MOLECULAR BIOLOGY & BIOTECHNOLOGY OF THE GRAPEVINE

MOLECULAR BIOLOGY & BIOTECHNOLOGY OF THE GRAPEVINE

MOLECULAR BIOLOGY & BIOTECHNOLOGY OF THE GRAPEVINE edited by KALLIOPI A. ROUBELAKIS-ANGELAKIS Professor of Plant Physiology and Biotechnology, Department of Biology, University of Crete, Heraklion, Greece and President of the Federation of European Societies of Plant Physiology SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

A C.I.P. Catalogue record for this book is available from the Library of Congress. ISBN 978-94-017-2310-7 ISBN 978-94-017-2308-4 (ebook) DOI 10.1007/978-94-017-2308-4 Printed an acid-free paper An Rights Reserved 2001 Springer SciencetBusiness Media Dordrecht Originally published by Kluwer Academic Publishers in 200 1 Softcover reprint of the hardcover 1 st edition 200 1 No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage and retrieval system, without written permis sion from the copyright owner.

To the Memory of my Parents Apostolos and Maria Roubelakis

PROLEGOMENA Research in Plant Biology, in the pre-molecular era, dealt mostly with work at the organismallevel. The molecular era has opened new avenues in our understanding of the secrets of life. Molecular Biology and Biotechnology have emerged as the crossing-point of basic biological sciences, such as Biochemistry, Cellular Biology, Genetics, Microbiology, and Physiology. The use of molecular techniques and other analytical instrumentation has increasingly contributed to further understanding' how, when and where' physiological phenomena occur in organisms. Non-molecular plant biotechnological methods, such as the plant tissue culture techniques, have been developed during the past decades whereas the advances in Plant Molecular Biology have been used for the development of molecular biotechnological application; they have been based upon the non-molecular developments.. Grapevine is one of the most widely cultivated plant woody species. As with most wooc(y plant species, and also some cereals and legumes, Molecular Biology and Biotechnology have had progressed at a slower pace, due to several obstacles, which have had to be overcome. In any case, it is now that substantial progress has been made and useful information has been accumulated in the literature. During the last decade, more than 100 genes have been characterized from grapevine and several genomic and chloroplastic microsatellite sequences have been deposited in the Genbanks. These genes encode for enzymes mediating synthesis and transport of sugars, poly phenols and pigments, organic acids, amino acids and polyamines, as well as for proteins related to biotic and abiotic stresses and to cell wall structure. Furthermore, protocols for non-molecular and molecular biotechnol-ogical applications for grapevine have been published. In an effort to collect and present the available information on Grapevine Molecular Biology and Biotechnology, 51 scientists from 10 countries jointly worked for the preparation of this Book. It is intended to be used as a reference-book by researchers, graduate and undergraduate students, viticulturists, biotechnological companies and any scientist, who is interested in the Molecular Biology and Biotechnology of Grapevine. Sincere thanks are due to all worldwide-leading scientists in their field, who have contributed and especially for their impeccable collaboration during the preparation

Vlll of this Volume; to Mrs Mary Papadakis-Savvopoulos for editorial assistance; to Miss Maria Mandelenakis for secretarial assistance; to Mr Nikolaos Papadoyannakis for his endless and devoted work during the preparation of the ready-to-camera material; to Kluwer Academic Publishers for the publication of the Book. Last but lot least to my husband, Andreas Angelakis, for his continuous encouragement and patience. Herak/ion, Crete, Greece January 2001 Kalliopi A. Roubelakis-Angelakis University of Crete, Greece

CONTENTS Contributing Authors xxi Chapter 1 MOLECULAR BIOLOGY OF SUGAR AND ANTHOCYANIN ACCUMULA TION IN GRAPE BERRIES 1 P.K. Boss and C. Davies 1. Introduction 1 2. The Molecular biology of sugar transport and accumulation in grape 2 2.1. Grape sucrose transporters 5 2.2. Grape monosaccharide transporters 6 2.3. Grape invertases 9 2.4. Future directions 12 3. Anthocyanins 13 3.1. Grape anthocyanins 13 3.2. The anthocyanin biosynthesis pathway 14 3.2.1. Introduction 14 3.2.2. The structural genes 14 3.2.3. Genes involved in pathway regulation 17 3.3. Grape anthocyanin gene expression 18 3.3.1. Anthocyanin gene expression in grapevine seedlings 18 3.3.2. Anthocyanin gene expression in berry skins during development 18 3.3.3. Anthocyanin gene expression in red and white grapes 21 3.5. ManipUlating grapevine anthocyanins 24 3.5.1. Total anthocyanins 24 3.5.2. Specific anthocyanins 25 4. Summary 27 Acknowledgments 28 References 28 Chapter 2 GRAPE BERRY ACIDITY N. Terrier and C. Romieu 1. Introduction 2. Changes in acidity during berry development 2.1. Evolution pattern of berry composition 2.2. Organic acid metabolic pathways in grape berries 35 35 36 36 38

x 2.2.1. Organic acid synthesis 2.2.2. The induction of malate respiration during ripening 2.2.3. Aerobic fermentation and malate breakdown 3. Compartmentation of organic acids in grape berries 3.1. Vacuolar proton pumps 3.1.1. Molecular structure 3.1.2. Thermodynamic properties 3.1.3. Enzymic properties 3.1.4. Two pumps on the same membrane 3.2. Organic acid accumulation 3.3. Vacuolar transport and ph variation 3.3.1. Proton pumps 3.3.2. Secondary transport 3.3.3. Vacuolar content efflux References 38 38 40 41 41 41 42 43 44 46 49 49 51 51 52 Chapter 3 NITROGEN ASSIMILATION IN GRAPEVINE 59 K.A Loulakakis and K.A Roubelakis-Angelakis 1. Introduction 59 2. Nitrogen assimilation 60 2.1. Reduction of nitrate 60 2.2. Ammonium assimilation 63 2.2.1. Glutamine synthetase 65 2.2.2. Glutamate synthase 68 2.2.3.Glutamate dehydrogenase 71 3. Regulation of ammonia assimilating enzymes in grapevine by nitrogen source 77 4. Future perspectives 80 References 80 Chapter 4 MOLECULAR BIOLOGY AND BIOCHEMISTRY OF PROLINE ACCUMULATION IN DEVELOPING GRAPE BERRIES R. van Heeswijck, AP. Stines, J. Grubb, I. Skrumsager Mpller and P.B. Hpj 1. Introduction 2. Amino acid composition of grape berries 3. The influence of grape berry proline on fermentation 87 87 87 91

4. Proline accumulation in plants 91 5. Pathways of proline biosynthesis 92 5.1. The glutamate pathway of proline biosynthesis 92 5.2. The Ornithine pathway of proline biosynthesis 94 5.3. Genes encoding P5CS and OAT are expressed in grape berry tissue 94 6. Vvp5cs gene expression during grape berry development 97 7. Other factors which could affect proline accumulation in grape berries 99 7.1. Ammonium and glutamine metabolism 99 7.2. Arginine metabolism and regulation of OAT 100 7.3. Proline degradation 101 7.4. Protein accumulation 102 8. Conclusions 103 Acknowledgments 104 References 104 xi ChapterS POLYAMINES IN GRAPEVINE 109 K.A. Paschalidis, A. Aziz, L. Geny, N.!. Primikirios and K.A. Roubelakis-Angelakis 1. Introduction 109 2. Biosynthesis of polyamines 110 3. Endogenous polyamines in grapevine organs 112 3.1. Polyamines in various grapevine organs ll2 3.2. Spatial and temporal free and conjugated polyamine distribution in grapevine leaves ll2 3.3. Polyamines and berry development 116 3.3.1. Polyamine oxidase activities and diaminopropane contents during floral development in grapevine 116 3.3.2. Hydroxycinnamic acid amines in flowers and berries of grapevine 118 4. ADC enzyme activity and transcript levels in developing grapevine organs 119 5. Polyamines and disorders of grape berry development 121 5.1. Polyamines and fruit set 121 5.2. Polyamines and abnormal development of berry (shot grape berries) 122 5.3. Polyamine metabolism in relation to flower and fruitlet abscission 122 5.3.1. Polyamines and abscission potential 124 5.3.2. Polyamines counteract abscission 125 5.3.3. Polyamine biosynthesis and abscission 128 5.3.4. Polyamine catabolism and abscission 129

xii 5.3.5. Photodependance of polyamine levels and abscission 130 5.3.6. Modulation of carbohydrate and amino acid levels by polyamines 130 6. Polyamines and stress 133 6.1. Free polyamines, ADC enzyme activity and transcript levels in grapevine cell suspension cultures under different treatments 133 6.2. Free polyamine titers and stress adaptation 136 6.3. Polyamines and potassium nutrition 140 6.3. Polyamines and biotic stress (Botrytis cinerea) 142 References 144 Chapter 6 PHYSIOLOGICAL ROLE AND MOLECULAR ASPECTS OF GRAPEVINE STILBENIC COMPOUNDS 153 L. Bavaresco and C. Fregoni 1. Introduction 153 2. Plant disease resistance mechanisms 153 3. Phytoalexins and biotic/abiotic elicitors 154 4. Grapevine induced stilbenes 155 4.1. First evidence of stilbenes in grapevine 155 4.2. Biotic elicitors 157 4.2.1. Botrytis cinerea 158 4.2.2. Plasmopara viticola 161 4.2.3. Phomopsis viticola 162 4.2.4. Rhizopus stolonifer 162 4.2.5. Bacteria 163 4.3. Abiotic elicitors 163 4.3.1. UV irradiation 163 4.3.2. Aluminum chloride 165 4.3.3. Ozone 165 4.3.4. Wounding 165 4.3.5. Fosetyl-Al 166 4.3.6. Other chemicals 166 4.4. Stilbene glycosides in Vitis 166 4.5. Cultural factors affecting induced stilbene synthesis 167 4.5.1. Fertilizer supply 167 4.5.2. Rootstock 168 5. Stilbenes in soft tissues of field grow grapevines 168 6. Grapevine constitutive stilbenes 169 7. Stilbenes in the wine 170 8. Molecular and biotechnological aspects of stilbene synthesis in grapevine 171

xiii 8.1. Grapevine stilbene synthesis 171 8.2. Transfer of stsy genes 173 8.3. Grapevine breeding and fingerprinting based upon molecular aspects of stilbene synthesis 174 Acknowledgements 176 References 17 6 Chapter 7 PATHOGENESIS RELATED PROTEINS-THEIR ACCUMULA TION IN GRAPES DURING BERRY GROWTH AND THEIR INVOLVEMENT IN WHITE WINE HEAT INSTABILITY. CURRENT KNOWLEDGE AND FUTURE PERSPECTIVES IN RELATION TO WINEMAKING PRACTICES 183 D.B. Tattersall, K.F. Pocock, Y. Hayasaka, K. Adams, R. van Heeswijck, EJ. Waters and P.B. H j 1. Introduction 183 2. The nature of unstable wine proteins 185 3. The major wine haze forming proteins are PR-like proteins 185 4. The synthesis of haze-forming PR-like proteins in grape berries is regulated in a developmental and tissue specific manner 187 5. The regulatory elements controlling PR-like protein synthesis at veraison are not known 188 6. Grape PR-like proteins show antifungal activity in vitro 190 7. The contribution of growing and harvesting methods to wine protein instability 191 8. Preventing visible haze formation with haze protective factors 192 9. The use of proteolytic enzymes to prevent protein haze formation 193 10. Use of PR-like proteins for varietal identification 194 11. Conclusions 195 Acknowledgements 196 References 196 Chapter 8 ALCOHOL DEHYDROGENASE: A MOLECULAR MARKER IN GRAPEVINE 203

XIV C. Tesniere and C. Verries 1. Introduction 2. Expression of Adhs in grape tissues 2.1. In developing fruit 2.1.1. ADH enzyme activity 2.1.2. ADH isoforms and biochemical properties 2.1.3. Adh gene expression 2.2. In response to an abiotic stress: anaerobiosis 2.3. In different tissues 3. Molecular characterisation of Adh genes from V. vinifera L. 3.1. Adh gene cloning 3.2. Structural organisation of V. vinifera Adh genes 3.3. Analysis of putative regulatory sequences 3.3.1. Initiation and transcription sites 3.3.2. Translation-initiation site selection 3.3.3. Processing sequences in 3'-ends 3.3.4. Anaerobic response elements (ARE) 3.3.5. Comparison of the encoded ADH polypeptides 4. Evolution of Adh multigene family 4.1. Among Adh from other species 4.2. Among other Vitis species 5. Conclusions References 203 204 204 204 207 208 209 210 211 211 212 212 213 214 214 215 215 216 216 218 218 219 Chapter 9 ENHANCEMENT OF AROMA IN GRAPES AND WINES: BIOTECHNOLODICAL APPROACHES O. Shoseyov and B. Bravdo 1. Free and glycosidic ally bound aroma compounds in grapes and wines 225 2. The role of terpenes as aroma compounds in must and wines 226 3. Terpenes cycle in leaves and fruit and their effect on aroma formation 227 4. Applications of glycosidases to enhance aroma of wines 229 5. Cloning and expression of recombinant A. niger beta-glucosidase in yeast 232 6. Purification of A. niger B-glucosidase 233 7. Proteolysis and N-terminal sequences of A. niger Bl B-glucosidase 234 8. Cloning of bgll cdna and genomic gene 234 9. Expression ofbgll cdna in Saccharomyces cerevisiae and Pichia pastoris 236 225

xv References 237 Chapter 10 WA TER TRANSPORT AND AQUAPORINS IN GRAPEVINE S. Delrot, S. Picaud and J.P. Gaudillere 1. Introduction 2. SoilfPlantl Atmosphere continuum in grapevine 2.1. Soil root conductivity 2.2. Radial root conductivity 2.3. Xylem conductivity 2.4. Stomatal control of transpiration 2.5. Water use by grapevine in the vineyard 3. Water management and grape quality 4. Phloem contribution to water traffic 5. Water traffic and aquaporins 5.1. Aq uaporins 5.2. Plant Aquaporins 5.2.1. TIPs 5.2.2. PIPs 5.3. Grapevine aquaporins 6. Summary Acknow ledgments References 241 241 242 242 242 243 244 244 245 246 248 248 250 251 252 253 257 257 257 Chapter 11 PLANT ORGANIZATION BASED ON SOURCE-SINK RELATIONSHIPS: NEW FINDINGS ON DEVELOPMENTAL, BIOCHEMICAL AND MOLECULAR RESPONSES TO ENVIRONMENT 263 A. Carbonneau and A. Deloire 1. Introduction 263 2. General biological model 263 2.1. A general basic biological organization exists, which assures functioning at each level 264 2.1.1. Classical model 264 2.1.2. Triptych model 264 2.2. A biological system is a complex network of triptychs and not only a complex association of the basic elements of the triptychs 265

XVI 2.3. Three modalities of connections between triptychs exist, which reveal the biological concepts of nutrition or "source-sink" relationships, growth and development 266 2.4. Water constraint does not equate precisely to water "limitation" 267 2.5. Feed back mechanism 267 2.6. Plant aging 267 2.7. Strategies of adaptation 268 2.8. Polyvalence 268 2.9. The role of genes 268 3. Recent developments of molecular biology applied to grapevine physiology 270 3.1. Genes involved in general berry development and maturation 270 3.2. Pathogenesis related proteins 271 3.3. Phenolic compounds 273 3.4. Biochemical and molecular responses to biotic stress 274 3.5. Biochemical and molecular responses to abiotic stress 274 References 278 Chapter 12 IN VITRO CULTURE AND PROPAGATION OF GRAPEVINE L. Torregrosa, A. Bouquet and P.G. Goussard 1. Introduction 2. Conditions of in vitro culture establishment 2.1. Choice of explants 2.2. Handling of stock plants 2.3. Production of sterile explants 2.4. Culture media and hormone requircmcnts 2.5. Browning of explants 3. Conditions of propagation and regeneration 3.1. Nodal and meristem tip culture 3.2. Axillary bud proliferation 3.3. Regenerative procedures 4. Physiological characteristics of in vitro cultures 5. Factors affecting success in producing plants 5.1. Stage I: In vitro culture establishment 5.2. Stage II: Regeneration and multiplication 5.3. Stage III: Pretransplantation 5.4. Stage IV: Transplant to soil 6. In vitro culture for grapevine improvement 6.1. Virus sanitation 6.2. Establishment of genetic repositories 281 281 282 283 283 284 284 284 285 286 286 287 289 293 293 293 294 295 297 297 300

xvii 6.3. In vitro embryo rescue 6.4. Haploid plant production and mutation breeding 6.5. Somaclonal variation 7. Other applications of in vitro culture 7.1. Callus culture 7.2. Cell culture 7.3. Organ culture 8. Conclusions References 303 305 306 309 309 310 311 312 313 Chapter 13 SOMA TIC EMBRYOGENESIS IN GRAPEVINE L. Martinelli and I. Gribaudo 1. Introduction 2. Protocols for somatic embryogenesis in grape 2.1. Induction and culture of embryogenic callus 2.2. Long-term embryogenic cultures 2.3. Somatic embryogenesis from embryonic tissues 3. Embryo teratology and low conversion rate 3.1. Somatic embryo teratology 3.2. Plant development 3.2.1. Dormancy 3.2.2. Morphological and physiological alterations 3.2.3. Culture conditions 3.2.4. Germination treatments 4. Towards a better understanding of grape somatic embryogenesis 4.1. Ontogenesis and differentiation of somatic embryogenesis 4.2. Molecular markers for somatic embryogenesis characterization 5. Conclusions Abbreviations Acknowledgments References 327 327 328 329 332 332 334 334 336 336 337 338 340 340 341 343 345 346 346 346 Chapter 14 PROTOPLAST TECHNOLOGY IN GRAPEVINE A. Papadakis, G. Reustle and K.A. Roubelakis-Angelakis 1. Introduction 353 353

XVIll 2. Recalcitrance 2.1. Plasma membrane functioning 2.2. Oxidative stress 2.2.1. Generation of Active Oxygen Species 2.2.2. Scavenging of active oxygen species 2.3. The role of polyamines 3. Isolation of grapevine protoplasts 3.l. Donor plant material 3.2. The isolation method 3.2.1. Enzymic treatment 3.2.2. Purification 3.2.3. Assessment of protoplast quality 3.2.4. Culture conditions 4. Progress in grapevine protoplast technology 5. Applications of protoplast technology 5.1. SomacIonal variation 5.2. In vitro selection 5.3. Somatic hybridization 5.4. Genetic transformation 5.5. Protoplasts as test system 5.6. Prospects Acknowledgements References 354 355 356 357 362 369 369 370 373 373 375 376 376 381 382 382 383 383 384 385 385 386 386 Chapter 15 GRAPEVINE GENETIC ENGINEERING J.R. Kikkert, M.R. Thomas and B.!. Reisch 1. Introduction 393 2. Application of Genetic Engineering to Grapevine Breeding and Genetics 394 3. Historical development of grapevine transformation systems 395 3.1. Early transformation work 395 3.2. Importance of embryogenic cultures 396 3.3. Successful transformation methods 399 3.3.l. Agrobacterium 399 3.3.2. Biolistics 400 3.4. Methods for selection and evaluation of transformants 401 4. Current status of grapevine transformation 402 5. Environmental release/commercialisation 403 5.1. Regulatory issues 403 393

xix 5.1.1. Europe 5.1.2 Australia 5.1.3 United States 5.2. Patenting 5.3. Naming 5.4. Public perception 6. Acknowledgments References 403 404 404 405 405 406 406 407 Chapter 16 GENETICALLY ENGINEERED GRAPE FOR DISEASE AND STRESS TOLERANCE 411 V. Colova-Tsolova, A. Perl, S. Krastanova, J. Tsvetkov and A. Atanassov 1. Introduction 411 2. Basic terms in genetics of host/pathogen interaction 412 3. Advantages and limitations of genetic transformation 414 4. Gene transfer in Grape for improved tolerance toward biotic and abiotic stress 417 4.1. Viruses 417 4.2. Fungal pathogens 421 4.3. Bacteria 423 4.4. Nematodes and insects 424 4.5. Abiotic stress 425 5. Co-transformation as an advanced approach for integration of multiple genes to confer for disease tolerance in grape 425 6. Concluding remarks 427 Acknowledgements 427 References 427 Chapter 17 MICROSATELLITE MARKERS FOR GRAPEVINE: A STATE OF THE ART K.M. Sefc, F. Lefort, M.S. Grando, K.D. Scott, H. Steinkellner and M.R. Thomas 1. Introduction 2. What are micro satellites? 433 433 436

xx 3. Development of microsatellite markers in Vilis 436 4. EST derived microsatellite markers: a new strategy 438 5. Identification of cultivars of Vitis vinifera and rootstocks from Vitis species 439 5.1 Source and quality of DNA used for PCR amplification 439 5.2. Analysis methods available and comparison between them 439 5.3. Identification of grapevine cultivars and rootstocks by using nuclear SSRS 440 5.4. Synonyms 443 5.5. Clonal lines and somatic mutants 445 5.6. Pedigree reconstruction 445 5.6.1 Methodology 445 5.6.2. Examples for the reconstruction of grapevine crosses 447 6. Genetic studies of the european Vitis vinifera germplasm 449 7. Chloroplast SSR markers 451 8. Use of SSR markers for genetic mapping of Vitis vinifera in combination with other markers 451 9. Computer programs for micro satellite data analysis 452 9.1. Introduction 452 9.2. Identity 1.0 453 9.2.1. Management of germplasm collections 453 9.2.2. Evaluation of micro satellite markers 453 9.3. Popgene 453 9.3.1. Evaluation of microsatellite markers 453 9.3.2. Characterisation of grapevine gene pools 454 9.3.3. Cluster analysis 454 9.4. Other computer programs 454 9.4.1. Other programs for cluster analysis 454 9.4.2. Other programs for the characterisation of grapevine gene pools 454 10. Genetic databases of SSR profiles 454 11. On the way to commercial certification of cultivars 455 12. Conclusion and prospects for the future 456 Acknowledgments 457 References 457 Author Index 465 Subject Index 467

CONTRIBUTING AUTHORS K. Adams Department of Horticulture, Viticulture & Oenology, Waite Campus, University of Adelaide, PMB 1, Glen Osmond, South Australia 5064, Australia. A. Atanassov Institute of Genetic Engineering, 2232 Kostinbrod-2, Bulgaria. A.Aziz Laboratory of Plant Biology and Physiology, UPRES EA 2069 URVVC, University ofreims, B.P. 1039, F-5l687 Reims Cedex 2, France. L. Bavaresco Institute ofpomology and Viticulture, Sacred Heart Catholic University, Via Emilia Parmense 84, 29100 Piacenza, Italy. P.K. Boss Commonwealth Scientific and Industrial Research Organisation, Plant Industry, Horticulture Research Unit, P.O. Box 350, Glen Osmond, South Australia 5064, Australia. A. Bouquet UMR Diversity and Genomes of Cultivated Plants, INRA, Grape Breeding Experimental Station "Le Chapitre", 34751 Villeneuve ii':s Maguelone, France. B. Bravdo The Hebrew University of Jerusalem, Faculty of Agriculture, Institute of Plant Sciences and Genetics, The Kennedy-Leigh Center for Horticultural Research, Jerusalem, Israel. A. Carbonneau Institut Superieur de la Vigne et du Vin, AGRO Montpellier-Viticulture, 2 Place P. Viala F, 34060 Montpellier Cedex 1, France. V. Colova-Tsolova Center for Viticultural Science, College of Engineering Sciences, Technology and Agriculture, Florida Agricultural and Mechanical University, Tallahassee, FL 32307, USA. C. Davies Commonwealth Scientific and Industrial Research Organisation, Plant Industry, Horticulture Research Unit, P.O. Box 350, Glen Osmond, South Australia 5064, Australia. A. Deloire Institut Superieur de la Vigne et du Vin, AGRO Montpellier-Viticulture, 2 Place P. Viala, 34060 MontpelIier Cedex 1, France. S. Delrot UMR CNRS 6161, Laboratoire de Physiologie et Biochimie Vegetales, University of Poi tiers, 40 Avenue du Recteur Pineau, 86022 Poitiers Cedex, France. C. Fregoni Institute ofpomology and Viticulture, Sacred Heart Catholic University, Via Emilia Parmense 84, 29100 Piacenza, Italy. J.P. Gaudillere Unite d'agronomie, BP 81, INRA, 33883 Villenave d'omon, France. L. Geny Faculty of Enology, University Victor Segalen Bordeaux II, 33405 Talence, France. P.G. Goussard Department of Viticulture and Oenology, University of Stell en bosch Private Bag Xl, 7602 Matieland (Stellenbosch), South Africa. M.S. Grando Istituto Agrario, Lab. Biologia Molecolare, Via Mach I, 38010 San Michele all'adige, Italy.

XXII I. Gribaudo Centro Miglioramento Genetico e Biologia del1a Vite - CNR, via Leonardo da Vinci 44, 10095 Grugliasco, Italy. J. Grubb Cooperative Research Centre for Viticulture, Glen Osmond, South Australia 5064, Australia. Y. Hayasaka The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, South Australia 5064, Australia. P.B. Hoj Department of Horticulture, Viticulture & Oenology, Waite Campus, University of Adelaide, PMB I, Glen Osmond, South Australia 5064, Australia. J.R. Kikkert Cornell University, New York State Agricultural Experimental Station, Department of Horticultural Sciences, Geneva, NY 14456, USA. S. Krastanova Cornell University, New York State Agricultural Experimental Station, Department of Plant Pathology, Geneva, NY 14456, USA. F. Lefort Department of Biology, University of Crete, P.O.Box 2208,71409 Hcraklion, Crete, Grecce. K.A. Loulakakis Department of Horticulture, Technological Educational Institution of Crete, 71500 HerakJion, Crete, Greece. L. Martinelli Laboratorio Biotecnologie, Istituto Agrario, 38010 San Michele all'adige (TN), Italy. A. Papadakis Department of Biology, University of Crete, P.O.Box 2208,71409 Heraklion, Crete, Greece. K.A. Paschalidis Department of Biology, University of Crete, P.O.Box 2208,71409 Heraklion, Crete, Greece. A. Perl Department of Fruit Tree Breeding and Molecular Genetics, Institute of Horticulture, Agricultural Research Organization, The Volcani Center, P.O. Box 6, 50250 Bet-Dagan, Israel. S. Picaud UMR CNRS 6161, Laboratoire de Physiologie et Biochimie Vegetales, University of Poitiers, 40 Avenue du Recteur Pineau, 86022 Poi tiers Cedex, France. K.F. Pocock The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, South Australia 5064, Australia. N.I. Primikirios Department of Biology, University of Crete, P.O.Box 2208, 714 09 Heraklion, Crete, Greece. B.I. Reisch Cornell University, New York State Agricultural Experimental Station, Department of Horticultural Sciences, Geneva, NY 14456, USA. G. Reustle 2 Centrum Grtine Gentechnik, SLF A Neustadt, Breitenweg 71, D67435 NeustadtlWeinstrasse, Germany. C. Romieu INRA, Unite de Recherche des Produits de la Vigne, Institut National de la Recherche Agronomique, 2 place Viala, 34060 Montpcllier Cedex 01, France. K.A. Roubelakis-Angelakis Department of Biology, Univcrsity of Crete, P.O.Box 2208,71409 Heraklion, Crete, Greece.

xxiii K.D. Scott Centre for Plant Conservation Genetics, P.O. Box 157, Lismore NSW 2480, Southern Cross University, Australia. K.M. Sefc Zentrum flir Angewandte Genetik, Universitat flir Bodenkultur, Wien Muthgasse 18, A-II90 Vienna, Austria. O. Shoseyov The Hebrew University of Jerusalem, Institute of Plant Sciences and Genetics, The Kennedy-Leigh Center for Horticultural Research, Jerusalem, Israel. I. Skrumsager Moller Department of Horticulture, Viticulture and Oenology, Waite Campus, University of Adelaide, Glen Osmond, South Australia 5064, Australia H. Steinkellner Zentrum flir Angewandte Genetik, Universitlit fur Bodenkultur Wien Muthgasse 18, A-I 190 Vienna, Austria. A.P. Stines Department of Horticulture, Viticulture and Oenology, Waite Campus, University of Adelaide, Glen Osmond, South Australia 5064, Australia D.B. Tattersall Department of Horticulture, Viticulture & Oenology, Waite Campus, University of Adelaide, PMB 1, Glen Osmond, South Australia 5064, Australia N. Terrier INRA Unite de Recherche des Produits de la Vigne, Institut National de la Recherche Agronomique, 2 place Viala, 34060 Montpellier Cedex 01, France. C. Tesniere INRA, Unite de Recherche sur les Produits de la Vigne, 2 Place Viala, 34060 Montpellier Cedex 1, France. M.R. Thomas CSIRO Plant Industry, P.O. Box 350, Glen Osmond, South Australia 5064, Australia. L. Torregrosa UMR Biology of Development of Cultivated Perennial Plants, ENSAM-INRA, 2 place Viala, 34060 Montpellier Cedex I, France. I. Tsvetkov Institute of Genetic Engineering, 2232 Kostinbrod-2, Bulgaria. R. van Heeswijck Department of Horticulture, Viticulture and Oenology, Waite Campus, University of Adelaide, Glen Osmond, South Australia 5064, Australia C. Verries INRA, Unite de Recherche sur les Produits de la Vigne, 2 Place Viala, 34060 Montpellier Cedex 1, France. E.J. Waters The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, South Australia 5064, Australia.