MOLECULAR BIOLOGY & BIOTECHNOLOGY OF THE GRAPEVINE

Transcription

1 MOLECULAR BIOLOGY & BIOTECHNOLOGY OF THE GRAPEVINE

2 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.

3 A C.I.P. Catalogue record for this book is available from the Library of Congress. ISBN ISBN (ebook) DOI / Printed an acid-free paper An Rights Reserved 2001 Springer SciencetBusiness Media Dordrecht Originally published by Kluwer Academic Publishers in Softcover reprint of the hardcover 1 st edition 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.

4 To the Memory of my Parents Apostolos and Maria Roubelakis

5 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

6 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

7 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 Grape sucrose transporters Grape monosaccharide transporters Grape invertases Future directions Anthocyanins Grape anthocyanins The anthocyanin biosynthesis pathway Introduction The structural genes Genes involved in pathway regulation Grape anthocyanin gene expression Anthocyanin gene expression in grapevine seedlings Anthocyanin gene expression in berry skins during development Anthocyanin gene expression in red and white grapes ManipUlating grapevine anthocyanins Total anthocyanins Specific anthocyanins 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

8 x Organic acid synthesis The induction of malate respiration during ripening Aerobic fermentation and malate breakdown 3. Compartmentation of organic acids in grape berries 3.1. Vacuolar proton pumps Molecular structure Thermodynamic properties Enzymic properties Two pumps on the same membrane 3.2. Organic acid accumulation 3.3. Vacuolar transport and ph variation Proton pumps Secondary transport Vacuolar content efflux References Chapter 3 NITROGEN ASSIMILATION IN GRAPEVINE 59 K.A Loulakakis and K.A Roubelakis-Angelakis 1. Introduction Nitrogen assimilation Reduction of nitrate Ammonium assimilation Glutamine synthetase Glutamate synthase Glutamate dehydrogenase Regulation of ammonia assimilating enzymes in grapevine by nitrogen source 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

9 4. Proline accumulation in plants Pathways of proline biosynthesis The glutamate pathway of proline biosynthesis The Ornithine pathway of proline biosynthesis Genes encoding P5CS and OAT are expressed in grape berry tissue Vvp5cs gene expression during grape berry development Other factors which could affect proline accumulation in grape berries Ammonium and glutamine metabolism Arginine metabolism and regulation of OAT Proline degradation Protein accumulation 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 Biosynthesis of polyamines Endogenous polyamines in grapevine organs Polyamines in various grapevine organs ll Spatial and temporal free and conjugated polyamine distribution in grapevine leaves ll Polyamines and berry development Polyamine oxidase activities and diaminopropane contents during floral development in grapevine Hydroxycinnamic acid amines in flowers and berries of grapevine ADC enzyme activity and transcript levels in developing grapevine organs Polyamines and disorders of grape berry development Polyamines and fruit set Polyamines and abnormal development of berry (shot grape berries) Polyamine metabolism in relation to flower and fruitlet abscission Polyamines and abscission potential Polyamines counteract abscission Polyamine biosynthesis and abscission Polyamine catabolism and abscission 129

10 xii Photodependance of polyamine levels and abscission Modulation of carbohydrate and amino acid levels by polyamines Polyamines and stress Free polyamines, ADC enzyme activity and transcript levels in grapevine cell suspension cultures under different treatments Free polyamine titers and stress adaptation Polyamines and potassium nutrition 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 Plant disease resistance mechanisms Phytoalexins and biotic/abiotic elicitors Grapevine induced stilbenes First evidence of stilbenes in grapevine Biotic elicitors Botrytis cinerea Plasmopara viticola Phomopsis viticola Rhizopus stolonifer Bacteria Abiotic elicitors UV irradiation Aluminum chloride Ozone Wounding Fosetyl-Al Other chemicals Stilbene glycosides in Vitis Cultural factors affecting induced stilbene synthesis Fertilizer supply Rootstock Stilbenes in soft tissues of field grow grapevines Grapevine constitutive stilbenes Stilbenes in the wine Molecular and biotechnological aspects of stilbene synthesis in grapevine 171

11 xiii 8.1. Grapevine stilbene synthesis Transfer of stsy genes 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 The nature of unstable wine proteins The major wine haze forming proteins are PR-like proteins The synthesis of haze-forming PR-like proteins in grape berries is regulated in a developmental and tissue specific manner The regulatory elements controlling PR-like protein synthesis at veraison are not known Grape PR-like proteins show antifungal activity in vitro The contribution of growing and harvesting methods to wine protein instability Preventing visible haze formation with haze protective factors The use of proteolytic enzymes to prevent protein haze formation Use of PR-like proteins for varietal identification Conclusions 195 Acknowledgements 196 References 196 Chapter 8 ALCOHOL DEHYDROGENASE: A MOLECULAR MARKER IN GRAPEVINE 203

12 XIV C. Tesniere and C. Verries 1. Introduction 2. Expression of Adhs in grape tissues 2.1. In developing fruit ADH enzyme activity ADH isoforms and biochemical properties 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 Adh gene cloning 3.2. Structural organisation of V. vinifera Adh genes 3.3. Analysis of putative regulatory sequences Initiation and transcription sites Translation-initiation site selection Processing sequences in 3'-ends Anaerobic response elements (ARE) 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 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 The role of terpenes as aroma compounds in must and wines Terpenes cycle in leaves and fruit and their effect on aroma formation Applications of glycosidases to enhance aroma of wines Cloning and expression of recombinant A. niger beta-glucosidase in yeast Purification of A. niger B-glucosidase Proteolysis and N-terminal sequences of A. niger Bl B-glucosidase Cloning of bgll cdna and genomic gene Expression ofbgll cdna in Saccharomyces cerevisiae and Pichia pastoris

13 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 TIPs PIPs 5.3. Grapevine aquaporins 6. Summary Acknow ledgments References 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 General biological model A general basic biological organization exists, which assures functioning at each level Classical model Triptych model A biological system is a complex network of triptychs and not only a complex association of the basic elements of the triptychs 265

14 XVI 2.3. Three modalities of connections between triptychs exist, which reveal the biological concepts of nutrition or "source-sink" relationships, growth and development Water constraint does not equate precisely to water "limitation" Feed back mechanism Plant aging Strategies of adaptation Polyvalence The role of genes Recent developments of molecular biology applied to grapevine physiology Genes involved in general berry development and maturation Pathogenesis related proteins Phenolic compounds Biochemical and molecular responses to biotic stress 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

15 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 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 Dormancy Morphological and physiological alterations Culture conditions 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 Chapter 14 PROTOPLAST TECHNOLOGY IN GRAPEVINE A. Papadakis, G. Reustle and K.A. Roubelakis-Angelakis 1. Introduction

16 XVIll 2. Recalcitrance 2.1. Plasma membrane functioning 2.2. Oxidative stress Generation of Active Oxygen Species 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 Enzymic treatment Purification Assessment of protoplast quality 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 Chapter 15 GRAPEVINE GENETIC ENGINEERING J.R. Kikkert, M.R. Thomas and B.!. Reisch 1. Introduction Application of Genetic Engineering to Grapevine Breeding and Genetics Historical development of grapevine transformation systems Early transformation work Importance of embryogenic cultures Successful transformation methods l. Agrobacterium Biolistics Methods for selection and evaluation of transformants Current status of grapevine transformation Environmental release/commercialisation Regulatory issues

17 xix Europe Australia United States 5.2. Patenting 5.3. Naming 5.4. Public perception 6. Acknowledgments References 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 Basic terms in genetics of host/pathogen interaction Advantages and limitations of genetic transformation Gene transfer in Grape for improved tolerance toward biotic and abiotic stress Viruses Fungal pathogens Bacteria Nematodes and insects Abiotic stress Co-transformation as an advanced approach for integration of multiple genes to confer for disease tolerance in grape 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?

18 xx 3. Development of microsatellite markers in Vilis EST derived microsatellite markers: a new strategy Identification of cultivars of Vitis vinifera and rootstocks from Vitis species Source and quality of DNA used for PCR amplification Analysis methods available and comparison between them Identification of grapevine cultivars and rootstocks by using nuclear SSRS Synonyms Clonal lines and somatic mutants Pedigree reconstruction Methodology Examples for the reconstruction of grapevine crosses Genetic studies of the european Vitis vinifera germplasm Chloroplast SSR markers Use of SSR markers for genetic mapping of Vitis vinifera in combination with other markers Computer programs for micro satellite data analysis Introduction Identity Management of germplasm collections Evaluation of micro satellite markers Popgene Evaluation of microsatellite markers Characterisation of grapevine gene pools Cluster analysis Other computer programs Other programs for cluster analysis Other programs for the characterisation of grapevine gene pools Genetic databases of SSR profiles On the way to commercial certification of cultivars Conclusion and prospects for the future 456 Acknowledgments 457 References 457 Author Index 465 Subject Index 467

19 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, 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", 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, 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, MontpelIier Cedex 1, France. S. Delrot UMR CNRS 6161, Laboratoire de Physiologie et Biochimie Vegetales, University of Poi tiers, 40 Avenue du Recteur Pineau, Poitiers Cedex, France. C. Fregoni Institute ofpomology and Viticulture, Sacred Heart Catholic University, Via Emilia Parmense 84, Piacenza, Italy. J.P. Gaudillere Unite d'agronomie, BP 81, INRA, Villenave d'omon, France. L. Geny Faculty of Enology, University Victor Segalen Bordeaux II, 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, San Michele all'adige, Italy.

20 XXII I. Gribaudo Centro Miglioramento Genetico e Biologia del1a Vite - CNR, via Leonardo da Vinci 44, 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, HerakJion, Crete, Greece. L. Martinelli Laboratorio Biotecnologie, Istituto Agrario, 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, Bet-Dagan, Israel. S. Picaud UMR CNRS 6161, Laboratoire de Physiologie et Biochimie Vegetales, University of Poitiers, 40 Avenue du Recteur Pineau, 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, 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, Montpcllier Cedex 01, France. K.A. Roubelakis-Angelakis Department of Biology, Univcrsity of Crete, P.O.Box 2208,71409 Heraklion, Crete, Greece.

21 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, Montpellier Cedex 01, France. C. Tesniere INRA, Unite de Recherche sur les Produits de la Vigne, 2 Place Viala, 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, 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, Montpellier Cedex 1, France. E.J. Waters The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, South Australia 5064, Australia.