance of resistance to downy mildew in Cucumis melo. Plant Dis., 72: 33-35. Thomas, C.E., Inaba, T., and Cohen, Y. 1987b. Physiological specialization in Pseudoperonospo- ra cubensis. Phytopathology, 77: 1621-1624. Wingler, A., Lea, P.J., Quick, W.P. and Leegood, R.C. 2000. Photorespiration: metabolic pathways and their role in stress protection. Philos. Trans. R. Soc. Lond. B Biol. Sci., 355: 1517-1529.

265 266 Disease impact and pathogenicity variation in Czech populations of Pseudoperonospora cubensis

A. Lebeda and J. Urban Palacký University, Faculty of Science, Department of Botany, lechtitelù 11, 783 71 Olomouc, Czech Republic; e-mail: [email protected]

Summary

Monitoring of Pseudoperonospora cubensis distribution and disease impact on cucurbits was conducted during 2001 (106 locations visited) and 2002 (134 locations visited) in the Czech Republic. Natural infection was observed only on cucumbers (Cucumis sativus), with other cucurbits were free of infection. On the majority of cucumber crops (63.2% in 2001, 64.6% in 2002) was observed a serious or very serious degree of infection (DI 3-4). A medium degree of infection was recorded on 25.5% of crops in 2001 and 32.8% in 2002. Other cucumber crops were characterized by low or no infection (11.3% in 2001, 2.6% in 2002). For determination of pathogenic variability were used a total of 96 P. cubensis isolates (42 isolates from 2001, 54 from 2002). Altogether, 43 different pathotypes were detected (33 in 2001, 16 in 2002). In both years, a large proportion of the screened isolates belonged to the group of highly pathogenic pathotypes (47.6% in 2001, 51.9% in 2002) and to the group of pathotypes with medium pathogenicity (47.6% in 2001, 48.1% in 2002). Only 2 isolates collected in the year 2001 expressed low pathogenicity. Race-specific interactions between Cucumis melo-P. cubensis and Cucurbita spp.-P. cubensis were observed.

Keywords: Cucurbits, cucumber, cucurbit downy mildew, host range, occurrence, disease severity, pathotypes

Introduction

Cucurbit downy mildew, caused by Pseudoperonospora cubensis (Berk. and Curt.) Rostov., is one of the most important diseases affecting field and glasshouse cucumbers and other cucurbits production around the world. Since 1984, there have been annual epidemics of this disease of high economic impact in the Czech Republic and the whole of Central Europe (Lebeda, 1990; Lebeda and Schwinn, 1994). Although downy mildew most often occurs in tropical, subtropical and warm temperate areas of the world, it is known to occur in cooler regions, too, such as Sweden (Forsberg, 1986) and Finland (Tahvonen, 1985), to where inoculum is transported by air flows. P. cubensis attacks a broad spectrum of cucurbitaceous plants. Palti and Cohen (1980) reported about 40 host species, however, more recently 60 species were reported to be affected (Lebeda, 1999). Cucumis sativus L. is the most often (ca 80 countries) and most seve- raly affected host, followed by C. melo L. (> 50 countries), Citrullus lanatus (Thunb.) Matsum. & Nakai., Cucurbita maxima Duchesne, and C. pepo L. (Lebeda, 1990). Interactions and host-parasite specificity between Cucurbitaceae and P. cubensis are heterogeneous and complex. Existence of P. cubensis pathotypes was observed in different countries around the world (Lebeda and Widrlechner, 2003), including the Czech Republic (Lebeda and Gadasová, 2002). Recently, some previously unknown

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 267 and highly virulent isolates were described from Israel (Cohen et al., 2003) and the Czech Republic (Lebeda and Urban, 2003). However, until now detailed information is lacking concerning the disease impact and pathogenic variation of P. cubensis populations in the Czech Republic and Central Europe. The main aims of this research were to study disease impact of cucurbit downy mildew and determine which pathotypes occur in the Czech Republic. Herewith is a summary of the results obtained in 2001 and 2002.

Material and methods

Origin of Pseudoperonospora cubensis isolates The occurrence of P. cubensis in the Czech Republic was monitored at 106 locations and at 134 locations in the growing seasons (July-September) of 2001 and 2002, respectively. Infected host plants were visually evaluated on a 0-4 scale (Lebeda and Køístková, 1994) modified for P. cubensis. Altogether, 181 isolates (77 in 2001, 104 in 2002) were collected from infected crops in ten regions and 32 districts of the Czech Republic.

Pathogen isolation and maintenance The infected leaf samples were on wet filter paper in plastic pots (110 x 85 x 45 mm). Inoculum was prepared by shaking small pieces of the infected leaves in distilled water and atomised over the abaxial surface of a leaf of C. sativus cv. Marketer 430 (susceptible) placed in a Petri dish on wet filter paper. Inoculated leaves were incubated in a growth chamber (Lebeda, 1986). The pathogen produced conidiosporangiophores with spores, usually after 7-8 days. Pure cultures of P. cubensis were stored in Petri dishes at -20°C. The spores were vital for about six months, after which it was necessary to renew the cultures by fresh inoculations.

Plant material The highly susceptible cucumber Marketer 430 was used for pathogen multiplication. Pathogenicity of isolates was screened on a differential set of 12 cucurbit taxa (Lebeda and Widrlechner, 2003) (Table 1). Plants were grown under optimal conditions (25°C/15°C day/night, daily watering, weekly fertilization) in a glasshouse and were not treated chemically. Discs were cut from leaves of 68-week-old plants (36- true-leaf stage).

Determination of pathogenic variation Altogether, 96 (42 collected in 2001, 54 in 2002) isolates of P. cubensis were screened for pathogenic variability. A leaf-discs method (Lebeda, 1986) was used for determination of P. cubensis pathotypes on the differential set (Lebeda and Widrlech- ner, 2003). Each differential genotype was represented by 5 leaf discs in 3 replicates (one replicate per plant). Discs were inoculated with a spore suspension (105 spores/ ml) by using a glass sprayer and incubated in a growth chamber under the conditions described by Lebeda (1991). Evaluations were made 6-14 days after inoculation by

268 using 0-4 scale (Lebeda, 1991). Differential genotypes with no or a low level of sporulation were considered to be resistant, and those with a medium and or high level of sporulation were considered to be as susceptible. Pathotypes were designed with tetrade numerical codes (Lebeda and Widrlechner, 2003).

Table 1. Differential set of cucurbit taxa for determination of pathogenic variability in Pseudoperonospora cubensis, after Lebeda and Widrlechner (2003)

No. Taxon Accession number Cultivar name Country of origin Donor EVIGEZ 1 Cucumis sativus H39-0121 Marketer 430 USA 2 C. melo subsp. melo PI 292008 H40-1117 Ananas Israel Yoqne´am 3 C. melo subsp. agrestis CUM 238/1974H40-0625 Baj-Gua Japan var. conomon 4 C. melo subsp. agrestis PI 200819 H40-0611 Myanmar var. acidulus 5 Cucurbita pepo PI 171622 H42-0117 Dolmalik Turkey subsp. pepo 6 C. pepo subsp. texana PI 614687 H42-0130 USA 7 C. pepo subsp. fraterna* PI 532355 H42-0136 Mexico 8 Cucurbita maxima H42-0137 Goliá Czechoslovakia

9 Citrullus lanatus H37-0008 Malali Israel 10 Benincasa hispida BEN 485 H15-0001 USA 11 Luffa cylindrica H63-0010 ? 12 Lagenaria siceraria H63-0009 ?

EVIGEZ - Czech genebank number * originally described as Cucurbita fraterna (Lebeda and Gadasová, 2002) ? unknown

Results and discussion

During the growing seasons of 2001 and 2002, natural infection of P. cubensis was observed only on Cucumis sativus, all other Cucurbitaceae (Cucumis melo, Cucurbita pepo, Cucurbita maxima, Cucurbita foetidissima, Citrullus lanatus) were free of infection. On the majority of C. sativus crops (63.2% in 2001, 64.6% in 2002) was recorded high or very high degree of infection (DI = 3-4). Medium degree of infection was observed on 25.5% of cucumber crops in 2001 and 32.8% in 2002. Other cucumber crops were characterized by low or no infection (11.3% in 2001, 2.6% in 2002). In 2002, a somewhat more severe infection was observed (DI = 4.27%) on C. sativus as compared to 2001 (DI = 0.9%). The 96 P. cubensis isolates belonged to 43 different pathotypes (Table 2), (examples in Table 4). In both years, a large proportion of the screened isolates could be

269 considered as belonging to highly pathogenic pathotypes (47.6% in 2001, 51.9% in 2002) with 9-12 pathogenicity factors, nearly all the rest could be considered as belonging to medium pathogenicity pathotypes (47.6% in 2001, 48.1% in 2002) with 5-8 pathogenicity factors. Only 2 isolates (pathotypes), both collected in the year 2001, expressed low pathogenicity. Isolates of P. cubensis from 2001 were characterized by higher pathogenic variability than those from 2002. In 2001, 33 pathotypes were detected whereas in 2002 only 16 pathotypes were detected. The pathotype 15.15.15., represented by isolate 39/01 originating from Vacenovice (district Hodonín, region of South Moravia), was the most virulent. Pathotypes 3.0.12. and 11.0.8., represented by isolates 65/01 (from Moravská Tøebová, district Svitavy, region Pardubice) and 38/01 (from Bzenec, district Hodonín, region of South Moravia), had low virulence, giving a susceptible reaction only on 4 differential genotypes (Cucumis sativus, C. melo subsp. melo or C. melo var. acidulus, Lagenaria siceraria and Luffa cylindrica). In 2001, the most frequent pathotype was 15.14.14., showing avirulence only on Cucurbita pepo subsp. pepo and Cit- rullus lanatus; in 2002, pathotype 15.10.10. had avirulence to Cucurbita pepo subsp. pepo, C. pepo subsp. fraterna, Citrullus lanatus and Luffa cylindrica.

Table 2. Summary of pathogenic variability of Pseudoperonospora cubensis isolates collected in 2001 and 2002 in the Czech Republic

Pathogenicity level (no. 2001 2002 of pathogenicity factors) No. of % of all No. of No. of % of all No. of isolates isolates pathotypes isolates isolates pathotypes Low pathogenicity (1-4) 2 4.8 2 0 0.0 0 Medium pathogenicity (5-8) 20 47.6 18 26 48.1 9 High pathogenicity (9-12) 20 47.6 13 28 51.9 7 In total 42 100.0 33 54 100.0 16

It is evident that Cucumis spp. were highly susceptible to Czech isolates of P. cubensis (Table 3). C. sativus and C. melo subsp. melo showed no resistance against the screened isolates. C. melo subsp. agrestis var. conomon and C. melo subsp. agrestis var. acidulus were susceptible to all P. cubensis isolates collected in 2002, however, in 2001 were recorded P. cubensis isolates with avirulence to these taxa (Table 3). Dif- ferentials of Cucurbita pepo (Table 1) exhibited large variation in susceptibility to different P. cubensis isolates. C. pepo subsp. pepo was one of the most resistant genotypes (susceptible only to 29% of the screened isolates in 2001 and to 11% of isolates in 2002). C. pepo subsp. fraterna was characterized also by a relatively high level of race-specific resistance, in contrast to C. pepo subsp. texana, which showed high susceptibility. C. maxima was susceptible to the majority of the isolates (60% in 2001, 80% in 2002). Benincasa hispida and Lagenaria siceraria were highly susceptible to Czech isolates of P. cubensis, however Citrullus lanatus showed a high level of resistance (compatible interaction with 10% of isolates from 2001 and 2% of isolates from 2002). Luffa cylindrica expressed susceptibility to 67% of the isolates from 2001, but only to 35% of the isolates from 2002 (Table 3).

270 The results show that there is extremely high variability of pathogenicity in the Czech population of P. cubensis. Until now, such a wide spectrum of variation was not reported from other countries (Bains and Sharma, 1986; Thomas et al., 1987; Cohen et al., 2003). Recent results demonstrate, that there is much higher variability in P. cubensis pathogenicity as was expected before (Lebeda and Gadasová, 2002). The constitution of the Czech P. cubensis population is probably not stable from the viewpoint

Table 3. Susceptibility of individual differential genotypes to P. cubensis isolates collected in 2001 and 2002 in the Czech Republic

Differential genotype* Frequency of compatible reactions 2001 2002 1 1.0 1.0 2 1.0 1.0 3 0.67 1.0 40.81 1.0 5 0.29 0.11 6 0.95 0.94 7 0.50 0.31 8 0.60 0.80 9 0.10 0.02 10 0.76 1.0 11 0.67 0.35 12 0.90 0.85 * see Table 1

Table 4. Examples of different P. cubensis pathotypes collected in 2001 and 2002 in the Czech Republic

Pathotype* No. of isolates 2001 2002 Low pathogenic 3.0.12. 1 0 11.0.8. 1 0 Medium pathogenic 15.2.10. 1 5 15.10.10. 1 10 High pathogenic 15.14.10. 1 8 15.10.14. 3 8 15.14.14. 4 6 15.15.14. 2 1 15.15.15. 1 0 * Tetrade codes according to Lebeda and Widrlechner (2003)

271 of pathogenic variation, as shown by comparing the isolates collected in the years 2001 and 2002. Some pathotypes observed in the Czech Republic could be considered to be unique from the global viewpoint, because they are characterized by complex of pathogenicity. Other data indicate, that the host-parasite specificity between Cucumis melo-P. cubensis and Cucurbita spp.-P. cubensis is controlled by race-specific R-factors (Lebeda, 1991, 1999; Lebeda and Gadasová, 2002; Lebeda and Widrlechner, 2003, 2004). Under laboratory conditions, a number of Czech P. cubensis isolates were able to infect cucurbit species that are not commonly cultivated in the Czech Repub- lic or elsewhere in Central Europe, including Citrullus lanatus, Benincasa hispida, and Lagenaria siceraria. These data suggest that at least part of the pathogen population occurring in this region might be transported from regions to the south and east where highly pathogenic isolates are known to occur (Cohen et al., 2003).

Acknowledgements

This research was supported by grants: QD 1357; MSM 153100010; National Pro- gramme of Genepool Conservation of Microorganisms and Small Animals of Econo- mic Importance.

References

Bains, S.S. and Sharma, N.K. 1986. Differential response of certain cucurbits to isolates of Pseu- doperonospora cubensis and characteristics of identified races. Phytophylactica, 18: 31-33. Cohen, Y., Meron, I., Mor, N. and Zuriel, S. 2003. A new pathotype of Pseudoperonospora cubensis causing downy mildew in cucurbits in Israel. Phytoparasitica, 31: 458-466. Forsberg, A.S. 1986. Downy mildew-Pseudoperonospora cubensis in Swedish cucumber fields. Växtskyddsnotiser, 50: 17-19. Lebeda, A. 1986. Pseudoperonospora cubensis. In: Lebeda, A. (Ed.), Methods of Testing Vege- table Crops for Resistance to Plant Pathogens. VHJ Sempra, Research Institute of Vegetable Crops, Olomouc, pp. 81-85. Lebeda, A. 1990. Biology and ecology of cucurbit downy mildew. In: Lebeda, A. (Ed.), Cucurbit Downy Mildew. Czechoslovak Scientific Society for Mycology by Czechoslovak Academy of Sciences, Praha, pp. 13-46. Lebeda, A. 1991. Resistance in muskmelons to Czechoslovak isolates of Pseudoperonospora cubensis from cucumbers. Sci. Hort., 45: 255-260. Lebeda, A. 1999. Pseudoperonospora cubensis on Cucumis spp. and Cucurbita spp. resistance breeding aspects. In: Abak, K. and Buyukalaca, S. (Eds.), Proceedings of the 1st International Symposium on Cucurbits. Acta Hort., 492: 363-370. Lebeda, A. and Gadasová, V. 2002. Pathogenic variation of Pseudoperonospora cubensis in the Czech Republic and some other European countries. In: Nishimura, S., Ezura, H., Matsuda, T. and Tazuke, A. (Eds.), Proceedings of the 2nd International Symposium on Cucurbits. Acta Hort., 588: 137-141. Lebeda, A. and Køístková, E. 1994. Field resistance of Cucurbita species to powdery mildew (Erysiphe cichoracearum). J. Plant Dis. Protect., 101: 598-603. Lebeda, A. and Schwinn, F.J. 1994. The downy mildews an overview of recent research progress. J. Plant Dis. Protect., 101: 225-254. Lebeda, A. and Urban, J. 2003. Distribution, harmfulness and pathogenic variability of cucurbit downy mildew in the Czech Republic. In: Hudec, K. and Huszár, J. (Eds.), XVI. Slovak and Czech Plant Protection Conference, Abstracts Supplement. Slovak Agricultural University, Nitra, Slovak Republic, pp. 119-120.

272 Lebeda, A. and Widrlechner, M.P. 2003. A set of Cucurbitaceae taxa for differentiation of Pseu- doperonospora cubensis pathotypes. J. Plant Dis. Protect., 110: 337-349. Lebeda, A. and Widrlechner, M.P. 2004. Response of wild and weedy Cucurbita L. to pathotypes of Pseudoperonospora cubensis (Berk. & Curt.) Rostov. (Cucurbit downy mildew). In: Spen- cer-Phillips, P.T.N. and Jeger, M. (Eds.), Advances in Downy Mildew Research, Vol. 2. Kluwer Academic Publishers, Dordrecht (in press). Palti, J. and Cohen, Y. 1980. Downy mildew of cucurbits (Pseudoperonospora cubensis): The fungus and its hosts, distribution, epidemiology and control. Phytoparasitica, 8: 109-147. Tahvonen, R. 1985. Downy mildew of cucurbits found for the first time in Finland. Växtskyddsnotiser, 49: 42-44. Thomas, C.E., Inaba, T. and Cohen, Y. 1987. Physiological specialization in Pseudoperonospora cubensis. Phytopathology, 77: 1621-1624.

273 274 Differential sensitivity to fungicides in Czech populations of Pseudoperonospora cubensis

J. Urban and A. Lebeda Palacký University, Faculty of Science, Department of Botany, lechtitelù 11, 783 71 Olomouc, Czech Republic; e-mail: [email protected]

Summary

During the growing seasons of 2001 and 2002, 42 isolates of Pseudoperonospora cubensis were collected from nine regions of Czech Republic and screened for tolerance or resistance to fungicides. The effectiveness of three frequently used fungicides (Previcur 607 SL /propamocarb/, Aliette 80 WP /fosetyl-Al/, Ridomil Plus 48 /metalaxyl/) was tested at five concentrations, using a floating disc bioassay. Fungicide effectiveness varied considerably. Propamocarb was the most effective as all isolates were sensitive to all concentrations (607-9712 µg a.i./ml). Fosetyl-Al was also effective, as most isolates were controlled by the optimal concentration (1600 µg a.i./ml). However, the occurrence of isolates collected in 2001 that sporulated at low concentrations (400 and 800 µg a.i./ml) of this fungicide could indicate that selection for tolerance is occurring in the population, but this was not re-observed on the P. cubensis isolates collected in 2002. Metalaxyl was ineffective, as 93% of the isolates showed resistance to the optimal concentration (200 µg a.i./ml) of this fungicide, and the other 7% of isolates were tolerant. Profuse and/or limited sporulation of 88% of the isolates was also observed on 400 and 800 µg a.i./ml solutions, i.e. concentrations 2x and 4x times higher than optimal. A substantial shift to highly resistant strains was evident in the Czech P. cubensis population in 2001 and 2002.

Keywords: cucurbit downy mildew, cucumbers, Czech Republic, metalaxyl, propamocarb, fosetyl-Al, fungicide tolerance/resistance, temporal shift

Introduction Cucurbit downy mildew, caused by Pseudoperonospora cubensis (Berk. et Curt.) Rost., has become an important, widespread disease of cucurbit crops in the Czech Republic and Central Europe. The first epidemic of P. cubensis in the Czech Repub- lic (former Czechoslovakia) occurred in 1984 (Lebeda, 1986a). Since then, epidemics have occurred almost every year, the source of inoculum derived primarily from eastern and southern parts of Europe (Lebeda, 1990). Cucumber (Cucumis sativus L.) and melon (C. melo L.) are the economically most important as well as the most susceptible cucurbits in the Czech Republic to this pathogen (Lebeda, 1999; Lebeda and Urban, 2003). The fungus is highly variable in its pathogenicity (Lebeda and Widrlechner, 2003) and disease control through the planting of resistant cultivars has not, as yet, been successful (Lebeda and Práil, 1994). Thus, control of the disease on field-grown crops often depends on the use of systemic fungicides. However, the appearance of P. cubensis strains resistant and/or tolerant to some fungicides has raised serious concern (Lebeda and Schwinn, 1994). Examples of the loss of effectiveness of several fungicides (e.g. metalaxyl, strobilurine, mancozeb) against P. cubensis has been reported elsewhere over the past 20 years (Samoucha and Cohen, 1984, 1985; O´Brien

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 275 and Weinert, 1995; Ishii et al., 2001). The potential existence of metalaxyl resistance was also hypothesized to occur in the Czech Republic (Ackermann, 1990) and the results of the first experiment in the Czech Republic confirming this were published recently (Urban and Lebeda, 2003). The aim of this research was to observe and monitor the possible occurrence of P. cubensis populations in the Czech Republic that are resistant or tolerant to several commonly used fungicides (metalaxyl, propamocarb and fosetyl-Al).

Material and methods

Origin of Pseudoperonospora cubensis isolates During the growing seasons (JulySeptember) of 2001 and 2002, samples of Cu- cumis sativus leaves infected by P. cubensis were collected. Altogether 42 isolates (15 in 2001, 27 in 2002) were collected in fields of nine regions and 29 districts of the Czech Republic.

Pathogen isolation and maintenance The infected leaf samples were placed in plastic pots (110 x 85 x 45 mm) on the wet filter paper. Inoculum was prepared by shaking small pieces of the infected leaves in distilled water and atomised over abaxial surface of a leaf of C. sativus cv. Marketer 430 (susceptible) placed in a Petri dish on the wet filter paper. Inoculated leaves were incubated in the growth chamber (Lebeda, 1986b). The pathogen produced conidiosporangiophores with spores, usually after 7-8 days. Pure cultures of P. cubensis were stored in Petri dishes at -20°C. The spores were vital for about six months, after which it was neccessary to renew the cultures by fresh inoculations.

Plant material The highly susceptible cucumber Marketer 430 was used for pathogen multiplication and for floating leaf-discs bioassays. Plants were grown under optimal conditions (25°C/ 15°C day/night, daily watering, weekly fertilization) in a glasshouse and were not treated chemically. Discs were cut from leaves of 6-8-week-old plants (3-6-true-leaf stage).

Fungicides and the floating leaf discs bioassay Three widely used fungicides (metalaxyl, source Ridomil Plus 48 WP, additional substance: oxychlorid Cu; fosetyl-Al, source Aliette 80 WP; propamocarb, source Previcur 607 SL) registered in the Czech Republic were used for the screening. Five concentrations of each fungicide were tested (Table 1), one recommended by the producer (i.e. optimal) and two others below and above the optimum. Treatment with distilled water served as the control. Leaf discs (15 mm in diameter) were floated, abaxial surface up, on fungicide solutions in Corning multiwell plates (Sigma-Aldrich) (Anony- mous, 1982). There were four leaf discs in three replicates for every concentration of each fungicide. After 24 h, each disc was inoculated with a spore suspension (1x105 spores/ml) using a glass sprayer. Inoculum was prepared from 2-3-day-old spores of P. cubensis. Leaf discs were incubated as described by Lebeda (1991).

276 Evaluations of the fungicide bioassay Evaluation was made 6th-14th day after inoculation by using the 0-4 scale and the degree of infection was expressed as a percentage (Lebeda, 1991). Three types of reactions were assigned: sensitive (degree of infection (DI) = 0-10%), tolerant (DI = 10.1- 34.9%) and resistant (DI ³ 35%). Values of ED 50 (fungicide concentration, which inhibits fungal growth by 50%) were determined for each screened isolate and expressed in ranges of fungicide concentrations.

Results and discussion

Considerable variation in the reaction of the 42 Czech P. cubensis isolates grown in vitro to the three fungicides was observed (Table 1). Propamocarb appeared as the most effective against the pathogen. All screened isolates were highly sensitive to this fungicide and were fully controlled by the lowest tested concentration (607 µg a.i./ml). Fosetyl-Al was also effective. This fungicide gave complete control of downy mildew at the optimal dosage (1600 µg a.i./ml (ED 50 values <400 µg a.i./ml)). Differences between isolates collected in the year 2001 and 2002 were detected. In 2001, 47% of the isolates collected were controlled even by the 400 µg a.i./ml solu-

Table 1. Reactions to several fungicides of some P. cubensis isolates collected from Czech populations in 2001 and 2002

Fungicide Concentrat. Isolates of P. cubensis/the type of reaction/the degree of infection (%) (µg a.i./ml) 53/02 17/01 16/01 101/02 3/01 24/02 10/1/02 28/01 35/01

Metalaxyl 0 +/100.0 +/96.0 +/75.0 +/100.0 +/100.0 +/100.0 +/100.0 +/100.0 +/100.0 50.4 +/100.0 +/95.8 +/100.0 +/100.0 +/100.0 +/100.0 +/100.0 +/100.0 +/96.0 100 +/100.0 +/81.3 +/100.0 +/100.0 +/83.3 +/100.0 +/100.0 +/87.3 (-)/29.2 200 +/100.0 +/79.3 +/75.0 +/100.0 +/72.8 +/87.5 +/100.0 (-)/31.3 (-)/20.8 400 +/79.0 +/77.0 +/81.2 +/100.0 (-)/27.2 (-)/23.0 -/0 -/0 -/2.1 800 +/79.3 +/45.8 (-)/23.0 -/6.25 (-)/10.4 -/0 -/2.1 -/0 -/0 Fosetyl-Al 0 +/100.0 +/100.0 +/100.0 +/87.5 +/100.0 +/100.0 +/85.3 +/56.2 +/89.7 400 -/0 +/41.8 (-)/10.5 -/0 -/0 -/0 -/6.25 (-)/14.7 (-)/29.3 800 -/0 (-)/29.8 -/0 -/0 -/0 -/0 -/0 (-)/10.4(-)/10.4 1600 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 3200 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 6400 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 Propamocarb 0 +/91.7 +/100.0 +/98.0 +/100.0 +/100.0 +/100.0 +/100.0 +/100.0 +/98.0 607 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 1214-/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 2428 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 4856 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 9712 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0 -/0

- = sensitive reaction (no sporulation), (-) = tolerant reaction (limited sporulation), + = resistant reaction (profuse sporulation) of P. cubensis.

277 tion, and 53% sporulated on concentrations lower than optimal (i.e. tolerant/resistant reactions on 400, resp. 800 µg a.i./ml). The isolate 17/01 (from Libìice, district Li- tomìøice) was the most resistant with the resistant reaction on 400 µg a.i./ml and tolerant reaction on 800 µg a.i./ml. P. cubensis isolates from 2002 were highly sensitive and without sporulation on all tested concentrations of the fungicide. Metalaxyl was practically ineffective and 93% of screened isolates showed resistance to the optimum concentration (200 µg a.i./ml) and 7% showed tolerance. A tolerant reaction on concentration lower than optimal was observed only on one isolate (35/01). Pro- fuse and/or limited sporulation was characteristic also on 400 and 800 µg a.i./ml solutions, i.e. concentrations 2x and 4x higher than optimal (88% of isolates). Four isolates (17/01, 43/02, 53/02, 74/02) were resistant at all tested concentrations of fungicide. A substantial shift to highly resistant strains is evident by comparing the isolates collected in 2001 and 2002 (Figs. 1 and 2). Whereas 20% of isolates collected in 2001 were defined by ED 50 values < 200 µg a.i. metalaxyl/ml, in 2002 there were no isolates with this attribute. On the contrary, 7% of isolates from the year 2002 had ED 50 values >800 µg a.i. metalaxyl/ml, and there were no isolates from 2001 with this characteristic. The majority of isolates (60%) collected in 2001 had ED 50 values 200-400 µg a.i. metalaxyl/ml. A substantial part (48%) of the isolates from the year 2002 had ED 50 values 400-800 µg a.i. metalaxyl/ml and only 45% 200-400 µg a.i. metalaxyl/ml.

Figure 1. Structure of P. cubensis isolates set collected in 2001 according to ED 50 values for metalaxyl.

Considerable differences were observed in the effectiveness of frequently used fungicides against cucurbit downy mildew in the Czech Republic (Table 1). Propamocarb appeared to be the most effective for application in the field, as no P. cubensis strains re-

278 Figure 2. Structure of P. cubensis isolates set collected in 2002 according to ED 50 values for metalaxyl. sistant or tolerant to it were found at any of the tested concentrations. In contrast, propamocarb-resistance of cucurbit downy mildew was described by Cohen and Sa- moucha (1984) in Israel, where this fungicide gave no control of downy mildew as a soil drench at a concentration of 420 µg a.i./ml. Fosetyl-Al was also effective. Howe- ver, the occurrence of isolates with tolerant/resistant responses to lower concentrations of this fungicide in 2001 could signal selection of more resistant strains, but this response did not reoccur in 2002. Cohen and Samoucha (1984) did detect P. cubensis isolates resistant to a soil drench of this fungicide at a concentration 3200 µg a.i./ml. Metalaxyl-resistance of P. cubensis was first reported by Samoucha and Cohen (1984). O´Brien and Weinert (1995) tested lower concentrations of metalaxyl and observed metalaxyl-sensitive (controlled by 0.1 µg/ml), metalaxyl-intermediate-sensitive (controlled by 10 µg/ml) and metalaxyl-resistant (sporulation on 100 µg/ml) isolates of P. cubensis. More recent results indicated that metalaxyl is completely ineffective and confirmed the gradual shift to resistant strains in the fungal population. his phenomenon is known from other oomycetes in other countries, too (Gisi, 2002), such as the high frequency of metalaxyl-resistance observed in Phytophthora infestans (e.g. Fisher and Hayes, 1984; Daayf and Platt, 1999; Shattock, 2002). Cohen and Samoucha (1984) also reported cross-resistance of metalaxyl-resistant P. cubensis strains to other systemic fungicides (vinicur, sandofan, propamocarb, fosetyl-Al), but this has not been confirmed, so far, in Czech isolates of P. cubensis. The chemical control of P. cubensis is the most effective strategy currently used. However, the presence of metalaxyl-resistance and possible starting of fosetyl-Al-tolerance in Czech pathogen population could make downy mildew control in cucurbit crops more difficult.

279 Acknowledgements

This research was supported by grants: QD 1357; MSM 153100010; National Pro- gramme of Genepool Conservation of Microorganisms and Small Animals of Econo- mic Importance.

References

Ackermann, P. 1990. Occurrence and protection against cucurbit downy mildew in Czechoslova- kia. In: Lebeda, A. (Ed.), Cucurbit Downy Mildew. Czechoslovak Scientific Society for Myco- logy by Czechoslovak Academy of Sciences, Praha, pp. 51-61. Anonymous, 1982. FAO Method No. 30. In: FAO Plant Protect. Bull., Vol. 30/2. Cohen, Y. and Samoucha, Y. 1984. Cross-resistance to four systemic fungicides in metalaxyl-resistant strains of Phytophthora infestans and Pseudoperonospora cubensis. Plant Dis., 68: 137-139. Daayf, F. and Platt, H.W. 1999. Assessment of mating types and resistance to metalaxyl of Cana- dian populations of Phytophthora infestans in 1997. Amer. J. Potato Res., 76: 287-295. Fisher, D.J. and Hayes, A.L. 1984. Studies of mechanism of metalaxyl fungitoxicity and resistance to metalaxyl. Crop Protect., 3: 177-185. Gisi, U. 2002. Chemical control of downy mildews. In: Spencer-Phillips, P.T.N., Gisi, U. and Lebeda, A. (Eds.), Advances in Downy Mildew Research. Kluwer Academic Publishers, Dord- recht, pp. 119-159. Ishii, H., Fraaije, B.A., Sugiyama, T., Noguchi, K., Nishimura, K., Takeda, T., Amano, T. and Hollomon, D.W. 2001. Occurrence and molecular characterization of strobilurin resistance in cucumber powdery mildew and downy mildew. Phytopathology, 91: 1166-1171. Lebeda, A. 1986a. Epidemic occurrence of Pseudoperonospora cubensis in Czechoslovakia. Temperate Downy Mildews Newsletter, 4: 15-17. Lebeda, A. 1986b. Pseudoperonospora cubensis. In: Lebeda, A. (Ed.), Methods of Testing Vege- table Crops for Resistance to Plant Pathogens. VHJ Sempra, VÚZ Olomouc, pp. 81-85. Lebeda, A. 1990. Biology and ecology of cucurbit downy mildew. In: Lebeda, A. (Ed.), Cucurbit Downy Mildew. Czechoslovak Scientific Society for Mycology by Czechoslovak Academy of Sciences, Praha, pp. 13-46. Lebeda, A. 1991. Resistance in muskmelons to Czechoslovak isolates of Pseudoperonospora cubensis from cucumbers. Scientia Hort., 45: 255-260. Lebeda, A. 1999. Pseudoperonospora cubensis on Cucumis spp. and Cucurbita spp. resistance breeding aspects. Acta Hort., 492: 363-370. Lebeda, A. and Práil, J. 1994. Susceptibility of Cucumis sativus cultivars to Pseudoperonospora cubensis. Acta Phytopath. Ent. Hung., 29: 89-94. Lebeda, A. and Schwinn, F.J. 1994. The downy mildews an overview of recent research progress. J. Plant Dis. Protect., 101: 225-254. Lebeda, A. and Urban, J. 2003. Distribution, harmfulness and pathogenic variability of cucurbit downy mildew in the Czech Republic. Acta fytotechn. et zootech. (in press) Lebeda, A. and Widrlechner, M.P. 2003. A set of Cucurbitaceae taxa for differentiation of Pseu- doperonospora cubensis pathotypes. J. Plant Dis. Protect., 110: 337-349. O´Brien, R.G. and Weinert, M.P. 1995. 3 metalaxyl sensitivity levels in australian isolates of Pseu- doperonospora cubensis (Berk. et Curt.) Rost. Austr. J. Exp. Agr., 35: 543-546. Samoucha, Y. and Cohen, Y. 1984. Differential sensitivity to mancozeb of metalaxyl-sensitive and metalaxyl-resistant isolates of Pseudoperonospora cubensis. Phytopathology, 74: 1437-1439. Samoucha, Y. and Cohen, Y. 1985. Occurrence of metalaxyl-resistant isolates of Pseudoperono- spora cubensis in Israel. EPPO Bull., 15: 419-422. Shattock, R.C. 2002. Phytophthora infestans: populations, pathogenicity and phenylamides. Pest. Manag. Sci., 58: 944-950. Urban, J. and Lebeda, A. 2003. Resistance to fungicides in population of cucurbit downy mildew in the Czech Republic. In: Hudec, K. and Huszár, J. (Eds.), XVI. Slovak and Czech Plant Protection Conference, Abstracts Supplement. Slovak Agricultural University, Nitra, Slovak Republic, pp. 157-158.

280 Disease impact and pathogenicity variation in Czech populations of cucurbit powdery mildews

A. Lebeda and B. Sedláková Palacký University, Faculty of Science, Department of Botany, lechtitelù 11, 783 71, Olomouc, Czech Republic; e-mail: [email protected]

Summary

Distribution, disease impact and pathogenic variability of cucurbit powdery mildew was monitored on cucurbitaceous crops in the Czech Republic (CR) during the years 2001 and 2002. Occurrence of powdery mildews, Golovinomyces cichoracearum s.l. (Gc) and Podosphaera xanthii (Px), was monitored at 84 locations in 2001 and 109 locations in 2002. The highest frequency of powdery mildew infection was observed on Cucurbita pepo (90% of locations) and Cucur- bita maxima (84%), but a low frequency was observed on Cucumis sativus (4%), and no infection was observed on Citrullus lanatus. A medium level of disease intensity (2550%) occurred on Cucurbita spp. The pathogens were identified on 237 samples of leaves with symptoms of powdery mildew originating from 9 regions and 30 districts of the CR. Gc occurred alone at 90% of the locations, Px occurred alone at only 2% and a mixture of both pathogens was found at 8% of locations. Pathogenic variability (pathotypes, races) was determined for 89 isolates (74 Gc, 15 Px) collected during the years 2000-2002. Altogether 12 different pathotypes (8 Gc and 4 Px) and 49 races (38 Gc and 11 Px) were found. Isolates virulent to C. melo line MR-1 and avirulent to C. melo Iran H were found in both powdery mildew species. Occurrence of some highly pathogenic strains of Gc were noted in 2001 and 2002 and a significant shift toward increased virulence was observed for both Gc and Px over the three-year period.

Keywords: cucurbits, Golovinomyces cichoracearum, Podosphaera xanthii, host range, distribution, disease severity, pathotypes, races

Introduction

Powdery mildew of cucurbits in Central Europe is caused by two ascomycete fungi, Golovinomyces cichoracearum s.l. (Gc) (syn. Erysiphe cichoracearum s.l.) and Podosphaera xanthii (Px) (syn. Sphaerotheca fuliginea). In general, Px occurs more frequently in warmer regions and on protected crops and Gc is more common in temperate cooler regions (Lebeda, 1983; Zlochová, 1990). However, Px became common in Europe (Bertrand et al., 1992; Vakalounakis et al., 1994) and spread to the northern areas of the Czech Republic (Køístková et al., 2002). Both genera are characterized by broad pathogenic variability represented by the existence of different pathotypes and races (Bertrand, 1991; Vakalounakis and Klironomou, 1995). Five races of Px and two races of Gc have been identified on melons, however recent results suggest that even more pathotypes and races exist (Lebeda et al., 2003). The purpose of this study was to observe the geographic distribution, host range and impact of powdery mildew on cucurbit crops and to survey and identify the powdery mildew species, pathotypes and races occurring in the Czech Republic. Herewi- th is a summary of the results obtained in 2001 and 2002.

Surveying of cucurbit powdery mildew and origin of isolates Occurrence of powdery mildew on cucurbits was monitored at 84 locations in 2001 and 109 locations in 2002 in the main production areas of the Czech Republic (CR). Infected host plants were evaluated visually on a 04 scale (Lebeda and Køístková, 1994). Altogether, 237 samples (104 in 2001, 133 in 2002) were collected from infected plants of Cucurbita pepo L., C. maxima Duchesne and Cucumis sativus L. in nine regions and 30 districts of the CR. The identification of both pathogens was carried out by microscopic examination of the morphological characters of dry conidia in a 3% KOH solution (Lebeda, 1983).

Pathogen isolation and maintenance Before isolation, the samples were microscopically examined. Isolates with a mixture of powdery mildew species were excluded. Conidia of pure cultures were transferred by tapping on primary leaves of highly susceptible cucumber (Cucumis sativus) Stela F . A total of 89 (74 Gc, 15 Px) isolates were obtained and used for deter- 1 mination of pathotypes and races. Isolates were cultured in plastic boxes (24°C/18°C day/night) for 12 h.

Determination of pathogenic variability Of the 89 isolates, three (2 Gc,1 Px) were collected in 2000; 34 (27 Gc, 7 Px) were collected in 2001 and 52 (45 Gc, 7 Px) were collected in 2002; they were screened for pathogenic variability (pathotypes, races) by a leaf-disc method (Lebeda, 1986). A set of six differential cucurbit taxa (Table 1) was used for determination of pathotypes (Bertrand et al., 1992). Races were identified by using 11 differential genotypes of Cucumis melo (Iran H, Védrantais, Solartur, PMR 45, WMR 29, Edisto 47, PI 414723, PMR 5, PI 124112, MR-1, Nantais Oblong) (Bardin et al., 1999). Each genotype was represented by 3 leaf discs (15 mm in diameter) in three replicates (one replicate per plant). Discs from true leaves (23-leaf stage) of cucumber plants were used for screening. Discs were inoculated by tapping a primary leaf of cucumber Ste- la F covered with 34-day-old sporulating mycelium and incubated under the con- 1 ditions described above. Evaluations were conducted 614 days after inoculation by using a 04 scale (Lebeda, 1984). Differential genotypes with little or no sporulation (degree of infection, DI = 01) were considered as resistant (R), genotypes with DI = 24 were scored as susceptible (S).

Results and discussion

There were not observed substantial differences between 2001 and 2002 in the distribution and degree of infection of cucurbit powdery mildew. The highest frequency of infection was observed on Cucurbita pepo (90% of locations) and C. maxima (84%), but there was a low frequency of occurrence on Cucumis sativus (4%). No powdery mildew infection was observed on Citrullus lanatus. The infections on most C. pepo plants were moderate to serious. Infections on C. maxima were mild to moderate; infections

282 , 1 ,,Diamant F CP(19), CM(9) CP CM CP CP Cucurbita pepo Cucurbita foetidissima. identified from 2000 through 2002 PMR 45, C = , CF = C. melo ,, = = 2 Cucurbita pepo Podosphaera xanthii Species/No. of isolates/Host Golovinomyces cichoracearum --- CS 1 -CM(3)CP(9), CS(1), 13 1 CPCM(5) CP(5), CS(1), 11 - - 2 CP(1), CM(1) CP 1 - 1 3 CP(1), CM(2) Podosphaera xanthii and , CP = Védrantais, B Védrantais, ,,Sugar Baby; C. melo ,, D -D CM 2 - CM 1 28 2 m m m m = = D - 4 CM(3), CF(1) 1 1 m m m CC CC C 1 CP 9 CP(6), CM(3) 3 CC CC CD - CP 1 CP 1 2 2 2 2 2 2 m Cucurbita maxima CC CC B B CCP 1 - CP 1 B CC B B B CD - CM 1 - 1 1 1 1 1 1 1 1 1 1 2 Citrullus lanatus , CM = Golovinomyces cichoracearum D 2000 2001 2002 ,,Marketer 430, B m ,,Goliá, D = CC Cucumis sativus 2 B . Pathotypes of 1 C. maxima Cucumis sativus = = m Differentialgenotype/reaction AB Pathotype of Year collection of isolates Host: CS = SSSSSS AB SRRSSRSS RSRR ACC SSSSRR AB AB SRSSRS AB SSRSSS AB SSSSSR AB Table Table 1 SSSSRS AB SSSSSS AB SSSSSR AB SSRSSR AB TotalA = 1 7 7 TotalSSRSSR AB 2 27 45 C

283 Table 2. Virulence of Golovinomyces cichoracearum and Podosphaera xanthii in 2000 through 2002

Pathogenicity level Year of collection of isolates (No. of virulence factors) 2001 2002 Species of pathogen No. of % of all No. of No. of % of all No. of isolates isolates races isolates isolates races

Golovinomyces cichoracearum Low virulence (1-4) 1 3.7 1 1 2.2 1 Medium virulence (5-7)* 12 44.4 12 4 8.9 4 High virulence (8-11) 1451.9 9 4088.9 13

Total 27 100.0 22 45 100.0 18

Podosphaera xanthii Low virulence (1-4) 1 14.3 1 1 14.3 1 Medium virulence (5-7)** 5 71.42 1 14.31 High virulence (8-11) 1 14.3 1 5 71.4 4

Total 7 100.0 47 100.0 6

In 2000: 3 screened isolates (2 Gc, 1 Px), all of them with medium level of virulence 2 isolates of Gc with 5 virulence factors, ** 1 isolate of Px with 6 virulence factors

Table 3. Frequency of compatible reactions on differential genotypes of C. melo after inoculation with cucurbit powdery mildew isolates originating from Czech Republic and collected from 2000 to 2002

Genotype of Species of pathogen/Year of collection Cucumis melo Frequency of compatible reactions

Golovinomyces cichoracearum Podosphaera xanthii 2000 2001 2002 2000 2001 2002

Iran H 0.50 0.89 0.98 1 0.86 0.67 Védrantais 1 0.93 1 1 1 1 Solartur 0.50 0.96 1 1 1 1 PMR 45 0.50 0.96 0.96 0 0.14 0.67 WMR 29 0.50 0.78 0.91 0 0.140.33 Edisto 47 1 0.740.87 1 0.141 PI 414723 0.50 0.70 0.96 1 0.86 0.83 PMR 5 0 0.59 0.80 0 0 0.67 PI 124112 0 0.26 0.71 0 0.14 0.33 MR-1 0 0.040.51 0 0.140.33 Nantais Oblong 0.50 0.67 0.87 1 1 1

284 on C. sativus were mild. Infections were first observed during the first half of July and the major disease spread occurred during the second half of July or early August. The limited powdery mildew infection of C. sativus could be explained by the earlier arrival and serious infection of Pseudoperonospora cubensis by the end of June or beginning of July. Cucurbit powdery mildew (CPM) was represented by a prevalence of Gc (89% of the samples in 2001, 91% in 2002); Px alone was recorded only in 3% of cases in 2001 and 1% in 2002. A mixture of both pathogens was found at 8% of locations (both in 2001 and 2002) (Lebeda et al., 2003). These results confirmed previous observations (Lebeda, 1983; Køístková et al., 2002) showing that the CPM species spectrum in CR is markedly different in comparison with some western and southern European countries, where Px prevails (Bertrand et al., 1992; Vakalounakis and Klironomou, 1995). Altogether 12 pathotypes (8 Gc and 4 Px) were identified in the set of studied isolates from 2000 through 2002 (Table 1). The Gc pathotypes AB B CC (48%) and 1 2 m AB B C (33%) prevailed in 2001, whereas in 2002 pathotype AB B CC D was the 1 2 1 2 m most frequent (62%) and pathotype AB B CC was rather common (24%). Px was 1 2 m characterized by predominance of pathotypes AB CC D (57%) and AB CC (29%) in 1 m 1 m 2001, but the AB B CC (43%) and AB B CC D (29%) pathotypes were more preva- 1 2 m 1 2 m lent in 2002. Evaluation of the pathogen population structure at the level of pathotypes showed that intermediate and highly pathogenic strains occur frequently. Vari- ation in pathotype spectrum and differences in predominance of pathotypes of both species among years were observed. Altogether, 49 races (38 Gc and 11 Px) were detected in the studied set of isolates during the years 20002002. In 2000, there were observed only races with medium virulence in both species. In 2001, there was a predominance of intermediate (44.4%) and highly virulent (51.9%) races of Gc, whereas in 2002, races with high virulence dominated (88.9%) (Tables 2 and 3). A similar situation occurred for Px, with prevalence (71.4%) of races with intermediate virulence in 2001 and prevalence of highly virulent races in 2002. Repeated occurrence of some highly virulent races of Gc (namely N, P, R, S) was observed in 2001 and 2002 (Table 4). Races S of Gc and F of Px were virulent on all C. melo differential genotypes. The most virulent race S of Gc was prevalent (78%) in 2002. These and other observations (Køístková et al., 2004) indicate that, in Czech pathogen populations, there occur races able to overcome the resistance of C. melo lines MR-1 and PI 124112. Five isolates of Gc and three isolates of Px were avirulent to C. melo Iran H, also consistent with the observations of Køístková et al. (2004), and until now, Iran H was considered to be susceptible to all races. These results indicate that even in Iran H there exist as yet unidentified race-specific resistance factor(s) to both CPM species. In conclusion, the results show that the Czech powdery mildew populations are highly heterogeneous, but that isolates of moderate and high virulence are prevalent. Furthermore, some pathotypes and races previously undescribed in Europe were observed in these Czech powdery mildew populations.

285 identified in 2001 2000 2001 2002 -- 2 -- 2 S S Podosphaera xanthii and Nobl Golovinomyces cichoracearum : IrH: Sol: Iran Solartur, P45: Védrantais, H, PMR Véd: 45, W29: WMR 29, E47: Edisto 47, C. melo Examples of highly virulent isolates of Golovinomyces cichoracearum SSSSSSSRRRSSSSSSSSSRRR NSSSSSSSRSRS - O 2 - N 2 2 - and 2002 Differential genotype/reaction patternIrH Véd Sol P45 W29 E47 PI41 P5 PI12 MR1 Race No. of isolates Table Table 4. SSSSSSSSRRSSSSSSSSSSRR PSSSSSSSSSRS - RSSSSSSSSRSS 42 -SSSSSSSSSSR Y 1 -SSSSSRSSSSS V - - 1 SSSSSSSSSSS R - 1 7 a - 4 - Podosphaera xanthii S - -SSSSSSSRRRS 1 2 SSSSSSSSRRS 14 2US- SSSSRSSSSSS GSSSSSSSSSSS - H - -Reaction: R = Fresistant, S -= susceptible; 2 -Genotypes of - 1 1 PI41: PI 414723, P5: PMR 5, PI12: PI 124112, MR1: MR-1, Nobl: Nantais Oblong.

286 Acknowledgements

Research was supported by grants: QD 1357; MSM 153100010; National Programme of Genepool Conservation of Microorganisms and Small Animals of Economic Im- portance.

References

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287 288 Variation in sensitivity to fungicides in Czech populations of cucurbit powdery mildews

B. Sedláková and A. Lebeda Palacký University, Faculty of Science, Departament of Botany, lechtitelù 11, 783 71 Olomouc, Czech Republic; e-mail: [email protected]

Summary

A survey was conducted of possible fungicide resistance or tolerance of cucurbit powdery mildews, Golovinomyces cichoracearum (Gc) and Podosphaera xanthii (Px), using 55 Czech isolates (43 Gc, 12 Px) collected at eight regions from 2000-2002. The effectiveness of three frequently used fungicides, Rubigan 12 EC (fenarimol), Karathane LC (dinocap), and Fundazol 50 WP (benomyl), at five concentrations, was observed using a modified leaf-disc bioassay. Discs were prepared from adult plants of Cucumis sativus cv. Stela F . Significant differences among 1 fungicides and even among years were observed. Fenarimol showed a high level of effectiveness and control of powdery mildew at dosage 36 µg a.i./ml (optimal concentration). Dinocap was 100% effective in the year 2000, however it was less effective in 2001 and 2002, when 5% of the isolates overcame the registered concentration (105 µg a.i./ml). Benomyl was the least effective of the fungicides as 94% of the isolates sporulated at every tested concentration and 48 of the isolates were resistant.

Keywords: Golovinomyces cichoracearum, Podosphaera xanthii, fenarimol, dinocap, benomyl, fungicide tolerance/resistance, geographical distribution, temporal shift

Introduction

Application of fungicides is the principal tool for managing powdery mildews (Hollomon and Wheeler, 2002). Fungicide resistance of cucurbit powdery mildews, Golovinomy- ces cichoracearum s.l. (Gc) (syn. Erysiphe cichoracearum s.l.) and Podosphaera xanthii (Px) (syn. Sphaerotheca fuliginea), is a serious worldwide problem, as new pathotypes and races overcoming resistance to fungicides are being increasingly reported (McGrath, 2001; McGrath and Shishkoff, 2001, 2003). The occurrence of fungicide resistance of powdery mildew on cucurbit crops in the Czech Republic was first reported recently in brief (Sedláková and Lebeda, 2003). The purpose of this research was to observe and monitor the possible occurrence of resistance or tolerance to several commonly used fungicides, specifically fenarimol, dinocap, and benomyl, in cucurbit powdery mildew populations in the Czech Republic.

Material and methods

Origin of cucurbit powdery mildew isolates During the growing seasons of 2000, 2001, and 2002, samples of cucurbit leaves (mostly Cucurbita pepo L. and C. maxima Duchesne, only a small number of samples originated from Cucumis sativus) infected by cucurbit powdery mildew (CPM) were

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 289 collected from July through September. Altogether 55 isolates (43 Gc, 12 Px) were collected in fields of nine regions and 20 districts of the Czech Republic and used for the fungicide-resistance/tolerance screening.

Pathogen isolation and maintenance The infected leaf samples were placed on wet filter paper in plastic pots (110 x 85 x 45 mm). For establishment of CPM cultures, seriously infected leaves of cucurbit host plants were used. Conidia of pure cultures were transferred by tapping on primary leaves of susceptible cucumber (Cucumis sativus) Stela F . Isolates were cultured 1 in plastic boxes in a growth chamber at 24°C/18°C day/night and 12 h photoperiod.

Plant material Highly susceptible Cucumis sativus Stela F was used for preparation of leaf discs. 1 Plants were grown in the glasshouse by 25°C/15°C day/night, with daily watering and weekly fertilization.

Fungicides and leaf-disc bioassay A modified leaf-disc bioassay (Anonymous, 1982; McGrath, 2001) was used for CPM resistance screening. Three widely used fungicides (fenarimol, source Rubigan 12 EC; dinocap, source Karathane LC; benomyl, source Fundazol 50 WP) registered in the Czech Republic were used for the screening. Five concentrations of each fungicide were tested (Table 1), one recommended by the producer (i.e. optimal*) and two others below and above the optimal. Treatment with distilled water served as the control. Leaf discs (15 mm in diameter) were prepared from leaves of adult plants of C. sativus Stela F . There were five leaf discs in three replicates for every concentrati- 1 on of each fungicide. Discs were deposited in plastic boxes (360 x 320 x 65 mm) with the fungicide solution and placed with the adaxial surfaces up on filter paper. Boxes were kept open for approximately 1 h to allow the discs to dry and then closed. The discs were inoculated 24 h later by tapping with the primary leaves of cucumber Stela F covered with 3-4-days-old and sporulating mycelium of the appro- 1 priate isolate of CPM. Incubation proceeded at the same conditions as the maintenance of isolates.

Evaluations of the fungicide bioassay Evaluations were conducted 6-14 days after inoculation by using the 0-4 scale (Lebeda, 1984) and the degree of infection was expressed as a percentage (Lebeda, 1991). Three types of reactions were assigned: sensitive (degree of infection, DI = 0- 10%; tolerant DI = 10.1-34.9%; and resistant, DI = ³35%).

Results and discussion

The response of the 55 powdery mildew isolates (43 Gc, 12 Px) to the three fungicides are summarized in Table 1. Rubigan 12 EC appeared to be the most effective as none of the screened isolates sporulated on the recommended concentration; howe-

290 ) ) ) ) ) ) ) ) ) Px Px Px Px Px Px Px Px Px 11 1 1 , 6 , 11 , , 12 , , 1 , 1 , 4 Gc Gc, Gc Gc Gc, Gc Gc Gc Gc, Gc Gc Gc Gc Gc Gc Gc (43

3 (2 ) 36 (32 )(34 45 )55 originating from the )(31 42 ) (4 8 Px Px Px Px Px , 3 6 6 , 6 , 6 , 3 Gc Gc Gc Gc, ) (1 3 ) 2 ) 2 ) 2 ) 1 ) (7 8 ) (1 2 ) 1 ) 3 ) 7 Gc Gc Gc Gc Gc Gc) Gc Gc Gc Gc Gc Gc ) 22 (16 ) 18 (15 ) 28 (22 ) 18 (12 ) ( 1 ) ( 1 ) 4 ( ) (2 5 Podosphaera xanthii Px Px Px Px Px Px Px Px 4 4 1 , 2 , 5 and , 1 , 1 , 1 Gc, Gc Gc Gc, ) ( 1 ) ( 1 ) ( 5 ) ( 1 Gc Gc Gc Gc, Px Gc Gc Gc 21 (17 ) 16 (14 ) 25 (20 ; Px Px Px) 1 , 1 , 1 isolates (pathogen) of isolates ) ( 1 ) 23 (19 Gc Gc Gc, Px Gc 2000 2001 2002 Golovinomyces cichoracearum Podosphaera xanthii = 250* 500 1000 105* 210 420 52.5 62.5 125 + + + + + + 2 (1 + + + + + (-) - (1 2 + + (-) (-) (-) (-) - ( 1 + (-) (-) (-) (-) (-) - ( 1 + (-) (-) - - - - - ( 2 + (-) ------( 1 + (-) (-) (-) - - - 2 (1 + (-) (-) - - - - 4 (3 + (-) - - - - - 3 (2 +---- -2 (1 (1 -2 +---- + + (-) - - - - ( 1 + (-) (-) - - - - - ( 1 + + ------( 3 Concentration: No. of + (-) - - - - 1( +---- -1 ( -1 +---- Effectiveness Effectiveness of fungicides against Golovinomyces cichoracearum, Px = = C = control (untreated by fungicide, characterized by profuse sporulation), *concentration recommended Reaction by of the CPM: producer; - = sensitive reaction (no sporulation), (-) = (profuse tolerant sporulation). reaction (limited sporulation), + = resistant reaction Gc Fundazol 50 WP benomyl C Karathane LC dinocap C 28 Czech Republic and collected in 2000-2002 FungicideEffective The year of collection of isolates Total no. Table Table 1. fenarimol C 9.6 18 36* 72 144 2 (1 substanceRubigan µg a. i. 12 /ml (p.p.m.) EC

291 ver, resistant reactions of some isolates on a lower concentration (9.6 µg/ml) were observed. In 2000 and 2001, Rubigan 12 EC gave control of 96% of the isolates at dosage 9.6 µg/ml, and only one isolate of Px expressed tolerant reaction on 18 µg/ml. In 2002, more isolates (especially Gc) had a tolerant or resistant reaction on lower concentrations than optimal. All tolled, 42 screened isolates (76%) were controlled even by the concentration 9.6 µg/ml, whereas 7 isolates of Gc (13%) were tolerant on this concentration and one isolate of Gc showed tolerance on concentration 18 µg/ml. Five isolates (9%) were resistant on 9.6 µg/ml and two of them expressed a tolerant reaction on 18 µg/ml (Table 1). Karathane LC proved completely effective in 2000, however in 2001 and 2002 it expressed a lower level of effectiveness. Altogether 36 isolates (65%) were controlled even by the concentration 28 µg/ml, 16 isolates (30%) sporulated on concentrations lower than optimal, and 3 isolates (5%) had a tolerant reaction on the optimal concentration of fungicide (105 µg/ml). Eight isolates sporulated on concentration 28 µg/ml (tolerant reaction) and the same number of isolates responded as tolerant also on 52.5 µg/ml (Table 1). Fundazol 50 WP showed the lowest effectivity (Table 1), as 52 isolates (94%) sporulated on every screened concentration. There were marked differences between the two powdery mildew species: while all tested isolates of Px were resistant, some isolates of Gc from the years 2001 and 2002 expressed a sensitive or a tolerant reaction. All tolled, 48 isolates (86%) were resistant, and three of these responded as tolerant on the highest concentration (1000 µg/ml). Two isolates of Gc (4%) expressed tolerant reactions at all concentrations and the same number of Gc isolates were resistant on 62.5 µg/ml, whereas on higher concentrations responded as tolerant. Three Gc isolates (6%) were controlled by the 250 µg/ml i.e. optimal concentration, one of them sporulated only on 62.5 µg/ml (tolerant reaction). These results indicate that considerable differences in effectivity occur among commonly used fungicides in the Czech Republic. Occurrence of resistant and/or tolerant isolates of both powdery mildew species in different localities was observed (Table 2). Ineffectiveness of benomyl (Fundazol 50 WP) could be explained by its frequent application in the past in the Czech Republic. Variation in efficacy of the other fungicides, fenarimol and dinocap (Rubigan 12 EC and Karathane LC), among isolates was also observed. The occurrence of resistance or decreased sensitivity of CPM populations to eight chemical groups of fungicides is widespread geographically and presents a major problem to cucurbit crop production in many regions (McGrath, 2001). The results presented herein are probably the most comprehensive concerning CPM resistance/tolerance to fungicides for Central Europe and show that the problem of fungicide resistance must be seriously considered in attempting to control CPM in this region, too. Further research will need to focus on acquiring more comprehensive data about the geographical distribution, spatial and temporal variability, and shift of fungicide resistance, including relationships with pathogenicity variation of CPM (Lebeda et al., 2003; Køístková et al., 2004).

292 250* 500 1000 Golovinomyces cichora- 62.5 125 and = race;

; R 105* 210 420 C Podosphaera xanthii Podosphaera xanthii = dinocap benomyl against some isolates of

()--- -++ + - --+++++ +(-)----+-- effective substance (concentration: p.p.m. = µg a.i./ml) Rubigan 12 ECfenarimol Karathane LC Fundazol 50 WP Golovinomyces cichoracearum, Px = = R Gc L e + + - - - - + (-) (-) - - - + + + + + + effectiveness effectiveness of fungicides

S---- -()-- - + - - --+++++ DS+-----+-- + - --+++++ - --+(-)(-)-- DS+-----+-- -D2US+-----+-- --+(-)(-)-- DS+-----+-- D Y + - - - - - + (-) - - - - + (-) (-) (-) (-) (-) m m m m m m of D B +D - B - + ------+ - (-) (-) - (-) + (-) - (-) - - + - + - + + + + + + + + + +

m m m CC ---- -++ + C - --+++++ A +-----+-- CC CC CC CC CC 2 2 2 2 2 2 2 CC B B CC CC B B B B B 1 1 1 1 1 1 1 1 1 1 AB AB AB AB AB AB AB AB AB specialization AB Comparison Px Gc Gc collected in 2001 and again in 2002 at five localities in the Czech Republic Px Px Gc Gc Gc Gc Gc 10/02 Table 2. Table 55/02 Uherské Hraditì (Uherské Hraditì) (Uherské Hraditì Uherské 19/01 Meziøíèí (Vsetín) Valaské 73/01 89/02 Nový Jièín-Kojetín (Nový Jièín) 100/01 Olomouc-Holice (Olomouc) 94/01 116/02 127/02 IS = isolate; P = pathogen, C = control (untreated by fungicide, characterized by profuse sporulation), *concentration recommended by Reaction the producer; of CPM: - = sensitive reaction (no sporulation), (-) = tolerant (profuse sporulation). reaction (limited sporulation), + = resistant reaction cearum Location(district) Physiological Fungicide (P) IS Lipovec (Blansko) 35/01 Pathotype R C 9.6 18 36* 72 144 C 28 52.5

293 Acknowledgements

This research was supported by grants QD 1357 and MSM 153100010, and by the National Programme of Genepool Conservation of Microorganisms and Small Ani- mals of Economic Importance.

References

Anonymous. 1982. FAO Method No. 30. FAO. Plant Protect. Bull., 30: 2. Hollomon, D.W. and Wheeler, I.E. 2002. Controlling powdery mildews with chemistry. In: Bé- langer, R.R., Bushnell, W.R., Dik, A.J. and Carver, T.L.W. (Eds.), The Powdery Mildews. A Comprehensive Treatise. APS Press, St. Paul, MN, pp. 249-255. Køístková, E., Lebeda, A. and Sedláková, B. 2004. Virulence of Czech cucurbit powdery mildew isolates on Cucumis melo genotypes MR-1 and PI 124112. Scientia Hort., 99: 257-265. Lebeda, A. 1984. Screening of wild Cucumis species for resistance to cucumber powdery mildew (Erysiphe cichoracearum and Sphaerotheca fuliginea). Scientia Hort., 24: 241-249. Lebeda, A. 1991. Resistance in muskmelons to Czechoslovak isolates of Pseudoperonospora cubensis from cucumbers. Scientia Hort., 45: 255-260. Lebeda, A., Sedláková, B. and Køístková, E. 2003. Distribution, harmfulness and pathogenic variability of cucurbit powdery mildew in the Czech Republic. In: Hudec, K. and Huszár, J. (Eds.), XVI. Slovak and Czech Plant Protection Conference, Abstracts Supplement. Slovak Agricultural University, Nitra, Slovak Republic, pp. 117-118. McGrath, M.T. 2001. Fungicide resistance in cucurbit powdery mildew: Experiences and challen- ges. Plant Dis., 85: 236-245. McGrath, M.T. and Shishkoff, N. 2001. Resistance to triadimefon and benomyl: dynamics and impact on managing cucurbit powdery mildew. Plant Dis., 85: 147-154. McGrath, M.T. and Shishkoff, N. 2003. First report of cucurbit powdery mildew fungus (Podo- sphaera xanthii) resistant to strobilurin fungicides in the United States. Plant Dis., 87: 1007. Sedláková, B. and Lebeda, A. 2003. Resistance to fungicides in cucurbit powdery mildews populations in the Czech Republic. In: Hudec, K. and Huszár, J. (Eds.), XVI. Slovak and Czech Plant Protection Conference, Abstracts Supplement. Slovak Agricultural University, Nitra, Slo- vak Republic, pp. 153-154.

294 Genetic variability in Sphaerotheca fusca as determined by AFLPs: the case of race 2 as a causal agent of powdery mildew in melon

T. Montoro1, M. Salinas2, J. Capel2, M. Gómez-Guillamón1 and R. Lozano2 1Estación Experimental La Mayora, CSIC. 29750 Algarrobo-Costa, Málaga, Spain 2Dept. Biología Aplicada, Universidad de Almería, 04120 Almería, Spain

Summary

Genetic differentiation of races of the most important causal agent of powdery mildew, Sphaero- theca fusca, is an important goal to design appropriate selection strategies for melon breeding. We have used AFLP markers to characterize nine isolates, seven of them identified as belonging to race 2 and two of race 5. Our results showed that AFLPs reveal a higher rate of polymorphism than other molecular markers (i.e. ITS, ISSRs and RAPDs) although AFLPs did not showed a good correlation between the molecular data and the physiological races probably due to the low number of markers analyzed. Interestingly, AFLPs were able to discriminate between isolates different geographical origins, what was not possible by means of other molecular markers. These results indicated the usefulness of the AFLPs as markers for the study of genomic variability in Sphaerotheca fusca.

Keywords: Cucumis melo, Cucumis sativus, resistance, molecular markers, AFLP, isolates, powdery mildew

Introduction Powdery mildew is an extensive disease affecting melon and other cucurbit species. Among the fungal species which originate this disease, Sphaerotheca fusca is the most important causal agent in greenhouse cultures of temperate regions (e.g. Bertrand, 1991; Kenigsbuch and Cohen, 1992; Vakalounakis et al., 1994). The different strains of S. fusca show host specificity being the concept of physiological race defined on the basis of the capacity to infect different cultivars of a given host species (Thomas et al., 1984). Besides to this, other specificity relationships have been reported among cucurbit species, allowing the identification of different pathotypes of powdery mildew (Bertrand, 1991; Bardin, 1997). In melon, five races of S. fusca have been reported (Thomas et al., 1984; Bertrand, 1991; Pitrat et al., 1998) although more recently Hosoya et al. (2000) have described up to nine physiological races. In all cases, identification of a given race depends on the response to inoculation of differential genotypes. Such response may give confused results since it depends on environmental factors such as inoculation method and developmental stage of plants (Cohen et al., 2002). Anyway, the identification of S. fusca only attending to phenotypic features showed by the plants after artificial inoculations may not reflect the genetic control underlying the plant-pathogen interactions. Thus, it seems to be needed complementary methods for a more accurate identification of S. fusca races on the basis of their genetic differences.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 295 Molecular markers have been proposed as useful tools for genotyping identification in many fields, among them, the analysis of genetic diversity in populations of S. fusca. Bardin et al. (1997) used ribosomal-RFLP and RAPD markers to analyse 28 isolates of races 0, 1 and 2. Unfortunately, restriction patterns of different ribosomal regions (ITS and 5,8 S) were the same among the isolates, and RAPD markers only showed a low DNA polymorphism level, what makes unfeasible a cluster differentiation of isolates or a association between morphological and molecular markers. The molecular characterization of 26 isolates of S. fusca from Israel has been also carried out by Katzir and Cohen (2000) by using RAPDs, ISSRs and fatty acid. Their molecular results also revealed a low diversity as well as a lack of coincidence with the biological races previously established. With the aim to analyse the molecular variability existing in the genome of S. fusca, we have employed high polymorphic AFLP (Amplified Fragment Lenght Polymor- phisms) markers (Vos et al., 1995) to characterize seven isolates classified as belonging to race 2, and two isolates of race 5. The usefulness of AFLPs to study genetic variation in several fungus species has been previously reported (Majer et al., 1996; Arenal et al., 1999; Baayen et al., 2000; Ciliers et al., 2000) but not in the main agent causing of powdery mildew in melon, S. fusca.

Material and methods

Fungal isolates Nine isolates of S. fusca were collected by Dr. Torés (E.E. La Mayora, CSIC) from mildewed plants, eight of them in Spain (Málaga and Almería) and one (P 44-3) in Sudan (Table 1). Isolates were maintained from single spores on axigenically growing melon cotyledons as described in Bertrand (1991).

Table 1. Host species, geographical origin and race of the nine isolates of Sphaero- theca fusca

Isolate Host Origin Racea

SF 29 Melon Málaga 5 SF 38 Melon Málaga 2 P 15-1 Cucumber Almería 2 M 32-1 Melon Almería 2 R2BO Melon Málaga 2 C-87 Melon Málaga 2 C-8 Melon Málaga 5 P 44-3 Melon Sudan 2 C-278 Melon Málaga 2 aAccording to virulence on differential cultivars (see Material and methods)

296 Pathogenicity test Race determination was performed on the basis of pathogenicity test in the following differential cultivars of melon (Pitrat et al., 1998): Iran H, Rochet, PMR 45, PMR 6, WMR 29, Edisto 47, PI 414723, PI 124112. Each isolate was inoculated in the second leaf of four plants as described by Ferriere and Molot (1998). After inoculation plants were kept in a growing chamber at 25°C, RH c.a. 70% and 16 h light /8h dark cycles.

DNA purification and AFLP analysis DNA isolation was performed from approximately 100 mg of spores collected from 20 infected squash cotyledons 10 to 12 days after inoculation. DNA from each isolate was extracted according to the method described by Möller et al. (1992) with the modifications proposed by Bardin et al. (1997). AFLP analysis was carried out essentially as described by Vos et al. (1995) using the enzyme combinations Eco RI / MseI. Preamplification reactions were performed with Eco RI and MseI primers both having a single selective nucleotide. However, amplification were done with three bases selective Eco RI primers radioactively labeled with [g33P]-ATP (3000 Ci/mmol) as described (Vos et al., 1995), and MseI primers with different number of selective nucleotides. The AFLP products were separated on 6% denaturing polyacrylamide gels using a Sequi-Gen (BioRad) sequencing equipment. Electrophoresis was carried out for two hours in 1 x TBE (50mM Tris/ 50mM Boric Acid/1mM EDTA). Gels were dried for 2 hour at 80°C, and exposed to a radiographic Kodak film over 7 days at room temperature. Genetic distances among isolates were calculated from the Jaccard similarity index (Jaccard, 1908). Cluster analysis (UPGMA) was performed by using the MEGA software (version 2.1; Kumar et al., 2001).

Results and discussion

Among the nine isolates collected in several locations of Spain and Sudan, two races could be distinguished on the basis of different virulence on eight melon differential cultivars. Most of the isolates (seven out of nine) were pathogenic to melon Iran H, Rochet and PMR45 and characterized as race 2. The other two strains were pathogenic towards the three previous accessions as well as WMR29 and Edisto47 cultivars and were classified as race 5. In order to determine whether DNA purified from S. fusca can be used in AFLP reactions, DNA from the nine isolates was digested with EcoRI and MseI and double stranded adapters were ligated as described by Vos et al. (1995). Preamplification reactions with selective primers Eco+A / Mse+G and amplification with a labeled Eco+AGT primer and six different MseI primers were performed in the same conditions described in the original paper of Vos et al. (1995). As a control, DNA from tomato (cv. Moneymaker) was also employed in all the reactions performed since we had successfully used it in previous analyses (Salinas et al., personal communication). The number of amplified fragments obtained with every primer combination ranged from 11 (EcoRI+AGT / MseI+GGT) to 37 (EcoRI+AGT / MseI+GAA) (Table 2).

297 Table 2. Number of AFLP fragments amplified with combinations of an EcoRI+AGT primer with six different MseI primers

MseI primer Amplified fragments Mildew Tomato

GAG 18 45 GGT 11 39 GAA 37 55 GTG 20 40 GCT 22 40 GTC 29 47

It should be noted that in every AFLP reaction the number of amplified fragment observed in tomato is two fold the number of fragment observed in the two fungal isolates. These results indicates that AFLP can be used in S. fusca but the number of fragments amplified is lower compared to tomato, probably due to the smaller size of the fungus genome. All the AFLP primers were polymorphic among the isolates here analyzed (Fig. 1), varying the polymorphism level from 3% ( EcoRI +AGT / MseI +AT) to 12% (EcoRI +AGT / MseI +GT). Thus, the levels of polymorphism revealed by AFLPs seemed to be higher than those displayed by RAPDs or ribosomal markers ITS, 5,8 S and ISSRs (Bardin et al., 1997; Katzir and Cohen, 2000).

Figure 1. AFLP fingerprints of the nine isolates of Sphaerotheca fusca generated with the primer combination EcoRI +AGT/ MseI +AG. The arrows indicate polymorphic bands.

298 In order to carry out a molecular characterization of the isolates, 42 AFLP polymorphic fragments were selected to estimate the genetic similarity among them. Va- lues of the Jaccard similarity index ranged from 0,143 (C-8 and C-87) to 0,450 (P 15- 1 and M 32-1). These data allowed us to perform an UPGMA cluster analysis and to obtain a dendrogram showing the genetic relationships among the different isolates. Results obtained from this analysis indicated the existence of a genetic variability among isolates of the race 2, in agreement with morphological analysis recently developed (Alvarez et al., 1998; Bertrand, 2002). It could be expected a similar variability in the remaining races of S. fusca. On the other hand, cluster analysis has also revealed that there was no correlation between molecular markers and the physiological races to which belong the different isolates. For example, isolates of race 5, SF29 and C-8, were included in different clusters, and similarly, C-8 seemed to be more genetically related with an isolate of race 2 (C-278) than with other isolates of the same race. Nevertheless, a high number of AFLPs and other markers homogeneously distributed along the S. fusca genome, would be needed to demonstrate the genetic relationships among isolates. Further- more, these results suggest that a more detailed analysis, which should include phenotypic (pathogenicity test, mating type, etc.), genetic (recombination, linkage maps) and molecular (distribution of DNA markers, sequencing of coding genes) data, is required to define a more accurate concept of physiological race in S. fusca.

Figure 2. Genetic relationships among the isolates of Sphaerotheca fusca.

It is noteworthy, however, that AFLP markers provided useful information concerning the geographical origin of the different isolates here analyzed. In fact, the isolate from Sudan (P 44-3) appeared clearly separated from those isolates from Spain. This is the first report about the usefulness of AFLPs as a tools to discriminate the origin of isolates of S. fusca. This capacity of AFLPs was not demonstrated for other molecular markers as RAPDs, RFLPs , ISSRs or ITS (Bardin et al., 1997; Katzir and Cohen, 2000) suggesting that AFLPs could reveal a higher rate of polymorphism in fungal genomes.

299 References

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300 A summary of eleven preliminary studies of greenhouse and field testing methods for resistance to gummy stem blight in watermelon

R. Song1, G. Gusmini2 and T.C. Wehner2 1Academy of Agricultural Sciences, Shanghai 201106, P.R. China 2North Carolina State University, Department of Horticultural Science, Raleigh, NC 27695-7609, USA; email: [email protected]

Summary

Gummy stem blight, caused by Didymella bryoniae (Auersw.) Rehm is a major disease of watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) in North America, Europe and Asia. In order to screen watermelon germplasm for resistance to the pathogen, a reliable screening method is needed. The objective of this study was to verify the conditions that may influence greenhouse and field tests. Best results were obtained using a pathogenic isolate grown for 2 to 3 weeks on potato dextrose agar under artificial light (12 hour photoperiod), a suspension of spores in water, a spore concentration of 105, the use of surfactant in the suspension, and use of high relative humidity during and after inoculation.

Keywords: gummy stem blight, Didymella bryoniae, watermelon, Citrullus lanatus, disease resistance, screening methods

Introduction

Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is a major vegetable crop, and gummy stem blight is one of the most important diseases of this crop. Gummy stem blight is caused by Didymella bryoniae (Auersw.) Rehm [=Mycosphaerella cit- rullina (C.O.Sm.) Gross. and Mycosphaerella melonis (Pass) Chiu & Walker] and its anamorph Phoma cucurbitacearum (Fr.:Fr.) Sacc. [=Ascochyta cucumis Fautrey & Roum]. Methods of seedling screening for resistance to gummy stem blight have been reported in watermelon (Boyhan et al., 1994; Dias et al., 1996), muskmelon (Zhang et al., 1997), squash (Zhang et al., 1995), and cucumber (Wehner and St. Amand, 1993; St. Amand and Wehner, 1995b; Wehner and Shetty, 2000). These studies shared a similar inoculation technique, based on spraying the seedlings with a water suspension of spores collected from in vitro cultures of the pathogen. Spore concentration differed among experiments: 105 spores/ml, 106 spores/ml, and 107 spores/ml. Different parts of the plants were rated for susceptibility after inoculation: true leaves only, true leaves and stems separately, and the whole plant. The main objective of this study was to understand the principal factors influencing an efficient test for field and greenhouse screening for use in breeding programs to develop gummy stem blight resistant watermelon cultivars.

A total of ten experiments (7 in greenhouse and disease chamber, and 3 in the field, 2 to 4 replications each) were run to investigate the effects of the following factors on testing watermelon for gummy stem blight resistance: 1) isolate of D. bryoniae (8 isolates from Florida, North Carolina and South Carolina, collected on Citrul- lus lanatus, Cucucmis melo and Cucumis sativus); 2) spore concentration (102-107 in 10-fold increments); 3) medium for pathogen growth and spore production (potato dextrose agar (PDA), 25% PDA, V-8 juice, oatmeal, or synthetic low nutrient agar (SNA)); 4) age of the pathogen at spore harvest (2, 4, or 6 weeks on growth medium); 5) spore carrier (dH O, dH O + 0.1% sucrose + 0.05% casein, dH O + 25% watermelon leaves); 2 2 2 6) inoculation method (cotton swab or a handheld artists airbrush); 7) presence of wounds on the leaves at inoculation (using a wooden stick shaped like an upside- down T); 8) temperature and relative humidity (using a disease chamber; T=26-34°C, RH=95-100%); 9) exposure time to humid conditions in greenhouse tests (0-5 days in one-day increments); and 10) frequency of irrigation in field tests. Finally, two detached-leaf test (75 and 321 watermelon cultigens, 4 and 5 replications, respectively) were performed and the results tested for correlation with the best seedling method. Cultigens of watermelon were chosen according to published differences in resistance to gummy stem blight. Charleston Gray (susceptible) was used in most tests. AU- Producer and Congo were used as slightly resistant cultivars when needed. D. bryoniae was cultured in Petri plates (100 mm) containing 25 ml of the medium required by the experiment (PDA being the default choice). Inoculated plates were incubated for two to three weeks at 24 ± 2°C under alternating periods of 12 h fluorescent light (40 to 90 mmol*m-2*sec-1 PPFD) and 12 h darkness. For all inoculations, spores from each plate were suspended in 10 ml of sterile distilled water. Immediately before inoculation, Tween 80 (0.06 g/l) was added to the inoculum. In the detached leaf test, agar cubes were excised from the cultures and placed with the pathogen in contact with the leaf-blade. The leaves were placed in Petri plates containing What- man filter paper and dH O. 2 Plants in seedling screenings and detached leaves were rated as published elsewhere (Gusmini, 2001; Gusmini et al., 2002). Data were analyzed using the MEANS, ANO- VA, and CORRELATION procedures of SAS-STAT Statistical Package (SAS Institute, Cary, NC).

Results and discussion Overall, the most effective method for screening watermelon for resistance to gummy stem blight appeared to be the seedling method with spray inoculation of the foliage. The detached-leaf method, which was a very powerful tool for testing disease resistance in several cases of hostpathogen interaction (Sharon et al., 1982; Randhawa and Civerolo, 1985; Tedford et al., 1990; Rhodes et al., 1992; St. Amand and Wehner, 1995b), in our experiments did not correlate with resistance to greenhouse and field epidemics of gummy stem blight (data not shown).

302 The variability among tests was high, but factors tested in different experiments within environment (field vs. greenhouse) had consistently significant effects in the analysis of variance by experiment (Table 1). The spore concentration in the greenhouse had an effect ranging from non significant to highly significant, which may be explained by its possible interaction with other factors such as temperature in the disease chamber and inoculum drift during or after inoculation. It appears that good results will be obtained by using spore concentrations of 104 to 106. In the field, the amount of overhead irrigation had an inconsistent effect. Natural rainfall and different level of relative humidity in different experiments might account for those differences. There were differences among environments in the effects of different isolates. The significant effects of isolate in the greenhouse were possibly due to differential adap- tability of the isolates to the high temperatures. D. bryoniae has not shown physiological specialization in cucumber or watermelon, and it was shown that breeders can use a single pathogenic isolate for testing (St. Amand and Wehner, 1995a). We recommend that a series of isolates be maintained for use in case of pathogenicity loss in the isolate being used for tests. In previous experiments, D. bryoniae grew better when leaves were damaged and exudates were present, or when sucrose and casein hydrolysate were added to the spore suspension (Svedelius, 1990). Gummy stem blight development was demonstrated to be enhanced from the emission of volatile compounds from the leaves in Cucumis spp. and Cucurbita spp. (Pharis et al., 1982). In our greenhouse and field tests, there was no difference in disease development due to spore carrier or leaf damage. The different results obtained were probably due to use of different hosts. This aspect of the host-pathogen interaction should be further investigated because the T-stick treatment is an easy way to get the leaves to produce exudates and is easily done, if needed, before each inoculation. After running many experiments, we would recommend using cultures grown for 2 to 3 weeks on PDA under artificial light for 12 hours per day, use of isolate mixtures to ensure pathogenicity, constant reisolation of new isolates from diseased plants in the field, suspension of spores in water for spray application, a spore concentration of 105, addition of surfactant to the suspension ([Tween 80]= 0.06 g/l), use of high relative humidity immediately following inoculation (with the presence of free-standing water on the leaves of test plants), and pre-inoculation damage of the leaves. Environmental variation requires that many plants, replications, seasons (spring, summer, fall), years, and test types (field and greenhouse) to evaluate resistance to gummy stem blight. If the objective were to develop resistant inbreds, we would recommend an experiment having 6 plants, 3 to 5 replications, 1 to 3 seasons (or planting dates), 1 year (for rapid progress), and 2 test types. Further research is needed in a large factorial experiment replicated in multiple years, environments (greenhouse vs. field), and locations (field test only) and with many plants per plot to verify the results presented herein. Our detached-leaf test adopted only one of many possible protocols. This technique, therefore, should not be discarded based only on our conclusions. Some factors that may be effective are direct injection of the pathogen into the leaf, or the use of a spore suspension instead of agar cubes (Sharon et al., 1982; Randhawa and Civero- lo, 1985; Tedford et al., 1990; Rhodes et al., 1992; St. Amand and Wehner, 1995b).

303 Table 1. Effects of factors tested in seedling screenings for watermelon resistance to gummy stem blight a

Category/ Tested Optimum Green- Factors tested levels level house b Field c

Spore production Medium PDA, 25% PDA, V-8, Oatmeal, SNA d PDA ** no data Age 2, 4, 6 weeks 2-4 NS no data

Inoculum Isolate 8 isolates e ** NS Concentration 102, 103, 104, 105, 106, 107 104-106 NS / ** NS Carrier dH O, dH O + additives (3 types) f dH O NS no data 2 2 2

Inoculation Chamber temp. T£34°C, T³34°C g T£34°C ** no data Time in chamber 0, 1, 2, 3, 4, 5 days 3 NS no data Pre-inoc. damage T-stick, none h T-stick NS NS Irrigation regime Every day, none i Every day no data NS / * Inoc. method Cotton-swab, hand-sprayer k Hand-sprayer NS no data

Cultigen Resistant and susceptible l no data **

NS, *, ** F ratio non-significant or significant at the 5% and 1% level, respectively. a Data from 9 experiments (split-plot RCBD, 2-4 replications, 2-6 plants/plot); b Disease chamber of clear polyethylene with humidifiers in controlled greenhouse; c Raised beds, bare-ground, overhead irrigation; data from 2 seasons/year (spring and fall); d Potato dextrose agar, 25% PDA, V-8 juice, oatmeal, synthetic low nutrient agar; e Isolated from Citrullus lanatus, Cucumis melo, and Cucumis sativus in Florida, North Carolina, and South Carolina; f 1) dH O, 2) dH O + 0.1% sucrose + 0.05% casein, 3) dH O + 25% watermelon leaves; 2 2 2 g T£34°C if disease chamber without top, T³34°C if disease chamber with top; h T-stick = 20 cm wood field stake mounted on a 60 cm handle; i 2 days before and 3 days after inoculation, none; k Hand sprayer = handheld artists airbrush; l Based on release publication and/or personal observations in other non-published tests.

References

Boyhan, G., Norton, J.D. and Abrahams, B.R. 1994. Screening for resistance to anthracnose (race 2), gummy stem blight, and root knot nematode in watermelon germplasm. Cucurbit Genet. Coop. Rep., 17: 106-110. Dias, R.D.C.S., Queiroz, M.A.D., Menezes, M. and De Queiroz, M.A. 1996. Identificao de fontes de resistencia em melancia a Didymella bryoniae. Hort. Brasil, 14: 15-17.

304 Gusmini, G. 2001. Cucurbit breeding - Gummy stem blight on watermelon [Online]. Available by Wehner, T.C., North Carolina State University, Raleigh, North Carolina; http://cuke.hort.ncsu.edu/ cucurbit/wmelon/gsbrating/gsbindex.html (verified Dec. 13, 2003). Gusmini, G., Wehner, T.C. and Holmes, G.J. 2002. Disease assessment scales for seedling screening and detached leaf assay for gummy stem blight in watermelon. Cucurbit Genet. Coop. Rep., 25: 36-40. Pharis, V.L., Kemp, T.R. and Knavel, D.E. 1982. Host plant-emitted volatiles as factor in susceptibility in vitro of Cucumis and Cucurbita spp. to the fungus Mycosphaerella melonis. Sci. Hort., 17: 311-317. Randhawa, P.S. and Civerolo, E.L. 1985. A detached-leaves bioassay for Xanthomonas campestris pv. pruni. Phytopathology, 75: 1060-1063. Rhodes, B., Zhang, X., Dean, R.A., Frick, J. and Zhang, J. 1992. Use of detached leaf assay for race 1 and race 2 anthracnose resistance in a diallel cross with Citrullus. Cucurbit Genet. Coop. Rep., 15: 84-86. Sharon, E., Okon, Y., Bashan, Y. and Henis, Y. 1982. Detached leaf enrichment: a method for testing small numbers of Pseudomonas syringae pv. tomato and Xanthomonas campestris var. ve- sicatoria in seed and symptomless leaves of tomato and pepper. J. Appl. Bacteriol., 53: 371-377. St. Amand, P.C. and Wehner, T.C. 1995a. Eight isolates of Didymella bryoniae from geographically diverse areas exhibit variation in virulence but no isolate by cultivar interaction on Cu- cumis sativus. Plant Dis., 79: 1136-1139. St. Amand, P.C. and Wehner, T.C. 1995b. Greenhouse, detached-leaf, and field testing methods to determine cucumber resistance to gummy stem blight. J. Am. Soc. Hort. Sci., 120: 673-680. Svedelius, G. 1990. Effects of environmental factors and leaf age on growth and infectivity of Didymella bryoniae. Mycol. Res., 94: 885-889. Tedford, E.C., Miller, T.L. and Nielsen, M.T. 1990. A detached-leaves technique for detecting Phytophthora parasitica var. nicotianae in tobacco. Plant Dis., 74: 313-316. Wehner, T.C. and Shetty, N.V. 2000. Screening the cucumber germplasm collection for resistance to gummy stem blight in North Carolina field tests. HortScience, 35: 1132-1140. Wehner, T.C. and St. Amand, P.C. 1993. Field tests for cucumber resistance to gummy stem blight in North Carolina. HortScience, 28: 327-329. Zhang, Y.P., Anagnostou, K., Kyle, M. and Zitter, T.A. 1995. Seedling screens for resistance to gummy stem blight in squash. Cucurbit Genet. Coop. Rep., 18: 59-61. Zhang, Y.P., Kyle, M., Anagnostou, K. and Zitter, T.A. 1997. Screening melon (Cucumis melo) for resistance to gummy stem blight in the greenhouse and field. HortScience, 32: 117-121.

305 306 Genetic analysis of resistance to Fusarium oxysporum f.sp. melonis race 1.2 in melon

L. Perchepied, C. Dogimont and M. Pitrat INRA, Génétique et Amélioration des Fruits et Légumes, BP 94, 84143 Montfavet cedex, France; e-mail: [email protected]

Summary

Fusarium oxysporum f.sp. melonis (FOM) is responsible for serious economic losses in melon (Cucumis melo). Two dominant resistance genes have been identified, Fom-1 and Fom-2, which provide resistance to races 0 and 2, and races 0 and 1 respectively. But race 1.2 overcomes these resistance genes. A partial resistance to FOM race 1.2 has been found in some Far-Eastern accessions. We have constructed a molecular linkage map of melon with a recombinant inbred line population, derived from a cross between resistant (Isabelle) and susceptible (Védrantais) lines. The linkage map consists of twenty two linkage groups, eighteen of which were assigned to ten linkage groups of the reference linkage map of melon. Artificial root inoculations on plantlets of this population were done using two strains, a wilting and a yellowing one. The inheritance study confirms that the resistance to FOM race 1.2 is polygenic. The combination of phenotypic and genotypic data allows to identify eight quantitative trait loci (QTL). These QTL were detected on six linkage groups by composite interval mapping controlling between 50 to 70% of the total variation. One major QTL explains 25% of the phenotypic variation and three others 12%. Colo- calizations were observed with resistance gene analogs and resistance genes like Fom-2. A strain specific QTL was detected.

Keywords: Cucumis melo, vascular wilt of melon, polygenic disease resistance, quantitative trait loci

Introduction

Fusarium oxysporum Schlechtend. f.sp. melonis Sny. & Hans. (FOM), the cause of vascular wilt of melon (Cucumis melo L.), is an important economic disease in the world. FOM is difficult to control because the fungus can survive in the soil as chla- mydospores during extended periods of time in the absence of the host roots. Althou- gh long crop rotations are used, FOM colonizes roots on a broad taxonomic range of plants. Chemical products, as methyl bromide, are effective to control Fusarium wilt but their use will be soon phased out. Alternative disease genetic control methods as grafting onto resistant Cucurbita or Benincasa roostocks can be used. Although this method is expensive and time consuming, it is used for greenhouse crop, but not under field condition. Risser et al. (1976) defined four races according to the host resistance genes over- comed by variants of pathogen. Two dominant resistance genes, Fom-1 and Fom-2, are known, which provide resistance to races 0 and 2, and races 0 and 1 respectively. Race 1.2 overcomes these two resistance genes and is further divided into two pathotypes: 1.2Y, inducing yellowing symptoms before the death of the plants, and 1.2W which produces wilting and death without yellowing symptom.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 307 Partial resistance to race 1.2 has been found in some Far-Eastern accessions like Ogon 9 and Kogane Nashi Makuwa (Risser et al., 1969). These exotic genotypes allowed breeding of tolerant lines to race 1.2, like Isabelle, and more recently two doubled- haploid lines Nad-1 and Nad-2 showing higher level of resistance than other genotypes (Ficcadenti et al., 2002). This study reports the mode of inheritance of resistance to FOM race 1.2. Moreo- ver a genetic map was developed using a recombinant inbred lines population to localize the genetic factors responsible for resistance to FOM race 1.2.

Material and methods

Plant material The population used in this study consisted of 120 recombinant inbred lines (RILs) derived by single seed descent from a cross between Védrantais (Fom-1) and Isabelle (Fom-1, Fom-2 and partial resistance to race 1.2).

Disease evaluations The 120 RILs, the parental lines (Védrantais and Isabelle) and Ogon 9 were evaluated for resistance by artificial infection. Two commercial F hybrids Manta (Clau- 1 se-Tézier) and Lunasol (ASL-Nunhems), tolerant to Fusarium race 1.2 and resistant to races 0, 1 and 2, were also evaluated in the same conditions. Two strains were used: TST (belonging to the yellowing pathotype) and DOléon 8 (belonging to the wilting pathotype). The experiments were carried out following 2 randomized complete blocks with 3 replications or 6 randomized complete replications design. Ten plants of each RIL and controls were evaluated per block. Inoculations were performed by dipping trays, containing 50 plants each, into 700 ml suspension of microconidia (2.106 ml-1). Trays were then placed in a growth chamber (18°C night, 24°C day, 12h of photoperiod). Symptoms first appeared 10-14 days after inoculation. As soon as a genotype (RIL or control) was infected, severity of symptoms was assessed using a semi-quantitative rating scale from 1 to 5 (1=no symptom, 5= death of plants). Plants were examined at 3- to 4-day intervals during three weeks. The phenotypic evaluations were realized by 5 private seed companies and INRA (6 experiments). The same method of inoculation was used for each experiment. The TST strain was evaluated in 4 experiments and the DOléon 8 strain in 2 experiments.

DNA extraction and molecular markers Genomic DNA was extracted from leaf tissue as described by Baudracco-Arnas (1995). The AFLP, SSR and IMA analysis protocols were described in Périn et al. (2002).

Data analysis Statistical analysis: 38 variables were studied. The disease score at the second scoring date (T2j), at an intermediate scoring date (TIj), at the final scoring date (TFj) and the area under the disease progress curve (AUDPC) were established for each experiment (j=1 to 6) and used for variance analysis. AUDPC was calculated for each RIL

308 using the following formula: AUDPC = S [(n +n -2)/2].(t t ) with i=1 to 5 or 1 to 6, i i i+1 i+1- i n = mean disease score of each plant at date i, n = mean disease score of each plant i i+1 at date i+1 and t t = number of days between scoring date i and scoring date i+1. i+1- i The evaluations by strain and for the two strains were calculated (W for the DOléon 8 strain, Y for the TST strain and WY for the two strains). Adjusted means of disease scores of RILs on replications and blocks (T2j, T2W, T2Y, T2WY, TIj, TIW, TIY, TIWY, TFj, TFW, TFY and TFWY) and adjusted values of AUDPC of RILs (AUDPCj, AUDPCW, AUDPCY, AUDPCWY) were estimated from variance analysis. Phenotypic correlations among the replications, the experiments and the two strains were calculated. Statistical analysis were performed with SAS (SAS Institute, 1988) software. ANO- VA of disease scores and AUDPC was performed using PROC GLM procedure of SAS. Linkage analysis between markers was performed using MAPMAKER v.3.0 computer program (Lander et al., 1987) with a minimum LOD score of 6.0. QTLs detection were realized by linear regression (LR), interval mapping (IM) (Lander and Bostein, 1989) and composite interval mapping (CIM) (Zeng, 1994) using QTL Carto- grapher 1.17 Software (Basten et al., 2002). For CIM, the 5 more informative markers were chosen as cofactors. Significance threshold was calculated by permutation for the LR detection method. The same significance threshold was used for IM and CIM method.

Results and discussion

Correlation between the disease scores and AUDPC, experiments and strains The correlations between the disease scores and AUDPC were highly significant (Table 1). The correlations between the six experiments were highly significant from 0.58 to 0.87, not only for AUDPC but also for the disease scores T2, TI and TF (data not shown). The two strains TST and DOléon 8 were also highly correlated (from 0.79 to 0.82) for T2, TI, TF and AUDPC.

Table 1. Correlations between the adjusted means of the resistance to race 1.2 of FOM in the 120 RILs derived from the cross between Védrantais and Isabelle on experiments, replications and blocks. The three disease scores T2, TI and TF are the disease scores at the second scoring date, at an intermediate scoring date and at the final scoring date respectively. AUDPC is the area under disease progress curve. All the correlation coefficients are highly significant (P<0.001)

T2 TI TF

TI 0.97 TF 0.840.91 AUDPC 0.92 0.99 0.97

Development of infection and behaviour of the recombinant inbred lines (RILs) Distributions of the RILs in classes for the AUDPC of two experiments are shown in Fig. 1. The parental lines Védrantais and Isabelle remained stable in susceptibility and resistance respectively. Manta, Lunasol and Ogon 9 remained among intermedi-

309 ate lines. Most of the RILs had intermediate reactions skewed toward susceptibility (experiments A and B). A few lines appeared more susceptible than the Védrantais parental line and very few lines were more resistant than the Isabelle parental line, indicating possible transgressive segregation. For simplicity we used only AUDPC to explain genetic analysis of resistance because of the highly significant correlations between the different disease scores and AUDPC and between the six experiments.

Figure 1. Distribution in classes of AUDPC (Area Under the Disease Progress Curve) of the 120 recombinant inbred lines from the cross between Védrantais and Isabelle under artificial infection by race 1.2 of FOM for the experiments A and B (TST strain and DOléon 8 strain respectively). Values of parental lines, controls and population mean are shown by arrows.

Genetic analysis of resistance Differences in disease scores and AUDPC among RILS were highly significant for all the variables. The replication effect was also highly significant for the experiments, and consequently this replication effect was significant for AUDPCW, AUDPCY and AUDPCWY. Interactions RIL*Replication, RIL*Block and RIL*Strain were also significant, but their F-values werer much lower than for replication and RILs effects. Low Genotype*Environment interactions and highly significant correlations between the experiments suggest highly heritable polygenic control of the resistance to FOM race 1.2.

Construction of the linkage map Only 9% of the AFLP markers were found to be polymorphic between Védrantais and Isabelle, which is a very low rate of polymorphism in comparison with that found by Périn et al. (2002) between Védrantais and PI 161375. The genetic map was obtained after linkage analysis of 28 ISSR, 37 SSR, 165 AFLP and 2 phenotypic markers (wt and Fom-2). Among 22 ordered groups, 18 were assigned to 10 linkage groups of the reference map of melon (Périn et al., 2002).

QTLs detection The localization of the QTLs is represented in Fig. 2. For the 38 variables studied (3 disease scores and AUDPC for the 6 experiments and for W, Y and WY), 8 QTLs

310 were detected on six linkage groups at a significance threshold of 2.7. Four QTLs are consistently identified for all the traits studied, fomIII.2, fomV.1, fomVI.1 and fom- XI.1. The resistance alleles of the QTLs originated from the resistant parental line, Isabelle, except for the QTL fomXII.1 which could explain transgression toward susceptibility.

Figure 2. Map locations of QTLs involved in resistance of FOM race 1.2. For each linkage group of the (Védrantais x Isabelle) map (Vedisa map), the linkage group of the reference map is represented by dotted lines. On the left of the linkage groups of the Vedisa map are represented QTLs detected for the yellowing strain (Y) and on the right QTLs detected for the wilting strain (W). QTLs are designated by the linkage group number followed by the number of the QTLs detected on this linkage group. Width of the QTLs bars indicates the percentage of phenotypic variation explained. Colours of QTLs indicate the frequency of detection of these QTLs on the 38 variables studied.

The QTL fomVI.1 is a major QTL (R2=29.9%), specific to the yellowing strain. Percentage of variation explained by the three other main QTLs were more important for the wilting strain than for the yellowing one.

311 Colocalization between QTLs and resistance genes and resistance genes analogs (RGAs) QTL fomXI.1 colocalized with the resistance gene Fom-2 which confers resistance to FOM races 1 and 0, and with an RGA, NBS3 (Brotman et al., 2002). QTL fomV.2 colocalized with the resistance gene Vat, which confers resistance to aphid colonization and virus transmission, and with three other RGAs, NBS2, NBS5 and NBS46-7.

In conclusion, resistance to FOM race 1.2 was shown to be under polygenic control and few QTLs seemed to be strain specific.

Acknowledgements

We thank ASL, Clause-Tezier, Gautier Graines, Seminis, Rijk Zwaan and Takii for their phenotypic evaluations and their financial support. L. Perchepied thank ANRT (French Ministry of Research) for financial support.

References

Basten, C.J., Weir, B.S. and Zeng, Z.B. 2002. QTL Cartographer, Version 1.17. A reference manual and tutorial for QTL Mapping. Department of Statistics, North Carolina State University, Raleigh (NC, USA). Baudracco-Arnas, S. 1995. A simple and inexpensive method for DNA extraction from Cucumis melo L. Cucurbit Genet. Coop. Rep., 18: 50-51. Brotman, Y., Silberstein, L., Kovalski, I., Périn, C., Dogimont, C., Pitrat, M., Klingler, J., Thompson, G.A. and Perl-Treves, R. 2002. Resistance genes homologues in melon are linked to genetic loci conferring disease and pest resistance. Theor. Appl. Genet., 104: 1055-1063. Ficcadenti, N., Sestili, S., Annibali, S., Campanelli, G., Belisario, A., Maccaroni, M. and Corazza, L. 2002. Resistance to Fusarium oxysporum f.sp. melonis in muskmelon lines Nad-1 and Nad- 2. Plant Dis., 86: 897-900. Lander, E.S., Green, P., Abrahamson, J., Barlow, J., Daly, M.J., Lincoln, S.E. and Newburg, L. 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics, 1: 174-181. Lander, E.S. and Bostein, D. 1989. Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics, 121: 185-199. Périn, C., Hagen, L.S., de Conto, V., Katzir, N., Danin-Poleg, Y., Portnoy, V., Baudracco-Arnas, S., Chadoeuf, J., Dogimont, C. and Pitrat, M. 2002. A reference map of Cucumis melo based on two recombinant inbred line populations. Theor. Appl. Genet., 104: 1017-1034. Risser, G., Mas, P. and Rode, J.C. 1969. Mise en évidence et caractérisation dune quatrième race de Fusarium oxysporum f. melonis. Annal. Phytopathol., 1: 217-222. Risser, G., Banihashemi, Z. and Davis, D.W. 1976. A proposed nomenclature of Fusarium oxysporum f.sp. melonis races and resistance genes in Cucumis melo. Phytopathology, 66: 1105-1106. Zeng, Z.B. 1994. Precision mapping of quantitative trait loci. Genetics, 136: 1457-1468.

312 Reduction of Monosporascus wilt incidence using different Galia-type melons grafted onto Cucurbita rootstock

R. Cohen, Y. Burger, C. Horev, A. Porat, U. Saar and M. Edelstein Department of Vegetable Crops, Agricultural Research Organization, Newe Yaar Research Center, Ramat Yishay, 30-095, Israel; e-mail: [email protected]

Summary

Galia-type melons Carrera, NUN-5554, 6003 and Arava were evaluated for their horticultural performance and for their response to Monosporascus wilt in two field experiments. The experiments were carried out during autumn and spring growing seasons in infested soil in the Arava region of southern Israel. Significant differences in disease incidence were found among cultivars, between grafted and non-grafted plants, and between growing seasons. Disease reduction and the beneficial effect of grafting on yield were more pronounced in the spring. The results indicate that the performance of grafted Galia-type melons is markedly dependent on the scion, which should be selected for adaptation to a particular growing area and season.

Keywords: Cucumis melo, Monosporascus cannonballus, grafting, Galia-type melon, rootstock

Introduction Grafting vegetables, including cucurbits, is common practice in Japan, Korea, the Mediterranean Basin, and some countries in Europe (Lee and Oda, 2003). The main purpose of grafting melons (Cucumis melo L.) is to control Fusarium wilt (Lee, 1994; Morra, 1998; Traka-Mavrona et al., 2000; Cohen et al., 2002; Nisini et al., 2002; Oda, 2002). However, grafting has the potential to prevent other diseases, also in field trials conducted in the Arava region of southern Israel, the incidence of Monosporas- cus wilt on grafted melon plants was significantly lower than on non-grafted plants (Edelstein et al., 1999). In the past, grafted cucurbits were not used in Israel because of the availability of methyl bromide for soil disinfestation, but this situation is rapidly changing because of the impending ban on methyl bromide use (Klein, 1996; Ristaino and Thomas, 1997; Cohen et al., 2000). The results of grafting melons onto Cucurbita rootstocks, in our previous studies (Edelstein et al., 1999) and elsewhere (Traka-Mavrona et al., 2000), varied. Performance of the grafted plants depends not only on their response to the disease. Compatibility of the rootstock with the scion, environmental conditions, and cultivation methods are important factors that may affect grafted-plant performance (Lee, 1994). In some cases, the vigorous rootstock root system enables grafted plants to absorb water and nutrients more efficiently than non-grafted ones, and it may also supply endogenous plant hormones. Thus, rootstock performance may lead to yield increases as well as contributing to disease reduction (Lee, 1994). On the other hand, poor rootstock- scion compatibility can lead to yield reduction, poor fruit quality, and even plant collapse (Andrews and Marquez, 1993; Lee, 1994; Traka-Mavrona et al., 2000).

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 313 The Galia F hybrid is an Israeli muskmelon cultivar released in 1974. Galia 1 became popular among growers and replaced other melon types that were common on the Israeli local market. By the 1980s, Galia melons had spread all over western Europe, and became a new, unique market class of melon (Karchi, 2000). The performance of grafted Galia melons has been not studied in Israel or elsewhere. During the past five years, numerous attempts by farmers and commercial nurseries to grow Galia-type melons grafted onto Cucurbita rootstocks, in various locations and growing seasons, gave in inconsistent results. These ranged from satisfactory disease suppression accompanied by profitable yields in the northern Arava (Edel- stein et al., 1999) to suppressed growth of the grafted melons in the central Arava during mid-summer (unpublished data). Therefore, the objectives of the present study were to examine the performance of various Galia-type melon cultivars grafted onto a Cucurbita rootstock in an attempt to elucidate the factors that determine success or failure of this unique crop.

Material and methods

Germplasm Four Galia-type cultivars, 6003, Arava, Carrera, and NUN-5554, were tested in two field trials. All were grafted onto the commercial Cucurbita maxima Duchesne × C. moschata Duchesne rootstock TZ 148 (Tezier, France).

Grafting procedure Seeds of the melon cultivars and of the rootstock TZ 148 were sown in seedling trays (Polyvid, Mishmar HaNegev, Israel) containing 128 cells of inverse pyramid shape, 35 × 35 mm at the top, 60 mm deep, filled with a 1:1 (v/v) mixture of peat and vermiculite. The plants were grown in an environment-controlled greenhouse at 25/20oC day/night, and grafted at the two-leaf stage. The true leaves, one cotyledon and the growing point of the rootstocks were removed with a razor blade. The roots and the lower part of the hypocotyls of the scions were also removed. Each scion was placed on the side of a rootstock (side grafted) and secured with a grafting clip (Sakata Seed Corp., Yokohama, Japan). Grafted plants were transferred to a mist chamber (relative humidity ³ 95%) for 8 days, after which the relative humidity was reduced gradually for acclimatization.

Field trials The effects of the Cucurbita rootstock TZ 148 on disease incidence and yields of grafted Galia-type melons (Arava, Carrera, 6003 and NUN-5554) were evaluated in two field experiments. The experiments were conducted in naturally Mono- sporascus cannonballus-infested soil at the Zohar Experiment Station, En Tamar (northern Arava). Grafted and non-grafted plants were transplanted in the autumn (19 Septem- ber 2002) and early spring (5 February 2003). Each treatment plot contained 8 plants, with within-row spacing of 40 cm. The bed centers were 190 cm apart. Standard cultural practices including drip fertigation were employed. There were four replicates per treatment in the two field experiments. Fruits were harvested when fully mature

314 (full slip). There were seven harvests at 3- and 4-day intervals. The fruits from each plot were counted and weighed.

Results and discussion

Marked differences were observed in the response of grafted and non-grafted Ga- lia-type melons to sudden wilt caused by the fungus Monosporascus cannonballus (Table 1). These differences were statistically significant for disease incidence among cultivars, between grafted and non-grafted plants, and between the growing seasons (Table 2).

Table 1. Wilt incidence of grafted and non-grafted Galia-type melons in Monospora- sus cannonballus-infested soil at En Tamar. Data recorded near the end of harvest

Wilt incidence (%) Melon cultivar and grafting Autumn 2002 Spring 2003

6003 non-grafted 100 65 6003/TZ 148 46 15

Arava non-grafted 75 47 Arava/TZ 148 93 45

Carrera non-grafted 75 57 Carrera/TZ 148 34 23

NUN 5554 non-grafted 50 40 NUN 5554/TZ 148 6 18

Table 2. The main effects of cultivar, grafting and season and their interactions on disease wilt incidence in Monosporascus cannonballus-infected soil, by three-way analysis of variance. Data were arcsin-transformed prior to analysis

Source df SS MS F P

Cultivar 3 12085.68 4028.56 4.77 < 0.01 Grafting 1 13110.25 13110.25 15.54< 0.01 Season 1 7267.56 7267.56 8.61 < 0.01 Cultivar × Grafting 3 7941.50 2647.16 3.13 0.03 Cultivar × Season 3 3860.68 1286.89 1.52 0.21 Grafting × Season 1 20.25 20.25 0.02 0.87 Cultivar × Grafting × Season 3 926.50 308.83 0.36 0.77 Residual 48 40479.00 843.31

315 Figure 1. Yield of grafted and non-grafted melons harvested in Monosporascus-infested soil at Zohar Experiment Station at En Tamar. Left column; autumn crop, transplanted on 19 September 2002; right column; spring crop, transplanted on 5 February 2003. Mean separations by Student-Newman-Keuls multiple-range test, 5% level.

316 In both experiments, grafting reduced plant mortality except for the case of Ara- va (Table 1). Plants of NUN-5554 (long shelf-life Galia-type melon), both grafted and non-grafted, exhibited the lowest disease incidence in both seasons. This cultivar has vigorous growth that may contribute to its good performance in infested soil. Disease incidence was lower in the spring than in the autumn crop for both grafted and non-grafted plants, and the effect of growing season on Monosporascus wilt incidence was significant. These results are consistent with earlier findings of greater disease severity in the short plant life cycle, characteristic of the autumn, as compared with the longer cycle in the spring (Pivonia et al., 2002). Yield response of the grafted and the non-grafted plants closely matched their disease- resistance response (Fig. 1). In the spring experiment, no difference in yield was found between grafted and non-grafted Arava and NUN-5554 melons, and grafted and non-grafted Arava plants had similar disease incidence (Table 1). Disease incidence in NUN-5554 was relatively low (40 and 18% for non-grafted and grafted, respectively) and the yield of the grafted plants was higher, but not significantly so. Grafted 6003 and Carrera had significantly higher yields than their non-grafted controls. Disease incidence (at harvest) of the non-grafted plants was higher in the autumn crop than in the spring one (Table 1). In spite of the observed major reductions in wilting of the grafted plants (except for Arava) during the autumn, no significant yield differences were evident. The contrasting between-season results can be explained by a combination of yield-ripening and disease-development rates. Early collapse may cause severe damage, while early ripening combined with collapse that occurs toward the end of harvest may result in escape from yield losses. The present study has highlighted the differing performances of different cultivars between two seasons of observations in the Arava. In general, the results of the present study indicate that the disease-prevention performance of grafted plants depends on scion response to diseases and not only on the contribution of the rootstock. A similar trend was observed previously for Ananas-type melons tested in soil infested with Fusarium oxysporum f.sp. melonis (Cohen et al., 2002). The results also suggest that the use of grafting for melons can be particularly valuable for the spring crop in the Arava, at least for the cvs. Carrera and 6003. The autumn season is more problematic, and emphasis should be placed on improving the grafting technology for this season.

References

Andrews, P.K. and Marquez, C.S. 1993. Graft incompatibility. In: Janick, J. (Ed.), Horticultural Reviews, vol. 15. John Wiley & Sons, New York, pp. 183-218. Cohen, R., Pivonia, S., Burger, Y., Edelstein, M., Gamliel, A. and Katan, J. 2000. Toward integrated management of monosporascus wilt of melons in Israel. Plant Dis., 84: 496-505. Cohen, R., Horev, C., Burger, Y., Shraiber, S., Hershenhorn, J., Katan, J. and Edelstein, M. 2002. Horticultural and pathological aspects of fusarium wilt management using grafted melons. HortScience, 37: 1069-1073. Edelstein, M., Cohen, R., Shraiber, S., Pivonia, S. and Shtienberg, D. 1999. Integrated management of sudden wilt in melons caused by Monosporascus cannonballus using grafting and reduced rates of methyl bromide. Plant Dis., 83: 1142-1145. Karchi, Z. 2000. Development of melon culture and breeding in Israel. In: Katzir, N. and Paris, H.S. (Eds.), Proceedings of Cucurbitaceae 2000. Acta Hort., 510: 13-17.

317 Klein, L. 1996. Methyl bromide as soil fumigant. In: Be, C.H., Price, N. and Chakrabarti, B. (Eds.), The Methyl Bromide Issue. John Wiley & Sons, New York, pp. 191-235. Lee, J.M. 1994. Cultivation of grafted vegetables. I. Current status, grafting methods, and benefits. HortScience, 29: 235-239. Lee, J.M. and Oda, M. 2003. Grafting of herbaceous vegetable and ornamental crops. In: Janick, J. (Ed.), Horticultural Reviews, vol. 28. John Wiley & Sons, New York, pp. 61-124. Morra, L. 1998. Potential and limits of grafting in horticulture. Informatore Agrario, 54: 39-42. Nisini, P.T., Colla, G., Granati, E., Temperini, O., Crino, P. and Saccardo, F. 2002. Rootstock resistance to Fusarium wilt and effect on fruit yield and quality of two muskmelon cultivars. Scientia Hort., 93: 284-288. Oda, M. 2002. Grafting of vegetable crops. Sci. Rep. Agric. & Biol. Sci., Osaka Pref. Univ., 54: 49-72. Pivonia, S., Cohen, R., Katan, J. and Kigel, J. 2002. The effect of soil temperature on disease development in melon plants infected by Monosporascus cannonballus. Plant Pathol., 51: 472- 479. Ristaino, J.B. and Thomas, W. 1997. Agriculture, methyl bromide and the ozone hole: can we fill the gap? Plant Dis., 81: 964-977. Traka-Mavrona, E., Koutsika-Sotiriou, M. and Pritsa, T. 2000. Response of squash (Cucurbita spp.) as rootstock for melon (Cucumis melo L.). Scientia Hort., 83: 353-362.

318 Response of cucumber to the broad mite (Polyphagotarsonemus latus)

M. Grinberg1,2, V. Soroker2, E. Palevsky2, I. Shomer3 and R. Perl-Treves1 1Faculty of Life Science, Bar-Ilan University, Ramat-Gan 52900, Israel 2Department of Entomology, ARO, Bet Dagan 50250, Israel 3Food Science Institute of Technology and Storage of Agricultural Products, ARO, Bet Dagan 50250, Israel

Summary

Broad mite is a minute herbivorous mite that attacks numerous plant crops from diverse families, including cucurbits, causing severe symptoms and yield loss. The interaction of broad mite (Polyphagotarsonemus latus) with host plants is poorly understood. We examined the effect of broad mite (BM) on cucumber morphology and growth patterns. BM caused a substantial decrease in growth and development of cucumber. Infested plants produced more ethylene than the non-infested controls, and their leaves were smaller and harder. Microscopical observations revealed that the mites were feeding on epidermis cells, which disappeared or collapsed. Infested leaves were thicker, and displayed additional layers of more compact cells which did not undergo typical mesophyll differentiation. We tried to identify possible signaling pathways that mediate plant responses to BM. Using Northern analysis, we tested the expression of genes that belong to known defense pathways: b-glucanase (BGL2), two cucumber lipoxygenase isoforms (LOX1 and LOX2), an ACC oxidase (ACO1) and a peroxidase (PRX). BGL2, LOX1, LOX2 and PRX were induced by mite feeding, with transcript levels increasing between 24 and 72 hrs post infestation. Mechanical wounding did not induce these transcripts, except for a slight induction of LOX1. ACO1, on the other hand, shows a rather uniform expression pattern. These results suggest that BM causes an induction of both the SA and the JA pathways.

Keywords: Cucumis sativus, broad mite, defense mechanisms

Introduction

Plant responses to herbivore attacks are complex, including a variety of direct and indirect defense mechanisms. Plants express constitutive as well as induced defenses, either locally or systemically throughout the plant (Kessler and Baldwin, 2002). Responses to chewing insects such as caterpillars differ from the response to phloem or cell-sap feeders (Walling, 2000); the latter, that include herbivorous mites, are less well studied. The broad mite, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) is a minute organism that feeds on the apical shoots and young leaves of many different species (Gerson, 1992). It has become an important pest in tropical and subtropical regions, including in greenhouse-grown cucurbits in Israel. Because of its size (0.2 mm) it escapes detection until the plant is severely damaged. Symptoms on cucumber include chlorotic, distorted shoots, brittle, curled leaves, growth inhibition and distorted fruits. Such symptoms could result from local damage to meristematic tissue in the apex, but could also be explained as a plant systemic response to mite feeding. Some authors suggest that P. latus releases a salivary toxin causing tissue malforma-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 319 tions; others implied an induction of changes in the plants hormonal balance, but these hypotheses have not been proven (Gerson, 1992). We set to characterize the interaction of broad mite (BM) with cucumber (Cucumis sativus L.). We intend to characterize the mites damage, identify plant genes that are induced by BM feeding and the signals that mediate the response. Here we describe the set-up of our experimental system and provide a preliminary characterization of the response.

Material and methods

Broad mite cultures were maintained on young potato plants in the growth chamber. Cucumber plants (cvs. M40 and Kfir) were inoculated by placing a heavily infested potato leaf on a pre-determined position on the cucumber host. At the time of sampling and at the end of an experiment, the tissue was rinsed with ethanol and the mites counted under the stereo-microscope. Infested cucumber plants and non-infested controls were grown in a growth chamber, individually isolated by plastic cages, topped with <2 m mesh nets. Ethylene evolution was measured using a Varian 3300 gas chromatograph: detached leaves were weighted and incubated in phosphate-citrate buffer with 50 mM sucrose (pH=5.2) in the dark to release wound ethylene. After two h the vials were sealed and incubated for 20 h, 5 ml gas samples were analyzed against ethylene standard (0.1 ppm). Leaf samples for light microscopy were fixed in 3.5% glutaraldehyde (pH=7.1), dehydrated in an alcohol series and in acetone, then embedded in epon and 2-3 m thick slices were prepared using a Pyramitom 1180 microtome. Sections were dyed with basic fuchsin and toluidine blue. Leaf rigidity was measured by a Stable Micro System texture analyzer and the resistance of a tissue to penetration was expressed using the maximum resistance values (Van Dijk et al., 2002). Total RNA was extracted using the Tri-reagent buffer (Molecular Research Center Inc.). Northern analysis followed standard procedures. Gene probes were made by PCR amplification from the following cloned genes: b-glucanase 2 (BGL2; Ando et al., 2001), GenBank Accession AB051372; lipoxygenase 1 (LOX1), accession BQ294486; Lipoxygenase 2 (LOX2; Ando et al., 2001), AB051385; ACC oxidase 1 (ACO1), AF033581. The BGL2 and LOX2 clones were kindly provided by Drs. S. Ando and S. Sakai, University of Tsukuba, Japan. Statistical analysis included ANOVA and Students test, followed by a-posteriori Fishers PLSD test, or Mann-Whitney U-test, using STAT VIEW 4.5.

Results

Broad mite effects on growth and morphology Plants at the 2-3 leaf stage were infested with 20 broad mites, and a week later with another 30 mites. We noticed a severe reduction in plant height (Fig. 1A) and in leaf number (not shown) beginning two weeks after infestation. The mites had a preference for the youngest leaves and apices and constantly moved upwards to feed on such regions. At the end of the 4-week experiment, control plants were 90 cm high and had 17 unfolded leaves, compared to the infested plants, which were severely stunted (26 cm), with only five well-developed leaves and many small, unfolded ones.

320 Figure 1. A. Growth inhibition in broad-mite infested cucumbers. Plants (2-3 leaf stage) were infested with 20 mites and a week later with another 30 mites. B. Effect of BM infestation on ethylene emission from cucumber apical leaves. Plants were infested with 200 mites at the 2-3rd leaf-stage. Results are means ±SEM of five replicates.

We asked whether infested plants evolved more ethylene. We took GC measurements of ethylene released by excised young leaves from BM-infested and control plants (Fig. 1B). Two days after infestation, plants already evolved more ethylene than the control; emission further increased after 11 days.

Figure 2. Structural changes in cucumber leaf following BM infestation. A. Infested leaves display a harder texture, as measured by a Texture Analyzer. Numbers above the columns indicate mean number of mites at the end of experiment. B. Light microscopy of leaf sections from BM-infested and control leaves. Top: 5th leaf, 11 d post infestation. Bottom: 5th leaf, 18 d post infestation. Left: control, right infested.

321 In another experiment, we applied a single dose of ca. 140 mites/plant. The new leaves that developed after infestation were smaller and harder, curled downwards, and their color was darker. We measured the mechanical resistance of the leaf using a Texture Analyzer, and prepared leaf sections for light microscopy observations. The leaves that developed in the presence of BM were mechanically more resistant already one week after infestation (Fig. 2A). When such leaves were examined under the microscope, we noticed that the infested leaves developed thicker and more compact tissues (Fig. 2B). Increased thickness resulted from additional mesophyll layers (7 layers compared to 5 in the control leaf), and the cells were larger. The epidermis was missing, but the mites feeding appendages apparently could not reach the inner cell layers. A layer of collapsed epidermis was sometimes apparent. The differentiation of the mesophyll was severely affected: the infested leaf-tissue was compact and the cells filled the inter-cellular spaces, while control leaves had large air spaces and smaller cells with a more regular shape.

Figure 3. Expression of stress- related genes after BM infestation. Total RNA (15 µg/lane) from BM-infested, mechanically wounded and untreated young apical leaves was subjected to Northern analysis and hybridized to radi- olabeled, cucumber gene probes. LOX1 - lipoxygenase-1, LOX2 - lipoxygenase-2, BGL2 - beta-glucanase-2, PRX - peroxidase, ACO1 - ACC oxidase-1.

322 Changes in specific gene transcripts following broad mite infestation We selected a few genes that belong to stress and pathogenesis related responses in model plants. Cucumis sequences were obtained form the GenBank, and primers were synthesized to amplify and clone gene-probes for Northern analysis. Young cucumber plants (3rd leaf stage) were infested with broad mites. Young leaves were sampled for total RNA extraction 24, 72 hrs and 1 week after infestation. Samples were also taken from mechanically wounded leaves (rubbed with carborundum), and from untreated controls. Fig. 3 shows the prominent induction of the LOX1, LOX2, BGL2 and PRX transcripts after 24 hrs, often peaking in the 72 hr samples and decreasing later. The ACO1 transcript levels remained constant. Mechanical wounding only slightly induced the LOX1 gene and perhaps the PRX gene.

Discussion

The severe reduction in shoot development in infested cucumbers could have resulted from direct result damage to the shoot apex. Alternatively, it could reflect a hormonal imbalance caused by the mite. The increase in leaf rigidity, that correlated with the formation of additional mesophyll cell layers, with a more compact structure, could be the result of auxin or ethylene production. It could reflect the arrest of normal expansion and differentiation, but may also have an adaptive value in prote- cting the leaf that was stripped from its epidermis by the mites. Northern analysis of a small number of selected transcripts suggests that both the salicylic acid and the jasmonic acid defense pathways could be induced by BM feeding: the BGL2 gene is a PR (pathogenesis related) protein, known to be induced by different plant pathogens via the salicylic acid signaling pathway (Walling, 2000). Lipoxygenases participate in the synthesis of jasmonic acid and are also induced by jasmonates and ethylene (Stotz et al., 2000; Walling, 2000). Aphid feeding induced both genes in Arabidopsis (Moran and Thompson, 2001). A peroxidase gene that may be related to either oxy-radical detoxification or lignification processes also responded to BM, but the ACC oxidase-1 transcript did not respond. The increase in ethylene production that we observed following infestation is probably mediated by other ethylene-synthesis enzymes. The fact that the same genes were not induced, or showed a smaller response to mechanical wounding, could suggest that they responded specifically to BM, as opposed to a general wounding response. Reymond et al. (2000) reported very different transcript profiles in mechanically wounded Arabidopsis, compared to plants infested with cabbage butterfly larvae. On the other hand, it is difficult to precisely mimic BM damage and it is possible that, in our hands, the wounding treatment was milder than the BM-inflicted damage. We have recently begun to test whether mites that were experimentally confined to a single leaf could induce growth alterations in mite-free organs. At present, our preliminary data cannot support such hypothesis. On the other hand, we saw that plant defenses were expressed systemically: some defense-related transcripts were induced in the mite-free organs of the infested plant. Transcript levels were, however, lower compared to the local response (not shown). Looking at additional defense genes both locally and systemically should yield a better description of the cucumber response

323 to this pest. On the long run, such studies could help in designing control strategies to improve plant resistance to BM.

References

Ando, S., Sato, Y. and Sakai, S. 2001. Cloning ethylene responsive genes from the apices of cucumber plants (Cucumis sativus L.). Plant Biotechnol., 18: 163-167. Gerson, U. 1992. Biology and control of the broad mite, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae). Exp. Appl. Acarol., 13: 163-178. Kessler, A. and Baldwin, I.T. 2002. Plant responses to insect herbivory: the emerging molecular analysis. Annu. Rev. Plant Biol., 53: 299-328. Moran, P.J. and Thompson, G.A. 2001. Molecular responses to aphid feeding in Arabidopsis in relation to plant defense pathways. Plant Physiol., 125: 1074-1085. Reymond, P., Weber, H., Damond, M. and Farmer, E.E. 2000. Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell, 12: 707-720. Stotz, H.U., Pittendrigh, B.R., Kroymann, J., Weniger, K., Fritsche, J., Bauke, A. and Mitchell- Olds, T. 2000. Induced plant responses against chewing insects. Ethylene signaling reduces resistance of Arabidopsis against Egyptian cotton worm but not Diamondback moth. Plant Physiol., 124: 1007-1017. Van Dijk, C., Fischer, M., Beekhuizen, J.G., Boeriu, C. and Stolle-Smits, T. 2002. Texture of cooked potatoes (Solanum tuberosum). 3. Preheating and the consequences for the texture and cell wall chemistry. J. Agric. Food Chem., 50: 5098-5106. Walling, L.L. 2000. The myriad plant responses to herbivores. J. Plant Growth Regul., 19: 195- 216.

324 Map-based cloning of the Vat gene from melon conferring resistance to both aphid colonization and aphid transmission of several viruses

J. Pauquet2, E. Burget1,2, L. Hagen1, V. Chovelon1, A. Le Menn1, N. Valot1, S. Desloire2, M. Caboche2, P. Rousselle1, M. Pitrat1, A. Bendahmane2 and C. Dogimont1 1INRA, Fruits and Vegetables Genetic and Plant Breeding Unit, Domaine St Maurice, BP94, 84143 Montfavet Cedex, France; e-mail: [email protected] 2INRA-Vegetal Genomics Unit, 2 rue Gaston Crémieux, CP 5708, 92057 Evry Cedex, France

Summary

Aphids cause serious damage to number of crops throughout the world, both directly through feeding damage and indirectly by transmitting several viruses. Here, we report the map-based cloning of the Vat gene from melon, which presents the unique feature to confer a double resistance, resistance to plant colonization by the melon/cotton aphid Aphis gossypii and resistance to A. gossypii transmission of unrelated viruses (CMV, potyviruses). Vat belongs to the coiled- coil (CC) nucleotide binding (NBS)/Leucin-rich repeat (LRR) type of disease resistance genes. It is approximately 6 kb long with three introns and encodes a large 1473-amino acid protein. It was shown to belong to a cluster of resistance gene analogs, including the Vat-like gene, which shares a high similarity with Vat but does not confer any known resistance. Functional validation was obtained by stable transformation of susceptible melons. The structure of the Vat gene is consistent with a role in the aphid recognition and activation of signaling cascade, which triggers non-specific plant defense responses.

Keywords: Cucumis melo, Aphis gossypii, insect resistance, virus transmission

Introduction

The aphid Aphis gossypii Glover is a serious pest of cucurbit crops worldwide. It causes direct feeding damage and indirect damage by transmitting plant viruses. In the seventies, a natural resistance to the melon aphid A. gossypii was identified in an Indian and a Far East melon variety (Kishaba et al., 1971; Lecoq et al., 1979). This resistance drastically reduces aphid longevity and fecundity, and alters their feeding behavior (Chen et al., 1996, 1997; Klinger et al., 1998). A major dominant gene, named Virus aphid transmission resistance symbol Vat (Pitrat and Lecoq, 1980) controls the resistance. Vat-mediated resistance is unique in that it confers, besides resistance to the aphid, resistance to non-persistently unrelated viruses transmitted by A. gossypii (Lecoq et al., 1979, 1980; Kishaba et al., 1992). The mechanisms by which melon plants protect themselves from both aphids and viruses are still unknown. More than 30 dominant resistance genes have been cloned from various plant species. Most of them belong to the nucleotide-binding/leucine-rich repeat (NB-LRR) family (Hammond-Kosack and Jones, 1997). Upon specific recognition, mediated directly or indirectly by pathogen avirulence (Avr) gene products, the resistance gene products

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 325 initiate an array of active defenses (Dangl and Jones, 2001; Hammond-Kosack and Parker, 2003). In the present work, we characterized by a positional cloning strategy a single melon gene that controls resistance to A. gossypii colonization and to virus transmission by this vector.

Material and methods

Plant material One pair of near isogenic lines except for Vat derived from the resistant accession PI 161375 on a Charentais melon background obtained after 16 successive back crosses was used to identify molecular markers closely linked to Vat. Aphid resistance was mapped on 180 recombinant inbred lines (RILs) from the cross Védrantais (susceptible) x PI 161375 (resistant) (Périn et al., 2002). The fine mapping of Vat relative to AFLP- derived PCR markers was performed on 6000 plants from the BC [F (Védrantais x PI 1 1 161375) x Védrantais]. Individual BC plants, presenting a recombination event within 1 a close genomic region encompassing Vat were selfed to obtain BC I progenies. 1 1

Aphid resistance tests A. gossypii strain Nm1 was maintained as previously described and the test adapted from Pitrat and Lecoq (1980). Five apterous adult aphids were deposited on each plant and left on the plants in a climatic chamber (16 h day 22°C, 8 h night 18°C). One week later, the number of adult aphids and immature larvae present on each plant and the presence of leaf curling were scored.

Virus transmission tests Virus transmission tests were performed as described in Pitrat and Lecoq (1980) by using five aphids per plant. Resistance to virus transmission was tested with zucchini yellow mosaic virus strain E9, to which both parental lines Védrantais and PI 161375 are susceptible.

Molecular markers AFLP markers were used to tag the resistance locus. AFLP analyses were performed with the enzyme pairs EcoRI-MseI and HindIII-MseI as previously described by Périn et al. (2002). The AFLP markers linked to Vat were cloned and sequenced. Se- quence-specific primers were designed to amplify the AFLP sequence from genomic DNA of both parents, Védrantais and PI 161375. When no sequence polymorphism was found between both parents, the AFLP sequence was extended by PCR walking using the Universal GenomeWalker Kit from Clontech.

High throughput PCR analysis Plants from the BC population were grown in 104 well plates (3 x 3 cm wells) for 2-3 1 weeks. Young leaves were collected in 96 well plates and lyophilized. Lyophilized leaves were ground and DNA was extracted as described in Burget et al. (submitted). The screening was performed with markers flanking the Vat locus, used in a multiplex PCR reaction.

326 BAC library A BAC library was constructed in pCUGIBAC1 using BamHI, HindIII and EcoRI from the homozygous resistant melon line PI 161375. It consists of 120 000 clones of 100200 kb and represents 23-melon genome equivalent. BAC clones were organized in DNA pools and superpools for a systematic PCR-based screening procedure.

Complementation experiments A genomic fragment of 11 kb, containing the Vat coding region and 2.5 kb of the 5 and 3 flanking sequences was cloned into pbin19. The binary vector was introduced in Agrobacterium tumefaciens strain C58C1. Leaves explants of 10-day-old seedlings of the susceptible melon cultivar Védrantais were transformed using a modified protocol of Guis et al. (2000).

Results and discussion

The Vat locus has been localized to the linkage group V of the melon reference map in a sub-terminal position (Périn et al., 2002). 25 AFLP markers were mapped in a 20 cM region spanning Vat on a 180 recombinant inbred line population (F -F ) 7 8 issued from the cross Védrantais x PI 161375. Flanking AFLP markers at 1.1 and 0.7 cM on either side of the gene were transformed into PCR specific markers by PCR walking and used to screen more than 6000 BC plants using a high throughput ge- 1 notyping system. The 140 recombinants were characterized regarding the aphid resistant phenotype. Using a systematic PCR-based procedure, the melon BAC library obtained from the resistant genotype PI 161375 was screened with markers tightly linked to Vat and positive clones were identified and isolated. New markers were derived from the BAC ends and mapped relative to Vat. This approach allowed physically delimiting Vat to a single BAC clone. This BAC clone was sequenced using a shotgun sequencing procedure and the sequence was assembled in a single contig of 120 kb. Using new markers derived from the BAC sequence, the Vat gene was delimited to an 11 kb DNA fragment. Sequence analysis of this 11 kb sequence identified Vat as a coiled-coil nucleotide binding/Leucin-rich repeat type of disease resistance genes. Recombinants presenting a recombination event on each side of the predicted gene and a susceptible intrage- nic recombinant allowed us to genetically identify the Vat gene. Moreover, phenotypic evaluation of recombinants indicated that the same gene confers resistance to A. gossypii colonization and virus transmission. The Vat gene was shown to be approximately 6 kb long with 4 exons and 3 introns. Introns cover in total 1474 nucleotides. Exons cover in total 4422 nucleotides and encode for a 1473-amino acid protein, which is predicted to be cytoplasmic. The C-terminal region of the protein is made up of 15 highly imperfect copies of a LRR motif, containing 20-30 amino acids per repeat, interrupted by 4 Leucin-rich repeated motifs of 65 amino acids, extremely conserved each other (66% to 90% identity). A structural and functional annotation of the sequenced BAC clone was performed. Thirteen open reading frames were predicted and among them, 5 resistance gene analogs from NBS-LRR type, indicating that Vat belongs to a cluster of resistance

327 genes. One of them, located 17 kb from Vat and named Vat-like, encodes a predicted 1410-amino acid protein. This gene was shown to be expressed in melon leaves but no phenotype was associated to him. In Vat-like 3 region, two ORFs also present a high homology with two distinct regions of the Vat gene. ORF potentially involved in integration of mobile elements were predicted in this region and could have had a role in the evolution of the gene cluster. A TIR-NBS-LRR gene was predicted at the 5 end of the BAC, 34 kb from Vat. It has been mapped previously at about 4 cM from Vat with a candidate gene strategy using degenerated primers (NBS-2) (Klinger et al., 2001; Brotman et al., 2002). A kinase gene, potentially involved in plant-pathogen interactions, was also predicted in the cluster. The LRR domain in other resistance genes was implicated as carrying determinants for specificity of pathogen recognition (Ellis et al., 1999). We observed also that the identity of the Vat and Vat-like genes is almost perfect in the NBS domain, while it is of 81 % in the LRR domain. Interestingly, the main difference is the lack, in Vat-like, of one of the four 65-amino acid motifs. Moreover in another study, we obtained by map based cloning (data not shown) the sequence of a natural allele of the Vat gene, which confers susceptibility to aphids and resistance to powdery mildew (Sphaerotheca fuliginea races 1, 2 and 3). This allele lacks two repeats of the 65 amino acid motif. Altogether, these observations strengthen the fact that this motif may have a major role in the specificity of recognition of resistance genes. Susceptible plants of the susceptible cultivar Védrantais were transformed with an 11 kb genomic fragment carrying the Vat gene under the transcriptional control of its own promoter. Transgenic melon plants were regenerated with the NptII system for in vitro selection. The introduced Vat gene was identified by PCR using different primers designed along the gene. Primary transformants were tested for resistance to A. gossypii colonization. While on all susceptible control plants, the aphids had reproduced, transformed plants displayed resistance. Further evaluations of transgenic plants are in progress. Vat-mediated resistance, like those controlled by many disease resistance genes, can be described in terms of an elicitor/receptor model involving a two stage process, the recognition and the response. During aphid early punctures, a specific A. gossypii elicitor interacts (probably indirectly according to current data (Hammond-Kossack and Parker, 2003)) with the receptor, likely encoded by the Vat gene, and activates plant responses, which include mechanisms that suppress not only the growth of A. gossypii but also of viruses belonging to different families transmitted by this vector. The Vat gene is the second cloned resistance gene that mediates resistance to an aphid; indeed, the nematode resistance gene Mi of tomato also confers resistance against a biotype of the potato aphid Macrosiphum euphorbiae (Kaloshian et al., 1995; Rossi et al., 1998). But the double phenotype activated by the recognition of an aphid elicitor is a unique feature of the Vat gene and has not been previously described.

Acknowledgements

We thank Nathalie Giovinazzo, Virginie Chareyron, Pascal Audigier and Didier Besombes for care of plants and aphids. This research was supported by GENOPLANTE.

328 References

Brotman, Y., Silberstein, L., Kovalski, I., Périn, C., Dogimont, C., Pitrat, M., Klinger, J., Thomp- son, G.A. and Perl-Treves, R. 2002. Resistance gene homologs in melon are linked to genetic loci conferring disease and pest resistance. Theor. Appl. Genet., 104: 1055-1063. Chen, J.Q., Delobel, B., Rahbé, Y. and Sauvion, N. 1996. Biological and chemical characteristics of a genetic resistance of melon to the melon aphid. Entomol. Exp. Appl., 80: 250-253. Chen, J.Q., Martin, B., Rahbe, Y. and Fereres, A. 1997. Early intracellular punctures by two aphid species on near-isogenic melon lines with and without the virus aphid transmission (Vat) resistance gene. Eur. J. Plant Pathol., 103: 521-536. Dangl, J. and Jones, J. 2001. Plant pathogens and integrated defense responses to infection. Na- ture, 411: 826-833. Ellis, J.G., Lawrence, G.J., Luck, J.E. and Dodds, P.N. 1999. Identification of regions in alleles of the flax rust resistance gene L that determine differences in gene-for-gene specificity. The Plant Cell, 11: 495-506. Guis, M., Ben Amor, M., Latche, A., Pech, J.C. and Roustan, J.P. 2000. A reliable system for the transformation of cantaloupe charentais melon (Cucumis melo L. var. cantalupensis) leading to a majority of diploid regenerants. Sci. Hort., 84: 91-99. Hammond-Kosack, K.E. and Jones, J.D.G. 1997. Plant disease resistance genes. Annu. Rev. Plant Physiol. Plant Mol. Biol., 48: 573-607. Hammond-Kosack, K.E. and Parker, J.E. 2003. Deciphering plant-pathogen communication: fresh perspectives for molecular resistance breeding. Curr. Opin. Biotech., 14: 177-193. Kaloshian, I., Lange, W. and Williamson, V. 1995. An aphid-resistance locus is tightly linked to the nematode-resistance gene, Mi, in tomato. Proc. Nat. Acad. Sci. USA, 92: 622-625. Kishaba, A.N., Bohn, G.W. and Toba, H.H. 1971. Resistance to Aphis gossypii in muskmelon. J. Econ. Entomol., 64: 935-937. Kishaba, A.N., Castle, S.J., Coudriet, D.L., McCreight, J.D. and Bohn, G.W. 1992. Virus transmission by Aphis gossypii Glover to aphid-resistant and susceptible muskmelons. J. Amer. Soc. Hort. Sci., 117: 248-254. Klinger, J., Powell, G., Thompson, G.A. and Isaacs, R. 1998. Phloem specific aphid resistance in Cucumis melo line AR5: effects on feeding behaviour and performance of Aphis gossypii. En- tomol. Exp. Appl., 86: 79-88. Klinger, J., Powell, G., Thompson, G.A. and Perl-Treves, R. 2001. Mapping of cotton melon aphid resistance in melon. J. Amer. Soc. Hort. Sci., 126: 56-63. Lecoq, H., Cohen, S., Pitrat, M. and Labonne, G. 1979. Resistance to cucumber mosaic virus transmission by aphids in Cucumis melo. Phytopathology, 69: 1223-1225. Lecoq, H., Labonne, G. and Pitrat, M. 1980. Specificity of resistance to virus transmission by aphids in Cucumis melo. Ann. Phytopathol., 12: 139-144. Périn, C., Hagen, L., de Conto, V., Katzir, N., Danin-Poleg, Y., Portnoy, V., Baudracco-Arnas, S., Chadoeuf, J., Dogimont, C. and Pitrat, M. 2002. A reference map of Cucumis melo based on two recombinant inbred line populations. Theor. Appl. Genet., 104: 1017-1034. Pitrat, M. and Lecoq, H. 1980. Inheritance of resistance to cucumber mosaic virus transmission by Aphis gossypii in Cucumis melo. Phytopathology, 70: 958-961. Rossi, M., Goggin, F.L., Milligan, S.B., Kaloshian, I., Ullman, D.E. and Williamson, V. 1998. The nematode resistance gene Mi of tomato confers resistance against the potato aphid. Proc. Nat. Acad. Sci. USA, 95: 9750-9754.

329 330 Scientific contributions

IV. Breeding and genetics

331 332 Performance of pickling cucumber cultivars presently on the Polish National List

K. Bartoszak Centralny Orodek Badania Odmian Rolin Uprawnych S³upia Wielka, Rzeczpospolita, Poland

Summary

The pickling cucumber is the most commonly grown group of cucumber in Poland. There are 42 pickling cucumber cultivars on the Polish National List at present. They were tested in the open field at experiment stations from 1996 through 2002. The newer cultivars tended to yield more and were more disease resistant, but not as early as their predecessors.

Keywords: Cucumis sativus, COBORU test, statistical analysis

Introduction

Pickling cucumber is an important vegetable crop in Poland and is grown on approximately 18,000 ha. A large number of cultivars are already listed and an increasing number have been newly submitted, indicative of the great economic importance of pickling cucumbers. Most of the cultivars were bred by domestic companies. Even though these companies offer a wide selection, the share of foreign-bred cultivars has increased substantially in recent years. The objective of this presentation is to scan the Polish National List of pickling cucumbers and compare trial results obtained for listed cultivars.

Material and methods

There are at present 36 large-wart and 6 small-wart cultivars of pickling cucumber on the Polish National List (NLI). These cultivars together with year of their entry into the NLI and their source are listed in Table 1. Results obtained from them in COBORU trials from 1996 through 2002 are presented in Tables 2 and 3. Each cultivar was grown in at least three of these years. Even though not all cultivars were grown in the same years, statistical analysis with REML (Restricted Maximum Likehood, from statistical package of GenStat) made it possible to compare cultivars using all pooled data. Least significant differences between cultivars at the 5% level of probability are given in Tables 2 and 3 to help in the practical interpretation of the results. The order of cultivars in the tables corresponds to their date of entry into the listing. Each year, trials were conducted at six experiment stations and each trial consisted of four replications. Seeds of all cultivars were sown in the middle of May on beds containing two rows each with 60 cm spacing; the distance between beds was 1.2 m. The plants were grown in accordance with best current commercial practices and the crop was harvested every 23 days from July to September. Early yield refers to the first four harvests from the beginning of harvest of the earliest cultivar. Data on early yield were tabulated only from 1998 to 2002.

No. Name Year Breeders Company listed country Large-wart cultivars 1 Polan H 1972 PL POLAN Kraków 2 Hela H 1979 PL PNOS Poznañ 3 Racibor H 1979 PL POLAN Kraków 4remski H 1998 PL SPÓJNIA Nochowo 5 Aladyn H 1993 PL IW Skierniewice 6 Cezar H 1993 PL PlantiCo Go³êbiew, IW Skierniewice 7 Parys H 1993 PL ZO Przyborów, IW Skierniewice 8 Wojan H 1993 PL POLAN Kraków 9 Krak H 1994PL POLAN Kraków 10 W³adko H 1994PL POLAN Kraków 11 Atlas H 1996 PL ZO Przyborów, IW Skierniewice 12 Natasja H 1996 NL Royal Sluis 13 Octopus H 1997 NL Syngenta Seeds 14Parker H 1997 NL Nunza 15 Atlantis H 1998 NL Bejo Zaden 16 Bolko H 1998 PL POLAN Kraków 17 Frykas H 1998 PL PlantiCo Go³êbiew 18 Kmicic H 1998 PL POLAN Kraków 19 Sander H 1998 PL POLAN Kraków 20 Cyryl H 1999 PL ZO Przyborów, IW Skierniewice 21 Prymus H 1999 PL PlantiCo Go³êbiew 22 Akord H 2000 NL Bejo Zaden 23 Alibi H 2000 NL Bejo Zaden 24Bazyl H 2000 PL ZO Przyborów, IW Skierniewice 25 Chrobry H 2000 PL POLAN Kraków 26 Hermes H 2000 PL PNOS O¿arów Maz., IW Skierniewice 27 Izyd H 2000 PL PNOS O¿arów Maz., IW Skierniewice 28 Opty H 2000 PL POLAN Kraków 29 Forum H 2001 PL POLAN Kraków 30 Gomes H 2001 PL PlantiCo Go³êbiew 31 Potomac H 2001 NL Asgrow Vegetable Seed 32 Soplica H 2001 PL PlantiCo Zielonki 33 Zag³oba H 2001 PL POLAN Kraków 34Kronos H 2002 PL PNOS O¿arów Maz., IW Skierniewice 35 Sonate H 2002 NL Rijk Zwaan Small-wart cultivars 36 Victoria H 1981 PL HR Snowidza 37 Fortuna H 1985 PL HR Snowidza 38 Othello H 1993 NL Syngenta seeds 39 Moro H 1994PL SPÓJNIA Nochowo 40 Stimora H 1997 NL Nunza 41 Evita H 1999 NL Royal Sluis

H = hybrid, NL = The Netherlands, PL = Poland

334 Table 2. Results for large-wart pickling cucumber cultivars

Yield Yield quality Length- Susceptibility to to Cultivar Total Early Too Too diame- Pickling long thick Deformed ter ratio AL DM PM (dt/ha) (% of total yield) (9-point scale)

Polan 241 94 64.8 4.0 7.3 23.4 2.8 3.9 4.2 6.4 Polan 241 94 64.8 4.0 7.3 23.4 2.8 3.9 4.2 6.4 Hela 185 76 59.6 6.46.5 27.0 3.0 3.5 4.2 6.0 Racibor 236 86 62.9 4.9 5.7 25.9 2.9 3.9 4.2 6.1 remski 262 88 68.4 5.2 6.5 19.6 2.9 4.2 4.3 6.2 Aladyn 305 72 70.5 3.9 5.7 19.6 2.9 5.7 5.8 6.8 Cezar 247 72 63.4 5.2 5.9 25.3 3.0 5.4 5.5 6.6 Parys 257 57 66.6 5.7 5.2 22.0 2.9 5.45.6 6.5 Wojan 187 68 62.5 5.1 5.0 27.1 3.0 4.0 4.5 6.0 Krak 249 82 62.8 6.1 6.9 23.8 2.9 5.2 5.46.6 W³adko 239 62 64.3 4.9 6.7 23.7 2.9 5.0 5.6 6.9 Atlas 289 58 72.0 3.8 6.3 17.5 2.9 5.7 5.7 7.0 Natasja 236 61 65.6 5.45.9 22.9 2.9 5.45.2 6.7 Octopus 285 69 70.3 6.5 5.3 17.7 3.0 5.7 5.5 7.0 Parker 275 87 67.6 6.0 4.2 21.9 3.0 4.6 4.7 6.5 Atlantis 279 83 66.7 6.6 5.8 20.7 3.0 5.1 5.46.5 Bolko 258 92 62.9 5.9 6.1 24.5 2.9 4.8 4.8 6.6 Frykas 272 64 64.8 5.3 5.5 24.1 3.0 5.5 5.0 7.3 Kmicic 26483 65.8 6.7 5.5 21.7 3.1 5.3 5.1 7.0 Sander 285 66 65.2 6.3 5.9 22.1 3.0 5.44.7 7.5 Cyryl 289 57 71.1 4.9 6.9 16.9 2.9 5.5 5.7 6.7 Prymus 275 64 65.3 5.8 5.9 22.6 2.9 5.1 5.0 6.4 Akord 252 72 68.2 5.1 5.8 20.6 3.0 5.0 5.2 6.6 Alibi 279 57 66.3 5.5 5.2 22.6 2.9 4.7 5.0 6.5 Bazyl 298 53 73.1 5.45.7 15.4 3.0 6.0 6.1 7.1 Chrobry 276 101 66.0 6.1 6.421.1 3.0 5.3 4.9 6.8 Hermes 301 74 72.0 3.8 6.6 17.3 2.9 5.3 5.5 6.9 Izyd 260 55 69.7 4.3 6.4 19.2 2.9 5.4 5.6 6.7 Opty 274 95 64.5 6.1 5.4 23.6 3.0 4.9 4.8 6.5 Forum 283 95 63.7 8.1 5.2 22.6 3.1 5.5 5.1 6.6 Gomes 241 54 64.8 6.9 5.5 22.3 3.1 5.2 5.0 6.3 Potomac 242 49 67.2 6.9 4.5 20.9 3.1 5.8 5.6 6.7 Soplica 244 78 59.8 8.2 4.4 27.2 3.1 4.8 4.5 6.5 Zag³oba 267 74 65.6 5.46.5 22.1 2.9 5.5 5.2 6.7 Kronos 319 75 70.5 3.0 8.9 17.2 2.8 5.7 5.6 6.9 Sonate 253 46 67.9 6.3 4.3 21.1 3.1 6.2 5.9 7.2 LSD 35 18 0.05 AL = angular leaf spot, DM = downy mildew, PM = powdery mildew; 9-point scale, from 9 = not susceptible to 1 = highly susceptible.

335 Results and discussion

Mean values for yield, yield quality, and some other characteristics for large-wart pickling cucumbers are presented in Table 2 and for small-wart pickling cucumbers in Table 3.

Table 3. Results for small-wart pickling cucumber cultivars

Yield Yield quality Length- Susceptibility to to Cultivar Total Early Too Too diame- Pickling long thick Deformed ter ratio AL DM PM (dt/ha) (% of total yield) (9-point scale) Victoria 212 84 59.8 9.4 4.5 26.1 3.0 4.9 4.8 5.5 Fortuna 195 62 59.7 9.1 5.4 25.5 3.1 4.7 4.8 5.3 Othello 277 108 69.5 6.9 5.417.9 3.0 4.95.2 5.7 Moro 258 88 71.7 5.47.8 14.9 2.9 4.2 3.9 5.1 Stimora 281 99 67.47.5 4.1 20.7 3.0 5.1 5.1 5.6 Evita 286 103 71.4 6.0 4.4 17.8 2.8 4.9 4.7 5.6 LSD 25 14 0.05

AL = angular leaf spot, DM = downy mildew, PM = powdery mildew; 9-point scale, from 9 = not susceptible to 1 = highly susceptible.

Statistically significant differences were observed among cultivars for total and early yields. Overall, small-wart cultivars yielded less than large-wart ones. Progress in breeding is evident, as the newer cultivars tended to have higher yields and lower susceptibility to disease than the older ones, but were not as early. The number of pickling cucumber cultivars is large, in accordance with market size and demands.

References

Godzik G. 1998. Metodyka badania wartoci gospodarczej odmian (WGO) rolin uprawnych. Ogórek (gruntowy). Wyd. COBORU, S³upia Wielka Lista Odmian Rolin Warzywnych 2003. Wyd. COBORU, S³upia Wielka. Bartoszak K., Kowalczyk B. and Litka M. 2003. Lista Opisowa Odmian. Roliny warzywne: ogó- rek i pomidor uprawa polowa. Wyd. COBORU, S³upia Wielka.

336 Suitability of new cucumber F hybrids for open-field 1 cultivation

U. Klosinska and E.U. Kozik Research Institute of Vegetable Crops, Skierniewice, Poland

Summary

Four newly developed F cucumber hybrids bred at the Research Institute of Vegetable Crops 1 at Skierniewice (Poland) were the objects of comparative investigations with the earlier released Aladyn F and Sremski F . The experiments were conducted during 2002 and 2003 in the open 1 1 field in order to evaluate suitability for cultivation. Results revealed that Odys F , Reja F and 1 1 Hermes F can be grown for pickling as dill cucumbers, while Rodos F is also recommended 1 1 for gherkin production. These hybrids are gynoecious with multiple resistance to diseases: downy mildew, scab, cucumber mosaic virus, powdery mildew, anthracnose, and angular leaf spot. Ro- dos F had the highest level of resistance to downy mildew among all hybrids tested, including 1 the resistant control, Aladyn F . 1

Keywords: Cucumis sativus, downy mildew, breeding, dill, fruit quality, gherkin, pickling, disease resistance

Introduction Most new cucumber (Cucumis sativus L.) cultivars are F hybrids. Obtaining pro- 1 spective parental lines is an important step of F hybrid breeding. Another is the se- 1 lection of parental components with improved combining ability. Faced with high and still-rising demands of the market, the cucumber breeding program at the Research Institute of Vegetable Crops (RIVC) in Skierniewice (Poland) has intensified, resulting in the development of 10 F hybrids that have been commercially released and 1 successfully grown in Poland as well as in some other European countries (Doruchowski and £¹kowskaRyk, 2000; Kubik et al., 2001). In registration process are two new hybrids, Reja F and Rodos F , and others are being tested (K³osiñska et al., 2003a,b; 1 1 2004). All of the new hybrids are recommended for open-field organic and integrated-management cultivation (Doruchowski and Robak, 1997). All F hybrids are gy- 1 noecious with multiple resistance to diseases: downy mildew, scab, cucumber mosaic virus, powdery mildew, anthracnose, and angular leaf spot. The aim of this study was to evaluate the horticultural value of new F hybrid 1 cucumbers bred at the RIVC in Skierniewice.

Material and methods The experiments were conducted in 2002 and 2003 in the field at Skierniewice, Poland using a randomised block design with three replications. The area of each plot was 10.8 m2. During dry spells, irrigation was provided and fertilization followed current recommendations.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 337 Four new pickling cucumber F hybrids, Rodos, Reja, Hermes, and Odys, bred 1 at the RIVC, were compared with earlier released Aladyn F (RIVC) and Sremski 1 F , which acted as controls. Seeds were sown in the middle of May. Fruits were har- 1 vested by hand twice a week. Quantity of total and marketable yields was weighted according to Polish standards, with separate categories based on fruit length (gherkin 4.06.0 cm, pickling 6.010 cm, dill 815 cm, and cull). Yield from the first three harvests was considered as early. Marketable yield consisted of pickling and dill fractions. The limited protection against downy mildew (only two sprays with a 2-week interval) was applied when 50% of the leaf area of the susceptible control, Sremski F , 1 was affected. The level of downy mildew incidence, quantified as percentage of leaf area infected by the pathogen, was observed three times during the growing season.

Results and discussion

All of the tested new hybrids had higher pickling, dill, marketable and total yields than the standard Sremski F (Table 1). The most productive were Reja and 1 Hermes, with all fractions of yield about twice as high as Sremski in 2002, whereas Odys was the best variety in 2003, except in earliness. The contribution of early crop to the total yield of the hybrids was smaller than of Sremski, which has been well-known as an early variety in Poland. Unfavorable weather conditions, especially drought, during the 2003 growing season resulted in lower yields and fruit quality than in 2002. The quality of yield, expressed as marketable yield proportion of total yield, was higher in all four of the new hybrids than in Aladyn and Sremski, in both years. Overall, a higher quality of yield was obtained in 2002 than in 2003 (Table 2). All of the hybrids had a higher proportion of pickling fruit than of dill fruit. It is worth noting that this tendency was the highest for Rodos F , which means that this par- 1 ticular variety may be recommended not only for pickling cucumbers, but even more so for gherkins.

Table 1. Yield of F cucumber hybrids 1

F hybrid Yield (kg/100 m2) 1 pickling dill marketable early total 2002 2003 2002 2003 2002 2003 2002 2003 2002 2003 Rodos 407.6* 226.6* 143.0** 115.1** 550.5 341.6 126.1 55.4 579.6 400.7 Reja 551.9 225.7 247.6 158.4 799.6 384.2 183.0 32.4854.0 4 38.7 Hermes 544.4 - 232.0 - 776.5 - 191.8 - 830.0 - Odys 486.4 272.6 203.4 196.5 689.8 469.1 176.9 36.3 732.7 535.8 Aladyn 421.9 195.4 182.6 117.2 604.6 312.5 161.4 39.5 680.6 428.0 remski 229.6 160.4109.4 120.0 339.0 280.4227.7 81.6 396.2 350.0

*gherkin, **pickling

338 Table 2. Yield quality of F cucumber hybrids 1

F hybrid Percent of total yield 1 pickling dill marketable cull 2002 2003 2002 2003 2002 2003 2002 2003 Rodos 70.3 56.5 24.7 28.7 95.0 85.2 5.0 14.8 Odys 66.450.9 27.8 36.7 94.287.6 5.8 12.4 Hermes 65.6 - 28.0 - 93.6 - 6.4- Reja 64.6 51.5 29.0 36.1 93.6 87.6 6.4 12.4 Aladyn 62.0 45.6 26.8 27.4 88.8 73.0 11.2 27.0 remski 58.0 45.8 27.6 34.3 85.6 80.1 14.4 19.9

In two years of testing, the new F hybrid cucumbers showed less downy mildew 1 infection than the resistant standard, Aladyn F (Table 3). Among them, Rodos F 1 1 had the highest level of resistance to downy mildew. All tested hybrids, including the two standards, had less disease incidence at the first observation in 2003 than in 2002. This means that the disease developed earlier and/or the pressure of the pathogen was stronger in 2002. However, in the subsequent two observations the opposite was observed, except for the control hybrids in the third observation. Nevertheless, at each time of observation in both years all hybrids remained in the same order with regard to downy mildew incidence.

Table 3. Disease incidence (in percent) of cucumber hybrids infected by downy mildew under field conditions

F hybrid Time of observation* 1 1st 2002 1st 2003 2nd 2002 2nd 2003 3rd 2002 3rd 2003

Rodos 1.0 0.6 4.3 8.3 7.6 10.3 Odys 1.3 0.7 6.6 10.0 10.0 7.8 Hermes 2.6 -0 9.3 -0 13.3 -0 Reja 3.3 0.8 11.6 16.7 14.0 17.3 Aladyn 4.3 0.8 16.6 18.3 19.0 13.1 remski 53.3 15.3 71.7 63.3 83.3 54.6

*1st = 24.07.2002; 22.07.2003 2nd = 31.07.2002; 30.07.2003 3rd = 07.08.2002; 06.08.2003

The new cucumber hybrids, as well as the others bred earlier by RIVC, showed high productivity and quality of fruit and can be grown with no chemical protection on organic farms or under limited chemical sprays using an integrated management program (Doruchowski and Robak, 1997). A big step forward was made in recent ge-

339 netic and breeding activities towards improving the yield, quality of yield, and increasing the level of resistance to diseases, especially to downy mildew, which has been the most destructive disease of field cucumbers in Central Europe.

References

Doruchowski, R.W. and £¹kowskaRyk, E. 2000. F hybrid pickling cucumbers developed for 1 increase yield, earliness and resistance to downy mildew (Pseudoperonospora cubensis). Acta Hort., 510: 45-46. Doruchowski, R.W. and Robak, J. 1997. Integrated control of field cucumber against downy mildew (Pseudoperonospora cubensis Berk & Curt) with the use of resistant F hybrids and limited 1 chemical treatment. In: Proc. All-Polish Conference: Improvements of Production of Vege- table Crops 1. IWARZ, Skierniewice, pp. 60-66. K³osiñska, U., Kozik, E.U. and £akowska-Ryk, E. 2003a. Evaluation of agronomic traits of the new pickling cucumber F hybrids bred at Research Institute of Vegetable Crops in Skierniewi- 1 ce. Folia Hort., Suplement 2003/1: 67-69. K³osiñska, U., Kozik, E.U. and £akowska-Ryk, E. 2003b. Nowy mieszaniec F ogórka gruntowe- 1 go SKW 1202 z przeznaczeniem do konserwowania i kwaszenia (New cucumber F hybrid 1 SKW 1202 for pickling and dill use). In: Mat. Ogólnop. Konferencji Upowszechnieniowej ,,Nauka praktyce. IWARZ, Skierniewice. pp. 3-5. (In Polish) K³osiñska, U., Kozik, E.U. and £akowska-Ryk, E. 2004. Nowy polski mieszaniec F ogórka gruntowego 1 SKW 1102 z przeznaczeniem na korniszony (New Polish cucumber F hybrid SKW 1102 for 1 gherkin use). In: Mat. Ogólnop. Konferencji Upowszechnieniowej ,,Naukapraktyce. IWARZ, Skierniewice: in press. (In Polish) Kubik, U., £akowska-Ryk, E. and Doruchowski, R.W. 2001. Progress in development of pickling cucumber F hybrids towards the horticulture traits. Folia Hort., 13/1A: 325-329. 1

340 The effect of ethephon on the sex expression of naked seeded oil pumpkin

J. Berenji1 and D. Papp2 1Institute of Field and Vegetable Crops, M. Gorkog 30, 21000 Novi Sad, Serbia and Montenegro; 2«DUDU Bt», Tessedik u. 171, 4032 Debrecen, Hungary

Summary

The effect of ethephon on the sex expression of the naked-seeded oil pumpkin Olinka was studied, with the goal of examining a possible method for commercial scale F hybrid seed pro- 1 duction. Ethephon was applied twice, at the 34-leaf stage and again 10 days later, at 300, 400, 500 and 600 ppm concentrations. The 500 ppm concentration was optimal, significantly delaying the appearance of the first male flowers and thereby providing a period of two weeks with female sex expression. This duration would be long enough to ensure sufficient cross-pollination of female flowers on maternal plants of a prospective hybrid by the pollen from male flowers on neighbouring untreated paternal plants.

Keywords: Cucurbita pepo, naked-seeded oil pumpkin, ethephon, sex expression, hybrid seed production

Introduction

Production of naked-seeded oil pumpkin (Cucurbita pepo L.) is increasing in Serbia and Montenegro and Hungary as well (Berenji, 2000), demanding improvement of the production practices, including more productive cultivars (Karloviæ et al., 2001; Sikora and Berenji, 2001). One of the promising directions of naked-seeded oil pumpkin breeding is creating F hybrid cultivars. Hybrid vigor in cucurbits is well-documented and exploited, 1 however little is known specifically for the naked-seeded oil pumpkin (Berenji, 1986; Bru- ce and Loy, 1994). Having in mind the assumed differential sensitivity of various pumpkin cultivars to ethephon (Edelstein et al., 1985), the aim of this study was to examine the effect of ethephon on the sex expression of the naked-seeded oil pumpkin Olinka, in an attempt to develop a method for the production of F hybrid seed on a commercial scale. 1

Material and methods

This study was conducted at the experimental field of the Institute of Field and Ve- getable Crops in Novi Sad (Serbia and Montenegro) during the season of 2003. The naked-seeded oil pumpkin Olinka was planted on May 12. Treatments consisted of 100 plants each, sprayed twice with clean water (control), or with 300, 400, 500 or 600 ppm of ethephon, first at the 34-leaf stage (June 2) and again 10 days later. Starting at the day of the opening of the first flowers in the experiment (June 20), the number of open male and female flowers was counted each day for a period of 70 days.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 341 Results and discussion Figure 1 shows the number of male and female flowers per plant in each treatment. The control plants of Olinka had a sex expression typical of C. pepo pumpkin plants. The average total number of flowers per plant was 151, with the ratio of male-to-female flowers being 10.8:1. Treatment with ethephon reduced significantly the total number of flowers per plant, but there were less obvious differences among the different ethephon concentrations (range of 83-103 flowers per plant). The ratio of male- to-female flowers gradually decreased with increased concentrations of ethephon from 10.8:1 to 3.4:1. These results are consistent with those of other reports (Borghi and Pironi, 1974; Robinson et al., 1979; Shannon and Robinson, 1979; Yongan et al., 2002). In Fig. 2, the typical appearance of a plant treated with ethephon with characteristic concentrated fruit set is shown. Treatment with ethephon results in shorte- ning of internodes and more concentrated fruit set. However, the open-pollinated cultivar Olinka has semi-bush growth habit and good productivity, bearing as many as 3-4 mature fruits per plant, and therefore these positive effects of ethephon would not be expected to be as obvious in this cultivar as in cultivars with viney growth habit.

Figure 1. Male, female, and total number of flowers per plant of the naked-seeded oil pumpkin Olinka, as affected by ethephon.

Figure 2. Typical appearance of a plant of the naked-seeded oil pumpkin Olinka treated with ethephon, showing characteristic fruit set.

342 The dynamics of flowering, that is, the occurrence of male and female flowers over time, of each treatment is presented in Fig. 3. In the control, anthesis of the first female flowers occurred 710 days after anthesis of the first male flowers (Fig. 3). Treatment with ethephon resulted in a delay of the onset of male flower occurrence, but the overall start of flowering remained unchanged due to an earlier start of female flowering. The longest period at the beginning of flowering characterised by female flowers exclusively was obtained using the 500 ppm ethephon treatment (Fig. 3). The indu-

Figure 3. Effect of different concentrations of ethephon on the occurrence of male and female flowers of the naked-seeded oil pumpkin Olinka.

343 ced two-week period of exclusive female-flower production has potential value for F 1 hybrid seed production. Plants of the prospective maternal parent could be treated to prevent selfing and sibbing, forcing pollination with pollen derived from untreated plants of the prospective paternal parent. The two-week duration would be adequate to ensure sufficient cross-pollination of female flowers on maternal plants of a prospective hybrid by the pollen from male flowers on neighbouring untreated paternal plants. Subsequent occurrence of female flowers on the treated plants, from the point when male flowers start opening, is undesirable from the point of view of F hybrid 1 seed production. All these fruits set from them would have to be discarded prior to harvesting of the F seed. 1 In the course of further research, long-internode, viney genotypes should be tested in order to obtain a more precise answer to the questions concerning the effect of ethephon on decreasing internode length and increasing the number of mature fruits per plant. Genotypes of naked-seeded oil pumpkin that tend to develop not more than 12 mature fruits from the female flowers as close to the base of the plant as possible might better fulfill the requirement of reliable cross-pollination of female flowers on the treated maternal plants during the period when male flowers are still under the suppressive effect of ethephon. In this case, fruit set would be terminated before selfing or sibbing could occur as a result of the disappearance of the suppressive effect of ethephon on male flowers of the maternal plants. Otherwise, it would be necessary to manually discard all of the fruits of the maternal plants except for the 12 fruits closest to the base of the plant, which would contain reliably F seed. 1

References

Berenji, J. 1986. Hibridna snaga kod uljane tikve-golice, Cucurbita pepo L. Uljarstvo, 23(3-4): 79-85. Berenji, J. 2000. Breeding, production, and utilization of oil pumpkin in Yugoslavia. Cucurbit Genet. Coop. Rep., 23: 105-109. Borghi, B. and Pironi, W. 1974. Evaluation of heterosis in Cucurbita pepo L. In: Janossy, A. and Lupton, F.G.H. (Eds.), Heterosis in Plant Breeding. Proceedings of the 7th Eucarpia Congress, Budapest, Hungary. Bruce, C.R. and Loy, J.B. 1994. Heritability of seed size in hull-less seeded strains of Cucurbita pepo L. Cucurbit Genet. Coop. Rep., 17: 125-127. Edelstein, M., Paris, H.S., Nerson, H., Karchi, Z. and Burger, Y. 1985. Differential sensitivity of Cucurbita pepo cultivars to ethephon. Cucurbit Genet. Coop. Rep., 8: 67-68. Karloviæ, Ð., Berenji, J., Recseg, K. and Kõvári, K. 2001. Savremeni pristup uljanoj tikvi (Cucurbita pepo L.) sa posebnim osvrtom na tikvino ulje (Oleaum cucurbitae). Zbornik radova 42. Save- tovanja industrije ulja Proizvodnja i prerada uljarica, pp. 177-182, Herceg Novi. Robinson, R.W., Whitaker, T.W. and Bohn, G.W. 1979. Promotion of pistillate flowering in Cucurbita by 2-chloroethylphosphonic acid. Euphytica, 19: 180-183. Shannon, S. and Robinson, R.W. 1979. The use of ethephon to regulate sex expression of summer squash for hybrid seed production. J. Amer. Soc. Hort. Sci., 104: 674-677. Sikora, V. and Berenji, J. 2001. Utilization of cucurbit (Cucurbita sp.) genetic resources. Book of Abstracts of the 1st International Symposium Food in the 21st Century, pp. 69-70, Sub- otica, Serbia and Montenegro. Yongan, C., Bingkui, Z., Enhui, Z. and Zunlian, Z. 2002. Chemical control of sex expression in summer squash (Cucurbita pepo L.). Cucurbit Genet. Coop. Rep., 25: 51-53.

344 Evaluation of some inbred lines of summer squash for plant, flower, fruit and seed properties

N. Ercan, M. Temirkaynak, F. ªensoy and A.S. ªensoy University of Akdeniz, Faculty of Agriculture, Department of Horticulture, Antaly, Turkey; e-mail: [email protected]

Summary

Five-generation inbred lines of summer squash (Cucurbita pepo), Everest 8(15), Whitebush 2(18), Whitebush 11(38), Gieda 8(27), Gieda 12(19), and Gieda 12(36), were developed by selfing and some of their plant, flower, fruit and seed characteristics were observed. Plants of all six inbred lines were uniform for bush growth habit. Everest 8(15) had many branches, while the others had no branches. The inbred lines differed markedly from one another in the number of seeds produced per fruit.

Keywords: Cucurbita pepo, inbreeding

Introduction Summer squash (Cucurbita pepo L.) are open pollinated and major pollinators are bees and other insects. Homozygous lines obtained by inbreeding are the first step toward developing hybrid cultivars. In plant breeding terms, inbreeding is defined as the result of self pollination of plants that are in nature cross-pollinating. Inbreeding leads to an increase in homozygosity of the population and can reduce plant vigour. Negative effects of inbreeding, as a whole, are referred to as inbreeding depression. The degree of inbreeding depression depends on the generations of selfing. Selfing results in little inbreeding depression in species of the Cucurbitaceae (Allard, 1960), but C. pepo and C. maxima have been reported to exhibit some degree of inbreeding depression (Abdel-al et al., 1973) and Ghaderi and Lower (1978) reported that self pollination did result in some deterioration of cucumber lines. The aim of this study is to evaluate some plant, flower, fruit and seed properties of five-generation inbred summer squash lines, in terms of their response to inbreeding depression.

Material and methods Everest, Whitebush and Gieda commercial cultivars were grown and selfed in both spring and fall, for two generations of inbreeding per year, until the S generation 5 was obtained. Seeds from the S lines were planted on March 15, 2001 using a rando- 5 mized block design with three replications. Male and female flowers to be used for pollination were secured prior to anthesis. After selfing, the female flowers were labelled and tied in order to prevent uncontrolled pollination. Inbred lines were evaluated for plant growth habit, branching, percent female flowers, number of fruit/plant, number of seed/fruit and 1000-seed weight.

Lines Female Number of Number of 1000-Seed flower (%) fruit/plant seed/fruit weight

Everest 8(15) 35.80 4.23 113.5 63.5 Whitebush 2(18) 40.95 3.93 84.0 109.2 Whitebush 11(38) 44.22 2.63 132.0 92.1 Gieda 8(27) 34.44 3.85 307.0 73.8 Gieda 12(19) 34.83 4.05 68.0 88.5 Gieda12(36) 43.24 2.90 106.0 115.3

Results and discussion

Plants of all six inbred lines were homogeneous for bush growth habit. Edelstein et al. (1989) reported that bush growth habit is controlled by a single incompletely dominant gene (Bu) in C. pepo and compact growth was advantageous for growers since different cultural operations were made easily. When branching was considered, it was observed that only Everest 8(15) had many branches. Results on flower, fruit, and seed properties of six S inbred lines are summarised 5 in Table 1. More male than female flowers were produced by all inbred lines. The inbreds differed considerably in the number of fruits produced per plant and even more so, by several-fold, in the number of seeds produced per fruit. Gieda 6(27) produced the greatest number of seeds per fruit, 307, while Gieda 12(19) produced the least, 68. For 1000-seed weight, Gieda 12(36) had the highest, nearly double that of Everest 8(15), which had the lowest. Self-pollination can be utilized to develop uniform strains from heterogeneous, open-pollinated cultivars. Number of seeds produced per fruit is an important consideration (Stephenson et al., 1988), especially for hybrid seed production. Although inbreeding can in some cases cause depression in seed yield, the results of this study suggest that inbreeding depression is genotype-dependent and that some of the inbred lines tested could be used as parents for F hybrid development. 1

References

Abdel-al, Z.E., Khalf-Allah, A.M. and Shenouda, G.S. 1973. Effect of visual selection and inbreeding on some quantitative characters of summer squash. Alex. J. Agric. Res., 21: 277. Allard, R.W. 1960. Principles of Plant Breeding. John Wiley and Sons, NewYork. Edelstein, M., Paris, H.S. and Nerson, H. 1989. Dominance of bush growth habit in spaghetti squash (Cucurbita pepo). Euphytica, 43: 253-257. Ghaderi, A. and Lower, R.L. 1978. Heterosis and inbreeding depression for yield in populations derived from six crosses of cucumber. J. Amer. Soc. Hort. Sci., 104: 564-567. Stephenson, A.G., Devlin, B. and Horton, J.B. 1988. The effects of seed number and prior fruit dominance on the pattern of fruit production in Cucurbita pepo (zucchini squash). Ann. Bot., 62: 653-661.

346 Evaluation of flower abscission and sex expression in different cultivars of zucchini squash (Cucurbita pepo)

P. Gómez1, A. Peñaranda1, D. Garrido2 and M. Jamilena3 1C.I.F.A. de Almería, Autovía del Mediterráneo s/n, La Mojonera, Almería, Spain 2Universidad de Granada, Departamento de Fisiología Vegetal. Facultad de Cienci- as, 18071 Granada, Spain 3Universidad de Almería, Departamento de Biología Aplicada, Área de Genética, Escuela Politécnica Superior, 04120 Almería, Spain; e-mail: [email protected]

Summary

Nine cultivars of zucchini squash were evaluated for flower abscission, sex expression and fruit production under spring/summer greenhouse conditions in Almería, Spain. Under the high temperatures of these experimental conditions, we found that many of the harvested fruits still retained their flowers, a developmental disorder that reduces the quality of the zucchini fruit. The percentage of fruits that showed this delay in flower abscission is cultivar dependent, and ranged from 0 to 70% among the analysed cultivars. Our results have also indicated that this disorder is associated with a promotion of maleness, mainly in cultivars in which a higher percentage of fruits with non-abscised flowers was observed. In fact, in those cultivars, female flowers that were delayed in abscission also showed an abnormal development of stamens, as well as a reduction in their rate of growth up to anthesis. Given that the sexual expression of squash is controlled by ethylene, and that flower abscission is also known to be controlled by this same hormone, data suggest that flower abscission disorder detected in greenhouse zucchini squash under high temperatures could be caused by a reduction of endogenous ethylene. By comparing auxins-treated and untreated plants of each cultivar, we have determined that neither the delay in flower abscission nor the maleness is caused by this hormone treatment that growers usually use for fruit set in greenhouse zucchini squash. Under these conditions, hormone treatments neither increased the quality of harvested fruits per plant, suggesting that at least during spring/summer season, treatments with synthetic auxins could be unnecessary in squash.

Keywords: Cucurbita pepo, zucchini, flower abscission, sex expression, fruit set

Introduction

Zucchini cultivars of summer squash (Cucurbita pepo) produce fruits to be harvested at an immature stage of development. Although there are some exceptions, in most of the zucchini cultivars, the abscission of the floral organs from the developing fruit occurs before harvest. Nevertheless, some environmental conditions may delay floral abscission, conditioning that the floral organs remain attached to the harvested squash fruit. This developmental abnormalities have constituted a problem for squash cultivation under greenhouse conditions in SE Spain, because manually remove of attached flower makes the fruit more susceptible to infection and rotting during its storage and transport, diminishing its shelf life and its commercial quality value. Little is known about the physiological or genetics factors that regulate the abscission of floral organs in Cucurbita pepo or other members of the Cucurbitaceae family. In other species, however, flower abscission is known to be a programmed develop-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 347 mental process that follows flower pollination and is promoted by endogenous ethylene (Brown, 1997; Van Doorn and Stead, 1997). It is well documented that ethylene accelerates the abscission of floral organs in a number of species (González-Carranza et al., 1998). Moreover, most of the mutant showing delayed flower abscission are deficient in ethylene perception. Thus, in comparison to wild type plants, ethylene insensitive mutants etr1 and ein1of Arabidopsis, as well as the Never ripe (nr) mutant of tomato, show delayed floral abscission (Bleecker et al., 1988; Lanahan et al., 1994; Grbic and Bleecker, 1995; Yen et al., 1995). Recent evidences, however, have indicated that ethylene is not the unique factor that influence floral organ abscission. Thus, Fernandez et al. (2000) reported that Arabidopsis plants that overexpress the MADs-box gene AGAMOUS LIKE- 15 (AGL15) are sensitive to ethylene but are delayed in flower abscission. Recently, it has been also suggested that the gene INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) participates in an ethylene independent pathway that controls floral organ abscission in Arabidopsis (Butenko et al., 2003). The involvement of ethylene in the abscission of C. pepo floral organs has not been studied. However, this hormone appears to be one of the most important factors regulating sex expression in this monoecious species, as well as other species in the Cucurbitaceae family. Thus, the exogenous application of ethylene relaxing agents such as ethephon inhibits the production of staminate flowers and increases the number of pistillate flowers in squash, being therefore used in the commercial production of hybrid seed (Robinson et al., 1970; Rudich et al., 1970). On the other hand, the application of AVG (aminoethyoxyvinyglycine), an inhibitor of ethylene biosynthesis, is able to induce a reduction of femaleness (Atta-Ally, 1992). Sex expression in cucurbits is also under the control of certain environmental conditions such as temperature and photoperiod. Long days and high temperatures promotes the producti- ons of male flowers, while short days and low temperatures favour the development of female flowers (Wien, 1997). In the present study we have analysed the delay in flower abscission, as well as sex expression and quality fruit production in eleven varieties of zucchini squash cultivated under spring/summer conditions in the Province of Almería, Spain. We found that cultivars responded differentially to these environmental conditions. While flower abscission in some of the cultivar was nearly normal, in three of the studied cultivars, more that 60% of the harvested fruits still retained the flowers, a feature that was found to be associated with an increasing of maleness in these cultivars. The effects of synthetic auxins, a treatment that growers usually carried out in order to increase fruit set and fruit production in greenhouse zucchini squash, have also been analysed.

Material and methods

A total of 9 commercial summer squash cultivars were compared for flower abscission, sex expression and fruit set when grown under high temperature conditions in greenhouses of the southeast of Spain. Experiments were conducted from March to July of 2003, under standard greenhouse conditions in La Mojonera, Province of Almería, Spain. The essay was carried out in absence of pollinating insects. The field plan was designed according to a randomised complete block design with four replicates. To

348 analyse the effect of auxin treatments on each cultivar, opened flowers of half of the plants were treated with the synthetic auxin at the concentration used by growers (ANA 0.45% + ANA-amida 1.2%). Fruit at the marketable stage were harvested twice a week. Flower abscission on each cultivar was estimated as the percentage of harvested marketable fruits with non-abscised flower per plant. The femaleness was calculated as the percentage of female flowers per plant in the first 30 nodes. For the estimation of quality fruit percentage, we considered those harvested fruits not showing delayed flower abscission, but neither any other developmental disorder. Differences among cultivars and treatments for either flower abscission, sex expression, and fruit production were tested by using analysis of variance.

Results and discussion

C. pepo is a monoecious species in which either male and female flowers are developed in each leaf axil of the plant. In most of the cultivars, plants go throughout two different developmental phases. In the first phase, the plant develops only male flowers. The second phase, initiated with the development of the first female flower (node 5 to 9 depending on the cultivar and environmental conditions), marks the period of fruit production, and is characterised by the development not only of female flowers but also of male flowers. Nevertheless, the aperture of the first male flowers can occur later than the first female flowers, because the growth rate of male flowers is slower than female flowers. We have measured the time that 2 cm male and female flower buds take to reach anthesis (Table 1) in two cultivars of zucchini squash. In both cultivars, it is apparent that the development of male flowers is much slower, reaching anthesis between 8-10 days later than female flowers (Table 1).

Table 1. Time to anthesis of female and male flowers, measured as the number of days that a flower bud of 2 cm takes to get anthesis, in two cultivars of zucchini squash grown under standard greenhouse conditions. Results are averaged of at least 20 flowers from 5 plants of each cultivar

Cultivar Days to anthesis ± SD Male flowers Female flowers

Mástil 22.00 ± 1.83 12.23 ± 2.07 Cora 20.28 ± 1.65 12.33 ± 1.75

Under certain environmental conditions, mainly high temperatures that can be reached in Almería greenhouses (Spain) even during certain periods in the cold season, farmers have observed a delay in flower abscission of zucchini squash, in such a way that the floral organs remain attached to the fruit even after harvesting. In order to analyse whether this disorder is dependent on the cultivar, we have made a essay with 9 cultivars of zucchini squash during March to July of 2002 in Almería, a season in which it is possible to reach very high temperatures (more than 30°C) in the greenhouses of this template region of Spain. Besides analysing the incidence of delayed

349 flower abscission in each cultivar, and given that high temperature may also promote changes in sex expression of squash (Wien, 1997), we have also studied whether this deficiency in flower abscission is associated with sex expression, and with the hormonal treatments that are normally used in this area for fruit set.

Table 2. Comparison of flower abscission, sex expression and quality fruit production in different cultivars of zucchini squash grow a high temperatures. Results are averaged from at least 8 plants of each cultivar. Plants were treated (+) or untreated (-) with synthetic auxins, a common treatment for fruit set in summer squash under greenhouse conditions. (G) Different letters within the same column indicate significant differences (p<0.05)

Fruits with Nodes to first Femaleness Quality fruits Cultivar non-abscissed female flower (percent) (percent) flower (mean) (percent) (-) (+) G (-) (+) G (-) (+) G (-) (+) G

1. Elite 26 17 b 7.6 7.4 bcde 43.7 43.7 abc 47.50 75.3 ab 2. Baccara 30 21 b 8.5 7.4 b 43.9 44.4 bc 33.00 49.0 bc 3. Cora 3 4c 7.2 6.6 de 37.6 38.6 cd 70.27 65.7 a 4. Tosca 2 8 c 8.7 7.5 b 37.9 40.9 cd 60.30 84.0 a 5. Balboa 1 0 c 6.5 7.0 e 52.8 47.1 a 74.30 66.0 a 6. Cavili 66 55 a 7.0 7.0 cde 43.6 39.4 ab 9.70 25.0 de 7. Xsara 58 73 a 8.2 6.6 bcde 40.2 45.5 a 0.00 1.7 e 8. Storr´s Green 7 1 c 6.8 6.7 e 39.2 40.2 bc 53.30 71.0 ab 9. Mastil 52 69 a 10.3 9.4a 34.535.2 de 10.80 12.0 de

Table 2 shows the proportion of fruits with delayed flower abscission, as well as the proportion of female flowers and quality fruits in each of the analysed cultivar. In many of the cultivars, the proportion of flowers in which abscission was delayed was very low, ranging from 0 to 30%. Nevertheless, in three of the cultivars (Cavili, Xsara and Mastil), more than 60% of the fruits still retained their flowers (Table 2). Many of these flowers were still closed when fruits were harvested, being difficult to separate the flower from the fruit. Indeed, a big scar remained in the fruit once the flower was manually removed. Cultivars have shown differences in either the initiation of female flower production or in the proportion of female flowers (Table 2). However, it is important to note that for example the cultivar Balboa, showing the lowest proportion of fruits with un-abscised flowers, and Mastil, showing this character as one of the biggest, were also the highest and lowest for femaleness (Table 2). In addition, we have observed that flowers that remain attached to the fruits in the cultivars with higher proportion of un-abscised flowers were hermaphroditic, and all of them developed stamens with different degrees of maturity (Fig. 1). Moreover, the growth rate of this hermaphroditic flowers resembled male flower development and was therefore much slower than that of female flowers (Fig. 1). All together, these results seems to indica-

350 te that the delay in flower abscission is associated with a process that also promotes maleness. Rylski and Aloni (1990) and Wien (1997) have also observed that high temperatures and long days promote the production of male flowers in squash pumpkin but the formation of perfect hermaphrodite flowers under those conditions has been never described. In squash, as well as in other species of the Cucurbitaceae family, it is known that sex expression is regulated by ethylene, being frequent the use of ethephon to inhibit male flowers in female lines used for hybrid seed production (Shanon and Robinson, 1979; Rudich,1990).

Figure 1. a) Zucchini fruits showing that non-abscised floral organs is associated with stamen development in female flowers. b) Female flower development in cultivars with delay in flower abscission. Note that, in comparison with normal flowers (arrowhead), the growth rate of the flower delayed in abscission (arrow) is much slow, and remains still closed although is the oldest.

Therefore, it is feasible that the observed delay in flower abscission in Zucchini squash can be caused by a decrease in internal ethylene level. Ethylene plays an essential role in floral organ abscission (Brown, 1997; Van Doorn and Stead, 1997), and ethylene insensitive mutants analysed in Arabidopsis and tomato show a considerable delay in flower abscission (Bleecker et al., 1988; Guzman and Ecker, 1990; Lanahan et al., 1994). Finally we have studied whether the observed disorder in flower abscission and sex expression could be caused by the hormonal treatments that are commonly used for fruit set in greenhouse crops of zucchini squash. Opened flowers from plants of the 9 tested cultivars were sprayed daily with the hormone solution during the whole cropping period, and compared with plants of the same cultivar that were not treated (Table 2). No significant differences have been found between treated and non-treated plants for either flower abscission, sexual expression, or production of quality fruits. Auxins are able to induce parthenocarpic fruits in C. pepo (Wong, 1947; Sanz, 1995), being also involved in sex determination of these species (Rudich, 1990). An interaction between auxins and ethylene was found by Chrominski and Kopcewics

351 (1972), who found that treatments of squash with ethephon may induce the production of female flowers but also reduce the activity of endogenous auxin. In our essay, we found that the treatment with auxins did not promote any change in the sexual expression, neither in the flower abscission in any of the analysed cultivars. Moreo- ver, no differences in the percentage of quality fruits per plant were detected between treated and untreated plants, indicating that at least under the experimental conditions, hormonal treatments of zucchini squash could be eliminated.

References

Atta-Aly, M.A. 1992. Chemical regulation of growth and sex expression in squash plants. Ann. Agric. Sci. Ain Shams Univ. Cairo, 37: 173-180. Bleecker, A.B., Estelle, M.A., Somerville, C. and Kende, H. 1988. Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science, 241:1086-1089. Brown, K.M. 1997. Ethylene and abscission. Physiol. Plant., 100: 567-576. Butenko, M.A., Patterson, S.E., Grini P.E., Stenvik, G.E., Mandal, A.A. and Aalen, R.B. 2003. INFLORESCENCE DEFICIENT IN ABSCISSION controls floral organ abscission in Arabidop- sis and identifies a novel family of putative ligands in plants. Plant Cell, 15: 2296-2307. Chrominski, A. and Kopcewicz, J. 1972. Auxin and giberellins in 2-chloroethyl phosphonic acid- induced femaleness in Cucurbita pepo L. Zeitschr. Pflanzenphysiol., 68: 184-189. Fernandez, D.E., Heck, G.R., Perry, S.E., Patterson, S.E., Bleecker, A.B. and Fang, S.C. 2000. The embryo MADS domain factor AGL15 acts postembryonically: inhibition of perianth senescence and abscission via constitutive expression. Plant Cell, 12: 183-197. González-Carranza, Z.H., Loyola-Gloria, E. and Roberts, J.A. 1998. Recent developments in abscission: Shedding light on the shedding process. Treds Plant Sci., 3: 10-14. Grbic, V. and Bleecker, A.B. 1995. Ethylene regulates the timing of leaf senescence in Arabidop- sis. Plant J., 8: 595-602. Guzmán, P. and Ecker, J.R. 1990. Exploiting the triple response of Arabidopsis to identify ethylene related mutants. Plant Cell, 2: 513-523. Lanahan, M.B., Yen, H.C., Giovannoni, J.J. and Klee, H.J. 1994. The Never ripe mutation blocks ethylene perception in tomato. Plant Cell, 6: 521-530 Robinson, R.W., Whitaker, T.W. and Bohn, G.W. 1970. Promotion of pistillate flowering in Cucurbita by 2-chroroethylphosphonic acid. Euphytica, 19: 180-182. Rudich, J. 1990. Biochemical aspects of hormonal regulation of sex expression in cucurbits. In: Bates, D.M., Robinson, R.W. and Jeffrey, C. (Eds.), Biology and Utilization of Cucurbitaceae. Cornell University Press, Ithaca, pp. 269-280. Rudich, J., Kedar, N. and Halevy, A.H. 1970. Changed sex expression and possibilities for F1 hybrid seed production in some cucurbits by application of Ethrel and Alar (B-995). Euphy- tica, 19: 47-53. Rylski, I. and Aloni, B. 1990. Parthenocarpic fruit set and development in Cucurbitaceae and Solanaceae under protected cultivation in a mild winter climate. Acta Hort., 287: 117-126 Sanz, M. 1995. Fitorreguladores para el calabacín. Hortofruticultura, 33: 46-48. Shannon, S. and Robinson, R.W. 1979. The use of ethephon to regulate sex expression of summer squash for hybrid seed production. J. Amer. Soc. Hort. Sci., 104: 674-677. Van Doorn, W.G. and Stead, A.D. 1997. Abscission of flowers and floral parts. J. Exp. Bot., 48: 821-837. Wien, H.C. (Ed.), 1997. The Physiology of Vegetable Crops. CABI, Wallingford, Oxon, U.K., pp. 345-386. Wong, C.Y. 1941. Chemically induced parthenocarpy in certain horticultural plant with special reference to watermelon. Botanical Gazete, 103: 64-84. Yen, H.C., Lee, S.Y., Tanksley, S.D., Lanahan, M.B., Klee, H.J. and Giovannoni, J.J. 1995. The tomato Never-ripe locus regulates ethylene-inducible gene expression and is linked to a homolog of the Arabidopsis ETR1 gene. Plant Physiol., 107: 1343-1353.

352 Susceptibility to sulfur dusting and inheritance in melon

L. Perchepied, C. Périn, N. Giovinazzo, D. Besombes, C. Dogimont and M. Pitrat INRA, Unité de Génétique et Amélioration des Fruits et Légumes, BP 94, 84143 Montfavet cedex, France; e-mail: [email protected]

Summary

Sulfur is commonly used to control powdery mildew, but it can induce severe necrosis on some melon accessions. Among 236 accessions tested for sulfur susceptibility, 111 exhibited no necrosis and 78 had severe necrosis. An inheritance study was conducted on two RIL populations obtained from the cross between Védrantais exhibiting no necrosis and PI 124112 and PI 161375 exhibiting severe necrosis. A major QTL explaining between 55 and 82 % of the total variance according to the populations and the method of detection (interval mapping or composite interval mapping) was located on linkage group I (LG I) on both populations. We propose to name this major QTL Sulfur resistance (symbol Sr) present in Védrantais. Sr was completely dominant in the Védrantais x PI 124112 cross but incompletely dominant in the Védrantais x PI 161375 cross. Two minor QTLs explaining between 8 and 14 % of the variance were detected only on the Védrantais x PI 124112 population on LG II and LG IV. The QTL on LG IV had a positive value for additivity indicating that the allel for necrosis was present in Védrantais.

Keywords: Cucumis melo, sulfur, resistance/susceptibility, inheritance, QTL

Introduction

Sulfur is commonly used to control powdery mildew in melon as well as in other crops such as Vitis vinifera. It is cheap, quite effective at least under low inoculum pressure, not harmful for the environment and effective against all strains of Sphae- rotheca fuliginea (Schlechtend: Fr.) Pollacci (= Podosphaera xanthii) and Erysiphe cichoracearum DC ex Merat (= Golovinomyces cichoracearum), the main causing agents of powdery mildew on cucurbits. But sulfur can induce severe damages on the foliage of some cultivars. This adverse effect has been documented mainly in the american cantaloupe type and for instance the cultivar PMR 45 is known to be sulfur susceptible (Johnson and Mayberry, 1980). We have tested a small collection of melon (Cucumis melo L.) for sulfur susceptibility and studied the inheritance in recombinant inbred line (RIL) populations between susceptible and resistant lines.

Material and methods

Plant material 236 accessions belonging to different cultigroups and from different geographic origins have been tested. RIL populations between Védrantais (sulfur resistant) and PI 161375 or PI 124112 (sulfur susceptible) were evaluated.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 353 Inoculation technique Seeds were sown in a potting soil mixture in order to obtain 25 plants in a 25 x 35 cm tray. When plantlets were at the first-second leaf stage they were dusted with sulfur. A transparent plastic cover was then placed on the tray for 4-5 days. Five to eight days after removal of the cover, a notation was made on a 0 to 4 scale (0 = no leaf necrosis, 4 = 2 leaves with necrosis).

Inheritance and QTL localization A genetic map with different molecular markers has already been developed on the RIL population Védrantais x PI 161375 (Périn et al., 2002). A new map on the RIL population Védrantais x PI 124112 using different types of molecular markers, mainly AFLP, was obtained. A total number of 136 and 118 RILs belonging respectively to the Védrantais x PI 161375 and Védrantais x PI 124112 populations were evaluated. The software QTL cartographer 1.16 was used for mapping the QTLs by linear regression, interval mapping (IM) or composite interval mapping (CIM) (Basten et al., 1994, 2002).

Results and discussion

Collection evaluation Among 236 accessions, 111 (47 %) exhibited no necrosis (score 0) and 78 (33 %) were severely damaged (scores 3 or 4) while 26 (11 %) were segregating (Table 1). The other accessions were homogeneous with intermediate scores. There was no clear cut grouping according to the geographic origin. It can nevertheless be noticed that all tested accessions from Italy, Turkey and North Africa are resistant. All the accessions belonging to the Charentais cultigroup are resistant. Some accessions in the american cantaloupe cultigroup are resistant (e.g. Topmark, King Henry, WMR 29) while others are susceptible (e.g. PMR 45, Edisto 47, Mainstream). These last results correspond to the observations of Johnson and Mayberry (1980).

Inheritance Védrantais, belonging to the Charentais cultigroup, is resistant while PI 161375 and PI 124112 are susceptible. One of the main interest of RILs is that they are immortalized progenies which can be used for different genotypic or phenotypic evaluations. The F Védrantais x PI 161375 exhibited necrosis (score 2) but was not as susceptible 1 as PI 161375 (score 3-4), indicating an intermediate dominance of sulfur resistance. On the RIL Védrantais x PI 161375 population, about 25% of the RILs exhibited no necrosis (Fig. 1). A major QTL (LOD score = 24.4 and R2 = 77% by IM; LOD score = 24.8 and R2 = 69% by CIM) with negative additivity was detected in telomeric position of linkage group I (LG I) between AFLP markers H36/M42_4 and E38/M43_2 (Fig. 2). The F Védrantais x PI 124112 exhibited no necrosis. In this cross, the sulfur re- 1 sistance seemed to be completely dominant. On the RIL population Védrantais x PI 124112, 37% of the RILs exhibited no necrosis (Fig. 1). A major QTL was detected by the three methods (ANOVA, IM and CIM) in a telomeric position on linkage group I (LOD score = 13.0 and 7.6, R2 = 83% and 55% by IM and CIM respectively). This QTL

354 Table 1. Behaviour of 236 melon accessions to sulfur dusting according to their geographic origin. Accessions were classified as resistant (score 0), intermediate (score 1 or 2), susceptible (score 3 or 4) or segregating (susceptible and resistant plants in one accession). Five plants per accession were evaluated

Resistant Intermediate Susceptible Segregating

France 26 46 0 Portugal & Spain 12 2 2 0 Italy 40 0 0 Other European countries 41 4 0 USSR 2 0 2 0 Turkey 7 0 0 0 Israel 2 0 0 0 Iran 10 1 42 Afghanistan, Uzbekistan & Pakistan 10 0 8 5 India & Sri Lanka 11 5 18 11 China, Japan & Korea 2 5 12 0 Morocco, Tunisia & Egypt 3 0 0 0 USA 11 1 10 1 Brasil & Colombia 2 2 3 0 Unknown 5 0 9 7 Total 111 21 78 26

Figure 1. Frequency of the RILs of two populations according to their susceptibility to sulfur dusting.

355 seemed to colocalize with the major QTL detected on the Védrantais X PI 161375 population (Fig. 2). A second QTL was detected on the gene andromonoecious (symbol a) (LOD score = 3.0 and 3.2, R2 = 14% and 8% by IM and CIM respectively). The gene a was mapped in telomeric position of LG II on the reference map (Périn et al., 2002). A third one was found on LG IV by ANOVA and CIM (LOD score = 3.3, R2 = 11%) but was not significant by IM. This last QTL has positive additive value which corresponds to the presence of the allele for sulfur resistance in PI 124112. In conclusion, susceptibility to sulfur is quite common in melon, as 53% of the tested accessions present necrosis after sulfur dusting. One must be careful not to introduce sulfur susceptibility when some accessions are used in breeding programs to introduce for instance disease resistance (Aphis gossypii or Cucumber Mosaic Virus or Me- lon Necrotic Spot Virus from PI 161375 or powdery and downy mildew from PI 124112). On both crosses, a major QTL on LG I explained a great part of the sulfur resistance. We propose to name it Sulfur resistance (symbol Sr), present in Védrantais. Its dominance was complete in the F Védrantais x PI 124112 but incomplete in the F Védrantais x 1 1 PI 161375. A second and a third QTLs with minor effects were detected only in the cross Védrantais x PI 124112 on LG II and LG IV. The allel for necrosis was present in Védrantais for this last QTL.

Figure 2. Localization on genetic maps of the QTL for resistance to sulfur dusting. A major QTL (Sr) was detected on Linkage Group I (LG I) on both RIL populations. Two minor QTLs were found only on the RIL Védrantais x PI 124112 population: one on LG II and one on LG IV. Black arrows on LG I and LG II indicates QTL with negative additive value (allel for sulfur resistance in Védrantais); white arrow on LG IV indicates QTL with positive additive value (allel for sulfur resistance in PI 124112).

356 References

Basten, C.J., Weir, B.S. and Zeng, Z.-B. 1994. Zmap-a QTL cartographer. In: Smith, C., Gavora, J.S., Benkel, B., Chesnais, J., Fairfull, W., Gibson, J.P., Kennedy, B.W. and Burnside, E.B. (Eds.), Proceedings of the 5th world congress on genetics applied to livestock production: computing strategies and software. Published by The Organizing Committee, 5th world congress on genetics applied to livestock production. Guelph (Ontario, CAN), 22, pp. 65-66. Basten, C.J., Weir, B.S. and Zeng, Z.-B. 2002. QTL Cartographer, version 1.16. Department of statistics, North Carolina State University, Raleigh (NC, USA). Johnson, H.J. and Mayberry, K.S. 1980. The effect of dusting sulfur on muskmelons. Hort. Sci., 15: 652-654. Périn, C., Hagen, L.S., de Conto, V., Katzir, N., Danin-Poleg, Y., Portnoy, V., Baudracco-Arnas, S., Chadoeuf, J., Dogimont, C. and Pitrat, M. 2002. A reference map of Cucumis melo based on two recombinant inbred line populations. Theor. Appl. Genet., 104: 1017-1034.

357 358 Distinctness, uniformity and stability testing of cucumber cultivars in Poland

B. Kowalczyk The Research Centre for Cultivar Testing, 63-022 S³upia Wielka, Poland

Summary

Testing for Distinctness, Uniformity and Stability (DUS) of cucumber cultivars is a statutory requirement before they can be entered into the Register of Cultivars (National List) and/or gran- ting of Plant Breeders Rights (PBR). The procedure and methods used for DUS testing of cucumber varieties are outlined. In Poland all activities connected with plant variety testing and the maintenance of the Register of Cultivars and the Register of Plant Breeders Rights are provided by the Research Centre for Cultivar Testing (COBORU). The DUS testing procedures for Polish requirements are based on the Union for the Protection of New Varieties of Plants (UPOV) guidelines.

Keywords: Cucumis sativus, DUS testing

Introduction

Poland, as a member of the International Union for the Protection of New Varieties of Plants (UPOV) since 1989, follows the UPOV Convention and acts according to UPOV regulations. According to the Polish Seed Act of 2003, every cultivar must pass complete Dis- tinctness, Uniformity, and Stability (DUS) statutory testing before entering the Re- gister of Cultivars and/or being granted protection. The administration of DUS testing and the maintenance of the Register of Cultivars and the Register of Plant Bre- eders Rights are provided by the Research Centre for Cultivar Testing (COBORU) located at S³upia Wielka. Entry of the variety into the Register of Cultivars is a cucumber cultivar seed marketing requirement. The variety testing is carried out at the Experiment Stations for Cultivar Testing (SDOO), which belong to COBORU. A breeder or his representative who wants a variety to be registered makes an application with completed Technical Questionnaire to COBORU. He has to pay registration fees for the National List and Plant Breeders Rights and has to provide seed for official tests and trials.

Methods

DUS testing of cucumber cultivars is carried out at two Experiment Stations for Cultivar Testing for a period of two growing seasons. DUS trials of cucumber cultivars are conducted under glass, under plastic cover and in the field. The cucumber cultivars in DUS testing in 2003 are categorized in Table 1.

Number of cultivars tested

Group of cucumber cultivars Total Domestic Foreign Candidates for Reference NL and PBR collection

In glasshouse (total) 68 30 38 12 56 short fruit 36 20 16 432 medium fruit 18 6 12 6 12 long fruit 14410 2 12 In plastic covers (total) 61 9 52 21 39 short fruit 35 8 27 7 28 medium fruit 22 - 22 11 11 long fruit 3 1 2 3 - In open field (total) 110 87 23 16 94 slicers 12 10 2 48 for natural fermentation 12 9 3 - 12 pickling 75 59 16 11 64 small pickling 11 9 2 1 10

Grand Total 239 126 113 49 189

The DUS testing procedures for Polish requirements are based on the Guidelines for Conduct of Tests for Distinctness, Homogeneity and Stability of Cucumber (TG/ 61/6). These guidelines, prepared by the UPOV Technical Working Party for Vege- tables, are recommended to the member States of UPOV. The DUS trials are carried out with at least two replications. Similar cultivars are placed close to each other. The varieties are randomised within each block and subgroup within the same DUS trials; plants from the seeds of two or three generations of the same cucumber cultivar are grown. Distinctness, Uniformity and Stability are judged on the basis of characteristics and their expression. The nature of the characteristics used in the technical procedure of DUS testing is an important element. Such characteristics used in DUS testing must be capable of precise recognition and description. There are a total of 49 different characteristics assessed to determine distinctness among cultivars (Table 2). They are recorded during the growing seasons. If a character is used for a crop to assess distinctness, it should also be checked for uniformity. The main statistical method used for continuous (measured) characters is analysis of variance.

360 Table 2. Characteristics of cucumber cultivars

UPOV National Characteristics State of expression No. No. 1* 2 Plant: - growth type determinate, indeterminate 2* 3 - vigor weak, medium, strong 3* 4- total length of first 15 internodes short, medium, long 4* 5 - length of internodes of side shoots short, medium, long G 12* 6 - sex expression male and female flowers approximately equally present, mainly female flowers, almost exclusively female flowers 13* 7 - number of female flowers per node one to three, more than three 5* 8 Leaf: - size of blade small, medium, large 6* 9 - intensity of green color light, medium, dark 7* 10 - blistering absent or very weak, weak, medium, strong, very strong 8* 11 - undulation of margin absent or very weak, weak, medium, strong, very strong 9* 12 - length of terminal lobe short, medium, long 10* 13 - width of terminal lobe narrow, medium, broad 11* 14- ratio length/width of terminal lobe less than 1, equal to 1, more than 1 15 - dentation of margin weak, medium, strong

14* 16 Young fruit: - type of vestiture hairs only, prickles only, hairs and prickles 15* 17 - density of vestiture sparse, medium, dense G 16* 18 - color of vestiture white, black 17* 19 - size of warts absent or very small, small, medium, large, very large G 19* 20 Fruit: - length very short, short, medium, long, very long 20* 21 - diameter small, medium, large 21* 22 - ratio length/diameter small, medium, large 23 - shape circular, tubby, club-shaped, serpentine 24- shape in cross sectionround, round-triangular, triangular, triangular concave 22* 25 - core diameter in relation small, medium, large to diameter of fruit 23* 26 - predominant shape of stem end at necked, acute, obtuse market stage 24* 27 - length of neck short, medium, long 25* 28 - shape of calyx end at market acute, obtuse stage G26* 29 - ground color of skin at market white, yellow, green stage

361 UPOV National Characteristics State of expression No. No.

27* 30 - intensity of ground color of skin light, medium, dark 28* 31 - ribs absent, present 29* 32 - prominence of ribs weak, medium, strong 30* 33 - coloration of ribs compared lighter, equal, darker to ground color 31* 34 - vestiture absent or very sparse, sparse, medium, dense, very dense 32* 35 - warts absent, present 33* 36 - stripes (ribs excluded) absent, present 34* 37 - length of stripes short, medium, long

35* 38 - mottling absent, present 36* 39 - predominant type of mottling small and round, large and irregular 37* 40 - intensity of mottling weak, medium, strong 41 - glossiness absent, present 42 - intensity of glossiness weak, medium, strong 38* 43 - length of peduncle short, medium long 39* 44 - thickness of peduncle thin, medium, thick 43* 45 - bitterness absent, present 40* 46 - ground color of skin at white, yellow, green, orange, physiological ripening brown 47 - tissue absent, present G18* 48 - parthenocarpy absent, present

41* 49 Time of development of female flowers early, medium, late

* = Characteristics that should be used every growing period for the examination of all cultivars and should always be included in the description of the cultivar, except when the state of expression of a preceding characteristic or regional environmental conditions render this impossible. G = Characteristics used for grouping of cultivars.

Distinctness The aim is to check if the candidate cultivar is different from each of all the commonly known cultivars in the reference collection. Two samples are considered to be distinct cultivars if the difference between them has been determined in at least one testing place, is clear, and is consistent.

Uniformity A candidate must have an acceptable level of uniformity among plants. A population standard of 1% and an acceptance probability of 95% is applied. The statistical

362 methods have been applied to determine the maximum acceptable number of off-types in samples of various sizes. In the case of a sample of 20 plants under covers and 50 plants in the open field, the maximum number of off-types allowed would be 1 under covers and 2 in open field.

Stability Generally, stability is a function of uniformity. If the cultivar is homogeneous, the biology of the plant and/or the breeding work will usually keep that uniformity through the multiplication process. Sometimes two or three generations of the same cultivar are compared. If there are no significant differences between them, the cultivar is considered stable.

Conclusions

There is close cooperation between UPOV-member countries to harmonize the methods used in DUS testing of cultivars. The experts of the UPOV Technical Working Party for Vegetables (TWV) are working on the improvement of guidelines for the conduct of tests on cucumbers. At meetings of the special technical working groups of UPOV are also discussed the statistical methods for interpretation of DUS trial data, use of disease resistance tests and modern methods, among them electrophoresis, methods of profiling DNA, and image analysis.

References

COBORU. Metodyka badania Odrêbnoci, Wyrównania i Trwa³oci Ogórka M (OGW) 12.94. TG/61/6 UPOV. Guidelines for the Conduct of Tests for Distinctness, Uniformity and Stability of Cucumber. Ustawa z dnia 26 czerwca 2003 r. o nasiennictwie. Dziennik Ustaw nr 137.

363 364 Generation means analysis of parthenocarpic characters in a processing cucumber (Cucumis sativus) population

Z. Sun1, R.L. Lower1 and J.E. Staub2 1Department of Horticulture, University of Wisconsin, and 2U.S. Department of Agri- culture, Agricultural Research Service, Vegetable Crops Unit, 1575 Linden Dr., Ma- dison, WI 53706, USA

Summary

The incorporation of genes for parthenocarpy (the production of fruit without fertilization) into processing cucumber (Cucumis sativus L.) can provide a means for increasing fruit yield and quality. The inheritance of parthenocarpy in this horticulturally important market class is not well understood. Thus, the inheritance of parthenocarpy was investigated in segregating generations derived from a line 2A (P , parthenocarpic) x Gy8 (P , non-parthenocarpic) mating. A Generation 1 2 Means Analysis was applied to yield data from six generations [P , P , F , F , BC (F x P ), and 1 2 1 2 1 1 1 BC (F x P )] collected at two locations [Arlington (greenhouse) and Hancock (open-field), WI] 2 1 2 in two years (1999 and 2000). The number of effective factors controlling parthenocarpy was estimated to be more than one. An additive-dominance model adequately explained the variation in generation means of the Arlington grown populations. A model that included additive x additive interaction and dominance x dominance interaction was necessary to explain the variation in generation means of the Hancock grown populations. Duplicate epistatic effects were detected at both locations. Data from the Hancock location indicated the presence of parthenocarpic genes in both parental lines. Narrow-sense heritability estimates ranged from 0.15 to 0.56 across locations and years. The direction of the dominance effect changed depending on environment. These results suggest that selection will likely be effective for increasing the degree of parthenocarpy in the environments tested, however, the amount of gain from selection for this trait will be environmentally dependent.

Keywords: Cucumis sativus, parthenocarpy, seedless fruit, vegetable breeding, generation means analysis

Introduction

Cucumber (Cucumis sativus var. sativus L.; n = 7) is an important and popular vegetable of worldwide economic significance. During the last 19 years (1984 ~ 2002), the average yield of processing cucumber in the United States has plateaued at 12.61 ± 0.15 tons per hectare (Agricultural Statistics Data Base, http://www.nass.usda.gov:81/ipedb/). The limitation of yield in seed-bearing cucumber is due to crown-fruit dominance or a first-fruit inhibition phenomenon (Tiedjens, 1928; McCollum, 1934; Uzcate- gui et al., 1979). Fruit with maturing seeds developed from the first pollinated flower inhibits the development of subsequent fruits. Parthenocarpy or the production of fruit without fertilization in gynoecious cucumber has been proposed as a mechanism for overcoming fruit-set inhibition (Den- na, 1973; de Ponti and Garretsen,1976). The phenomenon of parthenocarpy was first observed in the late 1800s (Sturtevant, 1890), and subsequently described in cucumber by Noll (1902).

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 365 Parthenocarpy has been found to be under genetic control. However, there is little agreement regarding the number and kind of gene action involved in parthenocarpic fruit development. Although Hawthorn and Wellington (1930) and Meshcherov (1974) suggested that a recessive single gene controls parthenocarpy, Kvasnikov (1970) believed that many incompletely recessive genes conditioned this trait. In contrast, Pike and Peterson (1969) and de Ponti and Garretsen (1976) proposed that parthenocarpy is controlled by a single dominant gene and three independent additive major genes, respectively. Given the controversial nature of genetic control of parthenocarpy and its potential economic impact for processing cucumber production, a study was designed to: 1) estimate the minimum number of effective factors controlling parthenocarpy; 2) determine types of gene action controlling parthenocarpy; and 3) estimate the broad- and narrow-sense heritability associated with its genetic control.

Materials and methods

Plant material Two processing cucumber inbred lines originating from the University of Wiscon- sin breeding program were intermated to produce F , F , and BC (P ) and BC (P ) 1 2 1 1 2 2 progenies for genetic analysis. Both the parthenocarpic inbred line 2A (designated P1) and the non-parthenocarpic inbred line Gy8 (designated P2) are gynoecious (gy), normal leaf (L), and indeterminate (De). Seeds of F progeny were produced in the greenhouse in Costa Rica during the 1 spring of 1998, and the initial production of F , BC (P ), and BC (P ) seeds was in 2 1 1 1 2 the Walnut Street Greenhouse, Madison, WI, Fall of 1998. A second seed production was completed in Costa Rica in the spring of 2000 employing same parental seeds as used in the first production.

Experimental design All generations (4) plus two parental lines were grown in a greenhouse at Arling- ton, WI in the spring of 1999 and in a field nursery at Hancock, WI in the summer of 2000. The experiment at each location was a randomized complete block (RCB) design with replication. The experimental units were two homogeneous plants of each two parental lines and hybrid F , six plants of each of two backcross generations and 1 twelve plants of the F generation (P :P :F :BC :BC :F = 2:2:2:6:6:12) totaling 30 2 1 2 1 1 2 2 plants per block (14 blocks at Arlington and 20 blocks at Hancock). At Arlington, seedlings of all generations were transplanted into 20 cm clay pots at the 2-3 true leaf stages. All fruits larger than 2.8 cm in diameter were harvested twice when about 15% of the fruits reached 5.0 cm in diameter in each harvest. At Hancock, the experiment was isolated 2.5 km from other cucumber fields to insure pollen free fruit set. Single plants (plot) were placed on 1.37 x 1.37 m centers, and harvested twice using the same criteria as that in Arlington.

Data analysis The data from both locations were analyzed independently using Proc Mixed Covtest procedure (SAS Version 8, SAS Institute Inc., Cary, NC, USA.) to obtain generation

366 least squares means (lsmeans) and variance estimates. Generations Means Analysis was used to obtain estimations of gene effects according to Mather and Jinks (1971, 1977). Scaling tests (Mather, 1949) were used to assess additive-dominance model fit among means. Joint scaling tests (Cavalli, 1952) were also used to test the adequacy of the additive-dominance model and estimate model parameters {m (mid-parent effect), [d] (additive effect), [h] (dominance effect), [i] (additive x additive interaction), [j] (additive x dominance interaction), and [l] (dominance x dominance interaction)} using generation (6) lsmeans. The three-parameter and six-parameter scaling tests were completed using the JNTSCALE software (Ng, 1990), and parameter significance was tested using the Students t test.

Estimation of heritability Broad (h2 ) and narrow (h2 ) sense heritability were estimated based on the formu- B N la h2 = (s2 + s2 ) / V and h2 = s2 / V , respectively, where s2 , s2 , V are the B A D F2 N A F2 A D F2 additive genetic variance, the dominance genetic variance, and the variance in F 2 generation, i.e. phenotypic variance, respectively. Standard errors (S.E.) of the heritability estimates were calculated using the methods similar to those of Hallauer and Miranda (1981) and Becker (1992), where S. E. (h2 ) = S. E. (s2 + s2 ) / V and S. E. B A D F2 (h2 ) = S. E. (s2 ) / V . N A F2

Estimation of least effective factors Estimates of least effective factors were obtained using the methods of Castle (1921) and Wright (1968).

Results and discussion Generation mean Least squares means (lsmeans) and their standard errors of the six generations for the number fruits per plant are given in Table 1. In both locations, progeny density distributions of F , BC (P ), and BC (P ) families were normal. Transgressive segre- 2 1 1 2 2 gation was not observed, and only in rare cases did F individuals reach values of the 2 parthenocarpic parent.

Gene effects The mean variation among generations (lsmeans) for parthenocarpy in the first and cumulative two-harvest yield at Arlington, WI was fitted to an additive-dominance model using scaling test (Table 1). However, a simple additive-dominance model was not adequate to explain the variation among generations for parthenocarpy in the first harvest at Hancock, WI. Nevertheless, epistatic interactions were detected. An additive-dominance model was adequate for genetically characterizing the cumulative two- harvest yield at Hancock, WI (Table 1). Joint scaling test results from a three-parameter model and six-parameter model are given in Table 2. The first and cumulative two- harvest yield variation among generations test at Arlington (1999) was fitted to a three-parameter model. Estimates of additive and dominance parameters deviated significantly from zero (µ = 0.01).

367 Chi-square tests of fit for the three-parameter model were not significant (µ = 0.05). Estimates of all six parameters, except for additive effect, were not significant from zero (µ = 0.05) when the six-parameter model was applied.

Table 1. Generation least squares means (lsmeans) and their standard errors, plant number (N) and scaling test results of first and cumulative two-harvest cucumber yield (fruits/plant) of 2A (P ) x Gy8 (P )-derived populations grown at Arlington, WI (1999) 1 2 and Hancock, WI (2000)

Population 2A x Gy8 (1999) Arlington 2A x Gy8 (2000) Hancock N First Cumulative N First Cumulative

P 23 3.07 ± 0.33 4.57 ± 0.39 28 1.78 ± 0.18n 4.66 ± 0.45 1 P 26 0.36 ± 0.141.19 ± 0.28 36 0.48 n ±1.14 0.12± 0.20 2 F 26 2.27 ± 0.26 4.04 ± 0.31 27 1.03 ± 0.20n 2.76 ± 0.34 1 BC (P ) 83 2.89 ± 0.21 4.52 ± 0.23 118 1.20 ± 0.12n 3.47 ± 0.23 1 1 BC (P ) 84 1.75 ± 0.19 2.94 ± 0.23 119 0.41 ± 0.10n 1.59 ± 0.21 1 2 F 166 2.14 ± 0.16 3.43 ± 0.20 239 1.05 ± 0.09n 2.97 ± 0.18 2 Scaling test1 A 0.45 ± 0.60 0.44 ± 0.68 -0.41 ± 0.36n 0.48 ± 0.72 B 0.87 ± 0.49 0.65 ± 0.62 -0.69 ± 0.31* -0.72 ± 0.57 C 0.61 ± 0.91 -0.12 ± 1.11- -0.14 ± 0.60n 0.58 ± 1.11

* indicates the term is significant at p < 0.05. 1 Mathers scaling test for additive-dominance model, where A = the mean of 2BC (P ) 1 1 -P -F , B = 2BC (P ) -P -F , and C = mean of 4F - 2F -P -P . 1 1 2 2 2 1 2 1 1 2

A Chi-square test of fit to the three-parameter model using first harvest from Han- cock (2000) was significant at µ = 0.01 (P= 0.0026), indicating the presence of epistatic gene action. And additive x additive and dominance x dominance epistasis were detected in a six-parameter model (µ = 0.01). The Chi-square test of three-parameter model assessing variation for cumulative two-harvest yield at Hancock (2000) was approached significance at µ = 0.05 (P = 0.0555) and the estimate of the dominance parameter was not significant from zero (µ = 0.05). When six-parameter model was applied, additive and dominance effects, as well as additive x additive and dominance x dominance interactions were significant at µ = 0.05. Fit to the six-parameter model indicated that there were positive dominance effect and negative dominance x dominance epistasis for first and cumulative two-harvest yield Arlington (1999). However, both terms were not significant at µ = 0.05. In contrast, the signs of the dominance and dominance x dominance epistasis effects were negative and positive, respectively at Hancock (2000).

Heritability estimates The broad- and narrow-sense heritability estimates ranged from 0.12 to 0.56 and 0.15 to 0.56, respectively, depending on growing environment.

368 Table 2. Estimates of the additive, dominance, epistatic effects, and their standard errors for first and cumulative two-harvest cucumber yield (fruits/plant) of 2A x Gy8- derived populations grown at Arlington, WI (1999) and Hancock, WI (2000)

Model1 2A x Gy8 (1999) Arlington 2A x Gy8 (2000) Hancock

First Cumulative First Cumulative

Three-parameter model m 1.77 ± 0.14** 2.94 ± 0.20** 1.11 ± 0.08** 2.91 ± 0.17** d 1.35 ± 0.14** 1.67 ± 0.19** 0.73 ± 0.07** 1.82 ± 0.15** h 0.78 ± 0.27** 1.23 ± 0.37** -0.35 ± 0.17*n -0.30 ± 0.32nn c2 3.875 2.095 14.227** 7.582

Six-parameter model m 0.99 ± 0.80nn 1.67 ± 1.00** 2.09 ± 0.34** 4.68 ± 0.72** d 1.35 ± 0.17** 1.69 ± 0.23** 0.65 ± 0.10** 1.76 ± 0.22** h 3.32 ± 2.05nn 4.66 ± 2.51nn -3.11 ± 0.88** -4.90 ± 1.88** i 0.72 ± 0.78nn 1.21 ± 0.97nn -0.96 ± 0.32** -1.78 ± 0.68** j -0.42 ± 0.64nn -0.22 ± 0.78nn 0.28 ± 0.29nn 0.24 ± 0.65nn l -2.05 ± 1.36nn -2.30 ± 1.64nn 2.06 ± 0.65** 2.98 ± 1.30**

*, ** indicates the term is significant at p < 0.05 and p < 0.01, respectively; 1 m = mid-parent effect, d = additive effect, h = dominance effect, i = additive x additive effect, j = additive x dominance interaction, and l = dominance x dominance interaction.

Heritability estimates The broad- and narrow-sense heritability estimates ranged from 0.12 to 0.56 and 0.15 to 0.56, respectively, depending on growing environment.

Number of effective factors Although estimates of the minimum number of effective factors (genes) controlling parthenocarpy ranged from 0.45 ~ 2.22 depending on growing environment (Table 3), it is likely that the number of genes controlling this trait is more than one.

Table 3. Estimation of the least number (n) of effective factors (genes) for first and cumulative two-harvest cucumber yield (fruits/plant) of 2A x Gy8-derived populations grown at Arlington, WI (1999) and Hancock, WI (2000)

No. effective 2A x Gy8 (1999) Arlington 2A x Gy8 (2000) Hancock

factors z First Cumulative First Cumulative n1 0.49 0.45 2.19 1.65 n2 0.53 0.55 2.22 1.65

Zn1 and n2 according to Castle (1921) and Wright (1968), respectively.

369 Distinct bimodal distributions were not observed in the F and two backcross ge- 2 nerations, suggesting that parthenocarpy is not conditioned by a single major gene in the parental lines. Epistatic gene interactions were empirically detected at Han- cock (2000), indicating that the inheritance of parthenocarpy is complex. Based on the direction of dominance effects and the dominance x dominance interaction, the principle types of epistasis detected are either predominantly duplicate or predominantly complementary (Kearsey, 1996). The contrasting signs between the estimated dominance and dominance x dominance interaction effects (Table 2) indicates the presence of predominantly duplicate gene interactions in this population. It is possible that these genetic effects are associated with the gynoecious character of the parental lines. This hypothesis is supported by the fact that de Ponti and Garretsen (1976) concluded that a gene for parthenocarpy and the F gene conditioning gynoecy are located on the same chromosome. Negative dominance effects were detected from the appraisal of plants in the open- field at Hancock (2000) at both harvests (Table 2). This finding is in contrast to estimates of dominance obtained under greenhouse conditions (Arlington, 1999). This suggests that the genes associated with parthenocarpy were present in both parental lines. The estimates of additive genetic variance and dominance genetic variance can be distorted due to the maternal effects, inter-allelic interactions, linkage effects, or genotype x environment interactions (Allard, 1956). A negative dominance variance was detected at Hancock (2000) for variation in fruit number per plant at first harvest. The sign and magnitude of this variation may be due to the significant additive x additive and dominance x dominance interactions detected. A three-parameter model adequately explains the variation differences recorded among generations at Arlington, WI in 1999. The importance of additive variance and narrowsense heritability may indicate that breeding methods, such as recurrent selection and pedigree selection will be effective for initial improvement of parthenocarpy under greenhouse growing environments. Nevertheless, the presence of epistatic gene interaction in open-field environments (e.g., Hancock, 2000) suggests that the selection for parthenocarpy may be difficult under such conditions.

References

Allard, R.W. 1956. Biometrical approach to plant breeding. Brookhaven symposium in biology, 9: 69-88. Becker, W.A. 1992. Manual of Quantitative Genetics. Pullman, Washington. Castle, W.E. 1921. An improved method of estimating the number of genetic factors concerned in cases of blending inheritance. Proc. Natl. Acad. Sci. USA, 81: 6904-6907. Cavalli, L.L. 1952. An analysis of linkage in quantitative inheritance. HMSO, London, pp. 135-144. Denna, D.W. 1973. Effects of genetic parthenocarpy and gynoecious flowering habit on fruit production and growth of cucumber Cucumis sativus L. J. Amer. Soc. Hort. Sci., 98: 602-604. Hallauer, A.R. and Miranda, J.B. 1981. Quantitative Genetics in Maize Breeding. Iowa State University Press, Ames. Hawthorn, L.R. and Wellington, R. 1930. Geneva, a greenhouse cucumber that develops fruit without pollination. Bul. N. Y. Sta. Agr. Expt. Sta., 580: 1-11. Kearsey, M.J. and Pooni, H.S. 1996. The Genetical Analysis of Quantitative Traits. Alden Press, Oxford. Kvasnikov, B.V., Rogova, N.T., Tarakonova, S.I. and Ignatova, I. 1970. Methods of breeding vegetable crops under the covered ground. Proc. Appl. Bot. Genet. Sel., 42: 45-57.

370 Mather, K. 1949. Biometrical Genetics, 1st edition. Methuen, London. Mather, K. and Jinks, J.L. 1971. Biometrical Genetics. Cornell University Press, Ithaca, New York. Mather, K. and Jinks, J.L. 1977. Introduction to Biometrical Genetics. Cornell University Press, Ithaca, New York. McCollum J.P. 1934. Vegetative and reproductive responses associated with fruit development in the cucumber. Cornell Memoi. Agric. Exp. Stn. (Ithaca) No. 163. Meshcherov, E.T. and Juldasheva, L.W. 1974. Parthenocarpy in cucumber. Proc. Appl. Bot. Plant Breed., 51: 204-213. Ng, T.J. 1990. Generation means analysis by microcomputer. HortSci., 25: 363. Noll, F. 1902. Fruchtbildung ohne vorausgegangene Bestaubung (parthenokarpie) bei der Gurke Sitzungaber. Niederrhein. Ges. Nat. Heilk. Bonn., pp. 149-162. Pike, L.M. and Peterson, C.E. 1969. Inheritance of parthenocarpy in the cucumber 9 (Cucumis sativus L.). Euphytica, 18: 101-105. Ponti, O.M.B. de and Garretsen, F. 1976. Inheritance of parthenocarpy in pickling cucumbers (Cucumis sativus L.) and linkage with other characters. Euphytica, 25: 633-642. Sturtevant, E.L. 1890. Seedless fruits. Mem. Torrey Bot. Club, 1: 141-185. Tiedjens, A.A. 1928. Sex ratios in cucumber flowers as affected by different conditions of soil and light. J. Agric. Res., 36: 720-746. Uzcategui, N.A. and Baker, L.R. 1979. Effects on multiple pistillate flowering on yields of gynoecious pickle cucumbers. J. Amer. Soc. Hort. Sci., 104: 148-151. Wright, S. 1968. Evolution and the Genetics of Populations. I. Genetic and Biometric Foundati- ons. University Chicago Press, Chicago.

371 372 Genetic analysis of branching in melon (Cucumis melo)

J.E. Zalapa1, J.E. Staub1 and J.D. McCreight2 1U.S. Department of Agriculture, Agricultural Research Service, Vegetable Crops Unit, Department of Horticulture, 1575 Linden Dr., University of Wisconsin, Madison, WI 53706 USA; e-mail: [email protected] 2U.S. Department of Agriculture, Agricultural Research Service, Agricultural research Station, 1636 East Alisal, Salinas, CA 93905 USA; e-mail: [email protected]

Summary

Plant improvement incorporating quantitatively inherited yield component traits is technical- ly difficult, time consuming, and resource demanding. In melon (Cucumis melo L.), the inheritance of yield components is poorly understood. Yield components include branching, flowering (sex expression and earliness), and fruiting habit (fruit position, fruit number and fruit weight). Because of the importance of yield to U.S. Western Shipping melon operations, a study was designed to characterize the inheritance of branching in this melon market class. Melon progeny derived (F , F , F , BC P1, and BC P ) derived from a cross between a U. S. Department of Agri- 1 2 3 1 1 2 culture line, USDA 846-1 (P1) and Top-Mark (P2) were used to assess segregating progeny for yield components. Estimates of components of variance, narrow- (h2 ) and broad- (h2 ) sense heritabilities, N B and the number of least effective factors for primary lateral branch number were calculated. Lateral branch numbers among 71 to 119 F families tested in three commercial growing environments 3 (California (1) and Wisconsin (2)) were significantly different (P £ 0.01). Covariance analyses among these F families indicates that branching is moderately heritable (h2 = 0.62 to 0.76, h2 3 B N = 0.43 to 0.48), and conditioned by several additive factors (perhaps 2 to 4) that are highly additive. Although environment plays an important role in lateral branch development, family rankings over environments were relatively consistent, indicating that effective selection for this trait could be performed in either Wisconsin or California. Because of the significant additive component underlying lateral branch number, selection of quantitative trait loci (QTL) conditioning this yield component will be likely enhanced by the identification of genetic marker-trait associations for subsequent use in marker-assisted selection.

Keywords: Cucumis melo, heritability, variance components, yield components, vegetable breeding

Introduction

Melon (Cucumis melo L.) is an economically important, cross-pollinated, diploid (2x = 2n = 24) vegetable species. In the United States, Arizona, California and Texas are the primary producers of melons for fresh market consumption (N.A.S.S., 1995, 1997). In 1997, U.S. farmers reportedly grew more than 120,000 acres of melons for a total production in excess of one million tons having a market value of over 500 million U.S. dollars (N.A.S.S, 1997). Worldwide, more than 18 million metric tons of melon was produced in 1999, with China, Turkey, Iran the United States, and Spain being the major producers (F.A.O., 1997, 1999). Yield of melon plants is affected by heritable components such as number of lateral branches, flowering characteristics (sex expression and earliness), and fruiting habit (fruit position, number, and weight). It has been proposed that higher lateral branch number in melon could affect source sink relationships to increase net photosynthe-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 373 tic capacity and hence yield potential (Davis et al., 1976; Paris et al., 1981, 1982; Nerson et al., 1983; Knavel, 1988, 1991; Rubatzky and Yamaguchi, 1997; Kultur et al., 2001). The differing growth habits of melon plants can result in a wide array of plant architectures. The sources of these architectural differences include differential gene expression for vining habit such as determinate and indeterminate plant forms possessing standard or shorten internodes on uni- and multi-lateral branching, e.g., birdsnest form, genetic backgrounds (Davis et al., 1976; Paris et al., 1981, 1982; Nerson et al., 1983; Knavel, 1988, 1991; Kultur el al., 2001). In the United States, only indeterminate vining types are grown commercially (Rubatzky and Yamaguchi, 1997; Kultur et al., 2001). United States Eastern Market and Western Shipping market types have relatively few branches (2-5), are typically andromonoecious, reach anthesis between 35 to 40 days after sowing depending on growing location, primarily set differentially maturing fruit distal to the crown, e.g., Top Mark, and often support a single fruit per branch (Davis et al., 1976; Paris et al., 1981, 1982; Nerson et al., 1983; Knavel, 1988, 1991; Kultur et al., 2001). In the U.S., melon harvesting operations are labor intensive and costly making machine harvesting an attractive alternative (Rubatzky and Yamaguchi, 1997). Mul- tiple branched genotypes with early, uniform flowering and concentrated fruit-setting ability might be a desirable plant ideotype for machine harvesting operations. Such an ideotype would set two to three fruit simultaneously (within a 1-2 day period of time) near the crown of the plant, i.e., concentrated setting. The creation of such high yielding ideotypes is complicated by the metric nature of architectural traits. Gain from selection during population improvement depends on the genetic variability available in the population, the heritability of the trait and selection differential imposed (Falconer, 1989). Thus, estimates of gene action, variance components, heritabilities and least number of effective factors conditioning yield components are critical for the development and characterization of plants possessing unique architectures. Quality traits in melon have been recently mapped in a Piel de Sapo derived population (Monforte et al., 2004). However, the inheritance and gene action associated with yield components is poorly understood. Our laboratories have performed a comprehensive two-year genetics study of yield components to include the branch number, flower earliness for pistillate and staminate flowering, sex expression, and fruit position, maturity, number and weight (unpublished). This study used a parental line derived from an exotic melon and Top Mark. We report herein information related to the inheritance of the multiple lateral branching habit unique to the parental line derived from exotic germplasm including estimates of least effective factors, trait heritabilities, and the effects of environment on trait expression.

Material and methods Line USDA 846-1 (P ; days to anthesis = 50; monoecious, 5 to 8 multiple lateral 1 branching, basal-concentrated fruiting habit) derived from wild free-living exotic germplasm originating in Costa Rica was crossed to Top-Mark (P ; days to anthesis 2 = 60; andromonoecious, 3 to 4 multiple lateral branching, diffuse-distal fruiting ha-

374 bit). A single F plant was subsequently self-pollinated to produce F individuals from 1 2 which 119 F families were derived. BC P , BC P progeny were also made by cros- 3 1 1 1 2 sing USDA 846-1 as the female and Top-Mark as the male parent. Seeds of the parental lines (USDA 846-1 and Top-Mark), their F , F , F , BC P , 1 2 3 1 1 BC P progeny and a control cultivar, Hales Best Jumbo (maturation at about 85 days 1 2 from transplanting), were sown in a greenhouse at the University of Wisconsin, Madi- son in 2000 and 2001. Hales Best Jumbo was used to provide a benchmark for maturation rate and harvest timing. Three-week old seedlings were transplanted in a randomized complete block design consisting of three replications with 10 plants per replication at two locations, Arlington and Hancock, Wisconsin. In Hancock 2000, plants were spaced at 0.35 m within rows that were positioned on 2 m centers. Becau- se of the results and management practice evaluation in 2000, plants were spaced 0.70 m within rows at 2 m centers at Arlington and Hancock in 2001. Data were collected on the number of primary lateral branches appearing between the cotyledonary node and 12.5 cm above node three 30 days after transplant. Analy- sis of variance of branch number in P , P , F , and F families (71 for Hancock 2000 1 2 1 3 and 119 for Arlington and Hancock 2001) was conducted (all effects considered random) using SAS Proc GLM (SAS Institute, 1990). Partitioning among and within entries was done according to Hallauer and Miranda (1981), and allowed for the estimation of the genetic variances and narrow-sense heritabilities. The estimation of additive (s2 ) and dominance (s2 ) variance was obtained using the variation among and A D within F progeny according to the following two equations: s2 + 1/4s2 and 1/2s2 3 A D A + 1/2s2 , respectively. The narrow sense heritability (h2 ) was estimated using h2 = D N N s2 /s2 , where s2 is the phenotypic variance, and is estimated from equation s2 = s2 A P P P A + s2 + s2 + s2 (over locations) or s2 = s2 + s2 + s2 (for each location), and s2 is D GL P A D error term. Estimation of least number of effective factor (least number of genes) or the minimum number of effective factors (n) (Lande, 1981) was estimated according to Wright (1968) using correction factor suggested by Cockerham (1986) as: n = [(P -P )2 (s2 + 1 2 P1 s2 )] / 8s2 ; where P and P are estimates of the mean phenotypic trait value of the P2 A 1 2 parents, s2 and s2 are the estimated variance of these means, and s2 is the additive P1 P2 A genetic variance for this trait. An analysis of variance was also performed on P , P , F , 1 2 1 F , BC P , and BC P (all effects considered random except the six generations which 2 1 1 1 2 were considered as fixed effects) using SAS Proc GLM (SAS Institute, 1990).

Results and discussion

The analysis of variance of P , P , F , and F families (Table 1) and of P , P , F , F , 1 2 1 3 1 2 1 2 BC P , and BC P progeny (Table 2) was performed by location and then in a combi- 1 1 1 2 ned analysis. The combined analysis was only performed for Arlington and Hancock 2001 since they were not significantly different (P ³ 0.10, Table 1 and 2) and included the same generations and experimental design. The coefficients of variation (C.V. %) for different locations ranged from 15.6 to 17.7 % for the F family analysis and from 3 15.15 to 17.96 % for the six-generation analysis. Significant variation (P £ 0.01) was detected among the families and generations (Tables 1 and 2) in all years and locations and also in the combined analysis.

375 Table 1. Analysis of variance and genetic parameters (± standard error) for lateral branching in a Cucumis melo L. Analysis performed on USDA-846 (P ) and Top Mark (P ), F 1 2 1 and F progeny grown in Wisconsin in 2000 and 2001 3

Hancock (00) Hancock (01) Arlington (01) Combined5

Source df F Pr > F1 df4 F Pr > F F Pr > F df F Pr > F

Location 1 3.03 n.s Rep 2 4.94 ** 2 0.94 NS 12.50 ** Rep (Loc) 46.65 ** Entry 73 3.69 ** 121 3.63 ** 03.62 ** 121 4.72 ** Among Gen2 3 7.31 * 3 41.49 ** 29.90 ** 3 7.81 ** Among F 70 3.41 ** 118 3.35 ** 03.28 ** 118 4.20 ** 3 Loc x Entry 121 1.28 * Among Gen 3 0.16 n.s. Among F 118 1.30 * 3 Error 144 242 484

Mean 5.26 5.51 5.345.42 C.V. (%) 17.72 17.12 15.57 16.38 s2 0.21 0.19 0.18 0.16 G s2 0.31 0.30 0.26 0.21 P h2 0.69 0.62 0.70 0.76 B s2 0.22 ± 0.040.23 ± 0.040.21 ± 0.040.20 ± 0.06 A s2 -0.02 ± 0.15 -0.15 ± 0.18 -0.13 ± 0.14-0.15 ± 0.09 D h2 0.43 ± 0.25 0.49 ± 0.23 0.48 ± 0.23 0.45 ± 0.28 N n3 3.50 ± 0.25 2.40 ± 0.25 3.90 ± 0.25 3.40 ± 0.25

1*, ** = significance levels at 0.05 and 0.01, respectively, and NS = not significant at the P = 0.05 level; 2Gen = generations; 3n = the number of least effective factors according to Wright (1968) using correction factor suggested by Cockerham (1986); 4df the same for Hancock and Arlington; 5Combined indicates Hancock and Arlington data taken collectively.

The effects of environment on the production of lateral branching in cucumber have not been remarkable (Serquen et al., 1997; Fazio, 2001). Likewise in melon, Kultur et al. (2001) reported that growing location and planting density did not influence branching in melon. Divergent selection (high and low) for branch number in a greenhouse environment performed at the University of Wisconsin Madison supports this observation (see companion paper this proceedings). In this case, recurrent selection through modified pedigree methodologies under relatively high density (stress) was successful for development of multiple lateral branching.

376 Analysis of branching in our experiment indicated that the Loc x Among F (genotype 3 x environment, GxE) interaction was marginally significant at P £ 0.05 (Table 1). The GxE detected might be explained by differential plant growth rates among the genotypes evaluated and/or planting density differences existing in 2000 and 2001. In Arlington 2001, the plants grew more rapidly (> 2 xs) and were larger (1m vs. 2m in diameter) than in Hancock in either 2000 or 2001. Moreover, the mean branch number over locations and generations (Tables 1 and 2) were consistently lower at Hancock 2000 (0.35 m plant spacing with slower plant growth) and Arlington 2001 (0.70 m plant spacing with accelerated plant growth) when compared to Hancock 2001 (0.7 m plant spacing with slower plant growth). However, branching habit (size) patterns did not change significantly (P ³ 0.10, Tables 1 and 2) for P , P , F , F , BC P , and BC P progeny across locations, i.e., no location among generation 1 2 1 2 1 1 2 2 interaction (Table 2). Moreover, branch number of most F families was similar in both lo- 3 cations and GxE was mainly due to differences in magnitude not changes in rank. Differen- ces were again likely related to planting density and growth rate. These results suggest that since lateral branch production is minimally affected by environment, highly branched genotypes could be identified and consequently selected in diverse environments.

Table 2. Analysis of variance, generation means and standard deviations (SD) of lateral branch number in a Cucumis melo L. lines USDA-846 (P1) and Top Mark (P2), and their F , F , BC P and BC P progeny grown in Hancock and Arlington Wiscon- 1 2 1 1 1 2 sin in 2000 and 2001

Hancock (00) Hancock (01) Arlington (01) Combined

Source df1 F Pr >F 3 F Pr > F F Pr > F df F Pr > F

Location 1 4.97 NS Rep 2 0.64NS 0.90 NS 1.62 NS 2 Rep (Loc) 41.20 NS Gen2 5 7.83 ** 12.77 ** 18.48 ** 5 194.57 ** Loc x Gen 5 0.16 NS Error 10 20

Mean 5.44 5.57 5.39 5.48 C.V. (%) 15.15 17.96 16.41 17.25

Mean SD Mean SD Mean SD Mean SD

P 6.63 0.88 6.77 1.01 6.70 0.88 6.73 0.94 1 P 4.00 0.61 4.37 0.61 3.92 0.56 4.16 0.63 2 F 5.540.87 5.63 1.07 5.81 1.03 5.71 1.04 1 F2 5.37 1.10 5.70 1.21 5.46 1.07 5.57 1.14 BC P 5.88 0.95 5.89 0.91 5.80 0.89 5.85 0.90 1 1 BC P 5.20 0.64 5.07 0.86 4.84 0.71 4.95 0.80 1 2 LSD (0.05) 0.92 0.640.63 0.41

1df the same for Hancock and Arlington locations taken individually for those presented; 2Gen = generations; 3 ** significance level at 0.01, and NS = not significant at the 0.05 level.

377 The mean branch number of line USDA 846-1 was significantly (LSD = 0.05) higher than Top Mark in all locations (Table 2). The progeny of both the F and F genera- 2 3 tions were normally distributed (data not shown). Transgressive segregation for branch number was observed in F and F progeny (data not shown). While the genetic vari- 2 3 ance among F progeny ranged from 0.21 to 0.16, the phenotypic variance ranged 3 from 0.31 to 0.21 (Table 1). Variance component analyses indicate that additive genetic variance is comparatively larger than dominance variance contribution in the populations examined (Table 1). While the additive variance ranged from 0.20 to 0.23, the dominance estimates were negative, ranging form -0.02 to -0.15. Such small, negative estimated values and the relatively large standard errors for these dominance estimates indicate that the dominance component of genotypic variation may be small (Hallauer and Miranda, 1981). This interpretation was confirmed by generation means analyses using a six- generation model (unpublished results). Broad-sense heritability was calculated as the proportion of genotypic over phenotypic variance and ranged from 0.62 to 0.76 (Table 1). Likewise, narrow sense heritability estimates ranged from 0.43 to 0.49. The number of least effective factors controlling this trait is relatively few, perhaps between 2 to 4, indicating that this trait is under polygenic control. The relatively large additive variance and narrow sense heritability for this trait, suggest rapid gain from selection for multiple lateral branching. Recurrent selection with single seed decent was most successful in increasing branch number in the first of three selection cycles (companion paper this proceedings). The variation for lateral branching in those populations may have been largely fixed after one cycle of selection. Genetic markers linked to quantitative trait loci (QTL) conditioned by additive genetic factors such as that identified for the branch number might be useful to increase gain from selection. Thus, a combination of recurrent population selection followed by inbreeding for line development and marker-assisted selection might be considered for accumulating favorable alleles during population development prior to line extraction.

References

Cockerham, C.C. 1986. Modifications in estimating the numbers of genes for a quantitative trait character. Genetics, 114: 659-664. Davis, D.W., Shehata, M.A. and Eide, C.J. 1976. Minnesota 266 muskmelon breeding line. Hort. Sci., 11: 273. F.A.O. 1997. FAOSTAT Agricultural Database, htpp://apps.fao.org. F.A.O. 1999. FAOSTAT Agricultural Database, htpp://apps.fao.org. Falconer, D.S. 1989. Introduction to Quantitative Genetics. 4th ed. Longman Scientific and Tech- nical, New York. Fazio, G. 2001. Comparative study of marker-assisted and phenotypic selection and genetic analysis of yield components in cucumber. PhD Diss., University of Wisconsin, Madison. Hallauer, A.R. and Miranda, J.B. 1981. Quantitative Genetics in Maize Breeding. Iowa State University Press, Ames. Knavel, D.E. 1988. Growth, development and yield potential of short internode muskmelon. J. Amer. Soc. Hort. Sci., 113: 595-599. Knavel, D.E. 1991. Productivity and growth of short internode muskmelon plants at various spacing and densities. J. Amer. Soc. Hort. Sci., 116: 926-929.

378 Kultur, F., Harrison, H.C. and Staub, J.E. 2001. Spacing and genotype affect fruit sugar concentration, yield, and fruit size of muskmelon. HortScience, 36: 274-278. Lande, R. 1981. The minimum number of genes contributing to quantitative variation between and within populations. Genetics, 99: 541-553. Monforte, A.J., Oliver, M., Gonzalo, M.J., Alvarez, J.M., Dolcet-Sanjuan, R. and Arus, P. 2004. Identification of quantitative trait loci involved in fruit quality traits in melon (Cucumis melo L.). Theor. Appl. Genet. (in press) N.A.S.S. 1995. (National Agricultural Statistics Service). USDA. Agricultural Statistics. US. GPO. IV-13. N.A.S.S. 1997. Farm resources, income, and expenses. http://www.usda.gov/nass. December 1997. Natl. Agr. Stat Serv. Nerson, H., Paris, H.S. and Karchi, Z. 1983. Characteristics of birdnest-type muskmelons. Scien- tia Hort., 21: 341-352. Paris, H.S., Karchi, Z., Nerson, H. and Burger, Y. 1982. On the compact appearance of birdnest type muskmelons. HortScience, 17: 476. Paris, H.S., Karchi, Z., Nerson, H., Govers, A. and Freudenberg, D. 1981. A new plant type in Cucumis melo L. Cucurbit Genet. Coop. Rep., 4: 24-26. Rubatzky, V.E. and Yamaguchi, M. 1997. World Vegetables. Principles, Production, and Nutritive Values. Melons. Chapman & Hall. New York, N.Y., pp. 322-326. SAS Institute. 1990. SAS/STAT users guide, release version 6.03. SAS Inst., Cary, N.Y. Serquen, F.C., Bacher, J. and Staub, J.E. 1997. Genetic analysis of yield components in cucumber at low plant density. J. Amer. Soc. Hort. Sci., 122: 522-528. Wright. S. 1968. Evolution and the Genetics of Populations. I. Genetic and Biometric Foundati- ons. University Chicago Press, Chicago.

379 380 Selection for lateral branch number in melon (Cucumis melo)

J.E. Staub1, J.E. Zalapa1, M.K. Paris1 and J.D. McCreight2 1U. S. Department of Agriculture, Agricultural Research Service, Vegetable Crops Unit, Department of Horticulture, 1575 Linden Dr., University of Wisconsin, Madison, WI 53706 USA; e-mail: [email protected] 2U. S. Department of Agriculture, Agricultural Research Service, Agricultural research Station, 1636 East Alisal, Salinas, CA 93905 USA; e-mail: [email protected]

Summary

Manipulation of plant architecture in melon (Cucumis melo L.) may provide for increased fruit yield. Recurrent selection through pedigree breeding was practiced in a melon population derived from a cross between line USDA 846-1 (days to anthesis = 50; monoecious, 5 to 8 primary lateral branches; basal-concentrated fruiting habit) and Top Mark (days to anthesis = 60; andromonoecious; 3 to 4 primary lateral branches; diffuse, distal fruiting habit). Thirteen low branching (LB; £ 4 branches) and 13 high branching (HB; ³ 6 branches) plants were selected from 200 greenhouse-grown F individuals. These selections were self-pollinated and then selected for two additional 2 cycles using the same methodology. Parental lines, their F , F , and selected high and low F , F , and 1 2 3 4 F families (13 low and 13 high/cycle) and controls were evaluated for primary lateral branch num- 5 ber, and fruit weight and number in a replicated open-field trial at Hancock, Wisc. during 2003. USDA 846-1 differed significantly (P ) from Top Mark for mean number of lateral branches 0.05 (5.8 versus 4.4), fruit number per plant (3.2 versus 1.6), and fruit weight (kg) per plant (1.7 versus 0.9). Mean branch number for HB families (5.8) was significantly higher (P ) than the compara- <0.05 tive means of USDA 846-1 (4.5 [4.4-see preceding statement]), commercial controls (Hales Best Jumbo = 4.7, Esteem = 5.2, Sol Dorado = 5.0), and the LB families (4.6). Major gains from selection for branching (low and high) were detected in the first cycle of selection, i.e., F to F , 2 3 with a decrease and increase in branching in LB and HB populations over the three cycles of selection (P ). No consistent significant differences were observed over cycles of selection for the <0.05 other traits examined. Differences were detected between HB and LB families for all traits (P ) £0.01 and for cycles of selection (high versus low) for branch and fruit number (P ) and fruit number £0.01 and fruit weight (P ). Positive and significant correlations were detected between the number of £0.05 lateral branches and fruit number (r = 0.39, P £ 0.01) and weight (r = 0.23, P ). Fruit number and £0.05 weight were significantly correlated (r = 0.54, P ). Selection was successful for developing HB £0.01 and LB lines with differing fruit setting potential.

Keywords: correlated response, gain from selection, pedigree breeding, recurrent selection

Introduction

Increasing early yield potential in melon (Cucumis melo L.) is important for obtaining premium prices (Roman, 1971), and may provide opportunities for opening new and/or underdeveloped market niches. Yield potential in melon is affected by several components that are defined by its architecture. One such yield component, lateral branch number, has been proposed as a yield-enhancing trait because of its physiological impact on source-sink relationships (Davis et al., 1976; Paris et al., 1981, 1982; Nerson et al., 1983; Knavel, 1988, 1991; Rubatzky and Yamaguchi, 1997; Kultur et al., 2001).

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 381 Two different plant ideotypes have been proposed for increasing yield potential in melon: short-internode (Davis et al., 1976; Knavel, 1988, 1991) and the bush or birdnest which is characterized by multilateral branching (Paris et al., 1981, 1982). These plant types may provide for increased fruits per plant or opportunities for modified cultivation (Nerson et al., 1983; Knavel, 1991). The short-internode types are indeterminate, but possess fewer nodes, shorter internodes, and amass less leaf area when compared to standard indeterminate melon plants (Knavel, 1991). It has been proposed that these small-statured melon types could be used to increase yield when planted at higher densities than conventional indeterminate types (Mohr and Knavel, 1966). Birdsnest, another highly branching indeterminate phenotype, possesses lateral branches of the same length, i.e., equilateral (Paris et al., 1981, 1982; Nerson et al., 1983). This plant type might be used to enhance yield potential since it sets early, uniform-sized fruit near the center of the plant (crown set). Qalya was among the first multiple lateral branching (4 to 6 primary branches) melon genotypes developed with relatively high fruit yield and quality. This cultivar originated from crosses between exotic Iranian germplasm (Paris et al., 1981) and Galia. Qalya is early flowering, and possesses a crown-setting fruiting habit. In contrast, typical U.S. Western Shipping market class melons possess two to four primary branches near base of the plant, and set fruit distally on primary and secondary branches. Introgression of early flowering and crown-setting fruiting habit genes into U.S. Wes- tern Shipping germplasm might allow for the development of cultivars with concentrated early fruit set. Therefore, a study was designed to develop melon phenotypes with contrasting branching habits (low and high) phenotypes in a U.S. Western Ship- ping genetic background. Recurrent selection was practiced through pedigree breeding to test: 1) whether gain from selection for high and low numbers of primary lateral branches could be made under greenhouse conditions, and; 2) if such selection would also result in changes in fruit number and weight over cycles of selection. Such information would allow for the development of breeding strategies for the incorporation of increased lateral branch number from exotic germplasm into U.S. Western Shipping market type melons to improve yield.

Material and methods

Plant material An exotic, feral monoecious, multiple lateral branching melon accession most likely C. melo L. subsp. agrestis (Naud.) Pangalo was received from Mr. Claude Hope, Cartago, Costa Rica in 1995 by the U.S. Department of Agriculture, Agricultural Research Service (USDA, ARS) melon breeding project, Madison, Wisc., and designated CR1. This highly branched (6 to 12 primary lateral branches), relatively early flowering, accession possessed small, lobed, watermelon-like leaves, and bore many small fruits (up to 100 fruits/plant) 3 to 6 cm in diameter (unpublished data). Plants of CR1 were evaluated for flowering date, sex expression, lateral branch number and fruit setting habit at Hancock, Wisc. in 1996, and a monoecious, early flowering selection having 12 lateral branches was designated CR1-1. This selection was crossed to an F plant 1 from a cross of USDA line FMR#8 x line SC#6. Line FMR#8 was an F line derived 3

382 from Galia x Qualya, and line SC#6 (S ) originated from the USDA, ARS melon 5 breeding project at Charleston, S.C. A randomly selected plant from this three-way cross was self-pollinated thrice to produce an S inbred line designated USDA 846-1. 3

Recurrent selection for lateral branching The monoecious line USDA 846-1 (P ) possessing high numbers of lateral bran- 1 ches (5 to 7 primary branches) and a basal-concentrated fruiting habit was crossed to the andromonoecous Top Mark (P ) which typically possesses between two to four 2 lateral branches and has a diffuse, distal fruiting habit typical of Western Shipping market types. A single F plant was subsequently self-pollinated to produce 200 F 1 2 individuals that were then grown for 30 days in a greenhouse in Madison, Wisc. prior to selection for lateral branching. Recurrent selection through pedigree breeding was practiced for primary branch number from 2000 to 2002 over three cycles of selection (F to F ) under greenhouse conditions. Twenty low branching (LB; £ 4 branches) 2 5 and 20 high branching (HB; ³ 6 branches) individuals from 200 F plants were selec- 2 ted and self-pollinated to produce F families; a 10% selection intensity (SI). In the 3 subsequent cycle (C) of selection (C2), this procedure was repeated on 400 plants [20 families x10 plants/family x 2 selection types (LB and HB)] of the previous cycle with a 10% SI. In the C3 (F to F ), 13 LB and 13 HB lines were selected for a 7% SI 4 5 because of a lack of sufficient variation to meet selection criteria set for HB families. Seed of P , P , F , F , three cycles of selected HB and LB families, and commercial 1 2 1 2 cultivars Esteem and Sol Dorado (Syngenta Seeds, Gilroy, Calif.), and Hales Best Jumbo (Excel Seeds, Chattanooga, Tenn.) designated collectively as controls, were sown in a greenhouse in Madison, Wisc. in 2002. The days to anthesis of Esteem, Sol Dorado, and Hales Best Jumbo under Wisconsin growing conditions are about 50, 44, and 55 days, respectively, and their maturity dates were used to determine experiment harvest date. Three-week-old seedlings were transplanted in a randomized complete block design consisting of four replications with 10 plants per replication at Hancock, Wisconsin. Plants were spaced 0.32 m apart in rows that were on 2 m centers, and received standard cultivation practices typical for Wisconsin Planefield loamy sand (Typic Udipsamment; sandy, mixed, mesic) soil conditions.

Data collection and analysis Data were collected on the number of primary lateral branches, and fruit number and weight. The number of branches for each plant was counted 30 days after transplant to include the main branch and branches originating no more than 12.5 cm above the third node. Fruit number and fruit weight (kg) data were collected on a per plot basis 80 days after transplanting and are reported herein on a per plant basis. Analysis of variance was performed using P , P , F , F , HB and LB families, and 1 2 1 2 controls data, where all effects were considered fixed using the SAS computer procedure Proc GLM (SAS Institute, 1990). Sources of variation were partitioned to test for differences among entries, families (HB, LB, and HB vs. LB), and cycles of selection (HB, LB, and HB vs. LB). Treatment means were calculated and then separated employing Fishers least significant difference test (LSD) at threshold of P using SAS Proc GLM. 0.05 Phenotypic correlations among traits were calculated by the Pearson Product-Moment method using SAS Proc Corr.

383 Results and discussion

Significant differences (< P ) were detected for all comparisons for all traits, ex- 0.05 cept selection in HB and LB families (ie., Cycles high only, Cycles low only) for fruit number and weight (Table 1). Selection for increased or decreased number of lateral branches was successful, and that experimental variation was adequately controlled by the design imposed.

Table 1. Analysis of variance of parental lines (USDA 846-1 and Top Mark), their F , F , and selected high and low families (three cycles of selection) and commercial 1 2 controls (Hales Best Jumbo, Esteem, and Sol Dorado) for numbers of lateral branches and fruit, and weight per fruit (kg)

Main effects for Branch number Fruit number Fruit weight (kg) sources of variation df F P > F2 F P > F F P > F

Rep 3 2.94* 2.10 N.S. 0.97 N.S. Entry with control1 8410.98 ** 9.33 ** 3.06 ** Entry without control 77 11.39 ** 9.90 ** 2.46 ** High vs. low families 1 143.57 ** 22.75 ** 5.34* High vs. high families 38 4.62 ** 9.56 ** 2.16 ** Low vs. low families 38 3.71 ** 9.82 ** 2.70 ** Cycles high vs. low 5 93.15 ** 4.96 ** 1.96 * Cycles high only3 2 15.46 ** 0.65 N.S. 0.14 N.S. High-families4 12 7.62 ** 19.72 ** 4.92 ** Cycles low only3 2 5.61 ** 0.46 N.S. 1.96 N.S. Low-families4 12 7.87 ** 20.71 ** 3.75 ** Mean 5.19 2.55 1.48 C.V. (%) 14.52 21.38 24.61

1Analysis perform including parental lines, F and F , high and low families and com- 1 2 mercial cultivars; 2*, ** significant at P and P , respectively. NS = not significant at P ; 0.05 0.01 0.05 3includes three cycles of all families; 4each family taken individually without regards to cycle.

The mean branch number of USDA 846-1and HB families were similar (~5.8), and significantly (P ) higher than the mean of Top Mark, controls (Esteem, Sol Do- 0.05 rado, and Hales Best Jumbo) and LB families (Table 2). The mean branch number of P , controls, and LB families were not significantly different. 2 The average fruit number per plant ranged from 3.2 (P ) to 1.7 (P ) (Table 2). The 1 2 mean number of per fruit plant of P and the F progeny were similar, and significant- 1 1 ly higher than all other entries examined, except for Esteem and the HB families. The mean fruit number of LB and HB plants was not significantly different.

384 The mean fruit weight of P was significantly greater than P , and the fruit weight of the 1 2 F and F progeny transgressed that of the high parent (Table 2). The fruit weight of HB and 1 2 LB families were similar and not different from the commercial cultivars examined. Line USDA 846-1 develops more lateral branches and bears more, but smaller fruit than Top Mark (Table 2). There are in fact significant source/sink relationships in melon that must be considered when improving yield-related traits. Based on HB family analysis, significant positive correlations were detected between branch number and fruit number (r = 0.39), branch number and fruit weight (r = 0.23) and fruit 0.05 0.05 number and weight (r = 0.54). These and other trait associations must be conside- 0.05 red when manipulating yield and quality traits in this HB melon population.

Table 2. Mean lateral branch and fruit number per plant and weight (kg) per fruit of parents, F , F , commercial melon cultivars and families selected for low and high branch 1 2 number over three cycles of recurrent selection by single-seed descent

Entry Branch number Fruit number Weight per fruit

P (USDA 846-1) 5.75 3.20 1.69 1 P (Top Mark) 4.54 1.63 0.88 2 F 5.43 3.13 1.92 1 F 5.18 1.81 1.81 2 Hales Best Jumbo 4.74 1.92 1.60 Esteem 5.15 2.70 1.68 Sol Dorado 4.95 2.30 1.91 Low families1 4.55 2.32 1.42 High families2 5.842.82 1.53 LSD 0.65 0.76 0.51 0.05

Cycle 1 low 4.66 2.42 1.51 Cycle 2 low 4.51 2.28 1.40 Cycle 3 low 4.49 2.23 1.35 LSD 0.11 0.37 0.16 0.05

Cycle 1 high 5.77 2.76 1.55 Cycle 2 high 5.93 2.77 1.52 Cycle 3 high 5.81 2.941.48 LSD 0.07 0.35 0.15 0.05

1Grand mean of families selected for low and high branch number over three cycles of selection; 2Mean of families selected for low and high branching taken collectively for each selection cycle.

The lateral branch number of the F was intermediate to the parents, and the num- 1 ber and weight of their fruit was most like USDA 846-1. If this is truly a heterotic

385 response and gene action conditioning these traits (fruit number and weight) is dominant, then selection for genotypes possessing high number of branches that bear comparatively more and larger fruit should be possible. Correlations among the traits examined herein would suggest that this is possible. In fact one line, HC2-4 (HB, cycle 2), was recovered that possessed seven branches and bore, on average, 4.8 fruits that weighed 1.7 (data not shown). Likewise, Line HC3-7 (HB, cycle 3), possessed an average of 5.7 branches, and bore about three fruits per plant weighing 1.92 kilograms. We have recovered unique monoecious genotypes that possess 11 branches and bear many (5 to 15 per plant), round fruit weighing between 0.9 to 1.7 kg (Line HC3-16, cycle 3). Selection for low and high numbers of lateral branches resulted in decreased or increased numbers of branches over selection cycles at P (Table 1). In general, se- 0.05 lection resulted in a lack of correlated response in both HB and LB families for fruit number and weight. These results and the correlations among traits in HB families suggest that selection for high lateral branch number will result in little change in fruit number and weight. Although there are rare, exceptional phenotypes of potential economic importance, the proper alignment of unique alleles for yield-related traits is complex. The judicious use of genetic mapping information regarding the location, gene action and interaction of quantitative trait loci (QTL) could assist in allelic alignments, and thus augment selection for yield components in melon (Monforte et al., 2004). We believe that yield in melon can be increased by increasing net photosynthetic potential through the remodeling of plant architecture, i.e., increasing the number of fruit-bearing branches. By its very nature this hypothesis requires an understanding and management of traits with often contrasting contributions to a plants source/sink allocations. Design of plant breeding strategies for increased yield in melon will require a knowledge of trait gene action, inherent epistatic interactions, and the imple- mentation experimental designs that seek to mitigate environmental effects during selection. With the increasing effort to characterize the genome of melon will come opportunities for the use of QTL-trait associations in marker-assisted selection (MAS) for increased breeding effectiveness and efficiency. Nevertheless, MAS is not without its methodological problems (see companion paper on cucumber in this proceeding), and it will require the continued varied use of conventional breeding procedures to assure the development of high-yielding genotypes with unique plant architecture.

References

Davis, D.W., Shehata, M.A. and Eide, C.J. 1976. Minnesota 266 muskmelon breeding line. HortScience, 11: 273. Knavel, D.E. 1988. Growth, development and yield potential of short internode muskmelon. J. Amer. Soc. Hort. Sci., 113: 595-599. Knavel, D.E. 1991. Productivity and growth of short internode muskmelon plants at various spacing and densities. J. Amer. Soc. Hort. Sci., 116: 926-929. Kultur, F., Harrison, H.C. and Staub, J.E. 2001. Spacing and genotype affect fruit sugar concentration, yield, and fruit size of muskmelon. HortScience, 36: 274-278. Monforte, A.J., Oliver, M., Gonzalo, M.J., Alvarez, J.M., Dolcet-Sanjuan, R. and Arus, P. 2004. Identification of quantitative trait loci involved in fruit quality traits in melon (Cucumis melo L.). Theor. Appl. Genet. (in press).

386 Mohr, H.C. and Knavel, D.E. 1966. Progress in the development of short-internode (bush) cantaloupes. HortScience, 1: 16. Nerson, H., Paris, H.S. and Karchi, Z. 1983. Characteristics of birdnest-type muskmelons. Scien- tia Hortic., 21: 341-352. Paris, H.S., Karchi, Z., Nerson, H., Govers, A. and Freudenberg, D. 1981. A new plant type in Cucumis melo L. Cucurbit Genet. Coop. Rep., 4: 24-26. Paris, H.S., Karchi, Z., Nerson, H. and Burger, Y. 1982. On the compact appearance of birdnest type muskmelons. HortScience, 17: 476. Roman, D.K. 1971. A statistical analysis of intraseasonal demand shifts in the cantaloupe industry. Ph.D. Thesis, St.Louis Univ. Rubatzky, V.E. and Yamaguchi, M. 1997. World Vegetables. Principles, Production, and Nutritive Values. Melons. Chapman & Hall., New York, pp. 322-326. SAS Institute. 1990. SAS/STAT users guide, release version 6.03. SAS Inst., Cary, N.Y.

387 388 Assortment of five gene loci in Cucurbita pepo

H.S. Paris, A. Hanan and F. Baumkoler Department of Vegetable Crops, Agricultural Research Organization, Newe Yaar Research Center, P. O. Box 1021, Ramat Yishay 30-095, Israel; e-mail: [email protected]

Summary

Although a few dozen loci have been identified in the genus Cucurbita, only one case of linkage of genes conferring phenotypic characteristics has been reported. A cross was made between an inbred of cocozelle squash and an inbred of crookneck squash (both C. pepo) in order to determine if any of five simply inherited traits, conferred by alleles of genes D, l-1, M, Wt, and Y, are linked. Results from the F generation suggested that the M and Wt loci are linked, appro- 2 ximately 27 map units apart.

Keywords: squash, pumpkin, genetic linkage

Introduction

The genetics of Cucurbita pepo L. is the best understood within the genus Cucur- bita L. (2n = 2x = 40), with 34 loci listed (Robinson and Paris, 2000). Only one case of linkage for genes affecting phenotypic traits has been reported so far. This linkage was observed between two genes affecting fruit color, D and mo-2, which were estimated to be 15 map units apart (Paris, 1997). Although much progress has been made in mapping the genome of the other important cucurbit crops, the same undertaking in Cucurbita is still in its infancy. Phe- notypic markers that are not subject to environmental variation, particularly those that occur relatively early in development, could be of great help for any future map construction. Furthermore, mapping of the genome of C. pepo could be optimized by a judicious choice of homozygous, highly disparate, well-characterized parental genotypes for population development. The results could be of great interest to the vegetable seed industry if these genotypes are representative of different economically important market types. Two genotypes meeting these criteria were chosen. Five of their differing phenotypic traits are controlled by each of five known genes. The purpose of the present study was to observe whether or not any two of these five genes are linked.

Materials and methods One of the genotypes was an S inbred, designated STIa-1-13-1-6, from the rather 4 variable Italian Striato dItalia (C. pepo subsp. pepo Cocozelle Group). The original seeds of this cultivar were obtained in 1989 by courtesy of S.A.I.S., Cesena, Italy. The other genotype was an S inbred, designated EYC-3-3-2-2-1-2-4-3, from the 8 American Early Yellow Crookneck (C. pepo subsp. texana (Scheele) Filov Crook-

Table 1. Gene designation and phenotypic description of the genes and the traits they confer that were tested for linkage

Gene (symbol) Dominant Recessive Cocozelle Crookneck

Dark stem (D) Dark stem Light stem d/d D/D light young fruit color (l-1) Striped fruit Light fruit l-1BSt/l-1BSt l-1/l-1 Mottled leaf (M) Silver mottled Non-mottled M/M m/m Warty fruit (Wt) Warted Non-warted wt/wt Wt/Wt Yellow fruit color (Y) Yellow Green y/y Y/Y

Plants of the two inbreds were reciprocally crossed. Some of the resulting F plants 1 were self-pollinated and others were backcrossed to the parents. Seeds of parental, F , 1 F , and BC generations were sown in flats on 03 March 2003 and transplanted to the 2 1 field 27 days later. They were scored for stem color and presence or absence of leaf mottling 25 30 days after transplanting. Presence or absence of striping was scored when the fruits were 2 5 days past anthesis and yellow versus green fruit color was scored when the fruits were 15 18 days past anthesis. Presence or absence of warts on the fruits was scored 30 40 days past anthesis. The results were tested for goodness-of-fit to a 9:3:3:1 ratio in the F (n = 226). Dominant-allele pairs in coupling 2 phase were also tested for goodness-of-fit to a 1:1:1:1 ratio in the backcrosses to the cocozelle (n = 115) and to the crookneck (n = 126).

Results and discussion

There were four combinations of dominant alleles in coupling phase: l-1BSt M from the cocozelle and D Wt, D Y and Wt Y from the crookneck. No linkage was found between any of these pairs of dominant alleles (Table 2). There were six combinations of dominant alleles in repulsion phase: D l-1BSt, D M, l-1BSt Wt, l-1BSt Y, M Wt, and M Y. Only in the M Wt combination was a significant deviation from the 9:3:3:1 expected F ratio found (Table 2). The deviati- 2 on, although significant, is not large, suggestive of weak linkage. Pending additional data, the estimated distance between these two loci can be interpolated as approximately 27 map units (Allard, 1960).

390 Table 2. Tests for possible linkage, using F and BC progenies, among 5 gene loci 2 1

Gene pair Generation Phase A/__ B/__ A/__ b/b a/a B/__ a/a b/b c2 P

D l-1 F Repulsion 132 45 35 14 1.634 0.66 2 D M F Repulsion 13443 37 12 1.383 0.71 2 D Wt F Coupling 131 46 42 7 4.026 0.26 2 D Wt BC Coupling 42 31 29 24 5.492 0.13 1 D Y F Coupling 131 46 33 16 2.751 0.44 2 D Y BC Coupling 41 32 22 31 5.746 0.12 1 l-1 M F Coupling 129 42 38 17 1.068 0.78 2 l-1 M BC Coupling 27 33 31 241.6960.64 1 l-1 Wt F Repulsion 121 52 46 7 6.385 0.09 2 l-1 Y F Repulsion 127 40 37 22 5.206 0.15 2 M Wt F Repulsion 122 49 51 4 10.26 0.01 2 M Y F Repulsion 123 48 41 14 0.926 0.84 2 Wt Y F Coupling 128 45 36 17 1.713 0.63 2 Wt Y BC Coupling 33 38 30 25 2.825 0.43 1

Dark stem (D) and Mottled leaves (M) are easily recognized on young plants, and thus are quite valuable as phenotypic markers. Linkage has now been observed for each, the former with one of the genes for mature orange fruits (mo-2) (Paris, 1997) and the latter with Warty fruits (Wt) (Table 2). Brown and Myers (2002) developed a partial map for Cucurbita based on a first- generation backcross of C. pepo (recurrent parent) with C. moschata Duchesne (do- nor parent). This map included 148 RAPD markers and loci for five phenotypic traits, including M, which was assigned to Linkage Group 6. However, as Brown and Myers pointed out, the silver-mottling trait in this population was derived from C. moschata, and it has not yet been proven that the gene conferring this trait in C. moschata (Coyne, 1970) is homeologous to the one in C. pepo. The two inbreds used for this study were derived from the two horticultural groups (Paris, 1986) that encompass the longest-fruited forms of each of the two cultivated subspecies of C. pepo. The cocozelles are classified within subsp. pepo, along with the pumpkins, vegetable marrows, and zucchinis, whilst the crooknecks are classified within subsp. texana, along with the scallops, acorns, and straightnecks. Long-frui- tedness apparently was selected as an adaptation to culinary use of young fruits (Pa- ris, 1989) and as the Cocozelle Group and the Crookneck Group are derived from different subspecies, selection toward longfruitedness apparently was conducted separately for centuries at widely separated geographic locations (Paris, 2001). There- fore, crossing representatives of each of these two horticultural groups would be expected to result in recombinants, a few of which would have considerably shorter fruits and a few others of which might have even longer fruits. Although plants bearing fruits that were considerably shorter than those of both parents were found in the F , 2 no plants were observed to bear fruits longer than those of both parents. Nonetheless, results obtained using different molecular marker systems (Ferriol et al., 2003; Paris

391 et al., 2003) have confirmed that the Cocozelle Group is highly disparate from the Crookneck Group. The population used for the present study, or a similar population derived from crossing a cocozelle with a crookneck, would be ideal germplasm for developing a genetic map for C. pepo.

References

Allard, R.W. 1960. Principles of Plant Breeding. John Wiley & Sons, New York, pp. 72-74. Brown, R.N. and Myers, J.R. 2002. A genetic map of squash (Cucurbita sp.) with randomly amplified polymorphic DNA markers and morphological markers. J. Amer. Soc. Hort. Sci., 127: 568-575. Coyne, D.P. 1970. Inheritance of mottle-leaf in Cucurbita moschata Poir. HortScience, 5: 226-227. Ferriol, M., Pico, B. and Nuez, F. 2003. Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP markers. Theor. Appl. Genet., 107: 271-282. Paris, H.S. 1986. A proposed subspecific classification for Cucurbita pepo. Phytologia, 61: 133-138. Paris, H.S. 1989. Historical records, origins, and development of the edible cultivar groups of Cucurbita pepo (Cucurbitaceae). Econ. Bot., 43: 423-443. Paris, H.S. 1997. Genes for developmental fruit coloration of acorn squash. J. Hered., 88: 52-56. Paris, H.S. 2000. Gene for broad, contiguous dark stripes in cocozelle squash (Cucurbita pepo). Euphytica, 115: 191-196. Paris, H.S. 2001. History of the cultivar-groups of Cucurbita pepo. In: Janick, J. (Ed.), Horticul- tural Reviews vol. 25. John Wiley & Sons, New York, pp. 71-170, 4 pl. Paris, H.S. and Nerson, H. 1986. Genes for intense fruit pigmentation of squash. J. Hered., 77: 403-409. Paris, H.S., Yonash, N., Portnoy, V., Mozes-Daube, N., Tzuri, G. and Katzir, N. 2003. Assess- ment of genetic relationships in Cucurbita pepo (Cucurbitaceae) using DNA markers. Theor. Appl. Genet., 106: 971-978. Robinson, R.W. and Paris, H.S. 2000. Cucurbita gene list update 2000. Cucurbit Genet. Coop. Rep., 23: 137-138. Scarchuk, J. 1954. Fruit and leaf characters in summer squash. J. Hered., 45: 295-297. Sinnott, E.W. and Durham, G.B. 1922. Inheritance in the summer squash. J. Hered., 13: 177-186.

392 Genetic compatibility between Cucurbita moschata and C. argyrosperma

L. Wessel-Beaver1, H.E. Cuevas1,2 and T.C. Andres3 1Department of Agronomy and Soils, University of Puerto Rico, P.O. Box 9030, Ma- yagüez, Puerto Rico, 00681-9030, USA; e-mail: [email protected] 2Former graduate student 3The Cucurbit Network, 5440 Netherland Ave., D24, Bronx, New York, USA; e-mail: [email protected].

Summary

The tropical pumpkins Cucurbita moschata Duchesne (MOS) and C. argyrosperma Huber are often found growing together in Central America. Various reports indicate that intermediate types occur. Also found is C. argyrosperma subsp. sororia (L.H. Bailey) (Merrick & Bates) (SOR), which appears to be the wild parent of domesticated C. argyrosperma subsp. argyrosperma (ARG). This research studied genetic cross compatibility between these species by evaluating progenies from various controlled pollinations. We observed compatibility between SOR and ARG, no matter the direction of the cross. There was a high degree of compatibility between ARG/SOR and MOS when ARG/SOR was used as the female parent, but incompatibility in the reciprocal cross where MOS was used as the maternal parent. Plants, fruits and seeds of F and backcross populations appeared 2 to be normal. Monogenic morphological traits had expected segregations in interspecific backcrosses, suggesting a high degree of homology between these two species. The high level of compatibility observed should make it easy to move traits of interest from one species to another.

Keywords: Cucurbita sororia, interspecific crosses, cucurbits, squash, pumpkin, gene flow

Introduction

A limited number of studies on phylogenetic relationships have been carried out in Cucurbita. Some studies have used morphological, isoenzyme and molecular data to establish relationships (Decker, 1985; Decker and Wilson, 1987; Wilson, 1989; Decker- Walters, 1990; Puchalski and Robinson, 1990; Wilson et al., 1992; Sanjur et al., 2002), with others have used a biological species approach, and attempted to establish relationships based on genetic compatibility (Bemis and Nelson, 1963; Whitaker and Be- mis, 1964; Whitaker and Cutler, 1965; Merrick, 1991; Wessel-Beaver, 2000a). A study by Merrick (1991) of C. argyrosperma (including the domesticated subspecies argyrosperma (ARG) and the wild or weedy subspecies sororia (SOR)) concluded that this species is the most closely related to C. moschata (MOS). She also concluded that SOR is likely the wild ancestor of ARG. Both SOR and ARG appear to have originated in Mexico (Nee, 1990; Merrick, 1991). Early authors placed the origin of MOS in Mexico or Central America as well (Whitaker and Davis, 1962), but this is now dis- puted. The ancestor of MOS has not been found, but Nee (1990) suggested that it might instead be in northern South American. Wessel-Beaver (2000b) observed that South American populations are morphologically distinct, especially for seed type, from Nor-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 393 th American MOS, and that both types are found in Colombia. This suggests that this northern South American country may be the center of origin of MOS. Landraces of MOS are found from the northern U.S. to Argentina. However, only at lower elevations in Mexico and Central America are MOS, ARG and SOR found cultivated (or growing as a weed) in close proximity (Whitaker and Davis, 1962; Whitaker, 1968; Merrick, 1988, 1991). Merrick (1991) found that when MOS was used as the paternal parent in crosses with either ARG or SOR, fertile F progeny were obtained. The reciprocal cross, with MOS 1 as the maternal parent, was unsuccessful. Merrick (1991) was able to produce F and 2 backcross progeny, but did not grow these plants to maturity. Her crosses were done in a temperature environment (California and New York). Most accessions used in her study originated in either Mexico or the U.S. The objectives of our study were to: 1) confirm Merricks (1991) results in a tropical environment using different accessions, including a diverse group of MOS from South America; 2) characterize hybrid plants; and 3) study F and backcross progeny. 2

Materials and methods

Experiment 1 Cross or sib pollinations were made within and among various species. The primary emphasis was on ARG, SOR and MOS, but other crosses and selfs were included for comparison (Table 1, 2). The pollinations were made in the field at the Isabela Substation (18 N latitude, 30 m above sea level) of the University of Puerto Rico at Mayagüez in 1997. Seed from each fruit was rated using a scale from 0 to 3 where 0 = cotyledons appear to be not developed; 1 = cotyledons slightly developed, seed less than half-filled; 2 = seed not completely filled; 3 = normal, plump seed with fully developed cotyledons. Values from each fruit were combined and averaged. Twenty seeds of each cross were planted in Promix® and percent emergence was recorded after 10 days.

Experiment 2 In an attempt to broaden the genetic pool sampled within MOS, additional crosses were made in 2001 and 2002 (Table 1, 3). It was assumed that MOS interspecific crosses using either ARG or SOR as the maternal parent would be successful. A limited number of these types of crosses, along with self or intraspecific sib pollinations, served as controls to verify that the crossing technique and field conditions were adequate for successful fruit and seed set. The following percentages were determined: fruit set, developed seeds, seeds with undeveloped cotyledons, and viable seeds. Seed viability was determined as above.

Experiment 3 Leaf length and width, fruit length and width, thickness of fruit flesh, fruit weight, width of the base and upper part of the peduncle, length and width of seeds, width of seed margin, and rind thickness on lignified fruits was measured in at least 10 plants and fruits from parents (ARG [Arg 182-2], SOR [sor 1(P)] and MOS [PRshortvine]) and F s. 1

394 Experiment 4 Reciprocal backcrosses between the interspecific F (Sor 177 x PRShortvine) and 1 MOS (PRShortvine) were planted at the Isabela Substation in 2002. Fruits were harvested and rated for flesh bitterness (Bi = bitter) and lignified rind (Hr = lignified rind). A chi-square test was performed. Several F s produced in 1997 were selfed and 1 the F populations grown out for observation. Fruits were harvested, photographed 2 and described.

Table 1. Accessions of Cucurbita moschata and C. argyrosperma used to test interspecific compatibility

Species Origin

C. moschata Expt. 1. Soler Puerto Rico, open pollinated cultivar Mos 166, Mos 17-1, Mos 168, Mexico (seed from T. Andres) Mos 51-1, Mos 130-5 Mos 2 (P), Mos 17-1 (P), Mos Panama (seed from T. Andres) 14-1 (P), Mos Hyb 15-2 (P) C. moschata Expt. 2. Bolivia 219, Bolivia 270-4Plant Ecogenetic Center, Pairumani, Bolivia Brazil 1 Public market, Salvador (Bahia), Brazil Colombia 10 Barsurto public market, Cartagena, Brazil Colombia 3 Public market, Santander de Qilichao (Valle), Colombia Nigerian Local Nigerian landrace Panama 2 Public market, Panama PRShortvine, PRLongvineSLR Open-pollinated cultivar; University of Puer- to Rico, Mayaguez C. argyrosperma subsp. argyrosperma Expt. 1. Same as in Expt. 2, plus the following: Green Striped Cushaw U.S. cultivar (seed from T. Andres) C. argyrosperma subsp. argyrosperma Expt. 2: Arg 51-5, Arg 46-3, Arg 182-1, Mexico (seed from T. Andres) Arg 182-2, Arg 46-1, Arg 51-6 C. argyrosperma subsp. sororia Expt. 1. Same as in Expt. 2, plus the following: Sor 50-3, Sor 80-1, Sor 72 Mexico (seed from T. Andres) Sor 18 (P) Panama (seed from T. Andres) C. argyrosperma subsp. sororia Expt. 2. Sor 50-2, Sor 50-1, Sor 19, Mexico (seed from T. Andres) Sor 149, Sor 177-1 (Sor 50-3 x Sor 18) F of cross between a Mexican and Panamanian 1 accession Sor 1(P) Panama (seed from T. Andres)

395 Results and discussion

Experiment 1 Selfed pollinations produced fully formed seed with good germination (Table 2). Within SOR, ARG and MOS, fruit set varied from 57% to 79%. ARG x MOS and SOR x MOS crosses had a slightly lower seed rating, but viability (emergence) was similar to that of selfs. Even within a species, fruit set upon hand pollination is often unsuccessful. In the experience of one of the authors (Wessel-Beaver, 2000), fruit set in selfs or crosses of MOS of 30% or less is not unusual. Of 58 MOS x ARG crosses, 17 (29%) produced fruit, but these fruit had undeveloped seeds. In the one fruit that produced a large amount of normal appearing seed (using MOS parent PRLongvineSLR), a germination test indicated that most of those seeds were not viable. Merrick (1991) observed a similar poor fruit set in MOS x ARG/SOR crosses. However, she reported that these fruits produced no seed. In our study, fruits produced seeds with a testa that was normal in appearance, but no embryo or cotyledons had developed inside. This suggests that fertilization occurred, and that the incompatibility we observed between species was post-zygotic.

Table 2. Fruit set and seed quality in fruits of various interspecific crosses in Cucur- bita (Experiment 1)

Cross No. of Fruit Seed Seed Seed pollinations set (%) rating1 emergence emergence (%) (min.-max. %)

C. argyrosperma Ä 1478.6 3.0 98.8 087-100 C. argyrosperma x C. sororia 19 47.4 2.4 68.1 080-100 C. argyrosperma x C. moschata 24 41.2 2.2 94.5 020-100 C. argyrosperma x C. pepo 6 16.7 0.0 7.0 7000 C. argyrosperma x C. fraterna 40.0 - - - 000 C. argyrosperma x C. ecuadorensis 1 0.0 - - -000 C. sororia Ä or sib 49 52.3 3.0 51.9 00-100 C. sororia x C. argyrosperma 26 46.2 2.4 52.1 13-100 C. sororia x C. moschata 46 47.8 2.4 67.9 20-100 C. sororia x C. pepo 8 25.0 0 0 0 00 C. sororia x C. fraterna 13 30.8 0 0 0 00 C. sororia x C. ecuadorensis 7 42.9 1.5 63.5 40-870 C. moschata Ä or sibbed 19 57.9 3.0 97.1 90-100 C. moschata x C. argyrosperma 2 0.0 - - -000 C. moschata x C. sororia 7 0.0 - - -000 C. moschata x C. ecuadorensis 1 0.0 - - -000 C. pepo x C. sororia 1 0.0 - - -000 C. pepo x C. fraterna 2 50 2.0 100 10000 C. pepo x C. maxima 1 0.0 - - -000 C. pepo x C. ecuadorensis 1 0.0 - - -000 C. fraterna x C. argyrosperma 5 20.0 3.0 100 10000

396 C. fraterna x C. sororia 8 0.0 - - -000 C. fraterna x C. moschata 3 0.0 - - -000 C. fraterna x C. pepo 3 33.3 3.0 90 9000 C. fraterna x C. ecuadorensis 20---000 C. maxima x C. ecuadorensis 10---000 C. ecuadorensis x C. argyrosperma 1 100 1.0 0 0 00 C. ecuadorensis x C. moschata 10---000 C. ecuadorensis x C. moschata 10---000 C. ecuadorensis x C. pepo 1 100 not tested C. ecuadorensis x C. fraterna 20---000 C. ecuadorensis x C. maxima 3 33.3 3.0 13 1300

1 Seed from each fruit was rated for seed fill using a scale from 0 to 3 (associated with development of cotyledons) where: 0 = cotyledons appear to be not developed; 1 = cotyledons slightly developed, seed less than half-filled; 2 = seed not completely filled or less plump than normal; 3 = normal, plump seed with fully developed cotyledons. Values from each fruit were combined and averaged.

Experiment 2 Compatibility of MOS x ARG/SOR crosses did not improve when we tested a more diverse group of MOS germplasm (Table 3). Few fruits set, and these fruits had almost all undeveloped seed.

Table 3. Fruit set and seed quality in fruits of Cucurbita moschata pollinated with C. argyrosperma subsp. argyrosperma and subsp. sororia (Experiment 2)

C. moschata No. of No. of Seed (%) accession crosses crosses that tested set fruit Developed Undeveloped Germinated

Bolivia 219 11 1 0.0 100.0 0 Bolivia 370-411 5 1.2 98.8 0 Brazil 1 3 0 - - - Colombia 10 6 1 0.0 100.0 5 Colombia 3 5 2 0.0 100.0 0 Nigerian Local 1 0 - - - Panama 2 3 3 0.0 100.0 0 PRLongvineSLR 2 1 51.5 48.5 18 PRShortvine 16 40.0 100.0 0

Experiment 3 Overall, we observed a great deal of hybrid vigor in vegetative growth among interspecific F plants. Seed set and development appeared normal. Of the 13 morpho- 1 logical traits studied, the F hybrid ARG x MOS was intermediate between (and sig- 1 nificantly different from) both parents for 6 traits, like ARG for 3 traits and like MOS for 4 traits (Table 4). F hybrids of SOR x MOS were intermediate for 8 traits and like 1

397 SOR for 4 traits. One trait was not polymorphic. The patterns of rind color and leaf shape were also observed, but were difficult to quantify. In general, when several morphological traits (leaves, fruit, peduncle and seed) are considered together, the F 1 plant or fruit can be distinguished from either parent, particularly if the parents are present as a point of reference. Still, the difference can be somewhat subtle, and the range of values can overlap, thus making it difficult at the field level to distinguish between inter- and intraspecific fruits. Leaf shape (data not shown), leaf size, and fruit size of the F hybrid ARG x MOS was not different from that of ARG. The morpholo- 1 gical differences between the F and ARG are associated with either peduncle or seed 1 characteristics, neither of which are obvious to the casual observer of the fruit. Typi- cal of most populations of ARG, Arg 182-2 has an enlarged, corky peduncle, not flared at the base. However, some populations of ARG have peduncles more characteristic of MOS (non-corky and flared at the base). The peduncle of SOR is typically like MOS, albeit thinner and more angular. An F hybrid SOR x MOS would be almost 1 impossible to distinguish from ARG if the later population was of the type without enlarged peduncles.

Table 4. Comparison of morphological traits of Cucurbita argyrosperma subsp. argyrosperma (Arg 182-2), Cucurbita argyrosperma subsp. sororia (Sor 1(P)), and C. moschata (PRShortvine) and their F hybrids 1

Trait LSDz C. argyro. F (subsp. argyro. C. mos. F (subsp. sor. C. argyro. 1 1 subsp. argyro. x C. mos.)x C. mos.) subsp. sor.

Leaf width (cm) 3.7 21.2 b 32.6 a 32.4a 24.4b 16.0 c Leaf length (cm) 2.5 16.4bc 22.4ab 23.3 a 18.2 c 12.6 d Fruit width (cm) 1.7 13.5 c 16.4b 18.9 a 12.0 d 7.6 e Fruit length (cm) 2.7 12.4bc 13.6 b 19.6 a 10.8 c 7.0 d Fruit weight (kg) 0.6 1.2 bc 1.7 b 3.4a 0.8 cd 0.2 d Flesh thickness 16.4 13.3 bc 24.0 b 41.2 a 19.6 bc 6.9 c (mm) Lignification (mm) 0.5 1.0 b 2.32 a nl 2.1 a 2.0 a Peduncle width at 2.1 20.7 c 24.4 b 29.8 a 13.0 d 8.9 e fruit attachment (mm) Peduncle width at 1.7 20.0 a 11.8 b 9.2 c 2.9 e 5.2 d stem attachment (mm) Stem width ratioy 1.41.1 b 2.1 ab 3.3 a 2.3 ab 2.2 ab Seed width (mm) 0.5 10.1 a 9.42 b 9.1 b 7.2 c 6.5 d Seed length (mm) 1.2 20.4a 17.4b 15.8 c 14.2d 12.4e Seed margin (mm) 0.1 1.8 a 1.9 a 1.6 b 1.4c 1.4c z Within a row, means followed by the same letter are not different according to the LSD multiple comparison test protected with an F test (a = 0.05). yRatio of peduncle width at fruit attachment/peduncle width at stem attachment. nl = not lignified.

398 Experiment 4 Segregations in the interspecific backcrosses were as expected for flesh bitterness and rind lignification, suggesting a high degree of homology between ARG/SOR and MOS (Table 5). The F ARG x MOS and SOR x MOS populations (no data shown) 2 produced plants and fruits combining characteristics from both parents. Plants set fruit and fruits had normally developed seed.

Table 5. Chi-square test of segregations of bitter and non-bitter fruits and lignified and non-lignified rinds in reciprocal interspecific backcrosses of Cucurbita moschata x (C. argyrosperma subsp. sororia x C. moschata) (F ) and F x C. moschata 1 1

Cross Observed Expected Observed X2 Prob. Bitter Non-bitter ratio ratio

C. mos. x F 1422 1 : 1 1 : 1 1.80 0.1824 1 F x C. mos. 20 18 1 : 1 1 : 1 0.11 0.7456 1 Lignified Non-lignified rind rind C. mos. x F 31 32 1 : 1 1 : 1 0.02 0.8997 1 F x C. mos. 42 42 1 : 1 1 : 1 0.00 1.0000 1

Conclusions Our results suggest that movement of genes from populations of C. moschata to populations of C. argyrosperma (either domesticated or wild/weedy) is possible. The reciprocal flow of genes from C. argyrosperma directly to C. moschata must be much more restricted due to the usual failure of that cross to produce viable progeny. In Central America it is possible C. argyrosperma fruits pollinated by C. moschata are at times harvested and intermixed with intraspecific fruits. Upon close inspection, it is possible to separate interspecific from intraspecific fruit. But it is doubtful that most farmers would notice these differences which would be especially subtle in a field planted to an already hetergeneous landrace. Nevertheless, both species continue to be quite distinct, perhaps mostly due to human management and selection. C. argyrosperma subsp. argyrosperma is mostly used for confections or snacks, while C. moschata is selected for its attractive flesh. There are several traits not found in one species that might be desirable in the other. One of the authors (Wessel-Beaver, 2000) is evaluating C. argyrosperma in her breeding program as a possible source of me- lonworm (Diaphania hylinata) resistance. The high degree of compatibility will make it easy to move desirable traits from one species to another.

399 Acknowledgements

This research was supported by the Tropical-Subtropical Agricultural Research (TSTAR) Program administered by the Caribbean Basin Administrative Group (CBAG) of the United States Dept. of Agriculture, by the Puerto Rico Agricultural Experiment Stati- on and by the Smithsonian Institute for Tropical Research. The authors are grateful for the technical assistance of Mr. Obed Román.

References

Bemis, W.P. and Nelson, J.M. 1963. Interspecific hybridization within the genus Cucurbita. I. Fruit set, seed and embryo development. J. Ariz. Acad. Sci., 2: 104-107. Decker, D.S. 1985. Numerical analysis of allozyme variation in Cucurbita pepo. Econ. Bot., 39: 300-309. Decker, D.S. and Wilson, H.D. 1987. Allozyme variation in the Cucurbita pepo complex: C. pepo var. ovifera vs. C. texana. Syst. Bot., 12: 263-273. Decker-Walters, D.S. 1990. Evidence for multiple domestications of Cucurbita pepo. In: Bates D. M., Robinson R.W. and Jeffrey C. (Eds.), Biology and Utilization of the Cucurbitaceae. Cor- nell University Press, Ithaca, New York, pp. 96-102. Merrick, L.C. 1988. Wild and cultivated cucurbits from the Sierra Madre Occidental of Northwest Mexico and the Río Balsas Valley of Southwest Mexico. Report to the International Board of Plant Genetic Resources, Rome. Plant Genet. Res. Newsl., 71: 45. Merrick, L.C. 1991. Systematics, evolution, and ethnobotany of a domesticated squash, Cucurbi- ta argyrosperma. Ph.D. Thesis. Cornell University, Ithaca, New York. 323 pp. Nee, M. 1990. The domestication of Cucurbita (Cucurbitaceae). Eco. Bot., 44 (Suppl. 3): 56-68. Puchalski, J.T. and Robinson, R.W. 1990. Electrophoretic analysis of isozymes in Cucurbita and Cucumis and its application for phylogenetic studies. In: Bates D.M., Robinson R.W. and Jef- frey C. (Eds.), Biology and Utilization of the Cucurbitaceae. Cornell University Press, Ithaca, New York, pp. 60-76. Sanjur, O.I., Piperno, D.R., Andres, T.C. and Wessel-Beaver, L. 2002. Phylogenetic relationship among domesticated and wild species of Cucurbita (Cucurbitaceae) inferred from a mitochondrial gene: implications for crop plant evolution and areas of origin. PNAS, 99: 535-540. Wessel-Beaver, L. 2000a. Cucurbita argyrosperma sets fruits in fields where C. moschata is the only pollen source. Cucurbit Genet. Coop. Rep., 23: 62-63. Wessel-Beaver, L. 2000b. Evidence for the center of diversity of Curcurbita moschata in Colom- bia. Cucurbit Genet. Coop. Rep., 23: 54-55. Whitaker, T.W. and Davis, G.N. 1962. Cucurbits: Botany, cultivation, and utilization. Leonard Hill [Books], Ltd. London. Whitaker, T.W. and Bemis, W.P. 1964. Evolution in the genus Cucurbita. Evolution, 18: 553-559. Whitaker T.W. and Cutler, H.C. 1965. Cucurbits and cultures in the Americas. Econ. Bot., 19: 344-349. Whitaker, T.W. 1968. Ecological aspects of the cultivated Cucurbita. HortScience, 3: 9-11. Wilson, H.D. 1989. Discordant patterns of allozyme and morphological variation in Mexican Cucurbita. Syst. Bot., 14: 612-623. Wilson, H.D, Doebley, J. and Duvall, M. 1992. Chloroplast DNA diversity among wild and cultivated members of Cucurbita (Cucurbitaceae). Theor. Appl. Genet., 84: 859-865.

400 Strategies for selection of multiple quantitative inherited yield components in cucumber

M.D. Robbins and J.E. Staub USDA/ARS, Vegetable Crops Unit, Department of Horticulture, 1575 Linden Dr., University of Wisconsin, Madison, WI 53706, USA; e-mail: [email protected]

Summary

Cucumber is an important vegetable crop whose yield has reached a plateau in the last 15 years. Factors, such as fruit-set inhibition, a narrow genetic base, and negative correlations among yield-related traits must be overcome to increase yields. A study has been designed and initiated to compare recurrent phenotypic (PHE) and marker-assisted (MAS) selection for relative efficiency. Selection was practiced on four yield-related traits over three cycles in each of four base populations originally created from the intermating of four inbred lines. The PHE selection strategy requires identification of unique plants in the open-field and then their recovery through meristem cuttings followed by cross pollination in a greenhouse. These plants are early flowering, gynoecious, multiple lateral branching, and develop relatively long fruit. The MAS strategy involves the selection of greenhouse-grown individuals by assessment of genotype using dominant (RAPD, SCAR, SNP, and AFLP) and codominant (SSR and SCAR) markers linked to QTL for the yield components described for PHE selection. There are several critical decision points for MAS including QTL selection, identification of markers most appropriate for MAS, and marker optimization. Decision-making requires compromises associated with QTL map position, marker efficiency maximization, and choice of marker type.

Keywords: Cucumis sativus, recurrent selection, gain from selection, marker-assisted selection, multiplexing

Introduction

The genetic improvement of a species through artificial selection depends on the ability to capitalize on genetic effects that can be distinguished from environmental effects. Phenotypic selection based on traits that are conditioned by additive genetic effects can produce dramatic, economically important changes in breeding populations. It has been well-recognized that genetic markers can theoretically be used by plant breeders as selection tools (Darvasi and Soller, 1994) in marker-assisted selection (MAS) to provide a potential for increasing selection efficiency by allowing for earlier selection and reducing plant population size used during selection. Cucumber (Cucumis sativus L.) is an economically important and popular vegetable whose production value surpasses snap bean in the U.S. (Staub and Bacher, 1997), and is the fourth most widely grown vegetable worldwide (Tatlioglu, 1993) by acreage. Yields of multiple hand-harvest U.S. pickling cucumber (process product value presently $1.5 billi- on) have increased dramatically since 1940 (~5% annual average increase; USDA Agricul- tural Statistics, 1998). Nevertheless, with the exception of perhaps some unique processing hybrids that are adapted to specific growing environments, average yield has reached a plateau in the past 15 years (~13,400 kg/ha 1997; USDA Agricultural Statistics, 1998).

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 401 Several biological factors limit cucumber yield (Gharderi and Lower, 1979; El- Shawaf and Baker, 1981). First fruit inhibition (first fertilized fruit inhibits subsequent fruits; Tiedjens, 1928; McCollum, 1934) and the narrow genetic base of cucumber (Meglic and Staub, 1996; Staub et al., 2002) are two such factors. Additionally, negative correlations exist between yield-related traits [i.e., multiple lateral branching (MLB), sex expression, days to anthesis (earliness), fruit length to diameter ratio (L:D)] in cucumber that hinder crop improvement efforts (Kupper and Staub, 1988; Staub, 1989). If such factors cannot be overcome or mitigated, then expected gain from either MAS or phenotypic selection can be compromised. The strength and direction of the correlations between yield-related traits in cucumber have been documented in a wide range of genetic backgrounds (Kupper and Staub, 1988; Serquen et al., 1997b; Fazio et al., 2003). Narrow sense heritabilities for these traits range from 0.14 (sex expression modifiers) to 0.48 (number of lateral branches), depending on genetic background and growing environment (Serquen et al., 1997b; Fazio et al., 2003). It is clear that physiological factors, genetic variance, and the genomic location of QTL associated with these yield-related traits are important for the creation and management of selection strategies in cucumber (Cramer and Wehner, 2000). We have designed an experiment that seeks to elucidate the comparative efficiency and effectiveness of MAS and phenotypic (PHE) selection (Robbins et al., 2002). Half- sib PHE and MAS recurrent selection for days to anthesis, sex expression, MLB, and L:D is being practiced in four diverse cucumber populations. In the case of MAS, we have attempted to optimize selection and develop selection procedures that consider source/ sink relationships and genetic factors (e.g., linkage relationships). We now report these strategies, and the implications that they have for decision-making during MAS.

Materials and methods

Experimental design In order to attain our goals, three types of selection were performed on four populations. Four lines (Table 1), which have contrasting mean values for several economically important traits (e.g., line 6632E vs. line 6823B for MLB), were intermated to recombine favorable alleles to achieve an optimum ideotype (i.e., a highly branched, gynoecious, early flowering, long-fruited plant type) for once-over and multiple harvest operations of processing cucumber (Robbins et al., 2002). The general crossing and selection procedure is outlined in Fig. 1. Intermating the four inbred lines produced four base populations. Simulta- neous selection is presently being made from these four populations using MAS and PHE over three cycles of selection. Each population is independently assessed for four yield component traits (MLB, gynoecy, days to anthesis, and L:D) during each cycle. The results of PHE and MAS will be compared to four random mating populations (RAN) as controls that were derived from the four base populations by the same mating scheme, but without selection. Three cycles of PHE, RAN, and MAS (protocol and markers of Fazio et al. (2003), and Fazio and Staub (2003)) have been completed. Population development over three cycles of selection has resulted in four populations generated by each of the selection methods (MAS, PHE, and RAN) for their replicated evaluation in one season and two environments in 2004 to characterize gain from selection (DG; Robbins et al., 2002).

402 Table 1. Inbred lines used to create four base populations for selection and their mean values for each trait

Line Population Branch number Sex expression Days to anthesis Fruit L:D

6632E 1 1.5 Gynoecious 45 2.80 6823B 2 3.5 Monoecious 56 3.20 6947A20 3 1.3 Gynoecious 47 2.96 6995C[B-122] 42.9 Monoecious 53 3.00

Table 2. List of markers from the molecular map of Fazio et al. (2003) including marker type, linkage group, and position, that are linked to QTL for earliness, multiple lateral branching (MLB), gynoecious sex expression (Sex), and fruit length to diameter ratio (L:D) in cucumber

Linkage Multiplex Marker Type Group Position Parent1 Group2 Earliness3 MLB3 Sex3 L:D3 Ideotype4

CSWCT28 SSR 1 5.0 G&H H H G H G&H L18-SNP-H19 SNP 1 7.4H 1 H H G H G CSWCTT14SSR 1 25.3 G&H H H G G&H OP-AG1-1 RAPD 1 31.8 G H H G H AJ6SCAR SCAR 1 61.4G 3 H H BC523SCAR SCAR 1 66.5 G 2 H H OP-AD12-1 RAPD 1 70.2 H G&H H H G G OP-W7-2 RAPD 1 87.8 H G&H H H G G AW14SCAR SCAR 3 3.9 G&H 1 G G OP-C1 RAPD 415.7 G G G CSWTAAA01 SSR 434.1 G&H 2 H H OP-AI4RAPD 5 101.0 G G G OP-AO12 RAPD 5 117.3 G G G OP-AI10 RAPD 6 22.5 H G G AK5SCAR SCAR 6 33.0 G 2 H H M8SCAR SCAR 6 39.1 H H H OP-W7-1 RAPD 6 83.4H G G L19-2-SCAR SCAR 6 115.0 H 1 G G G NR60 SSR 6 137.4G&H G G BC515 RAPD 7 0.0 H H H L19-1SCAR SCAR 7 9.9 H 3 H H

1 Marker amplifies allele from G = GY-7 parent, H = H-19 parent, or G&H = both parents (codominant). 2 Markers used in multiplex were placed in multiplexing groups (1, 2, or 3). 3 Markers are linked to QTL contributing to earlier harvest (Earliness), more lateral branches (MLB), gynoecy (Sex) and/or greater fruit length to diameter ratio (L:D) from G = GY-7, H = H-19, or G&H = both parents. 4 Plants were selected as an ideal genotype for the G = GY-7, H = H-19, or G&H = both allele(s) at each marker.

403 Marker identification and use in MAS Significant QTL (relatively high LOD scores over multiple environments) for earliness, MLB, gynoecious sex expression and high L:D were identified from Fazio et al. (2003). From the map location of these QTL, linked markers were identified for use in MAS (Table 2). Many factors were taken into consideration when selecting markers, such as marker type, genetic distance from QTL, number of QTL in proximity to the marker (see results and discussion). In general, dominant markers flanking QTL or codominant markers tightly linked to QTL were selected. The desired genotype (GY-7 allele, H1- 19 allele or both GY-7 and H-19 alleles) for each marker was determined depending on the QTL surrounding the marker, to create the ideal genotype, or ideotype (Table 2). To increase marker efficiency, some RAPD markers were converted to SCARs (e.g. OP-M8 was converted to M8SCAR) following Staub et al. (2003). In addition, SSR or SCAR markers that produce single bands and have similar PCR melting temperatures (empirically determined) were simultaneously run in the same PCR reaction to empirically determine which markers could be multiplexed. All single primer PCR reactions were set up and resolved on agarose gels as in Fazio et al. (2003), while multiplexing reactions were performed following Staub et al. (2003). Each individual in a population was genotyped at each marker locus. The individuals whose genotype most closely matched the ideotype at the greatest number of marker loci were selected and inter- crossed following Robbins et al. (2002).

Results and discussion

Practical application of MAS can only be justified when predicted benefits (long- and short-term gain from selection) outweigh the additional cost of MAS above traditional breeding methodologies (Gu et al., 1995). In some studies, MAS has been found to be more efficient and effective (Yousef and Juvik, 2001; Fazio and Staub, 2003), equivalent to (Wilcox et al., 2002), or less efficient and effective (Hoeck et al., 2003; Lu et al., 2003) than phenotypic selection. MAS may provide for increased efficiency over phenotypic selection when markers are tightly linked to target traits, where epistatic interactions are known, and source-sink relationships have been characterized (Staub et al., 1996). Cucumber is an ideal crop to use MAS because it has a low chromosome number (n = 7), a small genome (genetic map length = ~750 to 1,000 cM; DNA content approaching Arabi- dopsis thaliana (L.) Heyth., a rapid life cycle (4 cycles per year), and many economically important, simply inherited traits (Staub and Meglic, 1993). Fazio and Staub (2003) showed that MAS is effective and efficient for selection of MLB in a backcross breeding scheme. Their study compared PHE (selection for MLB in open-field environments), RAN (intermating without selection) and MAS (employing five markers for selection of greenhouse-grown plants) for MLB after three cycles of backcrossing in an H-19 x GY-7 population (GY-7 = recurrent parent). Five markers (two SSR , two RAPD and one SNP) were employed in MAS and no significant differences (p < 0.001) were detected between the means of PHE and MAS. However, both PHE and MAS populations were significantly higher that the RAN control. In this

404 case, MAS was more efficient than phenotypic selection since MAS BC populations were produced in one year and phenotypic selection required three years to complete. Given that MAS can be a valuable tool for selection of single yield traits in cucumber, it may also be useful in selection of multiple yield traits.

Figure 1. Schematic diagram of simultaneous selection of multiple quantitative traits using two selection methods and random mating control populations.

The physiological interactions (i.e., source/sink relationships) of yield-related traits have a dramatic effect on gain from simultaneous selection for multiple traits. Fazio et al. (2003) showed that MLB is negatively correlated with days to anthesis, gynoecy, and L:D, while gynoecy is positively correlated with days to anthesis. To overcome these effects, four distinct populations were created from four inbred lines, rather than one population from a pair of inbred lines. In addition, phenotypic selection was performed on all four populations in order to detect the ideotypes that resulted from the recombination of desired QTL.

405 Figure 2. Relative positions of QTL on linkage Groups 1 and 6 of cucumber (Cucu- mis sativus) which contain RAPD, SCAR, AFLP, and morphological markers (italici- zed). Linkage groups are designated by numbers (1 and 6) above Roman numerals and letters corresponding to linkage groups in maps by Bradeen et al. (2001) and Serquen et al. (1997a), respectively. RAPDs are identified by the preceding letters OP and BC according to Serquen et al. (1997a), SSR by the preceding letters CS, CM and NR, AFLP by E__M__, and SCARs by the designation SCAR according to Fazio (2001). The vertical bars to the left of each linkage group represent the QTL regions detected with their respective LOD score. Markers used in this study are in bold.

A total of 21 markers were identified and used in MAS (Table 2 and bold in Fig. 2). In many cases, especially on Linkage Groups 1 and 6, QTL were clustered such that several QTL from both parents were placed near a single marker. The marker ideotype in these instances was strategically determined, since selection could not be made for all QTL in a single cluster. For example, QTL for earliness (from H-19), high MLB (from H-19), gynoecy (from GY-7), and high L:D (from H19) are closely linked to CSWCT28 and L18-SNP-H19. The QTL for gynoecy from GY-7 is most likely the F locus (see Fig. 2), which is the most important locus for determining gynoecy in cucumber (Staub and Bacher, 1997). Even though there are QTL from H-19 near these markers, selection must be made for GY-7 alleles to obtain gynoecious plants. However, CSWCT28 is a codominant marker, which allows selection for both the GY-7 and H-19 alleles. Thus, the location of QTL (e.g., L19-2-SCAR and NR60 flank a QTL for MLB), the magnitude of the QTL (e.g., OP-AD12-1 and OP-W7-2 flank a QTL for MLB with a LOD of 32.9), the type of marker (i.e., codominant, ability to multiplex), and the proximity of

406 morphological traits (i.e., little leaf character, ll, and determinate character, de) on the linkage map determine the ideotype for each marker used in MAS (see Table 2 and Fig. 2). In many instances a sequencing apparatus is not available in laboratories for high- throughput analyses, necessitating the use of agarose gel electrophoresis for the characterization of genotypes. Among the 21 markers used in MAS in this experiment, 12 were SSR, SNP, or SCAR markers that produced one band per parent. These markers were empirically tested in multiplexing reactions and three multiplexing groups (Groups 1, 2, and 3) were created (Table 2). In general, SSR markers were difficult to multiplex because they required electrophoresis on 3% agarose for many hours to resolve the small size polymorphisms of <20 base pairs between alleles. However, CSWTAAA01 multiplexed well in Group 2 because the polymorphism is ~50 base pairs and can be evaluated on 1.6% agarose along with SCAR markers. All eight SCAR and SNP markers were amenable to multiplexing. However, one marker, M8SCAR, was not included in a multiplexing group so as not to exceed the optimal number of markers in a group. The largest number of markers that consistently worked well in a multiplexing group was three. Inclusion of four or more markers promoted competition among markers and required longer electrophoresis times to separate critical bands. Multiplexing of three markers greatly increased the efficiency of markers by reducing the amount of resources and time needed to evaluate individuals in MAS. By using both phenotypic and marker-assisted selection along with the strategies discussed herein, we hope to create the ideotypical highly branched, gynoecious, early flowering, long-fruited plant to increase the yield of U.S. processing cucumber. Deci- sion-making involving judicial compromises during MAS alone or in combination with PHE selection will play a major role in achieving maximum gain from selection.

References

Bradeen, J.M., Staub, J.E., Wyse, C., Antonise, R. and Peleman, J. 2001. Towards an expanded and integrated linkage map of cucumber (Cucumis sativus L.). Genome, 44: 111-119. Cramer, C.S. and Wehner, T.C. 2000. Path analysis of the correlation between fruit number and plant traits of cucumber populations. Hort. Sci., 35: 708-711. Darvasi, A. and Soller, M. 1994. Optimum spacing of genetic markers for determining linkage between marker loci and quantitative trait loci. Theor. Appl. Genet., 89: 351-357. El-Shawaf, I.I.S. and Baker, L.R. 1981. Combining ability and genetic variances of G x H F 1 hybrids for parthenocarpic yield in gynoecious pickling cucumber for once over mechanical harvest. J. Amer. Soc. Hort. Sci., 106: 365-370. Fazio, G., Staub, J.E. and Stevens, M.R. 2003. Genetic mapping and QTL analysis of horticultural traits in cucumber (Cucumis sativus L.) using recombinant inbred lines. Theor. Appl. Ge- net., 107: 864-874. Fazio, G. and Staub, J.E. 2003. Comparative analysis of response to phenotypic and marker- assisted selection for multiple lateral branching in cucumber (Cucumis sativus L.). Theor. Appl. Genet., 107: 875-883. Gharderi, A. and Lower, R.L. 1979. Heterosis and inbreeding depression of yield in populations derived from six crosses of cucumber. J. Amer. Soc. Hort. Sci., 104: 564-567. Gu, W.K., Weeden, N.F, Yu, J. and Wallace, D.H. 1995. Large-scale, cost-effective screening of PCR products in marker-assisted selection applications. Theor. Appl. Genet., 91: 465-470. Hoeck, J.A., Fehr, W.R., Shoemaker, R.C., Welke, G.A., Johnson, S.L. and Cianzio, S.R. 2003. Molecular marker analysis of seed size in soybean. Crop Sci., 43: 68-74. Kupper, R.S. and Staub, J.E. 1988. Combining ability between lines of Cucumis sativus L. and Cucumis sativus var. hardwickii (R.) Alef. Euphytica, 38: 197-210.

407 Lu, H.J., Bernardo, R. and Ohm, H.W. 2003. Mapping QTL for popping expansion volume in popcorn with simple sequence repeat markers. Theor. Appl. Genet., 106: 423-427. Meglic, V. and Staub, J.E. 1996. Inheritance and linkage relationships of allozyme and morphological loci in cucumber (Cucumis sativus L.). Theor. Appl. Genet., 92: 865-872. McCollum, J.P. 1934. Vegetative and reproductive responses associated with fruit development in cucumber. Cornell Univ. Agr. Expt. Sta. Memo. 163. Robbins, M.D., Staub, J.E. and Fazio, G. 2002. Deployment of molecular markers for multi-trait selection in cucumber. In: Maynard, D.M. (Ed.), Cucurbitaceae 2002. ASHS, Alexandria, VA, U.S.A., pp. 41-47. Serquen, F.C., Bacher, J. and Staub, J.E. 1997a. Mapping and QTL analysis of a narrow cross in cucumber (Cucumis sativus L.) using random amplified polymorphic DNA markers. Mol. Bre- eding, 3: 257-268. Serquen, F.C., Bacher, J. and Staub, J.E. 1997b. Genetic analysis of yield components in cucumber (Cucumis sativus L.) at low plant density. J. Amer. Soc. Hort. Sci., 122: 522-528. Staub, J.E. 1989. Source-sink relationships in cucumber. Cucurbit Genet. Coop. Rep., 12: 11-14. Staub, J.E. and Bacher, J. 1997. Cucumber as a processed vegetable. In: Smith, D.S., Cash, J.N., Nip, W. and Hui Y.H. (Eds.), Processing Vegetables: Science and Technology IV. Technomic, Lancaster, PA, U.S.A., pp. 129-193. Staub, J.E., Dane, F., Reitsma, K., Fazio, G. and Lopez-Sesé, A. 2002. The formation of test arrays and a core collection in Cucumis sativus L. using phenotypic and molecular marker data. J. Am. Soc. Hort. Sci., 127: 558-567. Staub, J.E. and Meglic, V. 1993. Molecular genetic markers and their legal relevance for cultigen discrimination: A case study in cucumber. Hort. Technol., 3: 291-300. Staub, J.E., Robbins, M.D., Chung, S. and Lopez-Sesé, A.I. 2003. Molecular methodologies for improved genetic diversity assessment in cucumber and melon. Proceedings XXVI Internatio- nal Horticultural Congress. Acta Hort. (in press). Staub, J.E., Serquen, F. and Gupta, M. 1996. Genetic markers, map construction and their application in plant breeding. Hort. Sci., 31: 729-741. Tatlioglu, T. 1993. Cucumber, Cucumis sativus L. In: Kalloo, G. and Bergh, B.O. (Eds.), Genetic Improvement of Vegetable Crops. Pergamon, Tarrytown, NY, U.S.A., pp. 197-234. Tiedjens, V.A. 1928. Sex ratios in cucumber flowers as affected by different conditions of soil and light. J. Agr. Res., 36: 721-746. U.S. Dept. of Agriculture. 1940, 1981 and 1998. Agricultural Statistics. U.S. Government Prin- ting Office, Washington, D.C. Wilcox, M.C., Khairallah, M.M., Bergvinson, D., Crossa, J., Deutsch, J.A., Edmeades, G.O., Gonzalez de Leon, D., Jiang, C., Jewell, D.C., Mihm, J.A. and Williams, W.P. 2002. Selection for resistance to southwestern corn borer using marker-assisted and conventional selection. Crop Sci., 42: 1516-1528. Yousef, G.G. and Juvik, J.A. 2001. Comparison of phenotypic and marker-assisted selection for quantitative traits in sweet corn. Crop Sci., 41: 645-655.

408 Scientific contributions

V. Tissue culture, biotechnology, molecular genetics and mapping

409 410 Cucumber (Cucumis sativus) haploids developed from parthenocarpic hybrids

J. Sztangret, J. Wronka, T. Ga³ecka, A. Korzeniewska and K. Niemirowicz-Szczytt Department of Plant Genetics, Breeding and Biotechnology, Warsaw Agricultural University, Nowoursynowska 166, 02-787 Warszawa, Poland; e-mail: [email protected]

Summary

As a result of cucumber flower pollination with irradiated pollen it was possible to obtain embryos and haploid plants. The embryos were rescued from the fruits of four hybrid varieties (three parthenocarpic ones: Pol10, Rubin and S³awko and the standard Izyd) and then transferred to in vitro culture. Some of the embryos (45-48%) were able to differentiate and become stable haploid plants. At this stage of experiments these haploids are subject to direct regeneration with the aim to obtain diploids.

Keywords: Irradiated pollen, embryo rescue, chromosome doubling

Introduction

For some 15 years the Department of Plant Genetics, Breeding and Biotechnology has carried out experiments with cucumber haploid induction with the use of irradiated pollen. In the process of parthenogenesis a certain number of haploid embryos can be developed (Faris and Niemirowicz-Szczytt, 1999) and some of them are capable of further growth after isolation and transfer to in vitro conditions (Niemirowicz- Szczytt and Dumas de Vaulx, 1989; Przyborowski and Niemirowicz-Szczytt, 1994; Niemirowicz-Szczytt et al., 2000). Since no spontaneous diploidization occurs, it is necessary to apply chromosome doubling methods, such as direct regeneration from leaf explants or colchicine treatment (Nikolova and Niemirowicz-Szczytt, 1996; Fa- ris et al., 2000). The aim of the study was to obtain haploid and doubled haploid plants from the Polish parthenocarpic cucumber hybrids.

Materials and methods

Out of four varieties selected for the experiment there were three parthenocarpic hybrids (Pol 10, Rubin and S³awko) and one standard (Izyd), which was known to give the best results during the previous experiments with haploid induction. Plants (40 of each variety) were grown in the greenhouse in two seasons (spring and summer-autumn). The pollen source was monoecious Borszczagowski line and the irradiation doses (gamma rays) applied amounted to 0.3 kGy. Pollination was continued until 3-4 well-developed fruits per plant were observed. After 3-5 weeks embryos were

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 411 isolated and transferred to artificial medium following the procedure already described (Przyborowski and Niemirowicz-Szczytt, 1994; Niemirowicz-Szczytt et al., 2000). In order to obtain diploids direct regeneration was applied. The first and second juvenile leaves of haploid plants growing in vitro were excised and cut into squares (2- 3 mm2). Then the explants were cultured on modified by Burza and Malepszy (1995) MS medium (Murashige and Skoog, 1962). Further culture and regeneration was carried out in accordance with the adopted protocol (Faris et al., 2000). At this stage of investigation we can only present the results of direct regeneration after one year of experiments. Further steps will include meristem colchicine treatment and its outcome will be known at the end of 2004.

Results and discussion

The numbers of isolated embryos and plants obtained from them are presented in Table 1. The comparison of the numbers of fruits grown in the spring and summer-autumn season shows that spring was more favorable (426 fruits as compared with 361).

Table 1. Haploid embryo and plants development (spring 2003; summer/autumn 2003)

Donor cultivar (F ) No. of fruits No. of embryos No. of plants % of plants 1 Spring 2003 Pol 10 120 58 23 45.5 Rubin 114 44 20 44.1 S³awko 82 3415 36.6 Izyd 110 79 43 54.4 Total 426 215 101 45.2 Summer/autumn2003 Pol 10 108 29 16 55.2 Rubin 93 44 20 45.5 S³awko 7423 12 52.2 Izyd 86 36 1438.9 Total 361 132 62 47.9

Similar seasonal variation was observed for the number of embryos and plants (215 and 132 embryos, 101 and 62 haploid plants). This situation could be brought about by the fact that spring climatic conditions are usually more favorable for cucumber growth and pollination. Moreover, the figures confirm our results (Niemirowicz-Szczytt et al., 2004) obtained previously with other cucumber varieties (Polan, Krak, Frykas and Izyd). This may indicate that parthenocarpic cucumber plants show similar response to irradiated pollen as the varieties which are not considered to be parthenocarpic. Similarly, the percentage of embryos capable of further development was approximately the same in both seasons (45% and 48% respectively). Table 2 shows the results of direct regeneration of plants from leaf explants of haploids obtained in the first experiment.

412 Table 2. Haploid plants possessing direct regeneration ability from juvenile leaf explant (2003)

Donor No of haploid Explant donor Regenerating explant donor cultivar (F ) plants plants plants 1 No. % No. %

Pol 10 23 21 91.3 5 21.7 Rubin 20 18 90.0 8 40.0 S³awko 15 11 73.3 7 46.7 Izyd 43 40 93.0 20 46.5 Total 101 90 86.9 40 38.7

Out of 101 plants 90 could be donors of proper juvenile leaves. This also means that the 90 plants showed regular development. During the three months of experiments plant regeneration was observed on 40 leaf explants. This number can be increased as the process of regeneration has not been completed yet. However, it is sure that not all the plants can be donors of suitable juvenile leaves and not all the explants are capable of plant regeneration.

Conclusions It was proved that haploid embryos and plants could be developed from parthenocarpic cucumber hybrids. Besides, there are no significant differences between parthenocarpic donors and the standard. All this requires further confirmation through at least one more year of experiments.

References

Burza, W. and Malepszy, S. 1995. Direct regeneration from leaf explant in cucumber (Cucumis sativus L.) is free of stable genetic variation. Plant Breeding, 114: 341-345. Faris, N.M. and Niemirowicz-Szczytt, K. 1999. Cucumber (Cucumis sativus L.) embryo development in situ after pollination with irradiated pollen. Acta Biol. Cracov. Ser. Bot., 41: 111-118. Faris, N.M., Rakoczy-Trojanowska, M., Malepszy, S. and Niemirowicz-Szczytt, K. 2000. Diploi- dization of cucumber (Cucumis sativus L.) haploids by in vitro culture of leaf explant. In: Bielecki, S., Tramper, J. and Polak, J. (Eds.), Food Biotechnology. Elsevier Science, pp. 49-54. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Niemirowicz-Szczytt, K. and Dumas de Vaulx, R. 1989. Preliminary data on haploid cucumber (Cucumis sativus L.) induction. Cucurbit Genet. Coop. Rep., 12: 24-25. Niemirowicz-Szczytt, K., Faris, N.M., Ruciñska, M. and Nikolova, V. 2000. Conservation and storage of a haploid cucumber (Cucumis sativus L.) collection under in vitro conditions. Plant Cell Rep., 19: 311-314. Niemirowicz-Szczytt, K., miech, M., Sztangret, J., Ga³ecka, T., Korzeniewska, A., Marzec, L., Ko³akowska, G. and Piskurewicz, U. 2004. Cucumber (Cucumis sativus L.) doubled haploid lines resistant to downy mildew (Pseudoperonospora cubensis). Plant Breeding (in press)

413 Nikolova, V. and Niemirowicz-Szczytt, K. 1996. Diploidization of cucumber (Cucumis sativus L.) haploids by colchicine treatment. Acta Soc. Bot. Pol., 65: 311-317. Przyborowski, J. and Niemirowicz-Szczytt, K. 1994. Main factors affecting cucumber (Cucumis sativus L.) haploid embryo development and characteristics. Plant Breeding, 112: 70-75.

414 Embryo and ovule cultures in Cucumis species and their utilization in interspecific hybridization

D. Skálová, A. Lebeda and B. Navrátilová Palacký University, Faculty of Science, Department of Botany, lechtitelù 11, 783 71 Olomouc-Holice, Czech Republic; e-mail: [email protected]

Summary

Cucumis sativus is one of the most economically important crops of the Cucurbitaceae. Recent cucumber cultivars display rather limited genetic variation and are susceptible to some serious diseases and pests (cucumber downy mildew, powdery mildew, nematodes, spider mites etc.). Sources of genetic resistance to these pathogens and pests are known to exist in certain wild Cucumis species. One possible method to introduce these resistance genes to cucumber cultivars is through interspecific hybridization. However, C. sativus is sexually incompatible with nearly all other Cucumis species, because of substantially different chromosome numbers (2n = 14 in C. sativus versus 2n = 24 in Cucumis melo and most wild Cucumis species). One way to overcome this crossability barrier is to use embryo rescue and/or ovule cultures. These techniques have been developed and sometimes used successfuly to obtain interspecific Cucumis hybrids. Recent progress and future trends in this area are summarized and discussed in this paper.

Keywords: Cucumis sativus, wild Cucumis spp., disease resistance, interspecific hybridization, sexual compatibility/incompatibility, embryo rescue and ovule cultures, embryogenesis, plant regeneration, cucumber, plant breeding

Introduction The Cucurbitaceae consists of about 118 genera and about 825 species almost equally divided between the New and Old World tropics (Jeffrey, 2001). Cucurbits are typically warm-season crops, given their tropical origins (Chadha and Lal, 1993). There are two Cucumis species of economic importance, cucumber (Cucumis sativus L.) and melon (Cucumis melo L). The genus Cucumis is represented by two distinct subgenera differing in their origins (Asia and Africa) and basic chromosome numbers (Singh, 1990; Chen and Kirkbride, 2001; Jeffrey, 2001). Both taxonomic groups are evolutionary isolated (Perl-Treves et al., 1985). To the Asian group (basic chromosome number generally n = 7) belongs subgenus Cucumis, which includes Cucumis sativus (represented by three varieties: var. sativus, var. hardwickii (Royle) Gabaev and var. xishuangbannanesis ined.) and C. hystrix Chakrav. Although C. hystrix morphologically and biochemically resembles C. sativus, its chromosome number is the same as C. melo (Chen et al., 1995). Other Cucumis species are included in subgenus Melo (Miller) C. Jeffrey (represented by six series and 30 species) and are primarily of Af- rican origin; their basic chromosome number is n = 12 (Køístková and Lebeda, 1995). A detailed review of Cucumis species and their systematics, origins, gene centres, and chromosome numbers has been summarized elsewhere (Jelaska, 1986; Kirkbride, 1993; Lebeda and Køístková, 1993; Køístková and Lebeda, 1995; Køístková et al., 2003). Genetic variability within C. sativus is rather limited (Knerr et al., 1989; Lebeda and Køístková, 1993; Lebeda et al., 1993), and this species is sexually incompatible

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 415 with most other Cucumis species because of cytogenetic differences (Raamsdonk et al., 1989; den Nijs and Custers, 1990; Lebeda et al., 1996). Cucumbers are attacked by many diseases and pests (e.g., cucumber downy mildew, powdery mildew, root- knot nematodes Meloidogyne spp.), which can reduce both yield and quality (Zit- ter et al., 1996). The identification and incorporation of resistance against various pathogens and pests are considered among the most important features of contemporary cucumber breeding (Lebeda, 1992a). Genes for resistance to several pathogens and pests that are not known to occur in cucumber have been found in some accessions of wild Cucumis species (Leppik, 1966; Chen et al., 2003). Accessions of C. anguria L., C. ficifolius A. Rich., C. metuliferus E. Mey ex Naudin and C. zeyheri Sond. have high levels of resistance to root-knot nematode (den Nijs and Custers, 1990; Wehner et al., 1990). And C. myriocarpus Naudin possesses resistance to Tetranychus urticae, Podosphaera xanthii (Sphaerotheca fuliginea), Fusarium oxysporum, Didymella bryoniae and cucumber green mottle mosaic virus (CGMMV) (Lebeda, 1984; Lhotský et al., 1991; Bordas et al., 1998). Very high levels of resistance to Tetranychus urticae were found in some accessions of C. africanus L. f., C. figarei and C. zeyheri (Lebeda, 1996). Cucumis metuliferus also displays resistance to squash mosaic virus and watermelon mosaic virus (McCarthy et al., 2001). However, for certain other diseases, such as cucumber downy mildew, it is very difficult to locate good sources of resistance in either- cultivated (Lebeda, 1992b; Lebeda and Práil, 1994) or wild relatives (Lebeda, 1992c). Nevertheless, C. melo (line MR 1 and some other accessions) has some resistance to cucumber downy mildew (Pseudoperonospora cubensis) (Lebeda et al., 1996; Lebeda, 1999). To incorporate these valuable resistance genes from wild species into cucumber breeding programmes, it is evidently necessary to use unconventional techniques, including various methods of in vitro cultures, because of hybridization barriers and embryo abortion early in embryo development (globular stage) (Ondøej et al., 2001). One of these techniques combines embryo rescue and ovule culture, presenting a possible way to obtain hybrids from interspecific crosses (Chen and Staub, 1996; Lebeda et al., 1993, 1996, 1999; Chen and Adelberg, 2000). This paper presents a review of all aspects of embryo rescue, ovule culture, and embryogenesis in the genus Cucumis, examining the development of methods, results, and potential utility of such methods in the interspecific transfer of useful traits to cucumber, including a review of the characterization of interspecific hybrids.

Embryo and ovule culture and embryogenesis in the genus Cucumis

Embryo and ovule culture Techniques for the in vitro culture of embryos are potentially useful in two important areas of cucumber breeding. Culture of immature embryos can be used: a) to reduce generation time and permit more rapid selection, and b) to obtain plants from normally inviable interspecific hybrids before embryo abortion (Wehner et al., 1990; Beharav and Cohen, 1995a,b; Chen and Staub, 1996; Lebeda et al., 1996). The culture of immature zygotic embryos is one of the oldest methods of in vitro cultures (Navrátilová, 1992; Sharma et al., 1996; Batygina, 1999; Raghavan, 2003).

416 In this method, female flowers are self- or crosspollinated and immature to mature embryos are dissected from surfacesterilized fruits and cultured on an agar medium with the addition of various growth regulators, sucrose, and the other components. Proembryos and immature embryos require a higher osmoticum (sucrose giving the best osmotic characteristics) and higher concentrations of growth regulators, especially cytokinins (Preová, 1995). Young embryos of C. sativus were cultured on media supplemented with IAA, KIN or on Norstogs medium (Jelaska, 1986). Coconut water in Goralski and Przywaras medium was also suitable for immature embryos (Goralski and Przywara, 1998). In some cases coconut milk can be replaced by yeast extract or casein hydrolysate (Niemirowicz-Szczytt and Wyszogrodzka, 1976). Mature embryos of C. sativus were cultivated on MS (Murashige and Skoog, 1962) medium without growth regulators or on media supplemented with IBA, BAP and ascorbic acid (Ond- øej et al., 2000) or casein hydrolysate (Ondøej et al., 2002a). On these media, both immature and mature embryos from C. melo, C. zeyheri and C. metuliferus were successfully cultured (Ondøej et al., 2000). GA had a positive influence on the development of mature embryos of C. melo (Beherav and Cohen, 1995a). Haploid embroys from immature fruits of C. melo were used for dihaploidization (plantlets developed in vitro were immersed in colchicine in vivo) (Yetisir and Sari, 2003). Custers (1982) used medium with IAA and KIN for C. zeyheri embryo culture. The frequency of regeneration of normal plants from cucumber tissue culture remains rather low (Lou and Kako, 1994). In addition, regeneration of abnormal embryos, recalusing or vitrification have been often observed (Bergervoet et al., 1989). In cucumber plants regenerated via organogenesis it was concluded that the decrease in regeneration competence corresponded with endopolyploidization (Colijn-Hooymans et al., 1994). Similar phenomenon, i.e. a close link between the polyploidization and the loss of totipotency in vitro, was observed during regeneration of cucumber (C. sativus) immature embryos (Kubaláková et al., 1996). Regeneration of plants was observed after a transfer to culture media either without growth regulators or supplemented with kinetin and NAA. However, callus cultures were mixoploid. The frequency of polyploid cells was increasing with the age of culture and the polyploidization was accompanied by a gradual loss of regeneration ability. Another method is represented by the culture of excised, intact ovules and their subsequent in vitro pollination. Excised ovules were pollinated in vitro in agar media (MS, NITSCH) with different concentrations of sacharose and with or without growth regulators and vitamins. The ovaries were surface sterilized. Pollen grains were obtained from male flowers isolated by cotton without sterilization and then manually pollinated in vitro (Niemirowicz-Szczytt and Wyszogrodzka, 1976). In vitro ovule cultures of C. sativus were established on a medium employing the osmotic effect of a doublelayer culture medium system. Dissected ovules (harvested two days after pollination) were transfered into the doublelayer culture media of Monnier (1995) and Goralski and Przywara (1998). After three weeks, cultures were supplemented by adding 0.4 ml of the medium of Ondøej and Navrátilová (2000) (Ondøej et al., 2002a,b). Some of the most important culture media used for embryo and ovule culture are summarized in Table 1.

417 Table 1. Media for embryo and ovule culture of Cucumis species

Species Stage Medium Reference of embryo development

C. sativus Immature Norstogs (1967) Jelaska (1986) Nitsch and Nitschs (1969) Jelaska (1986) Matured on MS (NAA 1 mg/l; Trulson and Shanin BAP 0.5 mg/l), devoloped (1986) on MS hormone-free medium LS (Linsmaier and Skoog, 1965), Malepszy (1988) N (Nitsch, 1951), MS Immature, Monnier (1973) Preová (1995) mature Goralski and Przywaras (1998) Goralski and Przywara (1998) MS (casein hydrolysate 1 g/l) Custers (1981) OK (sucrose 20 g/l; ascorbic acid Ondøej et al. (2000) 20 mg/l; IBA, BAP 0.01 mg/l) Mature MSC (sucrose 30 g/l; BAP 1 mg/l; Ondøej et al. (2000) NAA 2.5 mg/l) ON (sucrose 20 g/l; IBA, BAP 0.01 Ondøej et al. (2002a) mg/l; casein hydrolysate 1 g/l) C. melo Mature Vasils (1959) Jelaska (1986) MS (GA 0.5 mg/l ; KIN 0.5 mg/l) Beharav and Cohen 3 (1995a) Immature, OK (sucrose 20 g/l; ascorbic acid Ondøej et al. (2000) mature 20 mg/l; IBA, BAP 0.01 mg/l) C. zeyheri Immature, MS (IAA 0.01 mg/l; KIN 0.10 mg/l) Custers (1982) mature OK (sucrose 20 g/l; ascorbic acid Ondøej et al. (2000) 20 mg/l; IBA, BAP 0.01 mg/l) C. Immature, OK (ascorbic acid 20 mg/l; Ondøej et al. (2000) metuliferus mature IBA, BAP 0.01 mg/l)

418 A B

C D

E F

Figure 1. Embryo culture in the genus Cucumis. A Mother flowers of Cucumis sativus grown in the glasshouse. B Isolated embryos in the test-tubes in the cultivation room. C Germinated embryo of Cucumis melo, cv. Solatur 2 weeks after self-pollination. D Embryo of Cucumis melo after 3 weeks of cultivation. E In vitro pollination in the cross-pollination between Cucumis sativus (female flowers) and Cucumis melo (male flowers). F Germinated pollen tubes of Cucumis melo (coloured by FDA).

419 Embryogenesis

Somatic embryogenesis Somatic embryogenesis is the process of embryo formation from sporophytic tissues (Batygina, 1999; Raghavan, 2003). In the regeneration of Cucurbitaceae, it can occur either through a caulogenic or an embryogenic developmental pathway (Cus- ters, 1981; Jelaska, 1986). Somatic embryogenesis and plant recovery have been obtained from numerous explants, including protoplasts, but the best results have been observed from explants originating from seedlings (cotyledons and hypocotyls) (De- beaujon and Branchard, 1993). Somatic embryos from various explants, including cotyledons, hypocotyls, true leaves, shoot tips, stems, petioles and seeds have been cultured on various media supplemented with growth regulators, sucrose, agar, etc. (Debeaujon and Branchard, 1993; Lou and Kako, 1994). For C. sativus this process requires two media: a) an induction medium for embryogenic cell determination, and b) a maturation medium allowing embryogenic development (Debeaujon and Branchard, 1993). Lou and Kako (1994) successfully used the initiation medium for the first 3 weeks and then transfered calli to the induction medium for an additional 23 weeks. Higher sucrose concentrations in the initiation medium significantly increased the percentage of somatic embryo formation. Sucrose induced somatic embryogenesis, and, in the 200 mM sucrose treatment, somatic embryos were readily formed. Higher sucrose concentrations in the maturation medium decreased the total number of embryos and the number of normal embryos. Follow-up experiments (Lou and Kako, 1995) indicated that sucrose and glucose, at optimized concentrations in the initiation medium, gave high frequenceies of somatic hybridization of C. sativus and also determined that no embryos were formed when mannitol was included in the initiation medium. The influence of sugars, abscisic acid (ABA) and mannitol on somatic embryogenesis of C. melo was the same as the effects of these components on C. sativus embryogenesis. ABA and sucrose induced somatic hybridization and mannitol increased the number of somatic embryos (Nakagawa et al., 2001). Selected explant sources and culture media for somatic embryogenesis in Cucu- mis are summarized in the Table 2 .

Gametic embryogenesis Progress in the development of in vitro techniques has also led to increased emphasis on the utilisation of anther/microspore culture for genetic improvement. An- ther and microspore culture techniques of C. sativus are becoming important for haploid plant production and for generating de novo genetic variation (Ashok Kumar and Murthym, 2003). Selected culture media for gametic embryogenesis are summarized in the Table 3.

420 Table 2. Explant sources and culture media for somatic embryogenesis in Cucumis spp.

Species Explant source Medium Reference

C. sativus Leaf, stem MS (BAP, 2,4-D 0.4 mg/l) Debeaujon and Branchard (1993) Leaf MS (2,4 D 0.8 mg/l; Debeaujon and 0.8ip mg/l) Branchard (1993) Hypocotyl Liquid media with ABA, Ziv and Gadasi zeatin or activated charcoal (1986) Cotyledon, root MS (2,4 D, NAA 1 mg/l, Trulson and Shanin BAP 0.5 mg/l) (1986) Cotyledon, root MS (2,4 D, NAA 1 mg/l) Debeaujon and Branchard (1993) Cotyledon Initiation medium with 0.25 M Lou and Kako (1995) sucrose and 0.15 M glucose

C. melo Seed, leaf, stem MS (2,4 D 2 mg/l; NAA 3 Tabei et al. (1991) hypocotyl, mg/l; IAA 25 mg/l) cotyledon Seed N6 (2,4 D 3 mg/l ; Oridate et al. (1992) BAP 0.1 mg/l) Leaf, stem MS (2,4 D 1 mg/l; Debeaujon and NAA 2 mg/l) Branchard (1993) Leaf Growth regulator pretreatment Kintzios et al. (2002) 226.2 mM 2,4D for 48 h, transfer to an agar medium (without growth regulators) Hypocotyl, MS (IAA, KIN) Debeaujon and cotyledon Branchard (1993) Hypocotyl MS (2,4D 1 mg/l; NAA 2 mg/l; Ezura and Oosawa BAP 0.1 mg/l, 3% sucrose), (1994) growing callus transferred to MS hormone free medium Cotyledon MS (2,4 D, NAA 1 mg/l, Trulson and Shanin BAP 0.5 mg/l) (1986) C. melo Cotyledon MS (2,4D 0.05 mM; BA Kintzios and 0.26 mM), MS (2,4 D 9.0 mM; Taravira (1997) KIN 23.2 mM),for embryo maturation growing callus transferred to MS hormone-free medium Cotyledon MS (0.5 M ABA; 200 mM Nakagawa et al. sucrose) (2001) Cotyledon IK 1560 Moreno et al. (1985) N medium Curuk et al. (2002)

421 Table 3. Culture media for gametic embryogenesis in Cucumis sativus

Species Gamete type Medium Reference

C. sativus Anther pollen N 1 (modified from Nitsch, Malepszy (1988) 1969), MS (Murashige and Skoog, 1962); MS (Murashige and Skoog, 1962), Preová (1995) N6 (Chu, 1978); B5 (Gamborgs medium; Ashok Kumar Gamborg et al., 1968) (2,4 D; NAA and Murthym (2003) or 2,4 D; BAP/KN/TDZ) Different media (MS, B5, N6) Wehner et al. (1990) Ovules LS (Linsmaier and Skoog, 1965), Malepszy (1988) MS (Murashige and Skoog, 1962), N (Nitsch, 1951)

Interspecific hybridization of Cucumis species

The main aim of interspecific hybridization in cucurbits is to introduce disease resistance from wild species into cultivated ones (Jelaska, 1986). Most hybridization experiments have been focused on hybridization between C. sativus and C. melo, or between C. sativus and various wild Cucumis species (den Nijs and Custers, 1990; Lebeda et al., 1996, 1999; Chen and Adelberg, 2000). Research efforts in this area are strongly driven by the need to develop cucumbers resistant to various pathogens, especially Pseudoperonospora cubensis. Some valuable sources of resistance were detected in accessions of C. melo (Lebeda, 1999) and used for hybridization with C. sativus (Lebeda et al., 1996, 1999). In general, multiple crossing barriers prevent sexual hybridization between the two Cucumis subgenera. There are prezygotic barriers (factors hindering effective fertilization) and postzygotic barriers (occurring during or after syngamy). The characterization of these crossing barriers can help in the development of protocols to overcome these obstacles (Ondøej et al., 2001). One strategy to overcome these crossing barriers was to use irradiation pollen for crosspollination (Custers and Bergervoet, 1984). This IMP (irradiation mentor pollen) techniques efectively induced fruit set, but an increasing dose of radiation reduced fruit set. The seeds were flat and empty or their development stopped after few days (Beherav and Cohen, 1995b). In addition, Custers et al. (1981) exploited AVG (aminoethoxyvinylglycine) with positive effects on the success of reciprocal crosses between C. africanus and C. metuliferus. A few years later, the application of AVG to pollinated flowers improved crossability between other Cucumis species as well (Custers and den Nijs, 1986). Another approach to overcome hybridization barriers relies on the use of plant growth regulators to influence the receptivity of the maternal parent, as first established through the application of auxins in lanolin paste to cucumber floral stems and stigmata during pollination (Smith and Venkat Ram, 1954). This method

422 was also used by Lebeda et al. (1996, 1999) to promote the development of hybrid fruits, seeds and embryos. Wounds to the stigmatic surface were also shown to improve pollen tube growth (de Vaulx, 1979). Experiments with BA (benzyladenine) treatment and bud pollination were ineffective in overcoming the hybridization barriers between C. melo and C. metuliferus (no fruit set was observed) (Beherav and Cohen, 1995b). Embryo rescue, after in vitro pollination, has also been used to overcome crossing barriers (Ondøej et al., 2002a,c). The penetration of ovules of C. sativus and C. melo by pollen tubes of the same or another species was observed during in vitro pollination. The embryos (in globular stage) were observed in the immature seeds of interspecific and non-interspecific origin. However, the development mostly stopped in the globular stage. Some calli of interspecific and non-interspecific origin were obtained. Organogenesis of calli was not successfull because of high level of polyploidy. Their isozymes were analyzed (esterases, phosphatases) and confirmed hybrid origin (Ondøej et al., 2002c). Some key results of interspecific hybridization experiments in the genus Cucumis are summarized in Table 4.

Characterization of interspecific hybrids Some of the experiments listed in Table 4 were successful. Among them include interspecific crosses between C. sativus and C. melo (Lebeda et al., 1996, 1997, 1999) and hybrids between C. sativus and C. hystrix (Chen and Staub, 1996), which were described as a new nothospecies Cucumis x hytivus J. F. Chen & J. H. Kirkbr. (Chen and Kirkbride, 2001), and have been subjected to detailed morphological and caryo- logical characterization (Chen et al., 2003). In the period of 19921994 was realised the programme of interspecific crossing of C. sativus and C. melo with an aim to transfer resistance to Pseudoperonospora cubensis originating from melon (Lebeda et al., 1996). After a conventional pollination of cucumber with melon pollen grains there was observed fruit formation. Excis- sed young seeds and embryos had been cultivated in vitro on different media and regeneration of seven embryos was observed. Five embryos developed into the callus and two embryos developed into the flowering plants (Lebeda et al., 1996). These regenerated plants were compared morphologically, caryologically, and by isozyme and flow cytometry DNA analysis with their parents. The morphology of regenerated plants resembled to the maternal parent (C. sativus), and DNAflow cytometric analysis also showed that their DNA content was not significantly different from the maternal parent. However, the isozyme analysis supported a hybrid parentage, and levels of resistance to P. cubensis observed in these regenerants was higher than observed in C. sativus, leading to a conclusion that these hybrids were not symmetrical (Lebeda et al., 1997, 1999). The other successful experiment with interspecific hybridization was the cross between C. sativus and C. hystrix, a wild species from subgenus Cucumis, native to Asia, but with a basic chromosome number of n = 24 (Chen et al., 1995). Interspecific hybridization was made by conventional crossing of C. sativus (maternal parent) with C. hystrix (paternal parent). The 59 plants of F hybrid generation were obtained through em- 1 bryo rescue and were characterized as n = 19 (12 from C. hystrix and 7 from C. sativus), and were both male and female sterile (Chen and Staub, 1996). The restoration

423 Table 4. The review of interspecific hybridization between Cucumis species

Species Results Reference

C. sativus x C. melo Embryos in the globular stage Niemirowicz-Szczytt and Megurk 2n = 19 (fertile) Wyszogrodzka (1976); mixed pollen Ondøej et al. (2001) Embryos (5 callus formation, 2 van der Knaap et al. (1978) flowering plants) Lebeda et al. (1996, 1997, 1999) C. sativus x C. melo Embryos in the globular stage Niemirowicz-Szczytt and Kubicki (1979) C. sativus x C. metuliferus Fruit development, seed flat, Walters and Wehner (2002) (diploid, tetraploid) no viable seeds C. sativus x C. metuliferus Embryos Franken et al. (1988) C. sativus x C. hystrix Plants 2n = 19 (sterile), Cucumis Chen and Staub (1996); x hytivus 2n = 38 (amphidiploid,Chen and Kirkbride fertile) (2001); Chen et al. (2003) C. anguria x C. zeyheri Pollen tubes penetrated into the Visser and den Nijs (1983) ovules, fruits set, no viable seeds C. africanus x C. anguria Vigorous plants, some seedling den Nijs et al. (1981) death C. metuliferus x Fruit set with embryos Custers et al. (1981) C. africanus Embryos, plants Wehner et al. (1990) C. metuliferus x C. melo Embryos Fassuliotis (1977) C. metuliferus x C. melo Fertile F Norton and Granberry (1980) C. metuliferus x C. zeyheri Fruit set,1 some embryos Custers and den Nijs (1986) C. myriocarpus x Seeedling death den Nijs et al. (1981) C. africanus C. myriocarpus x Fruits with no seeds den Nijs et al. (1981) C. anguria C. melo x C. metuliferus Pollen tubes penetrated halfway de Vaulx (1979) down the style Embryos Kho et al. (1981), Soria et al. (1990) Embryos, plants Wehner et al. (1990) Pollen tubes penetrated into Beherav and Cohen (1994) the upper part of the style Fruit set (irradiation pollen, Beherav and Cohen high irradiation dose increases (1995b) fruit set) C. melo x C. zeyheri Pollen germination de Vaulx (1979) Unsuccessful experiment Chatterjee and More (1991) C. melo x C. myriocarpus Pollen tubes penetrated into de Vaulx (1979) the first quarter of the style Unsuccessful experiment Chatterjee and More (1991) C. melo x C. ficifolius Pollen tubes penetrated de Vaulx (1979) halfway down the style Embryos Kho et al. (1981) C. melo x C. figarei Unsuccessful experiment Chatterjee and More (1991) C. hystrix x C. sativus Fertile plants (4n) Chen et al. (1998) C. prophetarum x C. melo Fruit with inviable seeds Singh and Yadava (1984) C. sagittatus x C. melo Embryos Deakin et al. (1971) C. zeyheri x C. sativus Fruit with inviable seeds Custers and den Nijs (1986)

424 of fertility by chromosome doubling of the plants of F generation was carried out, 1 producing 62 plants with doubled chromosome numbers. Two primary amphidiploids (2n = 38) produced fertile flowers and fruit set with viable seeds. The fertile interspecific hybrid was described as a new nothospecies of Cucumis, Cucumis x hytivus J. Chen & J. H. Kirkbr. nothosp. nov. (Chen and Kirkbride, 2001). This nothospecies was morphologically and caryologically described, cytogenetic characterization of F generation plants and amphidiploid progeny was performed. Normal meioses were 1 observed to produce viable pollen grains, but meiotic anomalies were also noted (Chen et al., 2003).

Research progress in embryo and ovule cultures of Cucumis in the Department of Botany, Palacký University in Olomouc Research focusing on the improvement of embryo and ovule culture protocols has been carried out since 1992 (Lebeda et al., 1996). The influence of genotype and culture medium on the development of immature zygotic embryos of C. sativus and C. melo was studied, with two genotypes of cucumber and two genotypes of melon. Culture media were supplemented with ascorbic acid and growth regulators. Optimized protocols were used in interspecific hybridization experiments (Ondøej and Navrátilová, 2000). Immature seeds or embryos from C. sativus, C. melo, C. metuliferus and C. zeyheri were excised from fruits two days, and 1, 2, and 3 weeks after selfpollination and cultured on media with different concentrations of GA . Better development of em- 3 bryos was achieved on a medium lacking or with low concentrations of GA . Higher 3 concentrations of GA stimulated embryogenesis in C. sativus and proembryo cultu- 3 res (Ondøej et al., 2002b). Embryos from above-mentioned species and hybrid embryos from C. sativus x C. melo were cultured on four types of media. During the first three weeks the length of roots and shoots was higher on OK medium. Conversely, poor root and shoot development occurred on MS medium without growth regulators. Increased sucrose concentration (1C, MSC medium) promoted the highest frequency of callus formation (On- døej et al., 2000). In another experiment, ovules harvested two days after pollination were transferred into the doublelayer culture medium (Monnier, 1995; Goralski and Przywara, 1998) and after three weeks cultures were transferred to the medium developed by Ondøej and Navrátilová (2000) (Ondøej et al., 2002a). These experiments on embryo and ovule culture were used to help determine of the main crossing barriers between C. sativus and C. melo. Generally, both prezygotic and postzygotic barriers were identified, and protocols were designed to overcome them. Initial stages of embryo development were observed to investigate responses to treatments designed to overcome fertility barriers. Detailed observations of in situ and in vitro fertilization were conducted (Ondøej et al., 2001), and in vitro pollination and fertilization were used to overcome prezygotic barriers. Excised ovules and the pollen grains were transferred onto culture media in Petri dishes (pollen grains were placed on and around the ovules). After two days, fertilized ovules were transferred to four different types of media: 1) BC, used for cultivation of immature seeds); 2) OK, used for in vitro cultivation of immature embryos of C. sativus; 3) DI1c, used for cultivation of non-fertilized ovules of C. melo; and 4) DI1b. All embryos

425 aborted in the globular stage, except for those of C. melo on medium DI1b (Ondøej et al., 2002a). The experiments focused on interspecific hybridization between C. sativus and C. melo were partly successful and the results were described above (Lebeda et al., 1996, 1997, 1999).

Conclusions

1. Development of embryo and ovule cultures protocols permits the in vitro culture of immature embryos of various cultivated and wild Cucumis species, which increase the likelihood of successful interspecific hybridization. 2. Interspecific hybridization could be used to transfer valuable sources of disease and insect resistance from wild Cucumis species to C. sativus and can increase the bre- adth and diversity of the available gene pool for the improvement of C. sativus. 3. Further improvement of culture protocols and media for embryo and ovule cultures are needed to advance the practical use of these methods in varietal development.

Acknowledgements The authors thank Dr. Mark P. Widrlechner for valuable comments on this manuscript. Excellent cooperation with the USDA-ARS, North Central Regional Plant In- troduction Station, Iowa State University, Ames, Iowa (USA) in supplying cucurbit germplasm is acknowledged. This research was supported by grants: 1) NAZV No. QF 4108 Utilization of protoplast fusion technique in breeding of the important cultivated crops of the genera Brassica, Cucumis and Solanum, Ministry of Agriculture of Czech Republic (MA CR); 2) Ministry of Education of Czech Republic No. MSM 153100010.

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430 Somatic embryogenesis in a model cultivar, PI 161375 (Cucumis melo subsp. agrestis), of melon

H. Ezura1 and Y. Akasaka-Kennedy2 1Gene Research Centre, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan; e-mail: [email protected] 2Faculty of Agriculture, Iwate University, Iwate 020-8550, Japan

Summary

We investigated a plant regeneration using somatic embryogenesis in two melon cultivars: PI 161375 (Cucumis melo L. subsp. agrestis, chinensis group) and Vedrantais (C. melo L. subsp. melo, cantalupensis group). These model cultivars are used for genetic and development studies of melon. As explants, 1- to 4-mm2 sections of mature seeds were used, including the cotyledon and hypocotyl. To induce embryo formation, the explants were cultured in embryo-induction (EI) medium, which consisted of liquid MS medium containing 2 mg/l 2,4-D and 0.1 mg/l BA. After 4 weeks of culture, an average of 2.7 and 292.3 embryos per seed were produced for Vedrantais and PI 161375, respectively. After 8 weeks of culture, an increased number of somatic embryos were produced for PI 161375. Somatic embryos of Vedrantais were germinated in a higher frequency while those of PI 161375 were in a low frequency.

Keywords: melon, chinensis, cantalupensis, liquid culture, explants, somatic embryos

Introduction

The Korean melon accession Shongwan Charmi PI 161375 belongs to Cucumis melo subsp. agrestis, chinensis group (Pitrat et al., 2000), and has useful traits for melon breeding such as resistance to Fusarium wilt (Fom-1 and Fom-2), melon necrotic spot virus (nsv) and Ahips gossypi (Vat) (Baudracco-Arnas and Pitrat, 1996). This accession also has different traits of fruit quality to European and American cultivars, and should have the importance on improving the fruit quality of such cultivars (Monforte et al., 2003). PI 161375 have been used to develop a variety of maps in melon (Baudracco-Arnas and Pitrat, 1996; Danin-Poleg et al., 2000; Perin et al., 2002a). The loci of agronomically importance like Fom-2, nsv, fruits ripening and qualities have been mapped (Zhenget al., 1999; Morales et al., 2002; Perin et al., 2002b,c; Monforte et al., 2004). Therefore, PI 161375 is a model cultivar of melon study. Based on the above information, the related genes will be isolated in the near future. To confirm the function and the role of isolated genes, a reverse genetic approach is indispensable. Genetic engineering has already been used to improve fruit quality and introduce disease resistance and environmental tolerance in melon (Yoshioka et al., 1993; Ayub et al., 1996; Bordas et al., 1997). Unfortunately, in melon species, the transformation frequency is very low due to the production of escapes (Guis et al., 2000). In the previous studies, transgenic plants were generated using adventitious shoot organogenesis. To reduce the problem of escapes, an alternative regeneration system that can enable transformation is needed. We have succeeded in establishing

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 431 the transformation system via somatic embryogenesis in another model cultivar of melon, Vedrantais (Akasaka-Kennedy et al., 2004). Vedrantais is a commercial variety of the Charentais type (C. melo subsp. melo, cantalupensis group). To apply the method to PI 161375, plant regeneration via somatic embryogenesis is required. In this study, as a preliminary study for establishing genetic transformation system in melon cultivar PI 161375, we tested the methods of somatic embryogenesis for other cultivar Vedrantais. We succeeded in inducing somatic embryogenesis in PI 161375. This is the first report on somatic embryogenesis in a model melon cultivar PI 161375.

Materials and methods

Plant material Mature seeds of two melon cultivars, PI 161375 (Cucumis melo L. subsp. agrestis, chinensis group) and Vedrantais (C. melon subsp. melo, cantalupensis group) (kindly provided by Dr. M. Pitrat, INRA, Avignon, France), were harvested from our experimental field. The seeds were sterilized by soaking them in sterile distilled water for 6 hours, in accordance with Ezura et al. (1992). Then, they were cut in half lengthwise, and then crosswise, producing explants from 1 to 4 mm2 in size. Each seed yielded between twelve and twenty explants.

Somatic embryogenesis and germination The explants were cultured in liquid embryo induction (EI) medium at 25°C with a 16-hr photoperiod (30 µmol photons/m2/s) on a gyratory shaker (90 rpm). The liquid EI medium contained MS salts (Murashige and Skoog, 1962), MS vitamins, 3% sucrose, 2 mg/l 2,4-D, and 0.1 mg/l BA, as described in Akasaka-Kennedy et al. (2004). After 4 and 8 weeks in culture, the embryos were counted and then transferred onto 1.0% agar-solidified MS medium, lacking plant growth regulators, for germination. All the plates were sealed with surgical tape (Micropore surgical tape, 3M Health Care, St Paul, MN) and cultured at 25°C under a 16-hr photoperiod.

Results and discussion

Somatic embryos including globular, heart and torpedo stages and calli were observed when the explants from Vedrantais seeds were cultured in EI medium for 4 weeks (data not shown). Four weeks after transfer into solid MS medium lacking plant growth regulators, the somatic embryos were germinated and developed into plantlets. The average number of somatic embryos induced from each seed of Vedrantais was 292.3 (Table 1), coincident with our previous study (Akasaka-Kennedy et al., 2004). Whilst, after 4 weeks of culture in EI medium, friable calli were released from seed explants of PI 161375. Although a number of somatic embryos were low (2.7 per seed), somatic embryos were observed when the culture was transferred into solid MS medium (Table 1).

432 Table 1. Comparison of somatic embryogenesis in the two melon cultivars

Cultivars Number of somatic embryos per seeda Mean SD

PI 161375 2.7 3.1 Vedrantais 292.3 62.2 a Values are the Mean of three replications. The number of embryos was counted after 4 weeks of culture in liquid EI medium.

Figure 1. Somatic embryogenesis in the melon cultivar PI 161375. A. Embryogenic suspension induced 8 weeks after culture in EI medium. B. Globular stage embryos observed in the embryogenic suspension. C. Heart stage embryo observed in the embryogenic suspension. D. Rooting embryo on solid MS medium without plant growth regulators. E. and F. Cup-shaped embryos observed on solid MS medium without plant growth regulators.

433 Interestingly, when the liquid culture of PI 161375 was continued for additional 4 weeks, a number of somatic embryos, mainly at globular and heart stages were developed (Fig. 1A-C). Some embryos were further developed into cup-shaped embryos (Fig. 1E-F) and other embryos were rooted (Fig. 1D), when they were transferred into solid MS medium. However, the germination frequency of somatic embryos in PI 161375 is still low and we are improving the frequency for plant regeneration from somatic embryos. We are also testing the system of somatic embryogenesis for generating transgenic plant of PI 161375 via Agrobacterium-mediated transformation.

Conclusions

We tested the method of somatic embryogenesis, which was previously developed for the cultivar Vedrantais (Akasaka-Kennedy et al., 2004), to PI 161375, a model cultivar for genetic and molecular studies in melon. Embryogenic suspension cultures were induced by culturing the explants from mature seeds of PI 161375 in EI medium. Although the frequency was low, some of the embryos were further developed when transferred into solid MS medium without plant growth regulators. This is the first report on somatic embryogenesis in PI 161375.

Acknowledgements

This work was supported by, in part, by a Grant-in-Aid for Science Research (B) from the Japan Society for the Promotion of Science (No.15380002) to H.E.

References

Akasaka-Kennedy, Y., Tomita, K. and Ezura, H. 2004. Efficient plant regeneration and Agroba- cterium-mediated transformation via somatic embryogenesis in melon (Cucumis melo L.). Plant Sci., (in press). Ayub, R., Guis, M., Ben Amor, M., Gillot, L., Roustan, J.P., Latché, A., Bouzayen, M. and Pech, J.C. 1996. Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nature Biotech., 14: 862-866. Baudracco-Arnas, S. and Pitrat, M. 1996. A genetic map of melon (Cucumis melo L) with RFLP, RAPD, isozyme, disease resistance and morphological markers. Theor Appl Genet., 93: 57-64. Bordas, M., Montesinos, C., Dabauza, M., Salvador, A., Riog, A., Serrano, R. and Moreno, V. 1997. Transfer of the yeast salt tolerance gene HAL1 to Cucumis melo L. cultivars and in vitro evaluation of salt tolerance. Transgen. Res., 6: 41-50. Danin-Poleg, Y., Reis, N., Baudracco-Arnas, S., Pitrat, M., Staub, J.E., Oliver, M., Arus, P., deVi- cente, C.M. and Katzir, N. 2000. Simple sequence repeats in Cucumis mapping and map merging. Genome., 43: 963-974. Ezura, H., Amagi, H., Yoshioka, K. and Oosawa, K. 1992. Efficient production of tetraploid melon (Cucumis melo L.) by somatic embryogenesis. Jap. J. Breed., 42: 137-144. Guis, M., Roustan, J.P., Dogimont, C., Pitrat, M. and Pech, J.C. 1998. Melon biotechnology, Bi- otechnol. Genet. Eng. Rev., 15: 289-311. Monforte, A.J., Oliver, M., Gonzalo, M.J., Alvarez, J.M., Dolcet-Sanjuan, R. and Arus, P. 2004. Identification of quantitative trait loci involved in fruit quality traits in melon (Cucumis melo L.). Theor. Appl. Genet., (in press).

434 Morales, M., Luis-Arteaga, M., Alvarez, J.M., Dolcet-Sanjuan, R., Monfort, A., Arus, P., Garcia- Mas, J. 2002. Marker saturation of the region flanking the gene NSV conferring resistance to the melon necrotic spot Carmovirus (MNSV) in melon. J. Amer. Soc. Hort. Sci., 127: 540-544. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant., 15: 473-497. Pitrat, M., Hanelt, P. and Hammer, K. 2000. Some comments on infraspecific classification of cultivars of melon. In: Katzir, N. and Paris, H. (Eds.), Cucurbitaceae 2000, Seventh EUCARPIA Mee- ting on Cucurbit Genetics and Breeding. Maale Hahamisha (Israel.). Acta Hortic., 510: 29-36 Perin, C., Hagen, S., De Conto, V., Katzir, N., Danin-Poleg, Y., Portnoy, V., Baudracco-Arnas, S., Chadoeuf, J., Dogimont, C. and Pitrat, M. 2002a. A reference map of Cucumis melo based on two recombinant inbred line populations. Theor. Appl. Genet., 104: 1017-1034. Perin, C., Gomez-Jimenez, M., Hagen, L., Dogimont, C., Pech, J.C., Latche, A., Pitrat, M. and Lelievre, J.M. 2002b. Molecular and genetic characterization of a non-climacteric phenotype in melon reveals two loci conferring altered ethylene response in fruit. Plant Physiol., 129: 300-309. Perin, C., Hagen, L.S., Giovinazzo, N., Besombes, D., Dogimont, C. and Pitrat, M. 2002c. Genetic control of fruit shape acts prior to anthesis in melon (Cucumis melo L.). Mol. Genet. Genom., 266: 933-941. Yoshioka, K., Hanada, K., Harada, T., Minore, Y. and Oosawa, K. 1993. Virus resistance in transgenic melon plants that express the cucumber mosaic virus coat protein gene and in their progeny. Jap. J. Breed., 43: 629-634. Zheng, X.Y., Wolff, D.W., Baudracco-Arnas, S. and Pitrat, M. 1999. Development and utility of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLPs) linked to the Fom-2 fusarium wilt resistance gene in melon (Cucumis melo L.). Theor. Appl. Genet., 99: 453-463.

435 436 Regeneration ability of some Hungarian melon varieties

E. Kiss-Bába, S. Pánczél, V. Zarka, Gy.D. Bisztray and I. Velich Budapest University of Economic Sciences and Public Administration, Faculty of Horticultural Science, Department of Genetics and Horticultural Plant Breeding, Ménesi Street 44/A, Budapest, Hungary

Summary

Eight genotypes of Hungarian melon (Cucumis melo L.) were selected for investigation of regeneration compared to the popular Hales Best. The effects of combinations of BA, IAA, ABA, NAA, 2,4D in culture medium on shoot regeneration of cotyledon explants were tested. The age of cotyledons had a great effect on result of regeneration. The shoot regeneration response was significantly higher on those media that only contained BA as a growth regulator.

Keywords: Cucumis melo, in vitro, regeneration, cotyledon Abbreviations: 2,4 D - 2,4 dichlorophenoxyacetic acid, ABA - abscisic acid, BA - 6-benzylami- nopurine, IAA - indole-3-acetic acid, NAA - 1 naphthalene acetic acid

Introduction

Muskmelon (Cucumis melo L.) is an important vegetable crop that is cultivated all over the world. With the increase of consumption level, varieties cannot meet the ever growing requirements. The development of genetic transformation for melon offers the potential of introducing valuable traits into this crop, e.g. disease resistance, high sugar content and high protein content to improve its productivity and quality beyond the limits of conventional breeding. Regeneration of muskmelon has been reported from leaves (Kathal et al., 1988), cotyledonary explants (Moreno et al., 1985; Niedz et al., 1989), hypocotyl explants (Moreno et al., 1985) and protoplast-derived calli (Li et al., 1990; Debeaujon and Branchard, 1991). Genetic transformation has also been reported several times (Fang and Grumet, 1990; Dong et al., 1991). Regeneration ability of some Hunga- rian muskmelon cultivars was tested as first step for further transformation experiments. The tested varieties were those, which has special value from some respects. The purpose of this study was to screen the regeneration ability of Hungarian melon varieties.

Material and methods

Plant material Nine genotypes of Cucumis melo L.: Tétényi Csereshéjú, Magyar Kincs, Javított Zentai, Hógolyó, Muskotály, Fortuna, Topáz, Ezüst Ananász, Hales Best were selected for investigation of regeneration. Seeds were obtained from the National Institute of Agricultural Quality Control (Budapest, Hungary). The seed coat of melon was removed and surface sterilized for 20 minutes in solution of 15% (v/v) Clorox (5.25% sodium hypochlorite). After washing with sterile

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 437 distilled water, the seeds were sown on hormone-free MS medium (Murashige and Skoog, 1962) supplemented with 3% sucrose and 2.5 g/l Phytagel (Sigma). The media were sterilized by autoclaving at 120°C for 20 min. The seeds for germination were incubated in thermostatic box at 32°C for 2 days, than transferred to the growth room at 25°C, with 16h/8h light/dark photoperiod provided by cool-white fluorescent lamps. Approximate- ly four- or five-day old green, half-expanded cotyledons were used in the experiments.

Culture conditions The four- or five-day-old cotyledons were halved in transverse manner. Explants were put in 8 cm diameter Petri dishes (six explants per one Petri dish) with the abaxial side on MS medium supplemented with five kind of hormone combination (pH 5.6 5.8) in a growth chamber at 27°C, with 16 h photoperiod provided by cool-white fluorescent lamps. In the experiment five hormone combinations were used: A MS + 0.6 mg/l BA, 1 mg/l IAA and 0.24 mg/l ABA; B MS + 0.5 mg/l BA and 1 mg/l NAA; C MS + 0.5 mg/l BA and 1 mg/l 2,4 D; D MS + 0.5 mg/l BA; E MS + 1 mg/l BA. From every variety two hundred cotyledon segments were tested on each type of media in an experiment. Three independent experiments were made. As soon as shoots were well formed, explants were transferred to hormone-free MS medium to enhance shoot development. Then shoots were cut from explants and transferred to rooting medium (MS medium + 0.01 NAA) solidified with 6.5 g/l agar. Rooted plantlets were transferred to soil/vermiculite mixture (1:1) and incubated some days under 100% relative humidity at 23±3°C. After several days they were transferred to a greenhouse.

Results and discussion The age of cotyledons had a significant effect on the result of regeneration as it was found earlier by Niedz et al. (1989) for Hale Best Jumbo. Greater regeneration rate was obtained from explants of 4- or 5-day-old (half expanded) cotyledons than from explants from 6- or more day-old (fully expanded) cotyledons. On fully expanded cotyledons we could only detect callus development. Callus formation was easily obtained from all of the tested varieties. Some of these were embryogenic calli that produced shoots after few weeks (Table 1). From some calli (C) shoot buds (S) or leafy shoots (L) were formed. Direct shoot formation via organogenesis was observed on D or E media from Javitott Zentai, Hógolyó and Magyar Kincs. Regeneration via somatic embryogenesis (EC+L) also occurred but it was less effective (1-10%) than organogenesis (L) producing leafy shoots (15-35%). According to the results of three independent experiments from the tested varieties, the Hógolyó, Magyar Kincs, Javított Zentai and Hales Best performed the best ability for regeneration. The shoot regeneration response was significantly higher on those media that only contained BA (D and E). Results on E and D media were significant at the 5% probability level compared to A, B and C media.

438 Table 1. Regeneration ability from half expanded cotyledons of the tested varieties

Media Varieties A B C D E

Javított Zentai C C+L EC L* L* Muskotály C C C EC EC+S Topáz C C+S C C+S S Hógolyó C+S EC+L C L* L* Magyar Kincs EC+S EC+L C C L* Ezüst Ananász EC C+S C EC S Fortuna C EC C EC S Tétényi csereshéjú EC C C EC+L S Hales Best EC+L EC C EC+L* L*

C callus, EC embryo-like callus, S shoot buds, L leafy shoots, * significant (p<0.05)

When the leafy shoots were well formed they were cut and transferred to root induction media. After 16 weeks we observed well differentiated rooty plantlets. It has been found that the shoot forming was primarily obtained on the wounded surface or its surrounding tissues, which benefits an Agrobacterium-mediated transformation. This is the first report for successful plant regeneration from cotyledon segments of the Hungarian melon varieties: Javított Zentai, Hógolyó, Magyar Kincs.

References

Debeaujon, I. and Branchard, M. 1991. Somatic embryogenesis and organogenesis from protoplast-derived cultures of muskmelon (Cucumis melo L.). Acta Hort., 289: 225-227. Dong, J.Z., Yang, M.Z., Jia, S.R. and Chua, N.H. 1991. Transformation of melon (Cucumis melo L.), and expression from the cauliflower mosaic virus 35S promoter in transgenic melon plants. Bio/Technol., 9: 858-863. Fang, G. and Grumet, R. 1990. Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants. Plant Cell Rep., 9: 160-164. Kathal, R., Bhatnagar, S.P. and Bhojwani, S.S. 1988. Regeneration of plants from leaf explant of Cucumis melo cv. Pusa Sharbati. Plant Cell Rep., 7: 449-451. Li, R., Sun, Y., Zhang, L. and Li, X. 1990. Plant regeneration from cotyledon protoplasts of Xinjiang muskmelon. Plant Cell Rep., 9: 199-203. Moreno, V., Garcia-Sogo, M., Granell, I., Garcia-Sogo, B. and Roig, L.A. 1985. Plant regeneration from calli of melon (Cucumis melo L. cv. Amarillo Oro ). Plant Cell Tiss. Org. Cult., 5: 139-146. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 155: 473-497. Niedz, R.P., Smith, S.S., Dunbar, K.B., Stephens, C.T. and Murakishi, H.H. 1989. Factors influencing shoot regeneration from cotyledonary explants of Cucumis melo. Plant Cell Tiss. Org. Cult., 18: 313-319.

439 440 Protoplast cultures of Cucumis and Cucurbita spp.

J. Gajdová, A. Lebeda and B. Navrátilová Palacký University, Faculty of Science, Department of Botany, lechtitelù 11, 783 71 Olomouc-Holice, Czech Republic; e-mail: [email protected]

Summary

The aim of this paper is to present a critical analysis of the available information about protoplast cultures of Cucurbitaceae with the main focus on Cucumis and Cucurbita spp. Research on protoplast cultures in Cucurbitaceae began in 1975. Since that time, the regeneration of intact plants has been achieved in Cucumis sativus and Cucumis melo by using various isolation and culture protocols. Protoplast fusion led to the development of somatic hybrids of Cucumis melo with Cucurbita moschata x C. maxima, and of Cucumis melo with C. myriocarpus, however these hybrids were sterile. Future trends and progress in Cucurbitaceae protoplast culture research are discussed.

Keywords: Cucumis, Cucurbita, disease resistance, interspecific hybridization, protoplast isolation and fusion, plant regeneration, somatic hybridization

Introduction

The family Cucurbitaceae is represented by 118 genera and more tha 825 species (Jeffrey, 2001). In temperate regions, five economically important species (Cucumis sativus, Cucumis melo, Cucurbita pepo, Cucurbita maxima and Citrullus lanatus) are cultivated. Cucumis sativus originated from India, and its basic chromosome number is n = 7, which it shares only with its wild relative, C. sativus var. hardwickii. The other wild Cucumis species generally originated in Africa, and their basic chromosome number is n = 12, including the Asian species C. hystrix (Chen et al., 1995), which has been shown to be sexually compatible with C. sativus (Chen et al., 1997). Other wild Cucumis spp. differing in chromosome number are sexually incompatible with cucumber. All Cucurbita species originate from the New World, and they share a basic chromosome number of n = 20 (Køístková and Lebeda, 1995). Cucumis melo and Cucumis sativus belong to the most important vegetable crops in the Cucurbitaceae (Robinson and Decker-Walters, 1997). Melon and cucumber (like other cucurbits) are attacked by numerous diseases and pests (e.g., many viruses, Pseu- doperonospora cubensis, Podosphaera xanthii, Golovinomyces cichoracearum, root nematodes Meloidogyne spp., spider mites, etc.) that cause serious losses in the yield and quality of fruits (Zitter et al., 1996). One of the main objectives in cucurbits breeding is the introduction of a new and efficient sources of resistance (Lebeda, 1992). Application of classical breeding methods has been insufficient because of the lack of broad genetic variation and valuable sources of resistance among lines and cultivars of C. sativus (Knerr et al., 1989; Lebeda and Køístková, 1993). However, closely related wild species, for example several accessions of Cucumis anguria var. longipes are resistant to Golovinomyces cichoracearum, Fusarium oxysporum, Meloidogy-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 441 ne spp., Tetranychus urticae, cucumber green mottle mosaic virus (CGMMV) and yellowing disease (Dabauza et al., 1998). Cucumis ficifolius and Cucumis metuliferus have the highest levels of resistance to root-knot nematode (Wehner et al., 1990), however, they are also resistant to Didymella bryoniae and Trialeurodes vaporariorum (Láska and Lebeda, 1989; Lhotský et al., 1991). Cucumis metuliferus has resistance to squash mosaic virus and watermelon mosaic virus (McCarthy et al., 2001). In some accessions of C. africanus and C. zeyherii a high level of resistance to Tetranychus urticae was found (Lebeda, 1996). Cucumis myriocarpus has agronomically valuable traits such as resistance against Podosphaera xanthii, Fusarium oxysporum, Didymella bryoniae, CGMMV and Tetranychus urticae (Lhotský et al., 1991; Bordas et al, 1998). Cucu- mis melo (line MR 1 and some other genotypes) has resistance to cucumber downy mildew (Lebeda et al., 1996; Lebeda, 1999). Cucurbita species posses resistance to root-knot nematode and root elongation under low temperature conditions. Interspecific hybridization of Cucumis sativus with other species of the genus Cucumis and Cucurbita is very difficult because of their sexual incompatibility. Hybridizati- on of Cucumis sativus and C. melo with the wild related species is almost impossible by classical techniques (Lebeda et al., 1993). Intergeneric hybridization between Cucumis melo and Cucurbita has not yet been successfully achieved (Yamaguchi and Shiga, 1993). There is no available literature on intergeneric crosses between Cucumis and Cucurbita (Malepszy, 1988). It is obvious that new methods, especially biotechnolo- gical techniques, have to be used to overcome these obstacles. In vitro techniques (e.g. protoplast cultures) may be used to overcome the sterility barriers by fusing protoplasts of cucumber or melon with protoplasts of sexually incompatible species. Another application of tissue culture techniques is the genetic engineering of cucumber and melon. The increase in genetic variation that is frequently associated with tissue culture also may prove to be beneficial (Ezura, 1999). The aim of this paper is to summarize recently published information about protoplast cultures in Cucurbitaceae with the main focus on Cucumis and Cucurbita spp.

Protoplast cultures of Cucurbitaceae

Protoplast culture includes protoplast isolation from various plant tissues and their culture in special media. Under favourable conditions, the process is completed by regeneration of intact plants. The plant species, the conditions of plant growth, plant age, methodology of protoplast isolation and protoplast culture are often critical for sustained division of protoplasts (Fowke and Constabel, 1985). Achieving plant regeneration from protoplasts is necessary for the production of somatic hybrids through protoplast fusions. Products of protoplast fusion (cybrids) are represented by cells that contain a mixture of the organellar and nuclear DNA (nuclei, chloroplasts, and mitochondria) from both fusion parents (Yarrow, 1999). Protoplast isolation and culture techniques in Cucurbitaceae have been developed since 1975 (Coutts and Wood, 1975), when initial experiments led into regeneration of a cucumber callus. The plant regeneration from protoplasts was reported for the first time by Orczyk and Malepszy (1985). Later research efforts were focused on constructing somatic hybrids between cultivated species, especially involving Cucu-

442 mis melo and other Cucumis species. Many experiments demonstrated that the plant regeneration after fusion is very difficult (Table 1). Various protocols were developed and successfully used in protoplast cultures of Cucurbitaceae (Tables 1-3). They differed in the type of tissue (explant) used for protoplast isolation, and in isolation and culture techniques. Many of them led to regeneration of normal plants able to form roots in soil and produce fruits. In spite of that progress, fusion experiments are still problematic, because even when regeneration of hybrid plants was achieved, the features of one of the parents may gradually disap- pear (Yamaguchi and Shiga, 1993).

Plant material and source of protoplasts Most scientists have worked with Cucumis sativus and Cucumis melo. Protoplast regeneration has been accomplished by many authors (Orczyk and Malepszy, 1985; Jia et al., 1986; Roig et al., 1986b; Trulson and Shahin, 1986; Punja et al., 1990; Debeaujon and Branchard, 1992; Punja and Raharjo, 1993; Burza and Malepszy, 1995; Dabauza et al., 1998; and some others (Table 1)). Experiments with Cucumis anguria, Cucumis metuliferus, Cucumis myriocarpus, Cucumis zeyheri and Cucurbita pepo have also been reported, but plant regeneration was unsuccessful (Table 1). More research is needed to establish reliable protoplast culture procedures for these Cucurbitaceae species. In vitro grown plants are usually used for the protoplast isolation. Most frequently the protoplasts were isolated from cotyledons and true leaves (Punja and Raharjo, 1993), rarely from cell suspensions derived from callus (Burza and Malepszy, 1995; Fellner et al., 1996) or from calluses derived from different parts of the plant, e.g. cotyledons and hypocotyls (Fellner and Lebeda, 1998) or leaves (Burza and Maleps- zy, 1995). Cotyledons are excised after 47 days of cultivation of the seeds, leaves usually after 721 days. Plants have generally been grown in growth chambers with 1416 hours of light. The results of in vitro culture are strongly influenced by the protoplasts origin. Cotyledon tissue is younger and more metabolically active than is leaf tissue, but it is often tetraploid or octaploid which causes phenotypic instability, which explains why cotyledons provide lower quantities of normal shoots regenerated from protoplasts in comparison with cultures derived from leaves (Colijn-Hooymans et al., 1988; Punja et al., 1990). Callus protoplasts displayed a lower frequency of cell division than did those derived from mesophyl cells (Burza et al., 1992). Sutiojono et al. (1998) reported that hypocotyl protoplasts didnt divide, although cotyledon and leaf protoplasts underwent cell division. Genotype or cultivar also strongly influences the success of in vitro culture. For example, the best results were achieved in Cucumis melo with cvs. Charentais (Bo- kelman et al., 1991), Xinjiang (Li et al., 1990), Charentais T (Debeaujon and Branchard,1992), Green Delica (Sutiojono et al., 1998); in Cucumis sativus cvs. Hokus, Borszczagowski, Salty (Colijn-Hooymans et al., 1988) and genotypes 3672 and 3676 (Punja et al., 1990).

443 Table 1. Protoplast cultures of Cucumis spp. and Cucurbita spp.

Species Explant source Regeneration Reference

Cucumis C. anguria Cotyledon Minicallus Dabauza et al. (1998) C. melo Etiolated leaf Microcolonies Sutiojono et al. (1998) Leaf None Navrátilová et al. (2000) Leaf Callus Moreno et al. (1984) Leaf Intact plant Moreno et al. (1985); Debeaujon and Branchard (1992) Cotyledon Microcolonies Sutiojono et al. (2002) Cotyledon Callus Kantharajah and Dodd (1990) Cotyledon Intact plant Roig et al. (1986b); Debeaujon and Branchard (1990); Li et al. (1990); Bokelmann et al. (1991); Tabei et al. (1992); Dabauza et al. (1998) Hypocotyl None Sutiojono et al. (1998); Sutiojono et al. (2002) Cell susupension None Fellner et al. (1996) from callus Callus None Fellner and Lebeda (1998) C. Leaf None Punja and Raharjo (1993); metuliferus Navrátilová et al. (2000) Leaf Callus Debeaujon and Branchard (1990) Leaf Callus McCarthy et al. (2001a) Cotyledon None Sutiojono et al. (2002) Cotyledon Callus Dabauza et al. (1991) Callus with roots McCarthy et al. (2001a) Hypocotyl None Sutiojono et al. (2002) C. Cotyledon Minicallus Bordas et al. (1998) myriocarpus C. sativus Leaf Callus with roots Coutts and Wood (1977) Intact plant Orczyk and Malepszy (1985); Colijn- Hooymans et al. (1988); Punja et al. (1990); Punja and Raharjo (1993) Cotyledon Intact plant Jia et al. (1986); Trulson and Shahin (1986); Colijn-Hooymans et al. (1988); Punja et al. (1990) Callus Intact plant Burza and Malepszy (1992); Burza and Malepszy (1995) Suspension Intact plant Burza and Malepszy (1995) culture from callus C. zeyheri Leaf None Rokytová et al. (2001)

444 Cucurbita C. ficifolia Leaf None Navrátilová et al. (2000) C. pepo Cotyledon Callus Debeaujon and Branchard (1990) Leaf Callus Debeaujon and Branchard (1990) Leaf None Navrátilová et al. (2000) C. maxima Leaf None Navrátilová et al. (2000)

Methods of protoplast isolation Various protocols for protoplast isolation have been developed, differing in the application of certain treatments (pretreating explants on special cultivation media, abrading the lower epidermis, shaking the suspension), in the composition of enzyme and washing solutions, and in protoplast purification techniques. Various combinations and concentrations of enzymes have been used (Table 2), with the most common being a mixture of Cellulase Onozuka R-10 and Macerozyme R-10 (Bokelman et al., 1991; Debeaujon a Branchard, 1992; Jarl et al., 1995; Fellner and Lebeda, 1998; Navrátilová et al., 2000; Rokytová et al., 2001). Digestion usually proceeds for 16 h in the dark, at temperature of 23-26°C. The yield of protoplasts depends on the explant type and on the composition of the enzymatic solution (Jarl et al., 1995; McCarthy et al., 2001b).

Table 2. Enzyme solutions for protoplast isolation

Enzymes Concentration Explant Reference

Cellulase 1.5 % Cotyledon Dabauza et al. (1991); Bordas et al. (1998); Onozuka R-10 Dabauza et al. (1998) Pectolyase, 0.025 % Cotyledon Yamaguchi and Shiga (1993) Cellulase 1.0 % Onozuka R-10 Cellulase 1.5 % Cotyledon Colijn-Hooymans et al. (1988); Onozuka R-10, Debeaujon and Branchard (1990); Macerozyme R-10 0.3 % Debeaujon and Branchard (1992) Cellulysin, 1.0 % Leaf Punja et al. (1990); Punja and Raharjo (1993) Pectinase 0.5 % Cellulase 1.2 % Callus Burza and Malepszy (1995) Onozuka R-10, 1.2 % Fellner and Lebeda (1998) Macerozyme R-10, 0.3 % Driselase Cellulysin, 2.0 % Cotyledon McCarthy et al. (2001a,b) Macerase 0.5 % Pectinase, 2.0 % Callus Fellner and Lebeda (1998) Macerozyme R-10, 0.4 % Cellulase 2.0 % Onozuka R-10

445 Figure 1. Protoplast culture of Cucumis melo (line MR-1). A) Mesophyll protoplasts immediately after isolation. B) Viability of callus protoplasts after isolation (fluorescence staining with FDA). C) Dividing hybrid cell product of protoplasts fusion between Cucumis melo and Cucumis metuliferus, 7 days after fusion. D) Viability of mesophyll protoplasts (13 days after isolation, FDA staining).

Enzymes are often dissolved in a washing solution or in a simplier solution (MS medium with 0.25 M mannitol (Punja and Raharjo, 1993)). The washing solution usually consists of inorganic salts (often CPW salts (Frearson et al., 1973)), osmoticum, MES, glycine, and sometimes vitamins and growth regulators (Jia et al., 1986; McCarthy et al., 2001a,b) and 0.1 M glucose (Debeaujon and Branchard, 1990; Navrátilová et al., 2000; Rokytová et al., 2001). The most frequently used osmoticum is mannitol in concentration 0.15 – 0.5 M (Punja and Raharjo, 1993). Glycine is often added in order to stabilize protoplast membranes (Orczyk and Malepszy, 1985) and MES (2-N-morfolin-etansulfonic acid) minimizes the shifts in pH during the isolation process. The optimal pH of the enzymatic solution is between 5.6 and 5.8. After digestion in the enzyme solution, the protoplast suspension is filtered through a nylon membrane or steel sieve in order to remove extraneous tissues, then diluted by a washing solution, centrifuged and finally usually purified by means of a density gradient, comprised of sucrose or another saccharide. Protoplasts then concentrate in the interphase between sucrose and the washing solution, where they are repeatedly collected and centrifuged.

446 Culture medium Media for protoplasts are similar to those used for other plant tissue cultures, e.g. modified MS medium (Murashige and Skoog, 1962) was used by Orczyk and Maleps- zy (1985) and many others, modified DPD medium (Durand et al., 1973) by Jia et al. (1986), and B5 medium (Gamborg et al., 1968) by Dabauza et al. (1998). The content of inorganic salts can be reduced by half for macroelements (Colijn-Hooymans et al., 1988; Punja and Raharjo, 1993). The medium always contains growth regulators. Various combinations of auxins and cytokinins have been used, including 2,4-D (2, 4 - dichlor- fenoxyacetic acid) and kinetin (McCarthy et al., 2001), or NAA (naftylacetic acid) and BA (benzyladenin) (Punja et al., 1990). Glucose or sucrose serve as the energy source (Bokelmann et al., 1991). In some media, organic substances with undefined composition such as coconut milk (Dabauza et al., 1991) or casein hydrolysate (De- beaujon and Branchard, 1992) were added. Mannitol in concentration 0.250.36 M was used as the osmoticum in most studies. The osmotic potential is gradually lowered to zero by adding fresh medium without osmoticum during culture. Protoplasts are usually cultured at a density of 1×105 protoplasts/ml of culture medium, but successful regeneration of plants was also achieved at a somewhat lower density (4×104 protoplasts/ml) (Punja et al., 1990). Protoplast density has been demonstrated to influence division rates and microcallus production (McCarthy et al., 2001b).

Culture technique and conditions Both liquid media and those solidified by agarose have been used. Liquid media can be maintained in a thin layer on the bottom of a Petri dish (Dabauza et al., 1991; Rokytová et al., 2001) or in small drops on the bottom or lid of a Petri dish (hanging- drop culture; Orczyk and Malepszy, 1985). Solidified media can serve as a basal layer under liquid media containing protoplasts (Li et al., 1990), or they can contain embedded protoplasts (Orczyk and Malepszy, 1985). Agarose media can be cut into several pieces immediately after solidification or at a later stage and covered with liquid medium (bead culture; Orczyk and Malepszy, 1985; Colijn-Hooymans et al., 1988), or blocks of agarose can be put onto a solid substrate (Debeaujon and Bran- chard, 1992). In disc culture (Punja et al., 1990; McCarthy et al., 2001b), drops of agarose medium placed on the bottom of the Petri dish are covered with liquid medium. An advantage of the last two techniques is the possibility of changing of the liquid medium. In the co-cultivation method, dividing cells are placed around a central drop containing protoplasts (Li et al., 1990; Punja and Raharjo, 1993; McCarthy et al., 2001b). In a comparative study of liquid, agarose-embedded and droplet culture, Punja et al. (1990) reported that the most successful was agarose-embedded protoplast culture. In that experiment, development of calli from cucumber protoplasts was achieved within 8-10 weeks of culture (Punja et al., 1990). McCarthy (2001b) reported that agarose medium provided significantly higher division frequency than did a liquid medium. Colijn-Hooymans et al. (1988) achieved a plating efficiency of up to 50% by using agarose-solidified medium (either disc culture or bead culture) in contrast to less than 2% via liquid culture. However, liquid medium has also been used for successful plant regeneration (Dabauza et al., 1991). Protoplasts are cultivated in weak light or in the dark. McCarthy et al. (2001b) demonstrated that weak light can increase the frequency of second divisions of coty-

447 Figure 2. Protoplast culture of Cucumis sativus (line 6514). A) Mesophyll protoplasts immediately after isolation. B) First division and viability of mesophyll protoplasts, 11 days after isolation, fluorescence staining with FDA. C) Regenerated microcallus, 62 days after isolation. D) Leaf - derived callus, material for protoplast isolation. E) In vitro plants used as a source of protoplasts.

448 ledon protoplasts of Cucumis metuliferus, but had no effect on the first division. Cultures are generally maintained between 25 and 30°C. McCarthy et al. (2001a) reported that there was a significantly higher yield of microcalli regenerated from protoplasts at 30°C than at 25°C.

Regeneration of protoplasts The first precondition for regeneration is the isolation of viable protoplasts. After several days in culture, the regeneration of cell wall follows and is demonstrated by changes in protoplast shape. The first division of protoplasts can be observed after 2- 3 days of cultivation, second division after 48 days of cultivation (McCarthy et al., 2001b). Microcolonies of 1 mm size were visible after one month (Debeaujon and Branchard, 1992). After the callus stage, regeneration can progress to organogenesis or somatic embryogenesis. Developed shoots can be removed from the culture medium and rooted on a medium without hormones. The regeneration of plants took 4-6 months (Bokelmann et al., 1991; Debeaujon and Branchard, 1992). The most important factors influencing regeneration are: 1) type of the tissue used as the source of protoplasts, 2) composition of the culture medium (especially growth regulators), 3) culture procedures and conditions. The success of the work also depends on the genotype of the source plant (McCarthy et al., 2001b). The most difficult step is the induction of shoots or somatic embryos on the callus and then rooting the plants and their transfer to soil.

Protoplast fusion In Cucurbitaceae, production of somatic hybrids by protoplast fusion has been tried by several researchers, but reliable procedures for obtaining fertile hybrids remain elusive (Table 3). Most researchers have tried to produce interspecific cybrids involving Cucumis melo (Table 3). There are only two reports dealing with crossing of Cucumis sativus with Cucumis metuliferus (Tang and Punja, 1989; Punja and Raharjo, 1993). In those experiments, the regeneration of fused protoplasts stopped at the minicallus stage. The most common method is the electrofusion, sometimes polyethylene glycol (PEG) or high pH/Ca2+ was used to induce fusion (Roig et al., 1986; Punja and Raharjo, 1993). In most cases, no regeneration was achieved, or regeneration of normal plants was not observed. The most difficult step was the induction of shoots on the callus and their development into whole plants (Yamaguchi and Shiga, 1993). The regeneration of hybrid plants was achieved only in fusion of Cucumis melo with Cucurbita moschata x C. maxima (Yamaguchi and Shiga, 1993), and of Cucumis melo with C. myriocarpus (Bordas et al., 1998). In the first case, intergeneric hybrids did not retain Cucurbita features. In the second case, the resulting plants were unable to produce roots.

449 Reference (1998) Roig et al. Navrátilová et al. (1998) Branchard (1990) Roig et al. Roig et al. Punja and Raharjo (1993) Navrátilová et al. (1998) (1995) al. et Jarl Fellner et al. (1996) (1998) Method Electrofusion Debeaujon and Electrofusion Bordas et al. glycol (1986a) glycol glycolglycol (1986a) (1986a) glycol glycol Polyethylene Microcolonies Microcallus Polyethylene No Polyethylene No Electrofusion Cotyledon Callus losing hybrid features Shiga (1993) Shoots, no rootingIntact plant, badly rooting Intact plant, gradually Electrofusion Dabauza et al. Electrofusion Yamaguchi and inicallus Polyethylene Cucumis melo Source of protoplasts Leaf Source of Source protoplasts Leaf No No Leaf CotyledonSmall roots No No Polyethylene CotyledonCotyledon NoCallus NoLeaf No No M Polyethylene Polyethylene CotyledonHypocotyl, cell suspension CallusCotyledon Cotyledon Callus Cotyledon glycol . Protoplast fusion in Cucurbitaceae C. maxima Table Table 3 Species Cucumis metuliferus, Benincasa hispida Cucurbita martinezii Cucurbita pepo C. metuliferus x C. sativus C. anguria C. myriocarpus Cucurbita moschata

450 Research progress in protoplast cultures of Cucurbitaceae in the Department of Botany, Palacký University in Olomouc Our research has focused both on economically important species, such as Cucu- mis sativus, Cucumis melo, Cucurbita pepo and Cucurbita maxima, and on some wild or less important species, including Cucumis anguria, Cucumis metuliferus, Cucumis zeyheri, and Cucurbita ficifolia. Protoplasts have been successfully isolated from leaves, calli and cell suspensions. The yield per g of fresh mass and viability were compared in order to choose suitable genotypes and explant types for protoplast isolation and culture (Navrátilo- vá et al., 2000; Rokytová et al., 2001). Various culture media for initiating and maintaining calli and cell suspensions, and several enzyme solutions for protoplast isolation were tested (Fellner et al., 1996; Fellner and Lebeda, 1998). However, regeneration of all cultures stopped after the first cell division. Fusion between Cucumis sativus and Cucumis melo was facilitated by polyethyle- neglycol, and, after fusion, the formation of microcolonies was recorded (Fellner et al., 1996).

Conclusions Remarkable progress has been made in the recent development of protoplast culture methods for Cucurbitaceae. 1. There are suitable methods for isolation of viable protoplasts from various plant tissues. 2. Techniques for plant regeneration from protoplasts have been developed for the most important crops (Cucumis melo, Cucumis sativus) which can serve as a model for wild Cucumis spp. and Cucurbita spp. 3. Fusions of Cucumis sativus and Cucumis melo with wild (or less economically important) species of Cucurbitaceae has been accomplished through electrofusion and polyethylene glycol mediated fusion, but the regeneration of plants was achieved only from cybrids of Cucumis melo with Cucurbita moschata x C. maxima, and Cucu- mis melo with C. myriocarpus, and these have not been a complete success. 4. Future research should focus on improving regeneration techniques and identifying genotypes most suitable for protoplast fusion.

Acknowledgements

The authors thank Dr. Mark P. Widrlechner for valuable comments on this manuscript. Excellent cooperation with the USDA-ARS, North Central Regional Plant In- troduction Station, Iowa State University, Ames, Iowa (USA) in providing various Cucurbitaceae germplasm is acknowledged. This research was supported by grants: 1) NAZV QF 4108 Utilization of protoplast fusion technique in breeding of the important cultivated crops of the genera Brassica, Cucumis and Solanum, Ministry of Agriculture of Czech Republic (MA CR); 2) Ministry of Education of Czech Repub- lic No. MSM 153100010, and FRV 55/2004 (Protoplast Culture of Cucurbitaceae).

451 References

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454 Mosaic (MSC) cucumbers regenerated from independent cell cultures possess different mitochondrial rearrangements

G. Bartoszewski1, S. Malepszy1 and M.J. Havey2 1Warsaw Agricultural University, Faculty of Horticulture and Landscape Architectu- re, Department of Plant Genetics, Breeding and Biotechnology, Nowoursynowska 166, 02-787 Warsaw, Poland; e-mail: [email protected] 2Agricultural Research Service, U.S. Department of Agriculture, Vegetable Crops Unit, Dept. of Horticulture, 1575 Linden Dr., University of Wisconsin, Madison, WI 53706, USA

Summary

Cucumber plants regenerated from somatic cells occasionally produce progenies with a strong mosaic (MSC) phenotype on cotyledons and leaves. The MSC phenotype shows paternal transmission and the genetic bases of independently derived MSC lines are unknown. Because cucumber mitochondrial genome is paternally transmitted we hybridized fifteen cosmids covering the mitochondrial genome of Arabidopsis thaliana to DNA blots of independently derived MSC lines from tissue cultures and wild-type lines. Polymorphism was revealed near the rrn5/rrn18, nad5- exon2, rpl5 coding and JLV5 non-coding regions. Polymorphisms revealed by rrn18 and nad5- exon2 were due to one rearrangement bringing together these two regions. The polymorphism revealed by rpl5 gene was unique to MSC16 and was a rearrangement placing the rpl5 region next to the forward junction of the JLV5 deletion. Five different hybridization patterns were revealed between seventeen independently derived tissue culture lines. Passage of cucumber through cell culture may be a unique method to produce novel mitochondrial DNA rearrangements affecting mitochondrial gene expression.

Keywords: Cucumis sativus, paternal transmission, mitochondrial genetics, tissue cultures, somaclonal variation

Introduction

The highly inbred (>S ) cucumber line B originated from the Polish cultivar 22 Borszczagowski was selected by Kubicki (unpublished). Various protocols allowing to regenerate cucumber plants through organogenesis or somatic embryogenesis were established for this line (Malepszy and Nadolska-Orczyk, 1989; Burza and Malepszy, 1995a,b; Ladyzynski et al., 2002). Many plants were regenerated from somatic tissue using those protocols. Some of those plants segregated progenies with altered phenotype resulting from tissue culture induced somaclonal variation (Malepszy and Na- dolska-Orczyk, 1989; Burza and Malepszy, 1995a; Plader et al., 1998). One of the most frequent somaclonal variants is a paternally transmitted mosaic (MSC) phenotype that appears frequently after different regeneration procedures (Malepszy et al., 1996; Lilly et al., 2001; Ladyzynski et al., 2002). MSC phenotype is characterized by a strong mosaic on cotyledons and leaves, weaker growth and poorer germination. Mutant plants also possess smaller asymmetric flowers and smaller fruits with specific spotting. Seeds produced on MSC plants appear wrinkled or empty (Malepszy et al., 1996).

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 455 The mitochondrial (mt) genome of cucumber possesses two unique attributes. It is one of the largest known plant mt genomes at approximately 1500 kb (Ward et al., 1981), due to accumulation of short repetitive DNA motifs (Lilly and Havey, 2001; Bartoszewski et al., 2003). The second unique characteristic is that the cucumber mt genome shows paternal transmission (Havey, 1997). In this study we evaluated 17 lines (MSC and wild type) derived from different inbred line B regeneration experiments for structural rearrangements in the mtDNA.

Materials and methods

Plant material Wild-type inbred line B, cultivar Calypso and MSC lines 11, 16, and 19 have been described earlier (Malepszy et al., 1996; Lilly et al., 2001). Additional MSC and wild-type lines were produced after self pollination of plants regenerated from leaf callus (LC1, LCST1-2), protoplasts (DPR1, MSC9), cytokinin dependent embryogenic suspension (CDS1-CDS4), and liquid meristematic culture (MSC3, LMC1-2) (Burza and Malepszy, 1995a,b, 1998; Ladyzynski et al., 2002).

DNA isolation and blot hybridization Total DNA was isolated from all lines using CTAB extraction (Havey, 1997). For RFLP analysis, the genomic DNA of the five lines were digested with different restriction enzymes (BamHI, DraI, EcoRI, HindIII, SacI, XbaI AluI, HaeIII, CfoI, MspI, MboI, MseI), electrophorized through agarose gels, and transferred to nylon membranes (Ze- taprobe GT, Bio-Rad, Hercules, CA). Fifteen cosmids covering whole A. thaliana mt genome were provided by Prof. A. Brennicke (Klein et al., 1994). Cosmids were radi- olabelled by nick translations (Invitrogen, Carlsbad, CA) and hybridized to the cucumber blots. Hybridization conditions and autoradiography were performed according to Havey et al. (1998).

Results and discussion Initially we evaluated five accessions for polymorphisms in the mitochondrial genome (inbred line B, variety Calypso, and three tissue culture lines: MSC16 (with mosaic phenotype), LCST1 and LCST2 (wild type lines)). We revealed five structural polymorphisms in the mitochondrial genome among wild-type and MSC lines, all tracing back to the highly inbred line B. In addition to the previously described P1 and JLV5 regions (Lilly et al., 2001), new polymorphisms were revealed by rrn18, nad5-exon2, and rpl5. The polymorphisms revealed by rrn18 and nad5-exon2 were due to one rearrangement placing together these two coding regions. For the polymorphism revealed by rpl5, all accessions possessed a fragment corresponding to the functional rpl5 gene. Wild-type lines B and Calypso possessed a pseudo-rpl5 gene. MSC16 and the wild-type lines LCST1 and LCST2 did not possess this pseudo-rpl5 gene. MSC16 possessed a unique rpl5 6.2-kb SacI fragment due to a rearrangement placing a duplicated rpl5 gene next to the forward junction of the JLV5 region. JLV5 is a 15.1-kb

456 region missing in MSC16, present in wild-type line B, and sorts with the MSC phenotype (Lilly et al., 2001). We scored all mitochondrial polymorphisms among MSC and wild-type lines generated from independent cell-culture experiments, all started from the highly inbred line B, and revealed five unique patterns (Table 1). MSC16 and MSC19 were both regenerated from the leaf-callus experiment and shared all polymorphisms. MSC9 was regenerated after protoplast culture and shared all polymorphisms with MSC16 and MSC19 (pattern1). MSC3 and wild-type lines LC1 and CDS1 (pattern2); MSC11, MSC12, LCST1 and LCST2 (pattern 3); CDS2, CDS4, and DPR2 (pattern 4); and CDS3 (pat- tern5) possessed unique combinations of mitochondrial polymorphisms. Not all lines carried these rearrangements (LMC1, LMC2, DPR1), indicating that independent rearrangements may produce unique mitochondrial variants affecting mitochondrial gene expression.

Table 1. Origins of wild-type and mosaic (MSC) cucumber lines and polymorphisms detected in their mitochondrial genomes

Culture type Line Phenotype Polymorphism pattern

None B WT WT Calypso WT WT Direct regeneration from leaf callus MSC16 MSC Pattern 1 MSC19 MSC Pattern 1 LC1 WT Pattern 2 Regeneration from leaf callus after MSC11 MSC Pattern 3 selection for salt tolerance MSC12 MSC Pattern 3 LCST1 WT Pattern 3 LCST2 WT Pattern 3 Cytokinin dependent embryogenic CDS1 WT Pattern 2 suspension culture CDS2 WT Pattern 4 CDS3 WT Pattern 5 CDS4WT Pattern 4 Liquid meristematic culture MSC3 MSC Pattern 2 (shoot primordia culture) LMC1 WT WT LMC2 WT WT Direct protoplast regeneration MSC9 MSC Pattern 1 DPR1 WT WT DPR2 WT Pattern 4

457 References

Bartoszewski, G., Katzir, N. and Havey, M.J. 2003. Organization of repetitive DNAs and the genomic regions carrying ribosomal RNA, cob, and atp9 genes in the cucurbit mitochondrial genomes. Theor. Appl. Genet., (in press). Burza, W. and Malepszy, S. 1995a. Direct plant regeneration from leaf explants in cucumber is free of stable genetic variation. Plant Breeding, 114: 341-345. Burza, W. and Malepszy, S. 1995b. In vitro culture of Cucumis sativus L. XVII. Plants from protoplasts through direct somatic embryogenesis. Plant Cell Tiss. Organ Cult., 41: 259-266. Burza, W. and Malepszy, S. 1998. Cytokinin control of cucumber (Cucumis sativus L.) somatic embryogenesis. In: Proceedings of IX International Congress on Plant Tissue and Cell Culture, Jerusalem, June 14-19, 1998, p. 68. Havey, M.J. 1997. Predominant paternal transmission of the cucumber mitochondrial genome. J. Hered., 88: 232-235. Havey, M.J., McCreight, J., Rhodes, B. and Taurick, G. 1998. Differential transmission of the Cucumis organellar genomes. Theor. Appl. Genet., 97: 122-128. Klein, M., Eckert-Ossenkopp, U., Schmiedeberg, I., Brandt, P., Unseld, M., Brennicke, A. and Schuster, W. 1994. Physical mapping of the mitochondrial genome of Arabidopsis thaliana by cosmid and YAC clones. Plant J., 6: 447-455. Ladyzynski, M., Burza, W. and Malepszy, S. 2002. Relationship between somaclonal variation and type of tissue culture in cucumber. Euphytica, 125: 349-356. Lilly, J.W. and Havey, M.J. 2001. Small repetitive DNAs contribute significantly to the expanded mitochondrial genome of cucumber. Genetics, 159: 317-328. Lilly, J.W., Bartoszewski, G., Malepszy, S. and Havey, M.J. 2001. A major deletion in the cucumber mitochondrial genome sorts with the MSC phenotype. Curr. Genet., 40: 144-151. Malepszy, S., Burza, W. and Smiech, M. 1996. Characterization of a cucumber (Cucumis sativus L.) somaclonal variant with paternal inheritance. J. Appl. Genet., 37: 65-78. Malepszy, S. and Nadolska-Orczyk, A. 1989. In vitro culture of Cucumis sativus. VIII. Variation in the progeny of phenotypically not altered R1 plants. Plant Breeding, 102: 66-72. Plader, W., Malepszy, S., Burza, W. and Rusinowski, Z. 1998. The relationship between the regeneration system and genetic variability in the cucumber (Cucumis sativus L.). Euphytica, 103: 9-15. Ward, B.L., Anderson, R.S. and Bendich, A.J. 1981. The mitochondrial genome is large and variable in a family of plants (Cucurbitaceae). Cell, 25: 793-803.

458 Wounding by vortexing with carborundum facilitates Agrobacterium-mediated transformation of melon (Cucumis melo)

S. Curuk1,2, S. Cetiner2,6, C. Elman3, X. Xia3, Y. Wang3, A. Yeheskel3, L. Zilberstein4, R. Perl-Treves4, A.A. Watad5 and V. Gaba3 1Mustafa Kemal University, Faculty of Agriculture, Department of Horticulture, 31034 Antakya-Hatay, Turkey; e-mail: [email protected] 2Cukurova University, Faculty of Agriculture, Department of Horticulture, 01330 Balcali- Adana, Turkey 3Department of Virology, Agricultural Research Organization, Volcani Center, P. O. Box 6, Bet Dagan 50-250, Israel; e-mail: [email protected] 4Department of Life Sciences, Bar-Ilan University, Ramat Gan 52-900, Israel 5Department of Ornamental Horticulture, Agricultural Research Organization, Vol- cani Center, P. O. Box 6, Bet Dagan 50-250, Israel (deceased) 6Sabanci University, Faculty of Engineering and Natural Sciences, 81474 Tuzla, Is- tanbul, Turkey

Summary

Transformation of recalcitrant melon (Cucumis melo) cvs. Kirkagac 637 and Noy Yaroq was accomplished by wounding cotyledon explants by vortexing with carborundum prior to inoculation with Agrobacterium tumefaciens. Chimeric transgenic plants were produced after regeneration of putatively transformed callus, bud-like protuberances, buds and shoots on selective medium with the antibiotic kanamycin. Subculture every 15-20 days on fresh regeneration-selection medium containing 50 mg/l kanamycin, after either a relatively high or low level of kanamycin in the first regeneration-selection medium, was necessary for successful transformation of cv. Kirkagac 637. Continuous selection was effective in breaking chimeras using elongation medium with 50 mg/l kanamycin for up to 6 subcultures. The treatments producing the most buds or shoots after 30-40 days in culture were the most successful in the production of transgenic plants.

Keywords: genetic transformation, recalcitrant genotypes, carborundum wounding, Agrobacteri- um tumefaciens, chimera

Introduction

There have been several reports of the genetic transformation of melon (Cucumis melo L.) with marker genes and genes for virus resistance and fruit-quality attributes (Fang and Grumet, 1990, 1993; Gonsalves et al., 1994; Valles and Lasa, 1994; Ayub et al., 1996). We did not succeed in the transformation of several cultivars of melon (including Hales Best Jumbo, Kirkagac 637 and Noy Yaroq) with the methods of Fang and Grumet (1990), and Valles and Lasa (1994), presumably due to non-defined methodological or environmental differences, as well as genotype and/or seed batch dependence.

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 459 Several physical treatments that damage plant tissue to create sites for Agrobacte- rium infection have been reported to increase transformation frequency in different plant species, including cutting with a blunt scalpel blade (Fang and Grumet, 1990) and wounding tissues by vortexing with carborundum (Cheng et al., 1996). Transfor- mation of melon was attempted by particle wounding/Agrobacterium inoculation in preliminary experiments, but transgenic plants were not obtained. The objective of this study was to develop, using melon germplasm that appears to have good regene- rative ability, an efficient transformation method by Agrobacterium tumefaciens.

Materials and methods

Seeds of Kirkagac 637 were obtained from Cagdas Seeds Ltd. (Turkey) and seeds of Noy Yaroq were obtained from Hazera Seeds Ltd. (Israel). Seeds of these two casaba- type melon cultivars were prepared and sterilized as described by Curuk et al. (2003) and germinated on MS (Murashige and Skoog, 1962) medium, at 28±1°C, with 16h photoperiod and 90-120 mmol·m-2·s-1 cool white fluorescent light. Cotyledons were excised from 4-5-day-old seedlings and transversely cut to obtain three explants per cotyledon. N medium (MS salts and vitamins, 30 g/l sucrose, 8 g/l agar, 0.88 mg/l indole-3- acetic acid, 1.13 mg/l benzyladenine [BA], 0.26 mg/l abscisic acid) (Fang and Gru- met, 1990), NB00101 medium (BM3 medium [MS salts, 100 mg/l myo-inositol, 1 mg/ l thiamine-HCl, 30 g/l sucrose, 8 g/l agar] with 0.01 mg/l naphthaleneacetic acid, 0.1 mg/l BA) (Valles and Lasa 1994), and BM3 medium (Valles and Lasa, 1994) were used as regeneration, elongation, and rooting media, respectively. In vitro cultures were placed at 28 ± 1°C, with 16 h photoperiod/90 mmol·m-2·s-1 during regeneration-selection, elongation, and rooting. A. tumefaciens strain EHA 105 with the binary plasmid pME504 (Edelman et al., 2000) encoding neomycin phosphotransferase II (NPT II), phosphinothricin acetyltransferase (bar), and b-glucuronidase-intron (GUS) genes was used for transformation. From an overnight Agrobacterium culture, 200 µl was added to 20 ml of liquid LB without antibiotics and shaken for 2-3 h prior to inoculation. Carborundum pow- der (Fisher Scientific C192-500; 320 grit, 0.045 mm; Prolabo (Italy), 0.037 mm) was autoclaved (500 mg/30 ml distilled water). The slurry was poured into 50 ml sterile centrifuge tubes and explants from 16-23 seedlings added. Capped tubes were vortexed vertically at speed 7 on a 220V Heidolph REAX 2000. Then the contents were poured into a petri dish, and explants were transferred to Agrobacterium culture for 10 minutes, blotted dry, and 20-30 explants distributed per plate of N medium without antibiotics, and co-cultivated for 2-3 d. After co-cultivation, explants were washed, blotted, and placed adaxial side down in petri dishes on N-medium with carbenicillin and kanamycin (100 mg/l). After the regeneration-selection period, explants producing callus, bud-like protuberances (swellings that become buds), buds, and shoots were scored. Regenerating parts of Kirkagac 637 were excised and transferred to NB00101 elongation medium with 50-75 mg/l kanamycin and carbenicillin for 2 weeks, and subsequently at 2-3-weekly intervals up to 6 times. Shoots expressing GUS activity were rooted on BM3 with 50 mg/l kanamy-

460 cin. GUS expression was detected as per Jefferson et al. (1987). PCR detection of the NPT II gene followed DNA extraction as per Dellaporta et al. (1983). Transgenic plantlets were transferred to a greenhouse.

Results and discussion

Preliminary experiments were made to determine the effects of wounding by vortexing with carborundum (followed by selection with different kanamycin regimens) on gene transfer to cv. Noy Yaroq. As transformed plants were only obtained by vortex treatment, the duration of vortex treatment was examined as a factor in production of transgenic plants (Table 1). Here two putative transgenic lines of cv. Kirkagac 637 were obtained from 0.5 or 1.5 min vortex treatments, each with a transformation efficiency of 1%. However, the effect of vortex treatment was not significant (P < 0.05) in formation of buds or shoots, bud-like protuberances, or calli. Treatments producing most buds or shoots at this stage were the most successful in the production of transgenic plants (r = 0.89). The co-cultivation period (2 or 3 d) had no significant effect on formation of bud-like protuberances or callus, and silver nitrate had a negative effect on these processes (P < 0.05) (data not shown).

Table 1. Effect of vortex time on transformation and regeneration from cotyledon explants of cv. Kirkagac 637. Regeneration responses of cotyledon explants inoculated with A. tumefaciens after wounding by vortexing at speed 7 in carborundum, and co-cultivated for 2 days. The explants were scored after culture for 15-20 d on N- medium containing 100 mg/l kanamycin followed by 15-20 d on N-medium with 50 mg/l kanamycin. No significant difference was found in columns by Tukeys Honest- ly Significant Difference test at P < 0.05. Approximately 100 explants per treatment

Vortex duration (min.) Explants (%) forming callus bud-like buds or shoots transgenic protuberances shoots

0.0 97 9 1 0 0.5 79 15 41 1.0 76 9 2 0 1.5 78 7 3 1

In this and other experiments with Kirkagac 637 and Noy Yaroq, each transformation event resulted in a group of buds derived from bud-like protuberances or callus. The addition of 50 mg/l kanamycin to the elongation medium caused most control explants of Kirkagac 637 to bleach and die by the end of the first subculture. After initial selection, products of each transformation event were subcultured several times at 2-3-weekly intervals, during which the bud groups multiplied. Many of the bud groups produced from putative transformed lines bleached during subculture,

461 suggesting that these transgenic lines were chimeric. Each putative transgenic line was multiplied in vitro to provide rooted plants for characterization and fruit production. Putative transgenic plants rooted on rooting medium with 50 mg/l kanamycin, while control plants did not. The majority of GUS-negative buds and shoots were eliminated by continuous selection on medium with kanamycin of cultures from which samples stained partially positive for GUS. Continuous selection broke chimeras on elongation medium with 50 mg/l kanamycin for up to 6 cycles of subculture, and by increasing the kanamycin concentration up to 75 mg/l for a few cycles. Transgenic plants of Kirkagac 637 line 2/II/A were not affected by spraying with 1% Basta herbicide, which killed controls. PCR results showed that some GUS-positive lines contained the NPTII gene. Transgenic lines were tetraploid, mixoploid, or diploid. Three transgenic R plants of Noy Ya- 1 roq line 5/V/C showed the presence of a major BamHI fragment of the expected size (ca. 4 kkp) released from the T-DNA (Fig. 1). Two restriction enzymes, XbaI and SacI, cut only once in the T-DNA, and therefore should yield a different hybridization pattern in each transgenic line. The three plants analyzed here are the R progeny of a single 1 transformant, and therefore have the same SacI and XbaI patterns. The GUS gene in plant number 10 was silenced, without affecting the Southern blot pattern. Two major bands seen with the SacI digest suggests the presence of two insertions.

Figure 1. Southern blot (DNA) gel of transgenic R plants of line 5/V/C of Noy Yaroq. 1 DNA was prepared as per Baudracco-Arnas (1995), 5 µg restriction-digested, run on 0.8% agarose gels, blotted onto charged nylon membranes. Hybridization was at 60°C in 6% PEG, 5% SDS, 5× SSPE and 5 µg/ml denatured salmon DNA. The blots were hybridized with a labeled 3.8 kbp BamHI fragment (NPTII gene and 35S promoter) from pME504. After hybridization, blots were washed at the hybridization temperature with 1× sodium chloride and trisodium citrate buffer, 1% SDS, and exposed to a phospho-imager. DNA from three sibling plants was cut with 3 restriction enzymes, as indicated. Molecular weight markers are indicated. DNA from untransformed plants of Noy Yaroq (NY) did not react.

462 Conclusions

Wounding of cotyledon explants of melon cvs. Kirkagac 637 and Noy Yaroq by vortexing in water with carborundum enabled genetic transformation and the recovery of transformed plants from otherwise recalcitrant genetic material, as observed by Cheng et al. (1996) with papaya. The only treatments we found to produce transgenic melon shoots were where the plant material was vortexed with carborundum. Such treatments also created the most buds or shoots. In each case, vortexing with carborundum had to be accompanied by the correct selection procedure for that cultivar in order to obtain transgenic plants. Transformation of Noy Yaroq required subculture every 15-20 days onto fresh regeneration-selection medium with a high concentration of kanamycin (125 mg/l). However, successful transformation of Kirka- gac 637 required regeneration-selection for 15-20 days on high (100 mg/l) (similar to Dong et al., 1991) or low (50 mg/l) kanamycin level, followed by subculture every 15-20 days onto fresh regeneration-selection medium. Addition of silver nitrate to the regeneration medium had a negative effect on transformation, despite results to the contrary in melon (Ayub et al., 1996). All transgenic buds recovered were initially chimeric, as when putative transgenic buds were subcultured onto selection- elongation medium both kanamycin-sensitive and kanamycin-resistant shoots were produced. Additionally, the organ-specific pattern of GUS expression observed in the first or second subcultures disappeared during later subcultures. These results were consistent with the possibility of interconversion of shoot apex layers (Chris- tou, 1990). The transfer of three genes (bar, NPT II, GUS) to the melon genome was confirmed by growth on selection medium with kanamycin and/or glufosinate am- monium, expression of the GUS gene, PCR and Southern blot (DNA) detection of the transgenes, genetic transmission to the next generation (not shown), and Basta herbicide resistance tests.

Acknowledgements

Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 500/04. This work was supported by a fellowship and grant to S. Curuk from The Scientific and Technical Research Council of Turkey (TUBITAK/BAYG) and to V.G. by the Chief Scientist of the Ministry of Agriculture (Israel). We thank Dr. M.A. for providing pME 504.

References

Ayub, R., Guis, M., Amor, M.B., Gillot, L., Roustan, J.P., Lache, A., Bouzayen, M. and Pech, J.C. 1996. Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nature Biotechnol., 14: 862-866. Baudracco-Arnas, S. 1995. A simple and inexpensive method for DNA extraction from Cucumis melo L. Cucurbit Genet. Coop. Rep., 18: 50-51. Cheng, Y.H., Yang, J.S. and Yeh, S.D. 1996. Efficient transformation of papaya by coat protein gene of papaya ringspot virus mediated by Agrobacterium following liquid-phase wounding of embryogenic tissues with carborundum. Plant Cell Rep., 16: 127-132.

463 Christou, P. 1990. Morphological description of transgenic soybean chimeras created by the de- livery, integration and expression of foreign DNA using electric discharge particle accelerati- on. Ann. Bot., 66: 379-386. Curuk, S., Ananthakrishnan, G., Singer, S., Xia, X., Elman, C., Nestel, D., Cetiner, S. and Gaba, V. 2003. Regeneration in vitro from the hypocotyl of Cucumis species produces almost exclusively diploid shoots and does not require light. Hort Sci., 38: 105-109. Dellaporta, S.L., Wood, J. and Kicks, J.B. 1983. A plant DNA minipreparation revision II. Plant Molec. Biol. Rep., 1: 19-21. Dong, J.Z., Yang, M.Z., Jia, S.R. and Chua, N.H. 1991. Transformation of melon (Cucumis melo L.) and expression from the cauliflower mosaic virus 35S promoter in transgenic melon plants. Bio/Technol., 9: 58-63. Edelman, M., Perl, A., Flaishman, M. and Blumental, A. 2000. Transgenic Lemnaceae. European Patent Application 1021552. Fang, G. and Grumet, R. 1990. Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants. Plant Cell Rep., 9: 160-164. Fang, G. and Grumet, R. 1993. Genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene. Mol. Plant-Microbe Interact., 6: 358-367. Gonsalves, C., Xue, B., Yepes, M., Fuchs, M., Ling, K., Namba, S., Chee, P., Slingtom, J.L. and Gonsalves, D. 1994. Transferring cucumber mosaic virus-white leaf strain coat protein gene into Cucumis melo L. and evaluating transgenic plants for protection against infections. J. Amer. Soc. Hort. Sci., 119: 345-355. Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusion: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J., 6: 3901-3909. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bio-assay with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Valles, M.P. and Lasa, J.M. 1994. Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro). Plant Cell Rep., 13: 145-148.

464 Transformation of melon via PEG-induced direct DNA uptake into protoplasts

A. Atarés, B. García-Sogo, B. Pineda, P. Ellul and V. Moreno Instituto de Biología Molecular y Celular de Plantas, U.P.V., Valencia, Spain

Summary

Cotyledon protoplasts of melon (cv. Cantaloup Charentais) were transformed with plasmid DNA containing neomycinphosphotransferase (nptII) and green fluorescence protein (gfp) genes, using polyethylene glycol (PEG) to induce DNA uptake. Optimal experimental conditions for plasmid concentration were analyzed. A time course study of GFP expression from protoplasts, cells, aggregates and protoplast-derived calli was performed. Results showed that 100 µg plasmid DNA/ml of protoplasts gave the best transformation efficiency, allowing the acquisition of an average of 50 transgenic calli per million of treated protoplasts.

Keywords: Cucumis melo, protoplasts, direct transformation, polyethylene glycol, green fluorescence protein

Introduction

Breeding of melon by exploring extra-specific genetic variation has been handi- capped by sexual incompatibility barriers in interspecific crosses. Somatic hybridization via protoplast fusion and genetic transformation are the best alternatives to overcome the above-mentioned problems. Protocols for genetic transformation via Agrobacte- rium have been developed in our group (Bordas et al., 1993) and by other authors (Ben Tahar and De Both, 1988; Fang and Grumet, 1990; Dong et al., 1991; Toyoda et al., 1991; Valles and Lasa, 1994; Guis et al., 2000; Ezura et al., 2000; Galperin et al., 2003). Nevertheless, transformation efficiency is genotype-dependent and relatively low (usually 1-3%). An alternative would be the use of direct-transformation protocols. Biolistic methods have been used successfully in different species, but they usually lead to the integration of several copies in tandem. Direct transformation protocols based on electroporation or PEG-mediated DNA uptake into protoplasts could be of greater practical interest, provided that methods for plant regeneration of protoplasts are available. In this respect, we have previously developed efficient protocols for isolation and plant regeneration from melon protoplasts (García-Sogo, 1990). In this paper, a protocol for PEG-induced transformation of melon protoplasts is reported.

Material and methods

Protoplast isolation and regeneration The procedures for protoplast isolation, culture and plant regeneration were as previously described (Roig et al., 1986; García-Sogo, 1990). Protoplasts were isolated from 6- day-old plantlet cotyledons of Cucumis melo L. cv. Cantaloup Charentais. Explants

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 465 were pre-cultured in NB2510 medium (Moreno et al., 1984) for two days in darkness, cut into strips and incubated with 10 ml of a filter-sterilized enzyme solution at 50 rpm and 25°C. The enzyme solution was comprised of 1.5% cellulase Onozuka R10 (Yakult, To- kio) dissolved in WS washing solution [MS macronutrients (Murashige and Skoog, 1962), MES 0.51mM, Glycine 0.1M and Mannitol 0.4M pH 5.7]. Protoplasts were recovered after 12 hours of incubation, washed in WS solution, pelleted (180 × g) and then resuspended in FS floating solution [MS macronutrients, MES 0.51mM, Glycine 0.1M and Sucrose 0.6M pH 5.7]. After centrifugation (65 × g), the intact protoplasts formed a prominent band at the upper interface. Then protoplasts were collected, rewashed, resuspended in Sorbitol 0.5M and adjusted at a final concentration of 5·105 protoplasts/ml.

Protoplast transformation Plasmid p524EGFP-1 was kindly provided by Dr. Oscar Olivares (IVIA, Valencia). p524EGFP-1 (4.7 Kb) is a derivate of a Clontech Laboratories Inc. plasmid (pEGFP- 1) with the marker nptII and the report gfp genes. Different concentrations of circular plasmid DNA were added to protoplasts. 0.3 ml of PEG solution [30% PEG 6000 Sig- ma®, Glucose 0.25M, Glycine 0.1M and CaCl ·2H O 0.1M pH 5.7] were added in petri 2 2 dishes and 0.4 ml of protoplasts plus plasmid were added to the surface of the PEG solution. After incubation for 20 minutes at room temperature, 3 ml of DNB culture medium (Moreno et al., 1984) were added while 1.8 ml were soaked up simultaneously for PEG elimination. Protoplasts were then cultured as described in Roig et al. (1986).

Detection of transgenic cells Transient or stable GFP expression was visualized under a fluorescence Nikon-Diaphot inverted microscope with UV light and B2 (EX 450-490 / DM 510 / BA 520) Nikon filters. The number of protoplasts, cells, divisions and aggregates with GFP expression were counted 1, 7, 15 and 45 days after transformation. GFP expression in mini- calli was detected under a fluorescence Nikon SMZ800 stereomicroscope with UV light and GFP-L (EX 480-40 / DM 505 / BA 510) Nikon filters.

Results and discussion In the first experiment, several plasmid concentrations were used. Results showed that the greatest efficiency of transformation was obtained with the higher plasmid concentration (Fig. 1). According to those results, greater plasmid concentrations (200 and 400 mg of plasmid/ml of protoplasts) were tested in three experiments but no better results were observed (data not shown). In experiments of both electroporation and transformation via PEG-induced direct DNA uptake into protoplasts, the transformation efficiency was evaluated with respect to the concentration of DNA. Our results coincide with those previously obtained by other authors in that the increase of plasmid concentration improved the transformation efficiency until a point of saturation was reached. For example, Fromm et al. (1985) found a positive linear correlation in carrot protoplasts for up to 40 mg of plasmid / ml of protoplasts while Rathus and Birch (1992) observed a similar correlation in sugarcane protoplasts from 25 to more than 250 mg of plasmid/ml of protoplasts. These differences among genera probably depend mainly on the quantity of nucleases present in the cytoplasm (Joersbo and Brunstedt, 1996).

466 Figure 1. The effect of plasmid concentration on melon protoplast GFP expression one day after transformation. Bars indicate standard deviation.

In experiments of both electroporation and transformation via PEG-induced direct DNA uptake into protoplasts, the transformation efficiency was evaluated with respect to the concentration of DNA. Our results coincide with those previously obtained by other authors in that the increase of plasmid concentration improved the transformation efficiency until a point of saturation was reached. For example, Fromm et al. (1985) found a positive linear correlation in carrot protoplasts for up to 40 mg of plasmid/ml of protoplasts while Rathus and Birch (1992) observed a similar correlation in sugarcane protoplasts from 25 to more than 250 mg of plasmid/ml of protoplasts. These differences among genera probably depend mainly on the quantity of nucleases present in the cytoplasm (Joersbo and Brunstedt, 1996). Using a plasmid concentration of 100 µg/ml protoplasts, a more detailed time course study of GFP expression was performed (Table 1). From the first to the fifteenth day of cultivation, the quantity of GFP events was maintained although it changed its distribution among protoplasts, cells and first divisions. As was expected, the number of protoplasts with GFP expression decreased, mainly because they become cells after wall regeneration. GFP expression decreased from the cell to the first division stage. This could be due to the change from transient to stable expression. In fact, it appears that the cells that expressed the gfp gene during the first division stage were capable of maintaining it during the following stages of growth (Table 1).

467 Table 1. GFP expression in different cell stages and days after transformation. Mean of three experiments (standard error)

Days after Protoplasts Cells 1st division Aggregates transformation

1 652 0) 0) 0) (46) (0) (0) (0)

7 289 413 0) 0) (27) (66) (0) (0)

15 210 368 53 0) (76) (123) (18) (0)

450) 10 0) 51 (0) (7) (0) (12)

To the best of our knowledge, there is only one report on melon protoplast transformation (Nishiguchi et al., 1988). In that study, the authors used electrical dischar- ges as a DNA introduction method and obtained transient expression in protoplasts. On the other hand, some studies have reported that the use of PEG causes problems of toxicity (Kao and Saleem, 1986; Armstrong et al., 1990). However, with our method and plant material we did not detect any problems related to cellular viability. In fact, the protocol of transformation has turned out to be highly reproducible. In this research, we have obtained an average of fifty aggregates with GFP expression / million of protoplasts. The maintenance of GFP expression has been observed in subsequent phases of cellular development. Although the relative transformation efficiency (0.005%) is lower than that obtained through Agrobacterium-mediated transformation in melon (1-3%), the final number of transgenic calli is significantly higher. An average of 25 transgenic plants can be obtained from one million PEG-treated protoplasts using the protocol of plant regeneration from melon protoplasts previously developed (García-Sogo, 1990). It should be noted that this quantity of protoplasts may be isolated from just two plantlets. However, to obtain the same number of transgenic plants with the Agrobacterium-mediated transformation protocol we would need more than 200 plantlets.

References

Armstrong, C.L., Petersen, W.L., Buchholz, W.G., Bowen, B.A. and Sulc, S.L. 1990. Factors affecting PEG-mediated stable transformation of maize protoplasts. Plant Cell Rep., 9: 335-339. Ben Tahar, S. and De Both, M.T.J. 1988. Introduction of foreign genes into melon (Cucumis melo L.) using Agrobacterium tumefaciens. In: Risser, G. and Pitrat, M. (Eds.), Cucurbitaceae 88, Proc. 4th Eucarpia Meeting on Cucurbit Genetics and Breeding. Avignon-Montfavet, Fran- ce, pp. 209-216. Bordas, M., Dabauza, M., Salvador, A., Roig, L.A. and Moreno, V. 1993. Obtención de plantas transgénicas de melón. II Congreso Ibérico de Ciencias Hortícolas.

468 Dong, J.Z., Yang, M.Z., Jia, S.R. and Chua, N.H. 1991. Transformation of melon (Cucumis melo L.) and expression from the cauliflower mosaic virus 35S promoter in transgenic melon plants. Bio Technol., 9: 858-863. Ezura, H., Yuhashi, K.I., Yasuta, T. and Minamisawa, K. 2000 Effect of ethylene on Agrobacte- rium tumefaciens-mediated gene transfer to melon. Plant Breeding, 119: 75-79. Fang, G.W. and Grumet, R. 1990. Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants. Plant Cell Rep., 9: 160-164. Fromm, M., Taylor, L.P. and Walbot, V. 1985. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. U.S.A., 82: 5824-5828. Galperin, M., Patlis, L., Ovadia, A., Wolf, D., Zelcer, A. and Kenigsbuch, D. 2003. A melon genotype with superior competence for regeneration and transformation. Plant Breeding, 122: 66-69. García-Sogo, B. 1990. Morfogénesis en cultivo in vitro de melón: regeneración de plantas con alta eficacia a partir de células y protoplastos. Tesis Doctoral. Doctora en Ciencias Biológicas. Universidad de Valencia. Guis, M., Ben-Amor, M., Latche, A., Pech, J.C. and Roustan, J.P. 2000. A reliable system for the transformation of Cantaloupe Charentais melon (Cucumis melo L. var. cantalupensis) leading to a majority of diploid regenerants. Scientia Hort., 84: 91-99. Joersbo, M. and Brunstdet, J. 1996. Electroporation and transgenic plant production. In: Lynch, P.T. and Davey, M.R. (Eds.), Electrical Manipulation of Cells, pp. 201-222. Kao, K.N. and Saleem, M. 1986. Improved fusion of mesophyll and cotyledon protoplasts with PEG and high pH-Ca2+ solutions. J. Plant Physiol., 122: 217-225. Moreno, V., Zubeldia, L. and Roig, L.A. 1984. A method for obtaining callus cultures from mesophyll protoplasts of melon (Cucumis melo L.). Plant Sci. Lett., 34: 195-201. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Nishiguchi, M., Kohno, M. and Motoyoshi, F. 1988. Electroporation-mediated gene transfer into melon mesophyll protoplasts: transient expression of the chloramphenicol acetyltransferase (CAT) gene. Bull. Natl. Inst. Agrobiol. Resources Japan, 4: 177-187. Rathus, C. and Birch, R.G. 1992. Stable transformation of callus from electroporated sugarcane protoplasts. Plant Sci., 82: 81-89. Roig, L.A., Zubeldia, L., Orts, M.C., Roche, M.V. and Moreno, V. 1986. Plant regeneration from cotyledon protoplasts of Cucumis melo L. cv. Cantaloup Charentais. Cucurbit Genet. Coop. Rep., 9: 74-77. Toyoda, H., Hosoi, Y., Yamamoto, A., Nishiguchi, T., Maeda, K., Takebaiashi, T., Shiomi, T. and Ouchi, S. 1991. Transformation of melon (Cucumis melo L.) with Agrobacterium rhizogenes. Plant Tiss. Cult. Lett., 8: 21-27. Valles, M.P. and Lasa, J.M. 1994. Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro). Plant Cell Rep., 13: 145-148.

469 470 Stachyose to sucrose metabolism in sweet melon (Cucumis melo) fruit mesocarp during the sucrose accumulation stage

Z. Gao1, M. Petreikov1, Y. Burger2, S. Shen1 and A.A. Schaffer1 1Department of Vegetable Crops, Institute of Field and Garden Crops, Agricultural Research Organization, Volcani Center, P. O. Box 6, Bet Dagan, 50-250, Israel; e-mail: [email protected] 2Department of Vegetable Crops, Institute of Field and Garden Crops, Agricultural Research Organization, Newe Yaar Research Center, P. O. Box 1021, Ramat Yishay 30-095, Israel

Summary

Sugar content determines the taste and quality of sweet melon (Cucumis melo) fruit. The accumulation of the disaccharide sucrose accounts for practically all of the variability in total sugar content of the developing fruit. We studied the pathway of sugar metabolism, consisting of the activities of 13 enzymes that account for the metabolism of the translocated raffinose oligosaccharides and the subsequent steps leading to sucrose synthesis. Six of the enzymes (alkaline a- galactosidase I, galactokinase, UDPglu-4'-epimerase, phosphoglucose isomerase, phosphoglucomutase and sucrose phosphate synthase) had significantly increased activity concomitantly with the increase in sucrose levels; one enzyme, soluble acid invertase which hydrolyzes sucrose, decreased significantly in activity during the sucrose-accumulation period. These results suggest a coordinated control of fruit-sugar metabolism during the sucrose-accumulation period of fruit development.

Keywords: Cucumis melo, invertase, sucrose phosphate synthase, coordinated control

Introduction Sugar accumulation is responsible for the sweet taste and, hence, quality of the developing melon (Cucumis melo L.) fruit. Previous studies have shown that the melon fruit undergoes a shift during development where during the early stages imported carbohydrates are utilized primarily for growth and during the maturing stage the disaccharide sucrose is accumulated. The latter accounts for the increase in sugar content of the mesocarp during the final approximately two weeks of fruit development (Schaffer et al., 1996; Burger et al., 2000). Accounting for the increase in sucrose is a metabolic transition in which the early stage is characterized by high activities of soluble acid invertase, which functions in the breakdown of sucrose. The entry into the sucrose accumulation phase is characterized by the developmental decrease in acid invertase activity, allowing for sucrose to accumulate, together with the increase in activity of the enzyme sucrose-phosphate synthase (SPS), which catalyzes the synthesis of sucrose (Hubbard et al, 1989; Schaffer et al., 1996; Lester et al., 2001). However, the enzymes invertase and SPS are only two in a larger metabolic pathway that is responsible for the metabolism of carbohydrates translocated from the source leaves to the fruit sink (Schaffer et al., 1996; Feusi et al., 1999; Gao et al.,

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 471 1999). The pathway (Fig. 1) begins with the hydrolysis of the translocated galactosyl-sucrose oligosaccharides stachyose and raffinose by the enzymes a-galactosidase, of which there are three in the developing melon fruit (Gao and Schaffer, 2000). The released galactose is phosphorylated by a specific galactokinase and the gal-1-P product is subsequently transformed to glu-1-P by the concerted actions of a novel UDPgalactose pyrophosphorylase (UDPgalPPase), UDPglucose-4'-epimerase and UD- Pglucose pyrophosphorylase (UDPgluPPase). The resultant glu-1-P can undergo further transformations to glu-6-P and fru-6-P via the phosphoglucomutase (PGM) and phosphoglucoisomerase (PGI) reactions. The enzyme SPS catalyzes the synthesis of sucrose-P from the substrates fru-6-P and UDP-glucose. Sucrose synthase may also catalyze the synthesis of sucrose from fructose and UDP-glucose, or it can cleave sucrose to fructose and UDP-glucose in the reverse reaction. Finally, the invertase enzymes, of which there are acid and neutral pH forms, are responsible for the hydrolysis of sucrose, either from the initial remains of the translocated galactosyl-sucrose or of the newly synthesized sucrose.

Figure 1. Proposed metabolic pathway of raffinose family oligosaccharides in melon fruit.

472 The purpose of the present study was to characterize the complete sugar metabolism pathway described above in order to determine the relative importance of changes in enzyme activity that contribute to the phenomenon of sucrose accumulation in the fruit mesocarp. In a previous study (Gao et al., 1999), we reported the developmental changes in the activities of the pathway in young melon ovaries, from before anthesis to 20 days following pollination, in order to characterize fruit carbohydrate metabolism during fruit set and early developmental growth. The present report con- tinues this study and compares the metabolic pathway immediately prior to the onset of and during the sucrose accumulation phase.

Materials and methods

Plants of Revigal, a Galia-type melon cultivar, were grown using standard cultivation practices in a greenhouse in the spring season (fruit harvested April-May). Each plant was vertically trained to a single stem, except for the first internode and node of a single branch on which a fruit was allowed to develop, with a minimum of 20 leaves on the main stem. Ovaries were pollinated and tagged at anthesis and fruit were harvested at 11, 22, 30 and 44 days after pollination. Mesocarp from 30- and 44-day fruit was sampled, frozen in liquid nitrogen and stored at -78°C. Sugars were extracted and analyzed by HPLC and enzyme activities were assayed as described previously (Gao et al., 1999).

Results and discussion

The accumulation pattern of soluble sugars in the developing melon fruit is presented in Fig. 2. The fruit from 30 days past anthesis had not yet begun to accumulate sucrose, and the comparison of enzyme activities in these fruit to those of the sucrose accumulation stage of 44 days after anthesis can suggest physiologically significant differences associated with sucrose accumulation.

Figure 2. Changes in sugar content of developing C. melo Re- vigal fruit flesh.

473 The activities of the 13 enzymes assayed in this study are compared in Table 1. Acid invertase showed a decrease in activity accompanying sucrose accumulation, in accordance with previous reports (Schaffer et al., 1987; Hubbard et al., 1989; Schaf- fer et al., 1996). Six of the enzymes showed significant increases in activity during the sucrose accumulation phase, including SPS which has previously been reported (Hubbard et al., 1989; Schaffer et al., 1996). The additional enzymes include the novel alkaline a-galactosidase I which hydrolyzes both stachyose and raffinose (Gao and Schaffer, 2000). As such, this enzyme stands as the first in the pathway of metabolism of translocated photoassimilate in the fruit sink; its increase in activity during sucrose accumulation fits well with the generalization that metabolic pathways are frequently controlled by the initial and final enzymes in the pathway. As such, the alkaline a- galactosidase I together with SPS make up the first and last enzymes in the stachyose-to-sucrose metabolic pathway.

Table 1. Comparison of enzyme activities (nmol/g fresh weight/minute) in the fruit flesh of the sucrose-accumulating C. melo Revigal. Activity of each enzyme was measured on a minimum of three individual fruit. The onset of sucrose accumulation occurs later than 30 days after anthesis (DAA)

Enzyme Activity 30 DAA Activity 45 DAA

Alkaline a-gal I 108 147* Alkaline a-gal II 33 39* Acid a-gal 57 65* Galactokinase 123 214* UDPgal PPase 1293 766* Epimerase 447 594* UDPglu PPase 2296 2372* PGI 1166 2127* PGM 2765 3504* SPS 185 295* Sucrose synthase 103 99* Alkaline invertase 78 57* Acid invertase 186 80*

*Difference in activities at the two developmental stages significant at 5% level.

The four additional enzymes that show significant increases in activity during the sucrose-accumulation period are galactokinase and epimerase, as well as the PGI and PGM pair which are responsible for the interconversions of hexose-P in the cell. The increases in PGI and PGM activity are due particularly to increases in the cytosolic forms of these enzymes, as the chloroplastic forms are of low activity in the developing melon fruit (unpublished results). We cannot explain at present the significance of the rise in activity of these enzymes in particular. However, in a parallel study (not presented) we compared the enzyme activities of the pathway between two sucrose- accumulating C. melo genotypes and two genotypes which accumulate little, if any,

474 sucrose (C. sativus L. genotype as well as C. melo Flexuosus Group Faqqous; Bur- ger et al., 2002). Significantly, the enzymes that showed consistent differences in activities between the two sucrose accumulators and the two non-accumulators were the same enzymes that showed the significant developmental differences in activity herein reported (Table 1); these showed higher activities in the sucrose-accumulating genotypes than in the non-accumulating genotypes. Acid invertase showed lower activities in the sucrose-accumulating genotypes, as expected. Accordingly, the results presented in this study indicate that the onset of sucrose accumulation in developing melon fruit is accompanied by a coordinated transition of the sugar metabolism pathway in the fruit sink and not merely by the changes in activities of one or two of the enzymes in the pathway. Further study of this pathway at the level of gene transcription is presently being carried out in order to shed light on the important developmental phenomenon of fruit sucrose accumulation.

References

Burger, Y., Shen, S., Petreikov, M. and Schaffer, A.A. 2000. The contribution of sucrose to total sugar content in melons. In: Katzir, N. and Paris, H.S. (Eds.), Proceedings of Cucurbitaceae 2000. Acta Hort., 510: 479-485. Burger, Y., Yeselson, L., Sahar, U., Paris, H.S., Katzir, N., Levin, I. and Schaffer, A.A. 2002. A single recessive gene for sucrose accumulation in Cucumis melo fruit. J. Amer. Soc. Hort. Sci., 127: 938-943. Feusi, M.E.S., Burton, J.D., Williamson, J.D. and Pharr, D.M. 1999. Galactosyl-sucrose metabolism and UDP-galactose pyrophosphorylase from Cucumis melo L. fruit. Physiol. Plant., 106: 9-17. Gao, Z., Petreikov, M., Zamski, E. and Schaffer, A.A. 1999. Carbohydrate metabolism during early fruit development of sweet melon (Cucumis melo). Physiol. Plant., 106: 1-8. Gao, Z. and Schaffer, A.A. 2000. A novel alkaline a-galactosidase from melon fruit with a substrate preference for raffinose. Plant Physiol., 119: 979-987. Hubbard, N.L., Huber, S.C. and Pharr, D.M. 1989. Sucrose phosphate synthase and acid invertase as determinants of sucrose concentration in developing muskmelon (Cucumis melo) fruits. Plant Physiol., 91: 1527-1534. Lester, G.E., Arias, L.S.S. and Gomez-Lim, M. 2001. Muskmelon fruit soluble acid invertase and polypeptide profiles during growth and maturation. J. Amer. Soc. Hort. Sci., 126: 33-36. Schaffer, A.A., Aloni, B. and Fogelman, E. 1987. Sucrose accumulation and metabolism in developing fruit of Cucumis. Phytochem., 26: 1883-1887. Schaffer, A.A., Madore, M. and Pharr, D.M. 1996. Cucurbits. In: Zamski E. and Schaffer, A.A. (Eds.), Photoassimilate distribution in plants and crops. Marcel Dekker, New York, pp. 729- 757.

475 476 Consensus chloroplast primer analysis: A molecular tool for evolutionary studies in Cucurbitaceae

S-M. Chung and J.E. Staub U.S. Department of Agriculture, Agricultural Research Service, Vegetable Crops Unit, Department of Horticulture, 1575 Linden Dr., University of Wisconsin, Madison, WI 53706 USA; e-mail: [email protected]

Summary

Consensus chloroplast (cp) primers that possess hyper-variable cp DNA regions have been developed in several species. Although a limited number of such primers have been constructed from the tobacco cp genome, they may be of minimal value for studying the evolutionary relationships among closely related taxa in Cucurbitaceae. Therefore, a study was designed to provide additional genetic tools for the evolutionary assessment in this plant family by employing selected cp cucumber regions that were sequenced using consensus primers developed from Arabidopsis thaliana L., Nicotiana. tabacum L., and Spinacia oleracea L. Mono-nucleotide repeats (A or T ³ 7) in cucumber cp DNA sequences were used to select 24 consensus cp primer (CCP) pairs for evaluation in cucurbit species. All CCP pairs produced amplicons using cucumber (Cucumis sativus L.), melon (Cucumis melo L.), squash (Cucurbita pepo L.), and bottle gourd (Lagenaria siceraria (Molina) Standl.) DNA, except one pair (18086-F & 18935-R) that produced no bands in the bottle gourd accession sampled. Sequence analyses using four CCP primers (1571-F, 13876-F, 15358-R, and 31173- F) detected highly polymorphic sequences among these species. These results indicate that these CCP pairs, along with previously developed 23 consensus cp SSR (ccSSRs; Chung and Staub, 2003), will be useful for detection of hyper-variable cp DNA regions in the Cucurbitaceae, and, thus, will likely be of value for the evolutionary studies in the Cucurbitaceae.

Keywords: consensus, chloroplast, closely related taxa, sequence substitution, simple sequence repeats (SSR)

Introduction Preceding the discovery and elucidation of hyper-variable cp DNA regions, cp genome analysis was minimally useful for the examination of closely related taxa and/or for the characterization of intra-specific variation (McCauley, 1995). The relatively high level of cp DNA variation detected by simple sequence repeats (SSR) markers has provided opportunities for increased genetic resolution among closely related taxa (Powell et al., 1995b; Vendramin et al., 1996; Vendramin and Ziegenhagen, 1997), including Glycine (Powell et al., 1996), Hordeum (Provan et al., 1999), Oryza (Ishii and Mc- Couch, 2000), Pinus (Powell et al., 1995a,b), and Solanum (Bryan et al., 1999). Comprehensive molecular examination of many plant species is limited by the lack of sufficient organellar DNA sequence information. To overcome this limitation, consensus (synom. universal) markers have been developed which possess DNA sequences that are homologous to conserved regions of plant cp and mitochondrial genomes (Taber- let et al., 1991; Demesure et al., 1995; Dumolin et al., 1997). The development of consensus cp markers has been achieved through the use of a cp DNA sequence data-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 477 base (http://www.ncbi.nlm.nih.gov). For instance, Weising and Gardner (1999) developed 10 consensus cp microsatellite primers (ccmp) that span the genomic distance between base pair (bp) position 1 to 86,694 and from position 154,185 to 155,939 of the tobacco (Nicotiana tabacum L.) cp genome. The limited genetic variation among closely related taxa such as that found in the Cucurbitaceae can present a challenge for defining evolutionary relationships. Thus, 23 consensus cp simple sequence repeat (ccSSR) were developed markers which are uniformly distributed across the tobacco cp genome (Chung and Staub, 2003). These markers were successfully employed for the elucidation of genetic relationships at the tribe level in the Cucurbitaceae (Chung et al., 2003; Decker-Walters et al., in press). The number of ccSSR markers currently available for the genetic analysis of closely related taxa in Cucurbitaceae presently limits extensive assessment of evolutionary relationships in this family (Chung, unpublished data). For instance, some ccSSR markers do not detect nucleotide substitution variation among some Cucurbita pepo L. species. Moreover, only a few ccSSR markers (6 of 23) detect variation at a relatively low level. Thus, in order to allow for the detection of more genetic variation in Cucurbitace- ae, additional consensus cp primers were developed (bp position 1 to 51,025 according to the tobacco cp genome) for use as evolutionary tools and for sequencing of the cucumber cp genome. We report herein the construction and evaluation of 24 consensus cp primer pairs from cucumber cp DNA sequences that will likely detect hyper-variable cp DNA regions in the Cucurbitaceae.

Materials and methods

Primer design and construction To select candidate positions of consensus cp primers (CCP) for high variable sequence positions in Cucurbitaceae, N. tabacum cp sequences (accession number: CHNTXX, 155,939 bp) (Shinozaki et al., 1986) from bp position 1 to 55,000 were examined by BLAST searches (Altschul et al., 1990). BLAST results allowed for the choice of conserved sequence areas from the available GenBank databases for primer design. Candidate consensus primers were selected using Genetool software (BioTools Inc, Edmonton, Alberta, Canada), and modified according to the degenerations of mismatch nucleotides based on alignments with Arabidopsis thaliana L., Nicotiana tabacum L. and Spinacia oleracea L. cp sequences. Primers were then named according to bp positions of the tobacco cp genome. For instance, for 1571-F and 2328-R primers, 1571 and 2328 indicate that these primer sequences start at the tobacco cp 1571 and 2328 bp position, respectively, where F and R are abbreviations for forward and reverse priming direction, respectively (Table 1).

Sequencing of cucumber cp DNA and selecting CCP pairs Total DNA of cucumber line GY-14, melon TopMark, squash Orange Ball, and a bottle gourd accession designed #892 [The Cucurbit Network (TCN) number] was extracted according to Staub et al. (1996). Candidate consensus primer pairs (data not presented) possessing relatively short distances between forward and reverse pri-

478 mers (i.e., less than 3,000 bp) in relation to tobacco bp position were employed in polymerase chain reactions (PCR) with cucumber template DNA (50°C annealing temperature), and subsequently fractionized according to Chung and Staub (2003). Successfully amplified fragments were then direct sequenced using the consensus primers developed herein as described in Chung and Staub (2003). The consensus cp primer pairs that possess A or T mono-nucleotide repeats (n ³ 7) in cucumber cp sequences were chosen as final CCP pairs for evaluation (Table 1). To avoid duplication, ccSSRs motifs (Chung and Staub, 2003) were excluded from these final CCP pairs. These final CCP pairs were employed for the genetic analysis of cucumber, melon, squash, and bottle gourd using total DNA (Fig. 1). To investigate which CCP pairs effective in the detection of hyper-variable cp DNA regions in cucurbit species, obtained from the initial PCR amplicons were then sequenced using four primers (1571-F, 13876-F, 15358-R, and 31173-F), and aligned using GeneTool software (Fig. 2).

Figure 1. Amplifications of consensus chloroplast primer pairs (F = forward and R = reverse) with Cucurbitaceae species DNA where C = cucumber, M = melon, S = squash, B = bottle gourd, and Lamda = Lambda DNA EcoRI+HindIII size ladder.

479 Figure 2. The alignment of sequences in Cucurbitaceae species DNA as amplified by consensus chloroplast primer pairs, where C = cucumber, M = melon, S = squash, and B = bottle gourd. The mononucleotide repeat motifs targeted in cucumber chloroplast sequences are bold and underlined.

480 Results and discussion

Twenty-three ccSSR primer pairs were previously developed for an evolutionary study in plant species (Chung and Staub, 2003). Nevertheless, the effectiveness of these primer pairs was somewhat limited when used for the genetic analysis of closely related taxa in Cucurbitaceae. Therefore, an additional set of 24 CCP pairs were developed herein to increase effectiveness of this molecular tool for the detection of genetic variation in Cucurbitaceae. Four of these CCP primers were characterized by sequence analyses in cucumber, melon, squash, and bottle gourd. In order to improve the likelihood of detecting hyper-variable cp DNA regions in the Cucurbitaceae, CCPs were selected based on mono-nucleotide repeats identified in cucumber cp sequences. That is, instead of using tobacco cp sequence information as a source for mono-nucleotide repeats for primer pair construction (Chung and Staub, 2003), we sequenced candidate regions of the cucumber cp using consensus primers developed from Arabidopsis, spinach, and tobacco sequences. Using this strategy, 24 consensus primer combinations were chosen for the chloroplast analysis in the Cucurbitaceae (Table 1). All CCP pairs that were designed produced amplicons using cucumber, melon, squash, and bottle gourd DNA, except when primer pair 18086-F & 18935-R was used in PCR with bottle gourd DNA (Table 1 and Fig. 1). A single amplification was detected in all CCP pairs with cucumber DNA. In contrast, multiple amplifications were observed when several CCP pairs (i.e., 4281-F & 5276-R, 15802-F & 17040-R, 28344-F & 29566- R, 31934-F & 33544-R, 36140-F & 37160-R, and 43668-F & 44438-R) were employed using melon, squash, and bottle gourd template DNA (Table 1). This multiple amplification event in a single reaction is not desirable, and might be mitigated by the adjustment of annealing temperatures during PCR (i.e. Annealing Temperature Gradient PCR; Fazio et al., 2002). To prove that the selected CCP pairs are capable of the detection of hyper-variable cp DNA regions, three selected amplified fragments (pairs: 1571-F & 2328-R, 13876-F & 15358-R, and 31173-F & 31969-R) from cucumber, melon, squash, and bottle gourd were sequenced using 1571-F, 13876-F, 15358-R, and 31173-F primers. Most of sequenced regions detected sequence substitution variation and/or insertion/ deletion of nucleotide among the four Cucurbitaceae species examined (Fig. 2). The sequencing method employed herein produced 800 bp sequences (Chung, unpublished data). Both forward primer, 13876-F and reverse primer, 15358-R were employed for the sequencing of the amplified fragment from 13876-F & 15358-R pair whose expected bp lengths are 1483 bp. Although the targeted mono-nucleotide repeat motif for the CCP pair 13876-F & 15358-R was detected in the 15358-R sequence alignment (Fig. 2), the sequence alignments of 13876-F also detected hyper-variable cp DNA regions. This result suggests that franking regions of this and other mono- nucleotide repeats will likely have utility for the detection of hyper-variable DNA as well as mono-nucleotide repeat regions. Moreover, because of relatively large expected bp fragment lengths produced by the CCP pairs described herein (average of expected bp length, 1108) and their constituent targeted cucumber cp mono-nucleotide repeat motifs, these CCP pairs along with previously developed ccSSRs will likely be useful for evolutionary studies, especially for closely related taxa in Cucurbitaceae.

481 Spinacia , and Arabidopsis thaliana , Nicotiana tabacum Consensus chloroplast primers (CCP) developed from the chloroplast genomes Primers are named and expected location of primer pairs are given according to tobacco chloroplast genome bp positions B. gourd = Bottle gourd. Results of amplifications of CCP pairs with four Cucurbitaceae species DNA. Consensus chloroplast simple sequence repeats (ccSSRs) from Chung and Staub (2003). N/A = No amplification. (accession number = CHNTXX) (http://www.ncbi.nlm.nih.gov). CHNTXX) = number (accession 1 & 2 3 4 5 6 Table Table 1. oleracea

482 References

Altschul, S.F., Gish, W., Miller, W., Myers E.W. and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol., 215: 403-410. Bryan, G.J., McNicoll, J., Ramsay, G., Meyer, R.C. and De, J.W.S. 1999. Polymorphic simple sequence repeat markers in chloroplast genomes of Solanaceous plants. Theor. Appl. Genet., 99: 859-867. Chung, S-M. and Staub, J.E. 2003. The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa. Theor. Appl. Genet., 107: 757-767. Chung, S-M., Decker-Walters, D.S. and Staub, J.E. 2003. Genetic relationships within the Cucur- bitaceae as assessed by ccSSR marker and sequence analysis. Can. J. Bot., 81: 814-832. Decker-Walters, D.S., Chung, S-M. and Staub, J.E. 2003. Trends in Chloroplast Evolution: New Evidence from Nucleotide Substitutions in the Cucurbitaceae. J. Mol. Evol. (in press). Demesure, B., Sodzi, N. and Petit, R.J. 1995. A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Mol. Ecol., 4: 129-131. Dumolin, L. S., Pemonge, M.H. and Petit, R.J. 1997. An enlarged set of consensus primers for the study of organelle DNA in plants. Mol. Ecol., 6: 393-397. Fazio, G., Staub, J.E. and Chung, S-M. 2002. Development and characterization of PCR markers in cucumber. J. Amer. Soc. Hort. Sci., 127: 545-557. Ishii, T. and McCouch, S.R. 2000. Microsatellites and microsynteny in the chloroplast genomes of Oryza and eight other Gramineae species. Theor. Appl. Genet., 100: 1257-1266. McCauley. 1995. The use of chloroplast DNA polymorphism in studies of gene flow in plants. Trends Ecol. Evol., 10: 198-202. Powell, W., Morgante, M., Andre, C., McNicol, J.W., Machray, G.C., Doyle, J.J., Tingey, S.V. and Rafalski, J.A. 1995a. Hypervariable microsatellites provide a general source of polymorphic DNA markers for the chloroplast genome. Curr. Biol., 5: 1023-1029. Powell, W., Morgante, M., Doyle, J.J., McNicol, J.W., Tingey, S.V. and Rafalski, A.J. 1996. Ge- nepool variation in genus Glycine subgenus Soja revealed by polymorphic nuclear and chloroplast microsatellites. Genetics, 144: 792-803. Powell, W., Morgante, M., McDevitt, R., Vendramin, G. and Rafalski, A.J. 1995b. Polymorphic Simple Sequence Repeat Regions in Chloroplast Genomes: Applications to the Population Genetics of Pines. Proc. Natl. Acad. Sci. U.S.A., 92: 7759-7763. Provan, J., Russell, J.R. Booth, A. and Powell, W. 1999. Polymorphic chloroplast simple sequence repeat primers for systematic and population studies in the genus Hordeum. Mol. Ecol., 8: 505-511. Shinozaki, K., Ohme, M., Tanaka, M., Wakasugi, T., Hayashida, N., Matsubayashi, T., Zaita, N., Chungwongse, J., Obokata, J., Yamaguchi-Shinozaki, K., Ohto, C., Torazawa, K., Meng, B.Y., Sugita, M., Deno, H., Kamogashira, T., Yamada, K., Kusuda, J., Takaiwa, F., Kato, A., Toh- doh, N., Shimada, H. and Sugiura, M. 1986. The complete nucleotide sequence of the tobacco chloroplast genome: Its gene organization and expression. EMBO, 5: 2043-2049. Staub, J.E., Bacher, J. and Poetter, K. 1996. Factors affecting the application of random amplified polymorphic DNAs in cucumber (Cucumis sativus L.). HortScience, 31: 262-266. Taberlet, P., Gielly, L., Pautou, G. and Bouvet, J. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol. Biol., 17: 1105-1110. Vendramin, G.G., Lelli, L., Rossi, P. and Morgante, M. 1996. A set of primers for the amplification of 20 chloroplast microsatellites in Pineaceae. Mol. Ecol., 5: 595-598. Vendramin, G.G. and Ziegenhagen, B. 1997. Characterization and inheritance of polymorphic plastid microsatellites in Abies. Genome, 40: 857-864. Weising, K. and Gardner, R.C. 1999. A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms. Ge- nome, 42: 9-19.

483 484 Molecular mapping of the melon Fom-1/Prv locus

Y. Brotman1, I. Kovalski1, C. Dogimont2, M. Pitrat2, N. Katzir3 and R. Perl-Treves1 1Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel 2INRA, Station de Genetique et dAmelioration des Fruits et Legumes, BP 94, 84143 Montfavet Cedex, France 3Department of Vegetable Crops, Agricultural Research Organization, Newe Yaar Research Center, Ramat Yishay, Israel

Summary

The closely-linked Fom-1 and Prv resistance genes confer monogenic resistance against the soil borne fungus Fusarium oxysporum f.sp. melonis races 0 and 2, and against the papaya ring spot virus, respectively. Markers linked to these resistance genes were identified using a RIL population derived from a cross between Cucumis melo PI 414723 and C. melo Vedrantais. Using bulked segregant analysis, we developed two Random Amplified Polymorphic DNA (RAPD) and two Restriction Fragment Length Polymorphism (RFLP) markers linked to this locus. Two of the markers were converted to cleaved amplified polymorphic sequences (CAPS) markers. The RFLP marker-sequences bear homology to NBS-LRR resistance genes. These markers are the closest- linked markers published so far for this important locus and can now be used by breeders in marker-assisted selection.

Keywords: Cucumis melo, molecular markers, RAPD, CAPS, RFLP, NBS-LRR, Fusarium oxysporum, papaya ring spot virus (PRSV)

Introduction The soil-borne pathogen Fusarium oxysporum f.sp. melonis exclusively attacks melon, causing a severe wilt disease. There are four known races of the fungus, designated races 0, 1, 2 and 1,2 (Risser et al., 1976). Melon genotypes that are resistant to the different races have been identified, and monogenic dominant inheritance was described against races 0, 1 and 2. The Fom-1 gene, originally described in cultivar Doublon, confers resistance to races 0 and 2 (Risser and Mas, 1965). Another gene, Fom-3, controls resistance to races 0 and 2 in cultivar Perlita FR, but data about its possible allelism with Fom-1 is controversial (Zink and Gubler, 1985; Risser, 1987). The Fom-2 gene, found in PI 414723, confers resistance to races 0 and 1 (Perin et al., 2002). Potyviruses such as the zucchini yellow mosaic virus (ZYMV) and the papaya ring spot virus (PRSV, formerly called watermelon mosaic virus 1) form the largest and most economically important group of plant viruses (Riechmann et al., 1992). The papaya ring spot virus attacks different cucurbit species, and dominant monogenic resistance to this pathogen was described in PI 414723 (Webb, 1979; Pitrat and Le- coq, 1983). The Fom-1 and Prv genes are tightly linked to each other, and map to Linkage Group IX in the melon genetic map of Perin et al. (2002). In our previous study we isolated a set of melon sequences that share homology with R-genes of the NBS-LRR gene family. These were mapped as RFLP markers and several such markers were linked to the Fom-1/Prv, Fom-2 and Vat loci (Brotman et al., 2002).

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 485 In this study we focused on the Fom-1/Prv locus, in order to enrich the area with additional genetic markers. Using a RIL population derived at INRA from a cross between PI 414723 (McCreight et al., 1992) and cv. Vedrantais (Perin et al., 2002), we identified two tightly linked RAPD markers; one of them was then converted into a locus- specific CAPS marker. In addition, two NBS-LRR sequences were mapped as RFLP to the same locus, and one of them converted into a CAPS marker.

Materials and methods

RFLP and RAPD analysis Cloning and linkage analysis of NBS-1 and NBS-47-3 was described by Brotman et al. (2002). RAPDPCR was performed using random decamer sets (Operon Techno- logies, Alameda, Calif. and University of British Columbia, Vancouver, according to Williams et al. (1993) and Silberstein et al. (2003).

CAPS markers PCR bands were gel-eluted and cloned in the pGEM-Teasy plasmid vector (Pro- mega). Automated sequencing was carried out by Hylabs (Rehovot, Israel) using an ABI Prism 310 Genetic Analyzer and the BigDye Terminator Kit (Perkin-Elmer Corp., Foster City, CA). Oligonucleotides were from Hylabs (Rehovot, Israel). CAPS markers were amplified in a total volume of 25 ml, using an annealing temperature of 56°C and standard PCR cycles. The entire PCR reaction was digested for 3 hrs at 37°C, by adding 3 ml of the appropriate buffer and restriction enzyme, and the whole sample was separated on 2% TBE x 1-agarose gels. Inverse PCR reactions were according to Meissner et al. (2000).

Results and discussion

The RIL population used in this study consisted of 63 lines that segregated 1:1 between PRV-resistant and susceptible lines; 1:1 segregation was also observed for Fusarium race 2 resistance. About 5% of the RIL lines, that still segregated for these genes, were omitted from the analysis. A single pair of DNA mixes was prepared: the first mix included DNA from 25 plants that were PRV resistant and Fusarium race 2 susceptible, similar to the PI 414723 parent. The other mix contained 25 samples of Fusarium race 2 resistant, PRV-susceptible lines, like the Vedrantais parent. A total of 73 random decamer primers, shown to be polymorphic between the mapping parents, were screened to identify polymorphism between the two DNA bulks. On average, each primer amplified about 6-7 fragments that ranged in size from 120 bp to 4 Kb. Six of these detected a polymorphic band between the two bulks and were analyzed in the whole population. Four of the bands either did not segregate properly, or were unlinked. The remaining two bands were linked to the Fom-1/Prv locus. The primer coded 62 generated a 1235 bp fragment in PI 414723 and in the Fom- 1 susceptible, PRV resistant mix, but not in Vedrantais or in the opposite mix (Fig. 1A). The band was excised, cloned and sequenced, and two locus-specific primers were designed. These amplified a single DNA fragment from both parents. The sequenced fragment showed homology to dynamin sequences in the databases. Dynamin pro-

486 Figure 1. Markers linked to the Fom-1/Prv locus in melon, in the mapping parents and a sample of RIL lines. Arrows in A and C indicate the linked RAPD fragments. A. RAPD marker 62. B. CAPS marker derived from RAPD 62 by specific PCR amplification and cleavage with Alw26I. C. RAPD marker 53. D. RFLP marker NBS-47-3. Genomic DNA (3 mg) was digested with XbaI, blotted to charged Nylon membranes and hybridized with radio-labeled 250 bp NBS-47-3 fragment at high stringency. E. CAPS marker derived by specific amplification of NBS1 sequence, followed by digestion with NcoI.

487 teins are suggested to function as mechano-chemical proteins /regulatory enzymes (Song et al., 2003). A useful CAPS marker was obtained by cleaving the 420 bp product with restriction endonuclease Alw26I. In Vedrantais, the fragment is cleaved into 330 and 80 bp products, in PI 414723 it remains uncut (Fig. 1B). The CAPS marker, due to robust PCR conditions and single-band amplification product, is easier to score reproducibly, compared to the original RAPD marker from which it was derived. A second RAPD primer, coded 53, generates a 428 bp-fragment in PI 414723 (Fig. 1C). We cloned and sequenced the RAPD product from both parents, but failed to detect a single-base polymorphism between them. We screened the population with the 62-CAPS and the 53 RAPD markers and found that they flanked Fom-1/Prv from both sides, at distances of 2.8 and 4.3 cM, respectively (1 recombinant between Prv and 62, 4 recombinants between 53 and Fom-1; Fig. 2). In our previous study we isolated a set of 15 melon sequences that were homologous to disease and pest resistance genes of the NBS-LRR family (Brotman et al., 2002). Garcia-Mas et al. (2001) also found an NBS-LRR homologue that was likely to reside near the Fom-1/Prv locus. One of our clones, NBS 47-3, mapped at a distance of 4.8 cM (two recombinants) from Fom-1, and 2.8 cM (a single recombinant) from Prv (Fig. 1D). The NBS 47-3 sequence was 250 bp-long. Larger fragments, 2800 bp in total, were cloned by inverse PCR and read from both mapping parents, but no polymorphism was detected. A second NBS-like sequence, NBS-1, was recently mapped and found to be linked at the same locus. We detected a single-base polymorphism between the mapping parents, allowing to develop a robust CAPS marker for NBS-1. The Vedrantais allele was cleaved by restriction endonuclease NcoI into 45 bp and a 205 bp products, while the 250 bp-allele of PI 414723 remained uncut (Fig. 1E). Fig. 2 displays the present map of the Fom-1/Prv locus. We are expanding the analysis to additional populations, and the data so far, based on larger numbers of plants, indicate lower recombination fractions between the markers (not shown). Additional markers are being selected using the AFLP technique. The presence of two disease resistance genes and two NBS-LRR sequences in an interval spanning 4.8 cM indicates that this region harbors a cluster of R genes; such clusters are a common feature of plant genomes. The markers that we develop could assist breeders in their selection cycles, and provide starting points to map-based cloning of the locus.

Figure 2. Linkage map of the Fom-1/Prv locus. The map was derived using the MAPMAKER software (Lander et al., 1987) and is based on data from 63 RILS.

488 References

Brotman, Y., Silberstein, L., Kovalski, I., Perin, C., Dogimont, C., Pitrat, M., Klingler, J., Thompson, G. and Perl-Treves, R. 2002. Resistance gene homologues in melon are linked to genetic loci conferring disease and pest resistance. Theor. Appl. Genet., 104: 1055-1063. Garcia-Mas, J., Van Leeuwen, H., Monfort, A., de Vicente, M.C., Puigdomenech, P. and Arus, P. 2001. Cloning and mapping of resistance gene homologues in melon. Plant Sci., 161: 165- 172. Lander, E.S., Green, P., Abrahamson, J., Barlow, A., Daley, M.J., Lincoln, S.E. and Newberg, L. 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics, 1: 174-181. McCreight, J.D., Bohn, G.W. and Kinshaba, A.N. 1992. Pedigree P.I. 414723 melon. Cucurbit Genet. Coop. Rep., 15: 51-52. Meissner, R., Chague, V., Zhu, Q., Emmanuel, E., Elkind, Y. and Levy, A.A. 2000. High throughput system for transposon tagging and promoter trapping in tomato. Plant J., 22: 265-274. Perin, C., Hagen, S., De Conto, V., Katzir, N., Danin-Poleg, Y., Portnoy, V., Baudracco-Arnas, S., Chadoeuf, J., Dogimont, C. and Pitrat, M. 2002. A reference map of Cucumis melo based on two recombinant inbred line populations. Theor. Appl. Genet., 104: 1017-1034. Pitrat, M. and Lecoq, H. 1983. Two alleles for Watermelon Mosaic Virus 1 resistance in melon. Cucurb Genet. Coop. Rep., 6: 52-53. Riechmann, J.L., Lain, S. and Garcia, J.A. 1992. Highlights and prospects of potyvirus molecular biology. J. Gen. Virol., 73: 1-16. Risser, G. and Mas, P. 1965. Mise en evidence de plusieurs races de Fusarium oxysporum f.sp. melonis. Ann. Amelior. Plant., 15: 405-408. Risser, G. 1987. Controversy on resistance to fusarium wilt in Perlita (Cucumis melo L.). Cucurbit Genet. Coop. Rep., 10: 60-63. Risser, G., Banihashemi, Z. and Davis, D.W. 1976. A proposed nomenclature of Fusarium oxysporum f.sp. melonis races and resistance genes in Cucumis melo. Phytopathology, 66: 1105-1106. Silberstein, L., Kovalski, I., Brotman, Y., Perin, C., Dogimont, C., Pitrat, M., Klingler, J., Thompson, G., Portnoy, V., Katzir, N. and Perl-Treves, R. 2003. Linkage map of Cucumis melo including phenotypic traits and sequence-characterized genes. Genome, 46: 761-773. Song, B.D. and Schmid, S.L.. 2003. A molecular motor or a regulator? Dynamins in a class of its own. Biochemistry, 18: 1369-1376. Webb, R.E. 1979. Inheritance of resistance to watermelon mosaic virus in Cucumis melo L. HortScience, 14: 265-266. Williams, J.G.K., Hanafey, M.K., Rafalsky, J.A. and Tingey, S.V. 1993. Genetic analysis using random amplified polymorphic DNA markers. Methods in Enzymology, 218: 704-741. Zink, F.W., and Gubler, W.D. 1985. Inheritance of resistance in muskmelon to Fusarium wilt. J. Amer. Soc. Hort. Sci., 110: 600-604.

489 490 Isolation and characterization of fruit-related genes in melon (Cucumis melo) using SSH and macroarray techniques

Y. Yariv1, V. Portnoy1, Y. Burger1, Y. Benyamini1, E. Lewinsohn1, Y. Tadmor1, U. Ravid1, R. White2, J. Giovannoni2, A.A. Schaffer3 and N. Katzir1 1Department of Vegetable Crops, Agricultural Research Organization, Newe Yaar Research Center, P. O. Box 1021, Ramat Yishay 30-095, Israel; e-mail: [email protected] 2U.S.D.A. Plant, Soil and Nutrition Laboratory, Tower Road, Cornell University, Ithaca, NY 14850, USA 3Department of Vegetable Crops, Agricultural Research Organization, The Volcani Center, P. O. Box 6, Bet Dagan 50-250, Israel

Summary

Genes that are uniquely expressed in the course of the development and ripening of melon (Cucumis melo) fruit were isolated using suppression subtractive hybridization (SSH) and macroarray techniques. Subtractions were performed reciprocally between two developmental stages of the fruit as well as for flesh and rind. The libraries were printed on macroarrays and subsequently screened with cDNA subtracted populations for differentially expressed clones. Selected clones were subjected to sequence similarity search and to Northern blot analysis. The large fraction of fruit-related clones that were specifically expressed in the original developmental stage or fruit tissue demonstrates the efficiency of the method. This study is the first step towards the construction of a representative microarray for melon fruit.

Keywords: genomic tools, fruit development, ripening

Introduction

Fruit quality is determined by numerous traits that affect taste, aroma, texture, pigmentation, nutritional value and duration of shelf life (for review see Giovannoni, 2001; White, 2002). Recently, genomic tools, such as DNA microarrays, were applied to strawberry and tomato and provided large-scale information on patterns of gene expression throughout fruit development and maturation (Aharoni et al., 2000, 2002; Moore et al., 2002). A major advantage of this methodology is its capacity to detect unknown genes involved in processes of interest. Melon (Cucumis melo L.) is a highly polymorphic species that consists of a broad array of wild and cultivated genotypes that differ in fruit traits such as ripening physiology (climacteric and non-climacteric), sugar and acid content, and secondary metabolites associated with taste and aroma. A number of genes associated with the processes of fruit development and ripening in melon have been described. Among them are genes involved in ethylene biosynthesis and regulation (Balague et al., 1993; Ayub et al., 1996; Payton et al., 1996; Aggelis et al., 1997; Sato et al., 1999; Hadfield et al., 2000), cell wall modifications (Hadfield et al., 1998; Rose et al., 1998), and secondary metabolism (Karvouni et al., 1995). Suppression subtractive hybridization (SSH) is a powerful tool for selecting genes that are exclusively expressed in one tissue but not in another (Diatchenko et al.,

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 491 1996). The SSH method is based on the subtraction of genes expressed in two compared tissues/developmental stages. Consequently, the resulting libraries are enriched for differentially expressed genes. The combination of SSH and macro/microarrays has proved efficient in selecting genes specific to tissues or processes (Xiong et al., 2001). The aim of our project was to identify candidate genes that affect fruit quality in melon, for future marker-assisted selection and genetic modifications. A genomic approach, combining SSH and macroarray techniques, was applied in our study to identify genes that are uniquely expressed during fruit development in melon. The efficiency of this approach in isolating and characterizing fruit-related genes from melon is described.

Material and methods

Plant material Noy Yizreel, a parental line of Galia, was selected for the preparation of cDNA libraries. Noy Yizreel is a HaOgen-type green-fleshed cantaloupe (Group Cantalu- pensis) that is smooth-skinned, climacteric, aromatic and very sweet (Karchi, 2000). Other accessions studied were Arava, Védrantais, Dulce, Dudaim, Tam Dew, Faqqous, Rochet, and PI 414723.

SSH libraries Four cDNA libraries were constructed using the suppression subtractive hybridization (SSH, Diatchenko et al., 1996) PCR-SelectTM kit (Clontech Laboratories, Palo Alto, CA U.S.A.). RNA was extracted using a modification of La Claire II and Herrin (1997). RNA extracted separately from the fruit flesh (mesocarp) and from the fruit rind (exocarp) of young (25 days past anthesis, DPA) and mature (47 DPA) Noy Yiz- reel fruits was used for cDNA synthesis. The cDNA was used to construct four SSH libraries: (1) Mature flesh (young flesh cDNA subtracted from mature flesh cDNA); (2) Young flesh (mature flesh cDNA subtracted from young flesh cDNA); (3) Mature rind (young rind cDNA subtracted from mature rind cDNA); and (4) Young rind (mature rind cDNA subtracted from young rind cDNA)

Macroarrays Using a Q-bot robotic workstation at the Center for Gene Expression Profiling, Cornell University, 5000 clones from the four SSH libraries were printed, in duplicates, on Hybond-N+ membranes.

Clone selection and characterization Macroarrays were differentially screened with the uncloned forward and reverse subtracted cDNA mixtures (see PCR-SelectTM kit) as probes. Only clones hybridizing with either the forward or reverse subtracted probes were considered as differentially expressed. These clones were sequenced and similarities to known genes were identified using bioinformatic tools, including the National Center for Biotechnology Information BLAST algorithms (Altschul et al., 1990). Back hybridization to the macroarray with selected

492 clones as probes indicated their number of copies in each SSH library. Expression of selected clones in different tissue types and developmental stages was further analyzed on Northern blots. Northern blotting (4-5mg of mRNA/20-25mg of total RNA, per lane) and hybridization were performed as described by Sambrook and Russell (2001).

Enzymatic activity of alcohol acetyl transferase Fruit extractions and enzymatic activity assays were performed as described by Shalit et al. (2001).

Results and discussion

Construction of SSH libraries and printing of macroarrays The data presented here were derived from four SSH libraries developed from the fruit of Noy Yizreel. Subtractions were performed reciprocally between 25 DPA and 47 DPA fruits and separately for the fruit flesh and for the fruit rind. Five thousand colonies from the four libraries were printed in arrays, in duplicates, on nylon membranes (macroarrays, Figure 1). These macroarrays were differentially screened using probes of the uncloned forward and reverse subtracted cDNA mixtures. Approximate- ly 1000 clones, from all four libraries, with obvious, above-background duplicated signals, were considered positive. Positive clones, which hybridized with one cDNA population and not with its reciprocal, were considered as differentially expressed clones. The technique was found to be highly efficient in three libraries, where 88% to 94% of the positive clones hybridized with the tester cDNA subtracted mixture and not the driver. In the young rind library, however, only 48% of the clones were successfully hybridized with the young rind subtracted cDNAs. Back hybridizations of 25 selected clones to the macroarrays indicated that the number of copies of these clones in the four libraries ranged from one to 158 copies.

Identification and characterization of clones Of approximately 1000 differentially expressed clones, 227 were sequenced and analyzed by BLAST. Over 50% of the sequenced clones were annotated. The annotation was probably limited by the relatively small size of the clones (most of them in the range of 100-500 bp) obtained by the SSH technique. As expected, a large fraction of the annotated clones shared sequence or amino-acid similarities with genes known to be related to fruit maturation (28% of all sequences), such as genes involved in texture changes, especially modification of cell-wall ultrastructure (such as expansin, extensin, pectin esterase), synthesis of aroma volatiles (such as alcohol acetyl transferases, d-cadinene synthase, alchohol dehydrogenase), and ethylene synthesis and signal transduction (such as ACC oxidase, Ein3, ethylene responsive element binding protein) (Table 1).

493 AB

Figure 1. Magnification of equivalent regions of the macroarrays hybridized to probes of (A) young/rind population and (B) mature/rind population. Clones that demonstrate differential expression in the mature or young rind were marked with a circle or a square respectively.

Northern blots were performed to further verify the expression profiles of selected clones in different plant tissues and fruit developmental stages. Of the 57 clones analyzed by Northern hybridization, 35 showed fruit-specific expression profiles (Fig. 2), most of which were expressed during the expected developmental stage (such as clones selected from mature fruit libraries that were expressed mainly in mature fruits). Table 1 depicts several examples of these genes and their fruit specific expression pattern. Of special interest are regulatory genes (such as transcription/translation factors, Table 1) that were expressed uniquely during fruit development and maturation. Only half of the clones associated with fruit maturation (14%) have been previously described in melon. Further characterization of candidate genes included detailed expression profiling combined with enzymatic activity studies. For example, in the case of alcohol acetyl transferase (AAT, Shalit et al., 2001), at least four members of this family were detected. Northern blots demonstrated expression of AAT1 & 2 in mature aromatic but not in the non-aromatic genotypes (data not shown). This expression pattern fits well the composition of volatiles of these melons: the majority of the volatiles of non-aromatic and unripe melons were short- and medium-chain alcohols, while the majority of the volatiles of mature aromatic melons were acetates, mostly aliphatic. Cell-free extracts from aromatic melons contained AAT activity, enabling acetylation of several alcohol substrates, while non-aromatic melons did not contain significant AAT activity with any of the substrates tested (Fig. 3).

494 Table 1. Clones associated with functional categories (degree of homology amino acid = aa or base pair = bp, clone size in bp)

Function Best homology Degree of homology & clone size

Cell wall Expansin 77/83aa metabolism (92%) 250 bp

Cell wall Xyloglucan endo- 41/47aa metabolism glucanase (XET) (87%) 144 bp

Aroma Alcohol acetyl 523/532bp formation transferase (AAT) (98%) 574 bp

Aroma Alcohol 48/78aa formation dehydrogenase (61%) (ADH) 337 bp

Ethylene Aminocyclopropane- 354/356bp pathway 1-carboxylic acid (99%) oxidase (ACC360 bp oxidase)

Ethylene EIN3-ethylene 69/69bp pathway insensitive (100%) like protein 188 bp

Regulation Jasmonic acid 91/126aa regulatory protein (72%) 978 bp

Regulation Regulator of gene 31/53aa silencing (58%) w178 bp

495 AB

Figure 2. Differentially expressed clones: (A) development-specific expression and (B) tissue-specific expression.

Figure 3. AAT activity (picokatal/gram fresh weight) of cell-free extracts from various melon genotypes using phenethyl alcohol as substrate. AR = Arava, NY = Noy Yizreel, VED = Védrantais, DUL = Dulce, DUD = Dudaim, FAQ = Faqqous, ROC = Rochet, TAD = Tam Dew, and PI = PI 414723.

In summary, by combining SSH and macroarray approaches, we were able to identify genes that are uniquely expressed during fruit development and ripening in melon. The identification of a large fraction of fruit-related clones demonstrated the high efficiency of the method. Genes from major metabolic pathways that are typical to fruit maturation were isolated. This study is the first step towards the development of a melon microarray.

496 References

Aggelis, A., John, I. and Grierson, D. 1997. Analysis of physiological and molecular changes in melon (Cucumis melo L.) varieties with different rates of ripening. J. Exp. Bot., 48: 769-778. Aharoni, A., Keizer, L.C.P., Bouwmeester, H.J., Sun, Z., Alvarez-Huerta, M., Verhoeven, H.A., Blaas, J., Van Houwelingen, A.M.M.L., De Vos, R.C.H., Van der Voet, H., Jansen, R.C., Guis, M., Mol, J., Davis, R.W., Schena, M., Van Tunen, A.J. and OConnel, A.P. 2000. Identificati- on of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays. Plant Cell, 12: 647-661. Aharoni, A. and OConnel, A.P. 2002. Gene expression analysis of strawberry achene and receptacle maturation using DNA microarrays. J. Exp. Bot., 53: 2039-2055. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol., 215: 403-410. Ayub, R., Guis, M., Ben Amor, M., Gillot, L., Roustan, J.P., Latche, A., Bouzayen, M. and Pech, J.C. 1996. Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nature Biotech., 14: 862-866. Balague, C., Watson, C.F., Turner, A.J., Rouge, P., Picton, S., Pech, J.C. and Grierson, D. 1993. Isolation of a ripening and wound-induced cDNA from Cucumis melo L. encoding a protein with homology to the ethylene-forming enzyme. Eur. J. Biochem., 212: 27-34. Diatchenko, L., Lau, Y.-F.C., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D. and Siebert, P.D. 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA, 93: 6025-6030. Giovannoni, J. 2001. Molecular regulation of fruit ripening. Annu. Rev. Plant Physiol. Plant Mol. Biol., 52: 725-749. Hadfield, K.A., Rose, J.K.C., Yaver, D.S., Berka, R.M. and Bennet, A.B. 1998. Polyglactorunase gene expression in ripe melon fruit supports a role for polyglactorunase in ripening-associated pectin disassembly. Plant Physiol., 117: 363-373. Hadfield, K.A., Dang, T., Guis, M., Pech, J.C., Bouzayen, M. and Bennet, A.B. 2000. Characte- rization of ripening-regulated cDNAs and their expression in ethylene-suppressed Charentais melon fruit. Plant Physiol., 122: 977-983. Karchi, Z. 2000. Development of melon culture and breeding in Israel. In: Katzir, N. and Paris, H.S. (Eds.), Proceedings of Cucurbitaceae 2000. Acta Hort., 510: 13-17. Karvouni, Z., John, I., Taylor, J.E., Watson, C.F., Turner, A.J. and Grierson, D. 1995. Isolation and characterization of a melon cDNA clone encoding phytoene synthase. Plant Mol. Biol., 27: 1153-1162. La Claire II, J.W. and Herrin, D.J. 1997. Co-isolation of high-quality DNA and RNA from coeno- cytic green algae. Plant Mol. Biol. Rep., 15: 263-272. Moore, S., Vrebalov, J., Payton, P. and Giovannoni, J. 2002. Use of genomic tools to isolate key ripening genes and analyse fruit maturation in tomato. J. Exp. Bot., 53: 2023-2030. Payton, S., Fray, R.G., Brown, S. and Grierson, D. 1996. Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission. Plant Mol. Biol., 33: 1227-1231. Rose, J.K.C., Hadfield, K.A., Labavitch, J.M. and Bennett, A.B. 1998. Temporal sequence of cell wall disassembly in rapid ripening melon fruit. Plant Physiol., 117: 345-361. Sambrook, J. and Russel, D.W. (Eds). 2001. Molecular Cloning: A laboratory manual, pp. 7.21- 7.45. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, U.S.A. Sato, N.K., Yuhashi, K.I., Higashi, K., Hosoya, K., Kubota, M. and Ezura, H. 1999. Stage- and tissue-specific expression of ethylene receptor homolog genes during fruit development in muskmelon. Plant Physiol., 120: 321-329. Shalit, M., Katzir, N., Tadmor, Y., Larkov, O., Burger, Y., Shalekhet, F., Lastochkin, E., Ravid, U., Amar, O., Edelstein, M., Karchi, Z. and Lewinsohn, E. 2001. Acetyl CoA: alcohol acetyl transferase activity and aroma formation in ripening melon fruits. J. Agric. Food Chem., 49: 794-799. White, P.J. 2002. Recent advances in fruit development and ripening: an overview. J. Exp. Bot., 53: 1995-2000. Xiong, L., Lee, M.W., Qi, M. and Yang, Y. 2001. Identification of defense-related rice genes by suppression subtractive hybridization and differential screening. Mol. Plant-Microbe Interact., 14: 685-692.

497 498 Genetics of fruit quality in melon. Verification of QTLs involved in fruit shape with near-isogenic lines (NILs)

I. Eduardo, P. Arús and A.J. Monforte Departament de Genetica Vegetal, Laboratori CSIC-IRTA de Genetica Molecular Vegetal, Carretera de Cabrils s/n, 08348 Cabril, Spain

Summary

Three near isogenic lines (NILs), each one carrying a unique selected introgression from the Korean accession PI 161375 in the otherwise genetic background of a Piel de Sapo (PS) melon line were constructed by backcrossing and marker assisted selection. These NILs were used to verify the effects of the melon fruit shape Quantitative Trait Loci (QTL) fs1.1, fs9.1 and fs11.1, previously detected in F and Double Haploid Line populations developed from the same parents. 2 NILs carrying PI 161375 alleles at fs1.1 and fs9.1 genomic regions yielded more elongated fruits than NILs with PS alleles at those regions, demonstrating the effects of those QTLs. The study of components of fruit shape showed that fs1.1 acts only in the longitudinal direction, whereas fs9.1 acts only in the transversal direction. Concomitant effects were also observed in ovary shape, reinforcing that fruit shape is genetically determined at preanthesis in melon. On the other hand, the NIL with PI 161375 alleles at fs11.1 did not show differences in fruit shape compared with the NIL with PS alleles at that region. Some data suggest that the introgression of latter NIL might not include fs11.1, this QTL would have been lost by recombination during the development of the lines. These results demonstrate that NILs are a powerful genetic tool for the study of the genetic control of melon fruit quality traits.

Keywords: Cucumis melo, QTL, fruit shape, near isogenic line

Introduction

Melon (Cucumis melo L.) has a worldwide distribution with high phenotypic variation. For example, melon fruit shape ranges from oblong to extremely elongated, fruit weight from a few grams to several kilograms, and flesh taste from bitter to very sweet (Kirkbride, 1993; Stepansky et al., 1999). However, the genetic control of its phenotypic variation is largely unknown. Most of the variation could be due to a multiple allelic variability at a large number of quantitative trait loci (QTLs). Thorough analysis of QTLs requires detailed molecular marker linkage maps (Tanksley, 1993), which have been recently developed in melon (Oliver et al., 2001; Périn et al., 2002a). Périn et al. (2002a), using Recombinant Inbred Lines (RILs) and by Monforte et al. (2003), using F and Double Haploid Line (DHL) 2 populations, reported QTLs involved in melon fruit shape. QTLs detected in such populations must be verified in independent experiments, for example using Near-Isogenic Lines (NILs) with introgressions containing QTL candidate genomic regions (Tanksley et al., 1996). In the current report, three NILs developed from a cross between a Spanish Piel de Sapo (PS) line and Korean accession PI 161375 (Shongwan Charmi) with unique selected introgressions of PI 161375 in the PS genetic background were used to verify and characterize three fruit shape QTLs (fs1.1, fs9.1 and fs11.1) reported previously by Mon- forte et al. (2003).

Experimental populations DHLs developed from a cross between a Piel de Sapo (PS) line and the Korean accession PI 161375 (Gonzalo et al., 2003) were used as the base population for NIL construction. DHLs were backcrossed to PS. Several rounds of backcrossing and marker assisted selection, using as reference a saturated melon genetic map (Gonzalo, 2003), were made to obtain genotypes with unique introgression. Plants carrying a heterozygous introgression in the genomic regions including fruit shape QTLs (fs1.1, fs9.1 and fs11.1) were selected and self-pollinated. For each self-pollinated progeny, 10 plants homozygous for PI 161375 alleles and 10 plants homozygous for PS alleles at the target introgression, as assessed by molecular markers, were selected at seedling stage and transferred to the greenhouse for agronomic evaluation. NILs with PI 161375 introgressions were named NIL1.1, NIL9.1 and NIL11.1, respectively. With this approach, phenotypic differences between the plants carrying the PI 161375 alleles at target region and plants with PS alleles at that region can be attributed to genes or QTLs included in this introgression, because the rest of the genomic background is isogenic for both groups of plants.

Field experiments, traits and statistical analysis Plants were grown at the greenhouse in plastic bags by hydroponic culture during summer of 2003 in Cabrils (Spain). Flowers were hand pollinated and only one fruit was allowed to develop per plant. The agronomic evaluation included the following traits: fruit weight (FW) in g, fruit length (FL) in cm, fruit maximum diameter (FD) in cm, fruit and ovary shape (FS, OS) (ratio between length and maximum diameter) and soluble solids concentration (SSC), measured as °Brix with a hand refractometer from a homogenized of melon flesh. Means of NILs with PI 161375 and PS alleles at target genomic regions were compared by a t-test using SYSTAT 5.03 for Windows (SYSTAT Inc. Evanston, IL, USA).

Results and discussion Trait means for NILs with PS and PI 161375 alleles in the target introgressions in linkage groups (LG) 1, 9 and 11 (NIL1.1, NIL9.1 and NIL 11.1, respectively) are given in Table 1. Fruits of NIL1.1 and NIL9.1 were more elongated than fruits of the respective NILs with PS alleles at those regions, verifying the effects of fs1.1 and fs9.1 QTLs. However, FS of NIL11.1 was not different than the NIL with PS alleles at that region. Fs11.1 have been mapped in several independent experiments (Périn et al., 2002b, Monforte et al., 2003) close to the gene pentamerous (p) which controls the number of carpels (five in PI 161375 and three in PS) and it has been proposed as a candidate gene for this QTL. All fruits from NILs selected to verify fs11.1 had three carpels, indicating that the PI 161375 allele of p was not present in NIL11.1, probably due to recombination during the NIL development. NILs with the PI 161375 allele of p are currently being analysed to verify whether this locus can be considered a candidate gene for fs11.1.

500 Table 1. Means for the studied traits for NILs with PS and PI 161375 alleles in the target introgression at LG 1, 9 and 11 (NIL1.1, NIL9.1 and NIL 11.1, respectively)

Target LG1 Genotype at Fruit Fruit Fruit Fruit SSC2 Ovary QTL target length diameter shape weight shape introgression (cm) (cm) (g) (°Brix)

fs1.1 1 NIL1.1 24.89* 12.69* 1.98* 1818* 10.7* 2.61* PS 21.35* 12.75 1.68* 1595* 10.4* 2.19*

fs9.1 9 NIL9.1 23.01 12.63* 1.82* 1795* 9.4* 2.33* PS 22.7414.88*1.53* 2482* 11.4*1.98*

fs11.1 11 NIL11.1 21.71* 13.05* 1.67* 1797* 9.7* 2.04 PS 24.75* 16.09 1.65* 2763* 10.9* 2.18*

1 LG: Linkage Group, according to Oliver et al. (2001), where the target introgression is located. 2 SSC: Soluble Solid Content. * Significant difference between means (p< 0.05).

The selected introgressions also showed significant effect in other traits. OS differences were observed concomitant with the effects of fs1.1 and fs9.1 in FS, reinforcing that FS is genetically determined at preanthesis in melon (Périn et al., 2002b). Fruits from NIL1.1 showed larger FL, indicating that the effect of fs1.1 in FS was only through an increment in the longitudinal direction. On the other hand, the significant differences in FD observed in the NIL9.1 indicate that the effect of fs9.1 was only in the reduction of the fruit diameter. These results suggest that fs1.1 and fs9.1 control different processes of fruit development. Other effects not observed in previous experiments (Monforte et al., 2003) were also found in some NILs. NIL9.1 showed a reduction in FW, which may be due to the observed reduction in FD. In this NIL, effects on SSC were also observed. Simi- larly, NIL11.1 showed a reduction in FW. The detection of effects using NILs compared with populations as LDHs can be explained by the different genetic background and, more likely, to the higher power to detect QTLs of NILs compared with the more common mapping populations as LDHs, RILs, and F progenies (Eshed and 2 Zamir, 1995). In the current report, the suitability of the NILs constructed for verifying and characterizing QTLs involved in melon fruit traits was demonstrated. Altogether, the extent of the three introgressions represented in the studied NILs cover less than 15% of the melon genome. A collection of NILs covering the whole melon genome is currently under progress (Eduardo et al., 2003). This collection will represent an important genetic tool for the study of the genetic control of fruit quality in melon.

501 Acknowledgements

The authors thank N. Galofré, I. Marchal, A. Ortigosa, J. Adillón, P. Ramon, for excellent technical assistance. This work was supported in part by grants for the project AGL2000-0360 from The Spanish Ministry of Science and Technology. AJM was supported in part by a contract from Instituto Nacional de Investigaciones Agrarias (INIA), Spain. IE was supported by a fellowship from the Spanish Ministry of Science and Technology.

References

Eduardo, I., Arús, P. and Monforte, A.J. 2003. Development of a collection of Near Isogenic Lines (NILs) in melon. Proc. 7th International Congress of Plant Molecular Biology, Barcelo- na (Spain), 23-28 July. Eshed, Y. and Zamir, D. 1995. An introgression-line population of Lycopersicon pennelli in the cultivated tomato enables the identification and fine mapping of yield-associated QTLs. Gene- tics, 141: 1147-1162. Kirkbride, J.H. 1993. Biosystematics monograph of the genus Cucumis (Cucurbitaceae). Botani- cal identification of cucumbers and melons. Parkway, Boone, N.C. Gonzalo, M.J. 2003. Generación, caracterización molecular y evaluación morfológica de una población de líneas dihaploides en melón (Cucumis melo L.). Ph. D. Dissertation, Universitat de Lleida, Spain. Gonzalo, M.J., Claveria, E., Monforte, A.J. and Dolcet-Sanjuan, R. 2003. Generation and molecular characterization of a doubled haploid line population from a melon (Cucumis melo L.) hybrid by in situ induced parthenogenesis. ASHS Centennial Conference. Providence, Rhode Island (USA), 3-6 October, HortScience, 38: 751. Monforte, A.J., Oliver, M., Gonzalo, M.J., Álvarez, J.M., Dolçet-Sanjuan, R. and Arús, P. 2003. Identification of Quantitative Trait Loci involved in fruit quality traits in melon. Theor. Appl. Genet. (in press) Oliver, M., Garcia-Mas, J., Cardús, M., Pueyo, N., López-Sesé, A.I., Arroyo, M., Gómez-Pania- gua, H., Arús, P. and De Vicente, M.C. 2001. Construction of a reference linkage map for melon. Genome, 44: 836-845. Périn, C., Hagen, L.S., de Conto, V., Katzir, N., Danin-Poleg, Y., Portnoy, V., Baudracco-Arnas, S., Chadoeuf, J., Dogimont, C. and Pitrat, M. 2002a. A reference map for Cucumis melo based on two recombinant inbred line populations. Theor. Appl. Genet., 104: 1017-1034. Périn, C., Hagen, L.S., Giovinazzo, N., Besombes, D., Dogimont, C. and Pitrat, M. 2002b. Gene- tic control of fruit shape acts prior to anthesis in melon (Cucumis melo L.). Mol. Gen. Geno- mics, 266: 933-941. Stepansky, A., Kovalski, I. and Perl-Treves, R. 1999. Intraspecific classification of melons (Cu- cumis melo L.) in view of their phenotypic and molecular variation. Plant. Syst. Evol., 217: 313-332. Tanksley, S.D. 1993. Mapping polygenes. Annu. Rev. Genet., 27: 205-233. Tanksley, S.D., Grandillo, S., Fulton, T.M., Zamir, D., Eshed, T., Petiard, V., Lopez, J. and Beck- Bunn, T. 1996. Advanced backcross QTL analysis in a cross between an elite processing line of tomato and its wild relative L. pimpinellifolium. Theor. Appl. Genet., 92: 213-224.

502 Characterisation of simple sequence repeats (SSRs) and development of SSR markers in melon (Cucumis melo)

N. Fukino1, M. Kuzuya2, M. Kunihisa1 and S. Matsumoto1 1National Research Institute of Vegetable and Tea Science, 360 Kusawa, Ano, Mie 514-2392, Japan; e-mail: [email protected] 2Plant Biotechnology Institute, Ibaraki Agricultural Center, Iwama, Nishi-ibaraki, Ibaraki 319-0292, Japan

Summary

Genomic DNA of Cucumis melo L. was enriched for (CA)n, (GA)n, (AAG)n and (ATG)n repeats. Of the 1264 clones that were sequenced, 407 were determined to be unique, and 361 clones contained SSRs. In these clones, the number of repeats of uninterrupted CA, GA, AAG and ATG core motifs ranged from 3 to 100 (average 15), 39 (average 8), 13 (average 6) and 19 (average 4), respectively. 135 clones contained the CA motif, and these clones displayed the largest average repeat numbers. The clone sequences were used to design 129 primer pairs that were employed to identify polymorphisms between the melon varieties PMAR No. 5 and Harukei No. 3. Of the 102 marker loci that were amplified, 20 were polymorphic, and most of the 20 were co-dominant. The SSR markers exhibited a high level of polymorphism.

Keywords: Cucumis melo, simple sequence repeat, molecular marker

Introduction Simple sequence repeat (SSR) markers have proved to be powerful tools for applications such as genome mapping, trait mapping and marker-assisted selection, because they are greatly abundant, highly polymorphic and co-dominantly inherited. Only a small number of SSR markers from melon have been reported (Danin-Poleg et al., 2001). In the present study, 361 unique clones containing SSRs were isolated from melon genomic libraries enriched for (CA)n, (GA)n, (AAG)n and (ATG)n repeats. We also evaluated the characteristics of the SSR regions and tested their ability to detect polymorphisms between the melon varieties PMAR No. 5 and Harukei No. 3. The- se varieties were used to develop recombinant inbred lines.

Materials and methods

Plant material and DNA extraction Genomic DNA was extracted from fresh leaves of the melon varieties PMAR No. 5, Harukei No. 3 and their F hybrid, using the DNeasy Plant Mini Kit (QIAGEN). The 1 Harukei No. 3 DNA was enriched for (CA)n, (GA)n, (AAG)n and (ATG)n repeats. The melon breeding line PMAR No. 5, which was introduced into Japan from the Uni- versity of California in 1981 (Yoshida and Kohyama, 1986), is an orange-fleshed cantaloupe that possesses resistance to both powdery mildew and cotton aphid. Harukei No. 3

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 503 is a green-fleshed Earls Favorite type that is susceptible to both powdery mildew and cotton aphids. PMAR No. 5 and Harukei No. 3 were used as parents to develop recombinant inbred lines for the construction of a genetic linkage map.

Sequencing of SSR-enriched libraries and primer design Plasmids containing SSR-enriched sequences were transformed into the E. coli strain ElectroMAX DH10B (Invitrogen) by electroporation. After shaking at 37°C for 1 hr, the cells were spread on LB plates containing 100 mg/ml ampicillin, 20 mg/ml X-gal, and 10 ml/100 ml IPTG, and incubated for 18 hr at 37°C. White colonies were further cultured for 18 hr in Terrific Broth containing 100 mg/ml ampicillin, and DNA was extracted using the Wizard Plus SV kit (Promega) or MultiScreen 96-Well Filter Pla- tes (Millipore) and sequenced on an ABI PRISM 3100-Avant (Applied Biosystems) using the BigDye Terminator v. 3.1 cycle sequencing kit (Applied Biosystems). Sequences containing at least three uninterrupted di- or trinucleotide repeats were used for designing primers. To amplify the SSR loci, primers were designed according to the flanking regions of the repeat sequences.

PCR amplification and evaluation Using the above primer pairs, PCR reactions were carried out to detect polymorphisms between PMAR No. 5 and Harukei No. 3. Amplifications were performed using a GeneAmp PCR System 9700 according to the following protocol: (i) 94°C for 2 min; (ii) 25 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min; (iii) and 72°C for 7 min. PCR products were labelled with R110-ddUTP using a 3-terminal exchange reaction mediated by the Klenow fragment (Fukuoka, personal communication).

Results and discussion

SSR detection From the CA, GA, AAG, and ATG libraries, sequence data were obtained for 576, 288, 160, and 240 clones, respectively, and 158, 111, 57, and 81 clones were determined to be unique. SSRs were detected in 144, 109, 46 and 62 clones from these groups (Table 1). 218 primer pairs were designed to amplify the SSRs. The number of uninterrupted repeats of the CA, GA, AAG and ATG core motifs ranged from 3 to 100 (average 15), 39 (average 8), 13 (average 6) and 19 (average 4), respectively. Many clones with dinucleotide motifs contained more than 10 repeats, but the majority of clones with trinucleotide motifs contained less than 10 repeats (Table 2). It has been reported that GA and CT repeats are observed more frequently from the Cucumis melo genome than GT or CA repeats (Chiba et al., 2003). In this study, 135 clones were found to contain the CA motif, and these clones had the largest average repeat number. This suggests that the CA repeat is abundant in melon and can be used for developing SSR markers.

SSR length polymorphisms between PMAR No. 5 and Harukei No. 3 One hundred and twenty-nine SSRs from the 218 primer pairs were used to assess polymorphisms between PMAR No. 5 and Harukei No. 3. 102 marker loci were

504 Table 1. Frequency and type of the core motifs found in four enriched libraries

Core motif Library CA GA AAG ATG

CT/GA 32 97 16 18 GT/CA 115 15 3 2 TA/AT 43 12 4 1 CG/GC 5 2 2 1 GAA/CTT 47 241 ATT/TAA 3 2 0 1 GCC/CGG 3 1 1 0 GTT/CAA 7 0 0 2 TGG/ACC 2 3 0 0 TCC/AGG 446 2 AGT/TCA 1 1 1 25 ATG/TAC 1 1 0 15 AGC/TCG 1 3 0 0 TGC/ACG 2 1 0 0 Number of unique clones 158 111 57 81 Number of clones containing SSRs 144 109 46 62 amplified, and 20 of these were found to be polymorphic. Most of the marker loci were co-dominant (Fig. 1). Relatively few polymorphisms were found between PMAR No. 5 and Harukei No. 3. Previous reports on polymorphisms between these species revealed that 69 RAPD primers of the 1,200 used for screening (5.8%) detected polymorphisms (Fukino et al., 2002). We found that 20 loci of 129 detected using SSR markers showed polymorphisms (15.5%). It has been reported that a greater number of polymorphisms is associated with SSRs than with RAPDs (Katzir et al., 1996), which is consistent with our results.

Figure 1. Polymorphism between PMAR No. 5´ and Harukei No. 3´ detected by SSR markers 01-02 (A) and 53-05 (B). The amplified products were post-labeled by R110- ddUTP and detected by an ABI PRISM 3100-Avant (Applied Biosystems).

505 Table 2. Numbers of SSR repeats in 361 clones

Number of repeats(n) 3-5 6-10 11-15 16-20 >20

CT/GA 61 35 27 20 20 GT/CA 23 19 40 7 46 TA/AT 46 12 1 1 0 CG/GC 9 1 0 0 0 GAA/CTT 11 19 5 1 0 ATT/TAA 6 1 0 0 0 GCC/CGG 41 0 0 0 GTT/CAA 3 2 3 0 1 TGG/ACC 2 2 0 1 0 TCC/AGG 8 6 1 0 0 AGT/TCA 11 12 41 0 ATG/TAC 13 2 1 1 0 AGC/TCG 2 1 1 0 0 TGC/ACG 2 0 0 1 0

Acknowledgements

This work was supported by the Rice Genome Project (Grant DM-1601) of the Ministry of Agriculture, Forestry and Fisheries of Japan.

References

Chiba, N., Suwabe, K., Nunome, T. and Hirai, M. 2003. Development of microsatellite markers in melon (Cucumis melo L.) and their application to major Cucurbit crops. Breed. Sci., 53: 21-27. Danin-Poleg, Y., Reis, N., Tzuri, G. and Katzir, N. 2001. Development and characterization of microsatellite markers in Cucumis. Theor. Appl. Genet., 102: 61-72. Fukino, N., Taneishi, M., Saito, T., Nishijima, T. and Hirai, M. 2002. Construction of a linkage map and genetic analysis for resistance to cotton aphid and powdery mildew in melon. Acta Hort., 588: 283-286. Katzir, N., Danin-Poleg, Y., Tzuri, G., Karchi, Z., Lavi, U. and Cregan, P.B. 1996. Length polymorphism and homologies of microsatellites in several Cucurbitaceae species. Theor. Appl. Genet., 93: 1282-1290. Yoshida, T. and Kohyama, T. 1986. Mechanisms, genetics and selection methods of aphid resistance in melons, Cucumis melo. Bull. Veg. & Ornam. Crops Res. Sta. Jpn., Ser. C9: 1-12.

506 Genetic map for pumpkin Cucurbita pepo using random amplified polymorphic DNA markers

A. Zraidi1 and T. Lelley2 1University of Natural Resources and Applied Life Sciences, Vienna, Austria 2Department for Agrobiotechnology, Division Biotechnology in Plant Production IFA-Tulln, Konrad Lorenz Str. 20, A-3034 Tulln, Austria; e-mail: [email protected]

Summary

A genetic map for Cucurbita pepo (2n=40) would be a useful tool for immediate application in plant breeding and comparative genetic analysis. Using Random Amplified Polymorphic DNA (RAPDs), Simple Sequence Repeats (SSRs) and phenotypic/morphological traits, a molecular map for pumpkin was constructed. The map was developed using an F population obtained from a 2 cross between an Austrian oil-pumpkin inbred line (SZG1) and a zucchini genotype resistant to ZYMV. From a total of 280 loci, 254 RAPD markers, 24 of which are co-dominant, 3 SSRs and one qualitative trait could be mapped at a LOD score of 3 and maximum distance of 35; for the time being, 22 markers are unlinked. The map covers 1425 cM and contains 36 linkage groups with 17 of them including less than 4 markers. The average distance between markers is 5.5 cM. However, a number of gaps (>20 cM) are still to be filled. One-way ANOVA analysis revealed a significant association of RAPD markers with fruit length that accounts for more than 40% of the variation of this trait.

Keywords: RAPD, SSR, co-dominant RAPDs, oil pumpkin

Introduction

Appreciated primarily for the healing and nutritional properties of its seeds, oil pumpkin has a long-standing tradition and today increasing economic importance in Austria. Oil pumpkin belongs to the Pumpkin Group of Cucurbita pepo L. (Paris, 1986). This species is one of the most polymorphic in the plant kingdom, exhibiting wide variation among cultivar-groups with respect to fruit characteristics (Paris, 2001). Using molecular markers, several studies were conducted in order to better clarify relationships within C. pepo (Decker, 1988; Torres-Ruiz and Hemleben, 1991; Katzir et al., 2000; Ferriol et al., 2003; Paris et al., 2003). Genetic maps have become important tools in modern genetics and breeding. Until now, few and low density maps have been presented for the genus Cucurbita (2n=40). The first genetic map for Cucurbita was based on 11 isozyme loci in five linkage groups (Weeden and Robinson, 1986). Lee et al. (1995) developed a RAPD-based map, using a C. moschata Duchesne × C. pepo F population, that contained 28 markers 2 distributed into five linkage groups. Recently, Brown and Myers (2002) developed a map from a backcross generation to C. pepo of an interspecific cross between C. moschata and C. pepo; this map contained 148 RAPD loci and 5 phenotypic traits, distributed in 28 linkage groups covering 1945 cM. Compared to other important Cucurbitaceae, the genus Cucurbita lags far behind with respect to genetic mapping. To date, five maps for melon and three for cucumber

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 507 have been published (Serquen et al., 1997; Wang et al., 1997; Danin-Poleg et al., 1998; Liou et al., 1998; Brotman et al., 2000; Oliver et al., 2000; Park et al., 2000; Perin et al., 2000). Efforts are underway toward comparative mapping and map merging of the two species (Staub and Serquen, 2000). The pumpkin map presented here represents progress toward developing a saturated map for C. pepo.

Material and methods

The C. pepo mapping population consisted of 93 F -progeny taken from a single 2 F -fruit from a cross between an oil pumpkin breeding line (SZG1) developed by Sa- 1 atzucht Gleisdorf Austria, and a near-isogenic line True French (Zucchini Group), the seeds of which were kindly made available to us by Harry Paris, A.R.O., Newe Yaar Research Center, Israel. Originally, the parents were chosen by us to introduce resistance to ZYMV, present in the zucchini genotype, into oil pumpkin. Two qualitative traits, leaf mottling and hull-less seed coat, showing clear segregation in the F -population were scored. Leaf mottling, described as silver-gray areas 2 in the axils of the leaf veins (Scarchuk and Lent, 1965), was scored as present (Fig. 1a) or absent (Fig. 1b), disregarding the degree of silver appearance. The seed coat was scored as hulled (Fig. 2a) or hull-less (Fig. 2b), irrespective of an occasional residual lignification of the hull-less seeds. Hull-less seed coat is believed to be controlled by a single major gene (Schöniger, 1950; Grebenèikov, 1954). The dominant allele produces hulled seed coat, while seeds of plants having the recessive allele in the homozygous state are basically hull-less, although they may exhibit variation by having a small amount of lignin in the sclerenchyma of the testa, as histological observations revealed (Zraidi et al., 2003).

Figure 1. Leaves of the two parents used for crossing showing typical mottling (a) or complete absence of mottling (b).

Figure 2. Hulled (a) and hull-less seeds (b) of the two parents.

a b

508 Fruit length, fruit width and the number of locules per fruit were evaluated as quantitative traits. Oil pumpkin has nearly spherical, round fruits (Fig. 3a) and zucchini has a uniformly cylindrical fruits (Fig. 3b) (Paris, 1986). In the F , fruit shape showed a conti- 2 nuous variation. This trait was quantified by calculating length-to-width ratio, the latter being the average of measurements taken close to each end of the fruit. Seeds of C. pepo are normally contained in separate locules inside the fruits. The number of these chambers varies from 3 to 5 (Fig. 4a, and b) and was scored accordingly.

ab a b

Figure 3. Round shaped pumpkin (a) Figure 4. Fruit with 3 (a) and oblong zucchini (b). and 4 chambers (b).

DNA was isolated from fresh young leaves according to Promegas Wizard Genomic DNA Purification Kit (Promega Corp., Madison, U.S.A). DNA concentration was determined using a GenQuant RNA/DNA Calculator (Amersham Biosciences Europe, Germa- ny) according to the manufacturers instructions. Stock DNA was stored at 20°C. Parents were screened for polymorphism with 495 RAPD primers (Operon Techno- logy, Inc.). For this, DNA-pools of 10 plants from each parent were made. A 10µl reaction mixture contained 27 ng DNA, 0.10 mM dNTPs, 6 pmole primer, 0.4 units Taq DNA polymerase and 1× reaction buffer including 1.5 mM MgCl (Genecraft, Germa- ny). The PCR program for RAPD was 1 min at 94°C followed by 35 cycles of 1 min at 94°C, 45 s at 36°C, 30 s at 72°C, ending with 5 min at 75°C. Altogether 18 SSR primer pairs were tested on the parents, 13 of which were provided by Martin Pfosser, University of Vienna. The five remaining primer pairs were developed in our laboratory. Reaction mixture with a total volume of 10µl contained 27 ng DNA, 0.25 mM dNTPs, 10 pmole for each primer (rev and fwd), 0.35 units of Taq and 1× reaction buffer. The PCR program for SSR was 1 min at 94°C, followed by 32 cycles of 25 s at 94°C, 25 s at 50°C, 25 s at 72°C, ending with 5 min at 75°C. PCR products were loaded on 10% and 12% polyacrylamide gels for RAPD and SSR respectively, using 1× TBE buffer in a C.B.S. electrophoresis chamber (C.B.S. Scientific Inc., Del Mar, CA, U.S.A.). Electrophoresis conditions were set at a constant current of 400V and 10°C for two hours. The gels were stained with silver nitrate (Stift and Lelley, 2003) and dried on filter paper in a vacuum drier (Amersham Biosciences Europe, Germany). Using a chi-square (c2) test with P=0.05, the segregation of the markers was tested for goodness-of-fit to Mendelian ratios of 3:1 for dominant RAPD and of 1:2:1 for SSR and co-dominant RAPD. The map was constructed using MAPMAKER/EXP 3.0 (Lander et al., 1987; Lincoln et al., 1992) at a minimum LOD score of 3 and maximum distance of 35 cM. Linkage groups were established using the tow point group

509 command. The ordering and calculation of the distance between markers was made by the multipoint command order. In some cases, Mapmaker failed to automatical- ly find starting subsets using order command. Instead, lod table was used to choose the starting subsets, and the three point compare command to find the best order of these subsets. The remaining markers were placed in their relatively best order using the try command. To check the result of the previous analysis, ripple command was used. The error detection ratio was set at 5%. Single marker/trait associations were revealed by one-way ANOVA (SAS software). Associations between markers and phenotype were evaluated by F-test.

Results and discussion

From a total of 494 RAPD primers screened, 227 (46%) showed polymorphism between the two parents. Out of these 227 primers, 101 were selected for their relatively high level of polymorphism with an average of 2.7 polymorphic bands per primer. A total of 276 RAPD loci were scored, 24 of which were co-dominant. Brown and Myers (2002) although using an interspecific cross, found a far lower level of polymorphism, 0.7 polymorphic bands per primer. This difference could, however, be attributed to the use, in our case, of polyacrylamide instead of agarose for fragment separation (Stift and Lelley, 2003). Indeed, based on the 1:2:1 segregation pattern and the absence of the homozygous recessive allele in 93 F progeny, we consi- 2 dered 24 markers (10%) to be codominant, which would not have been visible on agarose gel (Fig. 5). Using MAPMAKER/EXP for linkage analysis at a LOD score of 3 and maximum distance of 35 cM, 258 loci were mapped, consisting of 254 RAPDs, 3 SSRs and one qualitative trait. Higher LOD scores resulted in an increase in the number of linkage groups, and increased the number of unlinked markers. Increase of maximum distance to 50 did not change the results of grouping of the data. The map (Fig. 6) includes 36

Figure 5. Part of a 96-well gel with RAPD fragments separated in polyacrylamide showing, besides three polymorphic dominant, two co-dominant markers (arrows). Missing fragments are indicated by dots.

510 L. containing Cucurbita pepo A genetic map of Figure 6. 254 RAPD markers, out of which 24 are co-dominant, 3 SSR markers and a morphological trait, seed coat. The map covers 1425 cM with an average of 5.5 cM between loci.

511 linkage groups (LG), with an average of 7 markers per LG and covers 1425 cM with an average of 5.5 cM between loci. The markers are not distributed evenly between and within the linkage groups, as 17 of them contain less than four markers and 14 gaps are longer than 20 cM. The map coverage is only three-quarters of that obtained by Brown and Myers (2000). Nonetheless, our pumpkin map includes a number of linkage groups in which the distance between markers is less than 0.5 cM. Of the 280 loci, only four (leaf mottle and three dominant RAPD markers) deviated significantly from the expected 3:1 segregation and one co-dominant RAPD did not fit the expected 1:2:1 ratio. Brown and Myers (2002) encountered segregation distortion for 21 out of 148 markers; this higher frequency could be attributed to sterility barriers in the interspecific cross. Out of 18 SSR markers tested, five (27%) were polymorphic and only three could be mapped on different linkage groups. SSRs are most useful loci and may serve as anchor markers for establishing a skeletal map, but they appear to be species specific. Out of 102 Cucumis SSR-markers tested in Jack Staub´s laboratory, Univ. Wiscon- sin at Medison, only one turned out to be polymorphic in a sample of four C. pepo genotypes. C. pepo SSRs are currently being developed in our laboratory. The seed coat (n) and leaf mottle (M) identified in Cucurbita (Hutton and Robin- son, 1992) were evaluated and included in the data analysis. The seed coat was placed in the map on LG4, flanked by two RAPD markers (AK11_340 and AB14_230), 4.4 cM and 24.1 cM far apart from the seed coat character, respectively. Leaf mottle is still unlinked. One way ANOVA analysis revealed associations between markers and quantitative traits, these are summarized in Table 1.

Table 1. Marker/phenotype associations revealed by one-way ANOVA analysis

Significance level (F-test)

Markers Linkage Fruit Fruit Length/width No. of fruit groups length width ratio chambers

AE07_165 Un 1.5.10-12 0.0003 5.10-13 - AC10_490 32 0.0002 - 0.00001 - AJ20_420 36 0.0002 0.008 1.1.10-6 - P13_750 16 0.001 - 7.2.10-6 - J01_600 32 0.001 - 5.3.10-6 - AO20_1200 1 0.005 - - - T08_460 1 0.008 - - - AB08_540 4 0.009 - - - AE09_1600 1 0.009 - - - AM10_950 24- 0.005 - - AG08_440 24 - 0.007 - - P13_950 8 - - - 0.003 AE08_470 11 - - - 0.009

512 Results presented here are a beginning toward a high-density map for pumpkin (C. pepo). More RAPD as well as other DNA markers could help fill the map. SSRs currently being developed in our laboratory together with successfully converted co- dominant RAPDs to STS-markers could provide anchor sites. More phenotypic markers, such as growth habit, stem color, and genes conferring resistance to ZYMV, could also be integrated into the map.

Acknowledgements

This work was financially supported by the Austrian Science Fund (FWF, No P 15773). The authors are most grateful to Dr. Jack E. Staub for his generous cooperation in testing cucumber SSRs on C. pepo.

References

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514 Developing a genetic linkage map for watermelon: polymorphism, segregation and distribution of markers

A. Levi1, C.E. Thomas1, J. Thies1, A. Simmons1, Y. Xu2, X. Zhang3, O.U.K. Reddy4, Y. Tadmor5, N. Katzir5, T. Trebitsh6, S. King7, A. Davis8, J. Fauve9 and T. Wehner10 1USDA, ARS, U.S. Vegetable Laboratory, 2700 Savannah Highway, Charleston, SC 29414, USA; e-mail: [email protected] 2China National Engineering Research, Center for Vegetables (NERCV), P.O. Box 2443, Beijing 100089, China 3Syngenta Seeds, Inc., 21435 Road 98, Woodland, CA 9569, USA 4Biotechnology Center, Alcorn State University, MS 39096-7500, USA 5Department of Vegetable Crops, Agricultural Research Organization, Newe Yaar Research Center, P.O. Box 1021, Ramat Yishay 30-095, Israel 6Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel 7Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843-2119, USA 8South Central Agricultural Research Laboratory, USDA, ARS, P.O. Box 159, Lane, OK 74555, USA 9Harris Moran Seed Cie, Centre de Recherche La Costiere, 30210 Ledenon, France 10Department of Horticulture, North Carolina State University, Raleigh, NC 27695- 7609, USA

Summary

A composite watermelon genetic linkage map is being constructed based on a testcross population and an F population. About 51.0% and 31.8% of the markers in the testcross and F popu- 2 2 lations are skewed form the expected segregation ratios. AFLP markers appeared to be clustered on linkage regions, while ISSR and RAPD markers are dispersed along the genome. AFLP markers also have greater genetic distances as compared with ISSR and RAPD markers, resulting in significant increase of map distance. An initial genetic map (based on the testcross population) that contains 27 ISSR and 141 RAPD markers has a total linkage distance of 1,166.2 cM. The addition of 2 ISSR, 8 RAPD and 77 AFLP markers increased the genetic distance of the map to 2,509.9 cM. Similar results with AFLP markers were also shown in mapping experiments with an F S recombinant inbred line (RIL) population that was recently constructed for watermelon. Al- 2 7 though the skewed segregation, marker order appeared to be consistent in linkage groups of the testcross and the F2 population. Experiments with SSR, SCAR, EST and RFPL markers are being conducted to saturate the linkage map of watermelon.

Keywords: Citrullus lanatus, genetic mapping, skewed segregation, AFLP, ISSR, RAPD

Introduction

Watermelon accounts for 2% of the world area devoted to vegetable crops (FAO, 1995). In the U.S. watermelon production has increased from 1.2 M tons in 1980 to 4.1 M tons in 2003, with a farm value of $310 million (U. S. Dept. of Agriculture,

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 515 2003). In recent years production of seedless (triploid) watermelons has increased considerably. During 2002, over 82% and 52% of the watermelons produced in Cali- fornia and in the Southeast of the United States, respectively, were seedless (U.S. Dept. of Agriculture, 2003). There is a continuous need to genetically improve watermelon, mainly for disease and pest resistance. Molecular markers are useful in identifying and isolating genes that affect fruit qualities, and in selecting superior triploid hybrid combinations of watermelon. Watermelon maps constructed so far are based on isozymes (Navot and Zamir, 1986; Navot et al., 1990) or RAPDs (Hashizume et al., 1996; Hawkins et al., 2001; Levi et al., 2001, 2002; Hashizume et al., 2003), and do not cover all regions of the watermelon genome. Many markers (including AFLPs, SSRs, and RFLPs) are still required for the construction of a dense map that can be used effectively in watermelon breeding programs and in isolating genes that control fruit quality or confer resistance to diseases and pests. In a recent study we attempted to construct a linkage map using an F population 2 (PI 296341; Citrullus lanatus var. citroides x NHM; C. lanatus var. lanatus). However, most of the RAPD, ISSR and AFLP markers used for mapping were skewed from the expected 3:1 segregation ratio among the F progeny, resulting in an unsaturated map. 2 Thus, we pursued the mapping using a testcross population {[Griffin 14113 (C. lanatus var. citroides) x NHM (C. lanatus var. lanatus)] x U.S. PI 386015 (C. colocynthis)} that was successfully used in our initial mapping of watermelon (Levi et al., 2002), and compared the mapping results with these to the markers that could be mapped using the F popu- 2 lation. Our objective is to extend and merge the maps constructed for watermelon using different types of molecular markers, and in a later stage map and isolate genes that control fruit quality and genes that confer disease and pest resistances in watermelon. This paper evaluates the efficiency and distribution of molecular markers on initial linkage maps for watermelon derived from testcross and F populations. 2

Materials and methods

Mapping populations Testcross population {[Griffin 14113 (C. lanatus var. citroides) x New Hampshire Midget (NHM) (C. lanatus var. lanatus)] x PI 386015 (C. colocynthis)}, and F po- 2 pulation [PI 296341 (C. lanatus var. citroides) x NHM] were used for mapping.

Isolation of DNA To avoid co-isolation of polysaccharides, polyphenols, and other secondary compounds that damage DNA, we used an improved procedure for isolation of DNA from young leaves of watermelon (Levi and Thomas, 1999).

Molecular procedures The ISSR and RAPD marker procedure were according to Levi et al. (2002). An AFLP procedure based on that developed by Vos et al. (1995) was modified using a commercially available kit (Plant Mapping Kit-Regular Plant Genome, Applied Bio- systems; Foster City, CA).

516 Marker scoring RAPD and ISSR markers were analyzed on 1.4 agarose gel. The AFLP markers were scored using Genescan/Genotyper software (Applied Biosystems; Foster City, CA).

Linkage analysis of markers Data were analyzed using Mapmaker version 3.0 (Whitehead Institute, Cambridge, Mass.; Lander et al., 1987; Lincoln et al., 1992).

Results and discussion

Marker polymorphisms and screening Although AFLPs are highly polymorphic among watermelon cultivars as compared with ISSRs and RAPDs (Levi et al., 2004), a relatively small number of AFLP markers (142 of 632 AFLP markers) were suitable for mapping in the testcross population (Tables 1 and 2). These markers were unique to one of the parents Griffin 14113 or NHM, present in the F hybrid, but absent in the testcross parent PI 386015. The AFLP mar- 1 kers in this study were highly reproducible from experiment to experiment, using the same reaction conditions, and DNA sequencer. Three hundred and thirty-six RAPD primers (10-mers) with 60 to 90% Guanine-Cytosine (GC) content and seventy ISSR primers were screened in amplification reactions against the parents Griffin 14113 and NHM, their F hybrid, and the testcross parent PI 386015. Of these, 243 RAPD and 57 1 ISSR primers resulted in DNA amplification, producing 1 to 14 RAPD bands (0.1-3 Kb), and 1-8 ISSR bands (0.1-2.6 Kb) per PCR reaction. Eighty-six RAPD and twenty-one ISSR primers produced 168 and 37 distinct bands (respectively) that were suitable

Table 1. Number of RAPD, ISSR, and AFLP markers used for mapping with the F and 2 testcross populations, the number of markers that could be mapped, and number of skewed markers (P<0.05) in each population

F Population Testcross Population 2

Markers Markers Marker used Mapped Skewed used Mapped Skewed for for Type mapping markers markers mapping markers markers

27 414934 RAPD 34 (71.1%) (11.8%) 168 (88.7%) (20.2%) 21 35 29 3 ISSR 108 (38.8%) (32.4%) 37 (78.4%) (8.1%) 25 92 77 73 AFLP 115 (21.7%) (80.0%) 142 (54.2%) (51.4%) 73 131 255 110 Total 257 (35.3%) (51.0%) 345 (74.0%) (31.8%)

517 for mapping. Using the procedure described by Levi et al. (2002) all RAPD and ISSR marker bands in the present study were highly reproducible from experiment to experiment, allowing accurate mapping of markers.

Marker segregation An average of 31.8% of all markers skewed away (P < 0.05) from the expected 1:1 ratio among testcross progeny (Table 1). In a recent mapping study using F S recom- 2 7 binant inbred lines (RILs) derived from a cross between U.S. PI 296341 (C. lanatus var. citroides) and the Chinese inbred line 97103 (C. lanatus var. lantus) 28.0% of mapping markers showed a segregation pattern that skewed away from the 1:1 ratio (P < 0.05) (Zhang et al., 2004). A large number of markers (47.5% and 48.0%) were also skewed in F and F mapping populations derived from a cross between PI 296341 2 3 and NHM (Hawkins et. al., 2001). In an initial linkage map constructed for watermelon using a BC population [(PI 296341 x NHM) x NHM] 25.7% of the markers were 1 skewed and clustered in their respective linkage group (Levi et al., 2001). Deviation from the expected Mendelian ratio is a common feature for interspecific crosses. They most probably arise from variation in genes that control reproduction processes (Za- mir and Tadmor, 1986). Skewed segregation may also be inferred from a meiotic drive where a chromosome with unique structural features or genetic properties render selective advantage or disadvantage to its respective gametes or zygotes (Lyttle, 1991; Bucler et al., 1999), as first shown in maize by Rhodes (1942). Highly skewed markers may contribute to overestimation of recombination frequency and larger linkage distances (loose linkage) between closely linked markers, or conversely cause pseudo linkage by merging of two linkage groups or two independent markers that skew to the same direction (Saliba-Colombani et al., 2000). Thus, the highly skewed markers (P < 0.01) were excluded from the linkage analysis. Skewed markers (P < 0.05) were not included in the initial linkage analysis with markers that segregated in a Mendelian ratio, but were included in a second mapping analysis. Marker order was fairly consistent in both maps (Fig. 1), indicating that the skewed markers (P < 0.05) in this study had little effect on marker grouping and ordering.

Map construction Of 343 markers suitable for mapping 255 markers (74.0%) could be ordered on the map (Table 1). The map consists of 32 linkage groups (Table 2) and marker order is consistent with these on linkage groups of the F population (Fig. 1). Also, a few markers 2 on the large linkage group in this study appeared to be in comparable linkage distances on the largest linkage group in the F S RIL-based map constructed by Zhang et 2 7 al. (2004).

518 Table 2. Distribution of molecular markers on linkage groups of the testcross population

Markers Linkage group Mean Distance Map Lenght RAPD ISSR AFLP Total (cm) (cm)

I 5 0 12 17 16.88 287.1 II 13 42 19 13.40 252.8 III 0 0 15 15 15.40 231.6 IV 16 5 1 22 8.20 181.7 IX 17 3 424 6.73 161.5 V 11 2 0 13 10.40 135.2 VI 0 0 6 6 20.90 125.8 VII 9 0 0 9 13.80 124.5 VIII 13 42 19 5.80 104.5 X 0 0 9 9 11.02 99.2 XI 12 1 0 13 7.10 91.2 XII 11 0 0 11 7.60 83.5 XIII 0 0 5 5 15.58 77.9 XIV 6 1 0 7 9.80 68.9 XIX 42 0 6 10.10 66.9 XV 2 0 3 5 10.60 53.2 XVI 41 0 5 10.50 52.6 XVII 3 0 1 4 13.10 52.3 XVIII 3 0 0 3 19.00 38.0 XX 0 0 3 3 11.63 34.9 XXI 9 0 2 11 3.10 34.2 XXII 0 0 3 3 11.06 33.2 XXIII 3 0 0 3 9.50 28.7 XXIV 0 1 1 2 14.05 28.1 XXIX 0 0 2 2 14.05 28.1 XXV 2 0 3 5 5.60 28.0 XXVI 1 2 0 3 8.70 26.0 XXVII 0 0 2 2 10.95 21.9 XXVIII 1 0 1 2 9.10 18.4 XXX 2 0 0 2 8.60 17.3 XXXI 1 2 0 3 3.70 11.1 XXXII 1 1 0 2 1.80 3.6 Total 149 29 77 255 11.20 2509.9

519 Figure 1. Alignment of a few linkage groups constructed in testcross (TC) and F mapping 2 populations.

The stringent linkage analysis criteria (a minimum LOD score of 3.0 and a maximum recombination value (q) of 0.3 in grouping and 0.25 in marker ordering) resulted in a high number of linkage groups in the present map. These criteria were used to avoid any possible mapping errors, making the map more valuable in merging experiments with other maps constructed for watermelon (Hashizume et al., 1996; Hawkins et al., 2001; Levi et al., 2001, 2002; Hashizume et al., 2003; Zhang et al., 2004). Most of the AFLP markers in this study were relatively distant from their neighboring markers as compared with RAPD and ISSR markers (Table 2, Fig. 1). The total

520 genetic distance of the testcross-derived map that contains 27 ISSR and 141 RAPD markers is 1166.2 with an average genetic distance of 8.1 cM between markers (Levi et al., 2002). However, in this study the addition of 2 ISSR, 8 RAPD, and 77 AFLP markers to the map increased the total mapping distance to 2,509.9 cM with an average genetic distance of 11.2 cM between markers. Similar results of large distances among AFLP markers as compared with RAPD and ISSR markers were also shown in mapping experiments using the F S RIL population (PI 296341 x 97103) (unpublished data). 2 7 A few large linkage groups (particularly in the BC -based map; Levi et al., 2001) 1 have regions with low recombination rates between markers (0 to 2.3 cM). These linkage regions might be chromosomal regions near a centromere, since fewer recombination events occur in the vicinity of a centromere than in regions distant from it (Mather, 1936; Dimitrov and Georgieva, 1994). Dense regions with low recombination rates also exist in linkage maps constructed for cucumber (Park et al., 2000; Bra- deen et al., 2001). Most of the AFLP markers were clustered on the certain linkage regions, indicating that certain regions of watermelon genome contain large number of MseI-EcoRI restriction sites. These results indicate that AFLP markers may cover linkage regions with DNA properties different than these detected by RAPDs or ISSRs.

Distribution of AFLP versus RAPD and ISSR markers There are sharp differences in segregation and distribution of AFLPs as compared with RAPD and ISSR markers. Most of the AFLP markers in this study were clustered in a few specific linkage regions (as shown for linkage groups F2-I, F2-II, and F2-IV in Fig. 1), in contrast with the RAPD and ISSR markers that randomly segregated on most of the linkage groups (as shown in the first linkage map Levi et al., 2002). The linkage groups containing AFLP markers appeared to be among the largest groups on the map (Table 2). Clustering of AFLP markers was shown in a mapping study of tomato genome where a large number of AFLP markers clustered on one linkage group (Saliba-Colombani et al., 2000). Perin et al. (2002) also showed clustering of AFLP markers on linkage regions of melon (Cucumis melo) genome. AFLP markers may cover linkage regions with DNA properties different than those detected by RAPDs or ISSRs. However, the possibility of pseudo linkage due to skewed segregation of many of the AFLP markers can not be excluded in this study.

Conclusions

A large number of markers in mapping populations (C. lanatus var. citroides x C. lanatus var. lanatus) are skewed away from the expected segregation ratio, hampe- ring the mapping analysis of watermelon genome. The number of skewed markers in the testcross population used in this study is relatively low (31.8%) as compared with the number of skewed markers in the F population (51.0%). Thus, mapping analysis 2 based on the testcross population is useful in complementing the F mapping data. 2 Although AFLPs produce high polymorphisms among watermelon cultivars, a relatively low number of these markers (21.7-56.4%) were useful for mapping. Furthermo- re, a large number (51.4-80.0%) of AFLP mapping markers are skewed from the ex-

521 pected segregation ratios, and are clustered on certain regions of the watermelon genome. Experiments with different marker types (including SSRs, RFLPs, SCARs and ESTs) are being conducted to further saturate this genome.

References

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523 524 Increased femaleness in transgenic cucumbers that overexpress an ethylene receptor

P.A. Rajagopalan, T. Saraf-Levy1, A. Lizhe2 and R. Perl-Treves Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel 1Present address: Department of Fruit Tree Genetics, Agricultural Research Organi- zation, Beth Dagan 50250, Israel 2Present Address: School of Life Sciences, Lanzhou University, Lanzhou, China

Summary

Ethylene is considered the main sex hormone in cucumber and its role in promoting femaleness is well established. In this study we explored the possible role of ethylene sensitivity in cucumber sex determination. We genetically engineered cucumber plants to over-express the an ethylene receptor, the cucumber ERS1 homologue, under the control of the 35S CaMV promoter. Seven independent, stably transformed plants were recovered and their T1 progeny examined for changes in sex expression. Five families had monoecious sex patterns similar to the non-transformed plants, but in two families we observed segregation between plants with a normal monoecious phenotype and plants with an enhanced-female phenotype. The latter plants formed their first female node at least four nodes earlier than the other plants and the proportion of female flowers was several folds higher. The enhanced-femaleness plants represented two thirds of the transgenic progeny, and displayed, at a young stage, higher levels of ERS transcripts, compared to control plants and to their phenotype-negative siblings. The feminized plants also exhibited accelerated yellowing of detached leaves, suggesting that the phenotype is related to the expression of the ERS transgene. Our data implicate ethylene perception in sex determination, and support the notion that receptor levels, and not only ethylene hormone levels, could be a limiting factor in bud sexual development.

Keywords: Cucumis sativus, ethylene, femaleness, transgenic cucumbers

Introduction

Ethylene plays a central role in controlling cucumber sex expression. When applied exogenously, it increases femaleness, whereas compounds that inhibit ethylene production or action increase maleness. Moreover, endogenous ethylene levels have been correlated with different sex phenotypes in cucumber (Perl-Treves, 1999). Local differences in ethylene levels along the shoot of monoecious cucumbers may be responsible for the sexual transition from maleness to femaleness. Interestingly, Trebit- sh et al. (1997) discovered that gynoecious cucumber lines possess a second copy of the cucumber ACS gene that co-segregated with gynoecious sex expression in genetic populations. The observed gradient in female tendency found in monoecious cucumber, and the differences in sex expression between male/female genotypes could result not only from modulation of ethylene synthesis, but also from local changes in ethylene sensitivity. Ethylene receptors have been extensively characterized in model plants (Wang et al., 2002). The ETR/ERS gene products bind ethylene and possess domains for ethylene sensing, signal transduction and response regulation. Focu-

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 525 sing on one ethylene-receptor homolog, the cucumber CS-ERS1 gene, we asked whether its expression could directly influence the plants sex. We over-expressed the gene in transgenic cucumbers and observed, in some transformed lines, marked changes in sex expression, implicating a role for ethylene sensitivity in cucumber sex determination.

Materials and methods

Cloning of the cucumber ERS1 gene Using degenerate primers, we cloned a cucumber genomic fragment homologous to tomato and Arabidopsis ERS; the latter fragment was used to screen cucumber floral bud libraries and a full-length, 2.28 kb-cDNA clone was isolated. The cDNA was sub-cloned into binary vector pME504 (Edelman et al., 2000) under the control of the 35S CaMV promoter. The binary construct was introduced to Agrobacterium tumefaciens strain EHA105 by triple mating.

Transformation of cucumber cotyledons Cucumber transformation was according to Tabei et al. (1998) with modifications (Rajagopalan and Perl-Treves, manuscript in preparation). C. sativus Poinsett 76 (Peto Seeds, USA) were partially germinated and cotyledon explants prepared by removing the embryo and adjacent tissue with a V-shaped cut. Selection during shoot differentiation was carried out in 100 mg/l kanamycin, followed by elongation in increasing kanamycin concentrations, up to 200 mg/l and rooting in 100 mg/l kanamycin.

PCR analysis, Southern and Northern hybridization PCR analysis was used to detect the trans-gene using nptII-gene primers. Putative transgenic plants were also tested by Southern blot hybridization using the nptII and ERS gene fragments as probes under high stringency conditions. Total RNA was extracted using the Tri-reagent Kit (Molecular Research Centre) from control plants and from T -generation transformed plants and standard Northern hybridization was performed. 1

Chlorophyll extraction from senescing leaves Leaf-discs were cut and placed on sterile, water-wetted filter paper in the growth chamber at 25°C, 16 hr light photoperiod. After 4-5 days, discs were inspected visually for their color and chlorophyll determination was carried out (Lichtenthaler, 1987).

Results and discussion

Cloning of a cucumber ERS1 homolog and production of transgenic cucumber We isolated a full-length, 2.28 kb cDNA clone of Cucumis sativus ERS, identical to the clone reported by Yamasaki et al. (2000). The ERS insert was subcloned under the control of the CaMV-35S promoter. We used the transformation-competent monoecious cultivar Poisett 76 (Chee, 1990) and a modified protocol based on Tabei et al. (1998). In our hands, transformation experiments starting with 100 explants (50 se-

526 eds) consistently yielded 1-2 independent transformants and a similar number of untransformed escapes. Seven independent 35S-ERS plants were produced and self- pollinated to obtain T seeds. 1

Figure 1. Poinsett 76 cucumber plants transformed with the 35S:ERS gene. Left: example of Type I and Type II transgenic plants: in Type I a single female node in the middle of several male nodes, in Type II, seven consecutive female nodes are apparent. On the right, schematic representation of nontransformed, Type I and Type II plants.

Sex expression of the transgenic plants and their progeny The T plants were morphologically similar to non-transformed Poinsett 76 plants. 0 We planted in the greenhouse samples of the T progeny of each plant, to observe 1 their flowering patterns in detail. PCR analysis identified those T individuals that 1 inherited the trans-gene and served to estimate the number of insertions in the genome. Of the seven families, one had a high ratio of transgenic to non-transgenic progeny (34:1) that could suggest 2-3 insertions, while the rest had lower ratios that were compatible, according to the chi-square test, with a single insertion. Samples of six T families were planted in the greenhouse over three growing seasons (Table 1), along 1 with control, non-transformed plants. PCR-negative plants were eliminated and we only kept the progeny that carry the transgene in homozygous or heterozygous com- plement. As simple indicators of the sex phenotype, we recorded the number of the first node bearing a female flower, as well as the total number of female flowers in the first 19 nodes of the main stem. The two parameters together provide a good estimate of the degree of femaleness of a plant. Non-transformed Poinsett plants are monoecious, with a prominent male phase: the first female flower only appears in the 13-16th node. Families 1-5 were similar to control plants and had only between one and three female flowers in the first 19 nodes.

527 As for families 6 and 7, we noted that their transgenic T progeny could be sorted 1 into two discrete phenotypes. Type I plants had sex expression patterns that closely resembled those of the control plants and families 1-5. However, the majority of the plants had a striking increase in femaleness. The female first node-number ranged between 5 and 8, instead of 10-15 in the phenotype-less siblings, while the total number of female flowers increased many folds, representing 6-13 female nodes in the recorded segment. Such sex pattern is typical of F-locus heterozygotes, or ethrel-treated plants, and is never encountered in untreated Poinsett 76 plants. The difference between the T families, and within families 6 and 7, was replicated consistently around the year. 1 Table 1 shows that femaleness increases in winter-grown plants, compared to spring and summer grown plants, where male tendency is strongest. Such effect was reported in cucumber (Perl-Treves, 1999). There was no clear seasonal effect on sex patterns in Type II plants (except for family 6 in winter): sex patterns seemed rather constant, as expected from an overriding effect of a constitutively expressed trans-gene. To further correlate the increased femaleness with an enhanced sensitivity to ethylene, we checked whether ERS-over-expressing plants displayed accelerated leaf senescence. Detached leaves or leaf discs left to senesce on moist filter paper differed according to the plants sex phenotype. Leaves from control Poinsett 76 plants, as well as plants from families 1-5 and Type I plants from families 6 and 7, looked green after 4-7 days. Those from Type II transgenic plants became yellow several days before the control plants. To quantify the effect, we extracted the leaf pigments and calculated the contents of chlorophylls and carotenoids. Table 1 shows a two-fold reduction in chlorophyll in Type II leaves. Carotenoids were also reduced (not shown). We asked whether ERS transcript levels were related to the sex and senescence phenotypes. A full correlation between ERS transcript level in the young leaf and the sex phenotype was apparent (Fig. 2). Plants from families 1-5 (not shown) and plants from families 6 and 7 that did not show a feminized phenotype, all had low transcript levels, similar to the endogenous levels of non-transformed plants. Plants 7, 8, 11 and 15 of family 7, and plants 2, 14, 17, 18 of family 6 are Type II plants, and they all displayed strong ERS signals.

Figure 2. Northern analysis of ERS transcripts in non-transformed and 35S:ERS transgenic plants of the T generation. All plants harbored the trans-gene. Bottom panel ethi- 1 dium bromide stain of the total RNA samples. Plants No. 7, 8, 11, 15 in Family 6, and plants 2, 7, 8 and 14 in Family 7 displayed Type II phenotype with increased femaleness, all others were designated Type I, i.e., similar to non-transformed Poinsett 76 plants.

528 NN NN Chlorophyll, Mg/gfw ± SE 5 plants NN NN Summer 1.0 ± 0.0 42.3 ± 0.2 NN ± 1.0 ± Spring 1.5 ± 0.5 1.5 ± 0.5 41.9 ± 0.3 2.1 ± 0.3 ± 0.3 1.3 ± 0.2 42.4 (first 19 nodes) leaf discs plants compared to Poinsett 76 non-transformed plants 1 ± 1.0 ± ± 1.0 ± 2.2 ± 0.2 1.6 ± 0.21.8 ± 0.2 Winter 4 NN NN Summer 13.8 ± 0.2 2.8 ± 0.3 1.0 NN 3 ± 1.0 ± Spring 12.3 ± 0.6 15.3 ± 0.8 1.0 13.9 ± 0.6 15.6 ± 0.8 3.0 Female Node ± SE Female Nodes Total ± SE Chlorophyll in senescing 2 st 1 ± 1.0 ± 1.0 ± Winter 1 plants Sex phenotype and senescence of detached leaves of transgenic T Total Total number of plants examined in three experiments. Winter season: November 02 to February 2003. Spring season: March-June 2003. Summer-fall season, August-November 2003. No. of plants assayed for chlorophyll content after 4 days detachment; discs from additional plants were inspected visually for Family No. Exp. 1 Exp. 2 Exp. 3 Exp. 1 Exp. 2 Exp. 3 No. Fam. 6 group IIFam. 7 group I 16 9 6.3 ± 0.9 12.0 8.1 ± 1.45.3 ± 0.9 12.6 ± 0.3 5.6 ± 0.7 8.8 ± 1.2 6 0.9 ± 0.1 Fam. 7 group II 15 5.3 ± 0.7 6.5 ± 0.3 5.3 ± 0.6 9.0 ± 0.6 9.0 ± 0.7 8.0 ± 1.2 41.0 ± 0.1 Wild TypeWild Fam. 2Fam. 3 16 13.0 18 12 11.6 ± 1.0 12.6 ± 0.5 13.1 ± 0.9 14.8 12.1 ± 0.8± 1.2 1.8 ± 0.2 1.4 ± 0.2 1.2 ± 0.2 3 2.0 ± 0.1 Fam. 4Fam. 5Fam. 6 group I 9 18 5 9.8 ± 0.8 11.0 ± 0.7 12.0 11.6 13.5 ± ± 1.01.1 13.8 ± 1.8 2 ± 0.0 1.4 ± 0.2 1.4 ± 0.2 4 2.0 ± 0.1 1 2 3 4 5 yellowing. Table 1. Table

529 Ethylene signals in particular zones along the shoot, or within the flower, must be locally perceived by receptors and communicated by signaling proteins, in order to inhibit stamens and promote carpel development. In the present study, we directly modulated ERS levels in transgenic cucumbers, to see whether receptor levels represent a limiting factor in sex expression. Ethylene receptors were shown to act as negative switches in Arabidopsis: the unoccupied receptor actively represses the ethylene response (Wang et al., 2002), while ethylene binding relieves such repression. Moreover, ethylene binding requires the supply of a copper co-factor to the receptor (Hirayama and Alonso, 2000). Such complex mode of action made it impossible to foresee the effect of increased ERS expression. Depending on the physiologically limiting factor(s), one could rationalize either an increased ethylene response or an inhibited response, depending whether or not the extra receptor protein is activated by copper ions and stimulated by endogenous ethylene. The strong feminization observed in two independently-derived T families argues 1 for ethylene receptors being important in sex determination, and for protein levels being limiting. Had ethylene been insufficient, extra unbound receptor could repress the response and decrease femaleness, but no such effect was observed, even in Type I transgenic plants. The phenotype observed in families 6 and 7 is likely an effect of ERS over-expression, as indicated by Northern analysis, and by the accelerated senescence of detached leaves. The fact that only two out of seven families exhibited the strong phenotype may be attributed to position effects. However, the segregation within these two families between phenotype positive and phenotype negative plants is less readily explained. We re-examined Type I and Type II plants by Southern blots and confirmed the presence of the trans-gene in both groups. A silencing effect triggered in part of the plants could be responsible for such variation. Another possibility is that the two groups represent trans-gene dose, e.g., the homozygous or heterozygous plants differ in phenotype. We are presently performing test-crosses to differentiate between these possibilities.

Acknowledgements This research was supported by Grant IS-3139-99 of the USA-Israel Binational Agricultural Research and Development Fund and by Grant I-682-166.12/2000of the German Israel Foundation for Scientific Research and Development.

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531 532 Authors index

Abak K. 129 Dias R.C.S. 157 Akasaka-Kennedy Y. 431 Dogimont C. 209, 307, 325, 353, 485 Alvarez J.M. 129, 203 Edelstein M. 63, 313 Andres T.C. 107, 113, 393 Eduardo I. 499 Aranda M.A. 209 Ekmekci Y. 57 Arús P. 209, 499 Ellul P. 465 Assis J.G.A. 157 Elman C. 459 Atarés A. 465 Ercan N. 345 Bakowska M. 245 Ezura H. 431 Barroso M.R. 135, 143 Fanourakis N. 101 Bartoszak K. 333 Fauve J. 515 Bartoszewski G. 455 Ferreira M.A.J.F. 157 Baumkoler F. 389 Frenich A.G. 69 Baysal G. 57 Fukino N. 503 Bendahmane A. 209, 325 Gaba V. 459 Ben-Hur M. 63 Gajdová J. 441 Benjamin I. 257 Galecka T. 411 Benyamini Y. 491 Galperin M. 257 Berenji J. 341 Gao Z. 471 Besombes D. 353 Garcia-Mas J. 209 Biriukova N. 51 Garcia-Sogo B 465 Bisztray Gy. D. 437 Garrido D. 347 Börner A. 129 Giovannoni J. 491 Brotman Y. 485 Giovinazzo N. 353 Burger Y. 63, 151, 313, 471, 491 Gómez P. 347 Burget E. 325 Gómez-Guillamón M.L. 129, 197, 295 Caboche M. 209, 325 González F.J.E. 69 Camero R. 197 Granero A.M. 69, 75 Capel J. 295 Grinberg M. 319 Carnide V. 129, 135, 143 Guner N. 223 Cetiner S. 459 Gusmini G. 301 Chen J.F. 189 Hagen L. 325 Chovelon V. 325 Hanan A. 389 Chung S-M. 477 Havey M.J. 455 Cohen R. 63, 313 Hirschberg J. 181 Cohen Y. 257 Hopkins D.L. 251 Corella P. 197 Horev C. 313 Cuevas H.E. 393 Jahn M. 189 Curuk S. 459 Jamilena M. 347 Daryono B.S. 213 Katzir N. 151, 181, 485, 491, 515 Davis A. 181, 515 Kenigsbuch D. 257 Desloire S. 325 King S. 181, 515

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 533 Kiss-Bába E. 437 Navrátilová B. 415, 441 Klonari E. 157 Niemirowicz-Szczytt K. 245, 411 Klosinska U. 337 Nieto C. 209 Korzeniewska A. 411 Nowaczyk L. 45 Koutsika-Sotiriou M. 163 Nowaczyk P. 45 Kovalski I. 485 Ohara T. 175 Kowalczyk B. 359 Olczak-Woltman H. 245 Kozik E.U. 337 Orjeda G. 209 Køístková A. 95, 119 Pachner M. 237 Køístková E. 21, 39, 95, 119 Palevsky E. 319 Kunihisa M. 503 Pánczél S. 437 Kuzuya M. 503 Papp D. 341 Lebeda A. 21, 39, 95, 267, 275, Paris H.S. 151, 389 281, 289, 415, 441 Paris M.K. 381 Lelley T. 237, 507 Pauquet J. 325 Le Menn A. 325 Pavlikaki H. 101 Levi A. 181, 515 Peñaranda A. 347 Lewinsohn E. 491 Perchepied L. 307, 353 Lizhe A. 525 Périn C. 353 López-Sesé A. 169 Perl-Treves R. 319, 459, 485, 525 Lower R.L. 365 Petreikov M. 471 Lozano R. 295 Pineda B. 465 Luis-Arteaga M.S. 129, 203 Pitrat M. 307, 325, 353, 485 Luný J. 39 Polák J. 231 Malepszy S. 455 Porat A. 313 Mallor C. 203 Portnoy V. 491 Marco C.F. 197 Puigdomenech P. 209 Martins S. 135, 143 Queiroz M.A. 157 Maslovskaya E. 51 Rajagopalan P.A. 525 Matsumoto S. 503 Ramos S.R.R. 157 McCreight J.D. 219, 373, 381 Ravid U. 491 Monfort A. 209 Ravina I. 63 Monforte A.J. 499 Reddy O.U.K. 515 Montoro T. 197, 295 Robbins M.D. 401 Morales M. 209 Romao~ R.L. 157 Moravec J. 21 Rousselle P. 325 More T.A. 81 Saar U. 151, 313 Moreno V. 465 Sáenz de Miera L.E. 135, 143 Moriarty G. 189 Sakata Y. 175 Moriones E. 129 Salinas M. 295 Morishita M. 175 Sánchez-Campos S. 197 Moura M.C.C.L. 157 Sanz J.M.G. 69, 75 Mpardas G. 163 Saraf-Levy T. 525 Mpeis A. 163 Sari N. 129 Natsuaki K.T. 213 Schaffer A.A. 151, 471, 491 Navarro C. Ponce 101 Schollenberger M. 245

534 Sedláková B. 281, 289 Traka-Mavrona E. 163 ªensoy A.S. 345 Trebitsh T. 515 ªensoy F. 345 Tsivelikas A.L. 163 Serrano A.R. 69, 75 Urban J. 267, 275 Seshadri V.S. 81 Valot N. 325 Shen S. 471 van Dooijeweert W. 91 Shomer I. 319 van Leeuwen H. 209 Silva M.L. 157 Velich I. 437 Silveira L.M. 157 Vences F.J. 135, 143 Simmons A. 515 Vidal J.L.M. 69 Skálová D. 415 Vinter V. 95, 119 Somowiyarjo S. 213 Wang Y. 459 Song R. 301 Watad A.A. 459 Soroker V. 319 Wehner T.C. 223, 301, 515 Souza F.F. 157 Wessel-Beaver L. 393 Staub J.E. 169, 365, 373, White R. 491 381, 401, 477 Wronka J. 411 Sugiyama K. 175 Xia X. 459 Sugiyama M. 175 Xu Y. 515 Sun Z. 365 Yariv Y. 491 Svoboda J. 231 Yeheskel A. 459 Sztangret J. 411 Yeselson Y. 151 Tadmor Y. 151, 181, 491, 515 Zalapa J.E. 373, 381 Taler D. 257 Zarka V. 437 Temirkaynak M. 345 Zhang X. 515 Thies J. 515 Zilberstein L. 459 Thomas C.E. 515 Zraidi A. 507 Tipirdamaz R. 57

535 536 List of participants

This list includes addresses of all participants who sent the final registration before 30 April 2004.

Armenia Tadevosyan Laura Scientific Center of Vegetable and Industrial Crop Breeding Darakert Aratat Region Masis Subregion 378 322, Armenia tel.: 036 40892585711 E-mail: [email protected]

Austria Kindler Alfred Landeskammer f. Ld.-u.Fw. Gartenbauabteilung Großmarktstr. 8a A-8020 Graz tel: +43-316-273186 E-mail: [email protected] Lelley Tamas Institute of Agrobiotechnology Department of Biotechnology and Plant Production Konrad Lorenz Str. 20 A-3430 Tulln tel: +43-227266280204 E-mail: [email protected] Lichtenecker Penelope Õkologik Schindlergasse 45 A-1180 Vienna tel.: +43-1-4701622 E-mail: [email protected] Vollmann Johann Eucarpia - Secretariat University of Agriculture Plant Breeding Department Gregor Mendel Str. 33 A-1180 Vienna Winkler Johanna SATZUCHT GLEISDORF Am Tieberhof 33 A-8200 Gleisdorf tel: +43-3112-210515 E-mail: winkler. [email protected]

A. Lebeda and H.S. Paris (Eds.): Progress in Cucurbit Genetics and Breeding Research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding. Palacký University in Olomouc, Olomouc (Czech Republic), 2004. 537 Zraidi Amine University of Natural Resources and Applied Life Sciences Department of Agrobiotechnology Konrad Lorenz Str. 20 A-3034 Tulln tel.: +43-227266280-281 fax: 0043227266203 E-mail: [email protected]

Brazil Queiróz Manoel, Abilio Migrantes 63A 56328-040 Petrolina PE E-mail: [email protected]

Bulgaria Dimova Dochka Agricultural University 12 Mendeleev Str. 4000 Plovdiv tel: +359-326126 Krasteva Lilia Institute of Plant Genetics Resources 4122 Sadovo tel: +395-88-585950 E-mail: [email protected] Panayotov Nikolay Agricultural University 12 Mendeleev Str. 4000 Plovdiv tel: +359-326126 E-mail: [email protected]

Czech Republic Dolealová Ivana Palacký University Faculty of Science Department of Botany lechtitelù 11 783 71 Olomouc-Holice tel.: +420 585 634 804 fax: +420 585 634 824 E-mail: [email protected] Gajdová Jana Palacký University Faculty of Science Department of Botany lechtitelù 11 783 71 Olomouc-Holice tel.: +420 585 634 820 fax: +420 585 634 824 E-mail: [email protected]

538 Holman Bohuslav Bzinská 1420 696 81 Bzenec tel.: +420 518 384 470 E-mail: [email protected] Holman Jiøí Bzinská 1420 696 81 Bzenec tel.: +420 518 384 370 E-mail: [email protected] Køístková Eva Research Institute of Crop Production Praha-Ruzynì Division of Genetics and Plant Breeding Department of Gene Bank, Workplace Olomouc lechtitelù 11 783 71 OlomoucHolice tel.: +420 585 209 966 E-mail: [email protected] Lebeda Ale Palacký University Faculty of Science Department of Botany lechtitelù 11 783 71 Olomouc-Holice tel.: +420 585 634 800 fax: +420 585 634 824 E-mail: [email protected] Luný Jan Za Potou 3 772 00 Olomouc Moravec Jiøí Sienkiewiczova 1 772 00 Olomouc Navrátilová Boena Palacký University Faculty of Science Department of Botany lechtitelù 11 783 71 Olomouc-Holice tel.: +420 585 634 811 fax: +420 585 634 824 E-mail: [email protected] Petrelová Irena Palacký University Faculty of Science Department of Botany lechtitelù 11 783 71 Olomouc-Holice tel.: +420 585 634 804 fax: +420 585 634 824 E-mail: [email protected]

539 Sedláková Boena Palacký University Faculty of Science Department of Botany lechtitelù 11 783 71 Olomouc-Holice tel.: +420 585 634 818 fax: +420 585 634 824 Sedláøová Michaela Palacký University Faculty of Science Department of Botany lechtitelù 11 783 71 Olomouc-Holice tel.: +420 585 634 804 fax: +420 585 634 824 E-mail: [email protected] Skálová Dagmar Palacký University Faculty of Science Department of Botany lechtitelù 11 783 71 Olomouc-Holice tel.: +420 585 634 820 fax: +420 585 634 824 E-mail: [email protected] Svoboda Jiøí Research Institute of Crop Production Praha-Ruzynì Division of Plant Protection Department of Virology Drnovská 507 161 06 Praha 6 E-mail: [email protected] Urban Jiøí Palacký University Faculty of Science Department of Botany lechtitelù 11 783 71 Olomouc-Holice tel.: +420 585 634 818 fax: +420 585 634 824 E-mail: [email protected] Vinter Vladimír Palacký University Faculty of Science Department of Botany lechtitelù 11 783 71 Olomouc-Holice tel.: +420 585 634 816 fax: +420 585 634 824 E-mail: [email protected]

540 Denmark Raeder Gert L. Daehnfeldt A/S Faaborgvej 248 B 5100 Odense E-mail: [email protected]

France Baudracco-Arnas Sylvie ASL Route de Graveson 13630 Eyragues tel.: +33-04-90242309 E-mail: [email protected] Bertrand Francois Seminis France Recherche Mas de Rouzel - Chemin des Canaux 30900 Nimes E-mail: [email protected] Buchwalder Vincent Seminis France Recherche Mas de Rouzel - Chemin des Canaux 30900 Nimes E-mail: [email protected] Dogimont Catherine INRA-UGAFL Domaine St. Maurice BP 94 84143 Montfavet Cedex E-mail: [email protected] Kol Bertrand Gautier Graines BP No. 1 Route dAvignon 136 30 Eyragues tel.: +33-04-90240260 fax: +33-04-90240261 Legnani Robert Takii France Petit Chemin de la Crau 13630 Eyragues E-mail: [email protected] Mention Philippe Centre Technique Interprofessionnel des Fruits et Légumes 30 127 Bellegarde E-mail: [email protected]

541 Moquet Frederic Gautier Graines BP No. 1 Route dAvignon 136 30 Eyragues tel.: +33-04-90240260 fax: +33-04-90240261 E-mail: frederic.moquet@gautiergraines Pitrat Michel INRA Génétique et Amélioration des Fruits et Légumes Domaine Saint Maurice 84143 Montfavet Cedex E-mail: [email protected] Perchepied Laura INRA Génétique et Amélioration des Fruits et Légumes Domaine Saint Maurice 84143 Montfavet Cedex fax: +330-432722702 E-mail: [email protected] Rocherieux Julien Gautier Semences BP No. 1 13630 Eyragues Roumanille Luc ASL 13630 Eyragues fax: +33-04-90928581 E-mail: [email protected] Tanaka Kazuyuki Takii France Petit Chemin de la Crau 13630 Eyragues E-mail: [email protected]

Great Britain Poostchi I. 97 St. Marks Road RG9 1LP Henley on Thames

Greece Fanourakis Nicholas Technological Educational Institute Stavromenos 71500 Heraklion / Crete tel.: +30-2810-369472 fax: +30-2810-311388 E-mail: [email protected]

542 Hungary Bisztray György Dénes Budapest University of Economic Sciences and Public Administration Faculty of Horticultural Sciences Department of Genetics and Horticultural Plant Breeding Ménesi 44/A H-1118 Budapest E-mail: [email protected] Kiss-Bába Erzsébet Budapest University of Economic Sciences and Public Administration Faculty of Horticultural Sciences Department of Genetics and Horticultural Plant Breeding Ménesi 44/A H-1118 Budapest Papp Dezso DUDU BT Tessedik Ut. 171 H-4032 Debrecen tel: +36-52-419225

India More Tukaram A. College of Agriculture (MPKV) Dhule, Maharashtra State 424004 Dhule E-mail: [email protected] or [email protected] Pandey S.N. Indian Council of Agricultural Research Krishi Anusandhan Bhavan-II New Delhi E-mail: [email protected] Rajan S. Kerala Agricultural University Directorate of Research 680 656 Thrissur tel: +91-487-2370497 E-mail: [email protected] Seshadri V.S. 15.A /12 W. E. A Karol Bagh 110 005 New Delhi tel: +91-011-25818617

543 Srivastava Umesh Indian Council of Agricultural Research Horticulture Division Krishi Anusandhan Bhawan -II Pusa Campus 110 012 New Delhi tel.: +91-011-25788405 E-mail: [email protected] or [email protected]

Indonesia Rokhman Fatkhu East West Seed Indonesia Desa Benteng Campaka 411 81 Purwakarta Jawa Bara tel: +62-0264-201871 fax: +62-0264-201875 E-mail: [email protected]

Iran Arzani Ahmad Isfahan University of Technology College of Agriculture Agronomy and Plant Breeding Department Isfahan E-mail: [email protected]

Israel Brotman Yariv Bar-Ilan University Faculty of Life Science 52900 RamatGan E-mail: [email protected] Cohen Ron Agricultural Research Organization Newe Ya´ar Research Center P.O. Box 1021 30095 Ramat Yishay tel.: +972-4-9539516 E-mail: [email protected] Davidi Haim Hazera Genetics Ltd. Mivhor Farm M.p. Lakish Darom 79354 Mivhor tel.: +972-08-6878119 E-mail: [email protected]

544 Edelstein Menahem Agricultural Research Organization Newe Yaár Research Center P.O. Box 1021 30095 Ramat Yishay E-mail: [email protected] Katzir Nurit Agricultural Research Organization Newe Yaár Research Center P.O. Box 1021 30095 Ramat Yishay tel.: +972-4-9539554 E-mail: [email protected] Nerson Haim Agricultural Research Organization Newe Yaár Research Center P.O. Box 1021 30095 Ramat Yishay tel: +972-4-9539508 E-mail: [email protected] Paris Harry S. Agricultural Research Organization Newe Yaár Research Center P.O. Box 1021 30095 Ramat Yishay E-mail: [email protected] Perl-Treves Rafael Bar-Ilan University Faculty of Life Science 52900 Ramat-Gan E-mail: [email protected] Vardi Eyal Hazera Genetics Ltd. Mivhor Mivhor Farm M.p. Lakish Darom 79354 Mivhor tel: +972-8-6878123 fax: +972-8-6814057 E-mail: [email protected] Wolf Shmuel The Hebrew University Faculty of Agriculture Institute of Plant Sciences 76100 Rehovot tel: +972-8-9489428 E-mail: [email protected]

545 Japan Daryono Budi S. Tokyo University of Agriculture Graduate School of Agriculture Laboratory of Tropical Plant Protection 1-1-1 Sakuragaoka, Setagaya-ku 156-8502 Tokyo tel: +81-3-54772412 E-mail: [email protected] Ezura Hiroshi Gene Research Center University of Tsukuba Tennodai 1-1-1, 305 8572 Tsukuba tel.: +81-298-537263 E-mail: [email protected] Fukino Nobuko National Institute of Vegetable and Tea Sciences 360 Kusawa 514-2392 Ano, Mie E-mail: [email protected] Sugiyama Mitsuhiro National Institute of Vegetable and Tea Sciences 360 Kusawa 514-2392 Ano, Age-gun, Mie E-mail: [email protected] Oladele Idowu Japan International Center for Agricultural Sciences 1-1 Ohawashi Tsukuba, Ibaraki 305-8686 Tsukuba E-mail: [email protected] Sakata Yoshiteru National Institute of Vegetable and Tea Sciences 360 Kusawa 514-2392 Ano, Age-gun, Mie E-mail: [email protected]

The Netherlands Bal Eric Rijk Zwaan Burgemeester Crecelaan 40 26782G De Lier E-mail: [email protected] De Jager Kitty Nunhems Zaden B.V. P.O. Box 4005 6080 AA Haelen E-mail: [email protected]

546 Degreef Paul Nunhems Zaden B.V. P.O. Box 4005 6080 AA Haelen tel.: +31-0475-599358 E-mail: [email protected] Den Nijs Ton Plant Research International P.O. Box 16 6700 AA Wageningen E-mail: [email protected] Den Hertog Maarten Rijk Zwaan Burgemeester Crecelaan 40 26782G De Lier E-mail: [email protected] Hertogh Kees Nickerson - Zwaan B.V. P.O. Box 28 4920 AA Made E-mail: [email protected] Mazereeuw Jacob Pieter Enza Zaden B.V. Haling 1e 1602 DE Enkhuizen tel.: +31-228-315844 E-mail: [email protected] Reuling Gerhard Nunhems Zaden B.V. P.O. Box 4005 6080 AA Haelen E-mail: [email protected] Segers Bart Nunhems Zaden B.V. P.O. Box 4005 6080 AA Haelen E.mail: [email protected] Suelmann Jos Nunhems Zaden B.V. P.O. Box 4005 6080 AA Haelen E-mail: [email protected] Van Dooijeweert Willem Centre for Genetic Resources Droevendaalse Steeg 1 6708 PB Wageningen tel.: +31-317-477083

547 Verbakel Henk Keygene N.V. Agro Business Park 90 6708 PW Wageningen

Pakistan Siddiqui Ahmed Mashood Environmental Protection Agency A-387 Talpur Colony Tandojam 70050 Sindh

Poland Antos Marta Krakowska Hodowla i Nasiennictwo Ogrodnicze POLAN Sp. Z o.o. ul. L. Rydla 53/55 30 130 Krakow tel.: +48-12-6370377 E-mail: [email protected] Bartkowska Zofia Zaklad Ogrodniczy Przyborow Przyborow123 39 217 Grabiny tel.: +48-146703336 E-mail: [email protected] Bartkowski Krzysztof Zaklad Ogrodniczy Przyborow Przyborow123 39 217 Grabiny tel.: +48-146703336 E-mail: [email protected] Bartoszak Katarzyna Centralny Osrodek Badania Odmian Roslin Uprawnych 63 022 Slupia Wielka E-mail: [email protected] or [email protected] Bartoszewski Grzegorz Warszaw Agricultural University Faculty of Horticulture Department of Plant Genetics Nowoursynowska 166 02 787 Warszaw tel.: +48-22-8439041 fax: +48-22-8430982 E-mail: [email protected] Galecka Teresa Warszaw Agricultural University Department of Plant Genetics Breeding Nowoursynowska 166 02 787 Warszaw tel.: +48-22-8430982 E-mail: [email protected]

548 Gleñ Miroslawa Zaklad Ogrodniczy Przyborow Przyborow 123 39 217 Grabiny tel.: +48-146703336 E-mail: [email protected] Korzeniewska Aleksandra Warszaw Agricultural University Department of Plant Genetics Breeding Nowoursynowska 166 02 787 Warszaw tel.: +48-22-8430982 E-mail: [email protected] Kowalczyk Bogna Centralny Osrodok Badania Odmian Roslin Uprawnych 63 022 Slupia Wielka E-mail: [email protected] or [email protected] Kozik Elzbieta U. Research Institute of Vegetable Crops Konstytucji 3 Maja 1/3 96 100 Skierniewice tel: +48-46-8334193 E-mail: [email protected] Lasocka Joanna Przedsiebiorstwo Nasiennictwa Ogrodniczego i Szkolkarstwa Zeromskiego 3 05 850 Ozarów Mazowiecki tel.: +48-22-7223075 E-mail: [email protected] Niemirowicz-Szczytt Katarzyna Warszaw Agricultural University Faculty of Horticulture Department of Plant Genetics and Breeding Nowoursynowska 166 02 787 Warszaw tel.: +48-22-8430982 E-mail: [email protected] Nowaczyk Pawel University of Technology and Agriculture Department of Genetics and Plant Breeding Bernardynska 6 85 029 Bydgoszcz tel: +48-52-3749524 E-mail: [email protected] Olczak-Woltman Helena Warszaw Agricultural University Faculty of Horticulture Department of Plant Genetics and Breeding Nowoursynowska 166 02 787 Warszawa tel.: +48-22-8430982 E-mail: [email protected]

549 Parkot Justyna Przedsiebiorstwo Nasiennictwa Ogrodniczego i Szkolkarstwa Zeromskiego 3 05 850 Ozarów Mazowiecki tel.: +48-22-7223075 E-mail: [email protected] ¯uradzka Izabela Krakowska Hodowla i Nasiennictwo Ogrodnicze POLAN Sp. Z o.o. ul. L. rydla 53/55 30130 Krakow tel.: +48-12-6370377 E-mail: [email protected]

Portugal Barroso Maria Rosário Centro Experimental da Terra Quente Direcçao Regional de Agricultura de Trás-os-Montes, DRATM Quinta do Valongo 5370-087 Carvalhais, Mirandela tel.: +351-278-260965 E-mail: [email protected] Carnide Valdemar University of Trás-os-Montes and Alto Douro (UTAD) Department of Genetics and Biotechnology 5000-911 Vila Real tel.: +351-259-350501

Russia Biriukova Nina All Russian Research Institute for Vegetable Crops Novomitishinskyi Prospect 82 141 018 Mitishi, Moscow reg. tel.: 095-5828791 E-mail: [email protected]

Slovakia Nôková Janka Slovak Agricultural University Institute of Biodiversity Conservation and Biosafety Tr. A. Hlinku 2 949 01 Nitra E-mail: [email protected]

South Africa Swanepoel Cobus Pannar P.O. Box 19 3250 Greytown tel: 27-33-4139644 E-mail: [email protected]

550 Spain Checa Alguacil Pilar Syngenta Seeds, S.A. Autovia E-15, km. 417,5 04700 El Ejido / Almería tel: +34-950-606403 fax: 629970351 E-mail: [email protected] Álvarez José Mario Centro de Investigación y Tecnología Agroalimentaria de Aragón Apdo. 727 50080 Zaragoza E-mail: [email protected] Alvarez Casanueva Jose Syngenta Seeds, S.A. Autovia E-15, km 417.5 04700 El Ejido / Almeria Ignacio tel: +34-950-606403 Fax: 629385021 E-mail: [email protected] Garcia-Mas Jordi Institut de Recerca i Tecnologia Agroalimentaries Carretera de Cabrils s/n 08348 Cabrils E-mail: [email protected] Gómez-Guillamón Marisa Consejo Superior de Investigaciones Científicas Málaga Experimental Station ´La Mayora ´ 29750 Algarrobo-Costa, Málaga E-mail: [email protected] López-Sesé Ana Isabel Consejo Superior de Investigaciones Científicas Málaga Experimental Station ´La Mayora´ 29750 Algarrobo-Costa, Málaga E-mail: [email protected] Marisol Luis-Arteaga Centro de Investigación y Tecnología Agroalimentaria de Aragón Apdo. 727 50080 Zaragoza E-mail: [email protected] Mallor Cristina Centro de Investigación y Tecnología Agroalimentaria de Aragón Apdo. 727 50080 Zaragoza E-mail: [email protected]

551 Mena Ángeles Centro de Investigación y Formación Agraria Aut. del Mediterráneo/Sal. 420/Paraje S 04745 La Majonera-Almería Email: [email protected] Monforte Antonio J. Institut de Reserca i Tecnologia Agroalimentaria Carretera de Cabrils s/n 08348 Cabrils E-mail: [email protected] Montoro Teresa Consejo Superior de Investigaciones Científicas Málaga Experimental Station ´La Mayora ´ 29750 Algarrobo-Costa, Málaga E-mail: [email protected] Niclos M. Jose Diez Polytechnic University of Valencia Department of Biotechnology Camino de Vera 14 460 22 Valencia tel: +34-96-3879421 E-mail: [email protected] Peòaranda Ruiz Ascension Cifa Almeria (Junta de Andalucia) Consejeria de Agricultura y Pesca ATVIA. Mediterraneo Salida 420 04745 El Ejido / Almería E-mail: [email protected] Roldán Serrano Ana AGROBÍO, S.L. Ctra. Nacional 340 km.419 E1 04745 La Majonera / Almería tel.: +34-950-558030 E-mail: anaroldan77ayahoo.es

Sudan Ahmed Elamin Abu Haraz Fac. of Agric. University of Gezira Wad Medani tel.: +249-51350551 E-mail: [email protected]

Sweden Lehmann Louis Louie´s Pumpkin Patch Brinkgatan 6 SE 26832 Svalöv E-mail: [email protected]

552 Tunisia Jebari Hager Institut National de la Recherche Agronomique de Tunisia / INRAT Rue Hedi Karray 2049 Ariana

Turkey Ayar ªensoy Funda Akdeniz University Dumlupinar Bulvari 07049 Antalya E-mail: [email protected] Ellialtioglu ªebnem Ankara University Faculty of Agriculture Department of Horticulture 06110 Ankara tel.: +90-0532-6520980 E-mail: [email protected] Ercan Nurgül Akdeniz University Dumlupinar Bulvari 07049 Antalya E-mail: [email protected] Gökçen Baysal University of Gazi Faculty of Science and Arts Department of Biology Teknikokullar/Beºevier 06500 Ankara E-mail: [email protected] Kuºvuran Sebnem Yüzüncü Yil University Faculty of Agriculture Department of Horticulture Zeve Kampüsü 65080 Van E-mail: [email protected] ªensoy Ahmet Sirri Akdeniz University Dumlupinar Bulvari 07049 Antalya E-mail: [email protected] Temirkaynak Meliha Akdeniz University Dumlupinar Bulvari 07049 Antalya E-mail: [email protected]

553 Yalcin-Mendi Yesim University of Cukurova Faculty of Agriculture Department of Horticulture Laboratory of Biotechnology Balcali / Adana tel: +90-322-3386615 fax: +90-322-3386615 E-mail: [email protected] Yaºar Fikret Yüzüncü Yil University Faculty of Agriculture Department of Horticulture Zeve Kampüsü 65080 Van E-mail: [email protected]

USA Andres Thomas C. The Cucurbit Network 5440 Netherland Ave. D24, Bronx, New York E-mail: [email protected] Chen Jin Feng Cornell University Department of Plant Breeding 317 Bradfield Hall, 14853 Ithaca, New York E-mail: [email protected] Cook Kevin L. Syngenta Seeds 7500 Olson memorial Highway Golden Valley, Minnesota Gabor Brad Seminis Vegetable Seeds Inc. 37437 State Highway 16 95695 Woodland, California tel.: +1-530-6696233 E-mail: [email protected] Goldman Amy P. 164 Mountain View Road 12572 Rhinebeck, New York tel.: +1-845-266-4545 fax: +1-845-266-5232 E-mail: [email protected] Havey Michael J. USDA - ARS and University of Wisconsin Department of Horticulture 1575 Linden Drive 53706 Madison, Wisconsin tel: +1-608-2621830 E-mail: [email protected]

554 Holmes Gerald J. North Carolina State University Department of Plant Pathology Campus Box 7616 27695 Raleigh, North Carolina tel.: +1-919-5159779 E-mail: [email protected] Hopkins Donald L. University of Florida IFAS Mid-Florida Res. And Educ. Center 2725 Binion Road 32703-8 Apopka, Florida tel: +1-407-8842034 fax: +1-407-814-6186 E-mail: [email protected] Jahn Molly Kyle Cornell University Department of Plant Breeding 312 Bradfield Hall 14853 Ithaca, New York tel: +1-607-2558147 E-mail: [email protected] Johnson William Seminis Vegetable Seeds Inc 37437 State Highway 16 95695 Woodland, California tel: +1-530-6696260 E-mail: [email protected] Juarez Benito Seminis Vegetable Seeds Inc Woodland Research Center 37437 State Highway 16 95695 Woodland, California tel.: +1-530-6696264 fax: +1-530-6665759 E-mail: [email protected] Knerr Larry D. Shamrock Seeds Company 3 Harris Place 93901 Salinas, California tel: +1-831-7711500 E-mail: [email protected] Levi Amnon USDA, ARS U.S. Vegetable Laboratory 2700 Savanah Highway 29414 Charleston, South Carolina E-mail: [email protected]

555 Martyn Ray D. Purdue University Department of Botany and Plant Pathology 915 West State Street 47907-2054 West Lafayette, Indiana tel.: +1-765-494-4615 fax: +1-765-494-0363 E-mail: [email protected] McCreight James D. U.S. Department of Agriculture Agricultural Research Service 1636 E. Alisal St. 93905 Salinas, California Nunez-Palenius Hector University of Florida P.O. Box 110690 32611 Gainesville, Florida tel: +1-352-392-9905 E-mail: [email protected] Shetty Nischit V. Seminis Vegetable Seeds 432 TyTy Omega Rd 31794 Tifton, Georgia tel: +1-229-3868701 E-mail: [email protected] Shirmohamadali Asghar Harris Moran Seed Company 9241 Mace Blvd 95616 Davis, California tel.: +1-530-756-1382 fax: +1-530-756-1016 E-mail: [email protected] Schultheis Jonathan North Carolina State University Department of Horticultural Science Box 7609 27695-7 Raleigh, North Carolina tel: +1-919-5151225 E-mail: [email protected] Staub Jack E. USDA - ARS and University of Wisconsin Department of Horticulture 1575 Linden Drive 537 06 Madison, Wisconsin tel.: +1-608-262-0028 E-mail: [email protected] Taurick Gary Harris Moran Seed Company 1255 15th St N, Suite 6, 34142 Immokalee, Florida E-mail: [email protected]

556 Thomas Claude E. USDA, ARS U.S. Vegetable Laboratory 2700 Savannah Highway 29414-5334 Charleston, South Carolina tel.: +1-843-5540840 E-mail: [email protected] Tolla Greg E. Seminis Vegetables Seeds 432 TyTy Omega Rd. 31793 Tifton, Georgia tel: +1-229-3868701 E-mail: [email protected] Wang Gang Haikou Essence Agrobiotech & Development Co., Ltd. 1206 Rahway Avenue 07001 Avenel, New Jersey tel.: +1-732-7504874 E-mail: [email protected] Wehner Todd C. North Carolina State University Department of Horticultural Science 222 Kilgore Hall 27695-7609 Raleigh, North Carolina Wessel-Beaver Linda University of Puerto Rico Department of Agronomy and Soils P.O. Box 9030 00681 Mayaguez, Puerto Rico Zalapa Juan University of Wisconsin 1575 Linden Drive 53706 Madison, Wisconsin E-mail: [email protected] Zhang Xingping Syngenta Seeds Inc. 21435 Rd 98 95695 Woodland, California tel: +1-530-6660986 fax: +1-530-6665273 E-mail: [email protected]

Yugoslavia Berenji Janos Institute of Field and Vegetable Crops Novosadski Put B.B. 21470 Backi Petrovac tel: +381-21-780365 E-mail: [email protected]

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