Horticultural Reviews (Volume 28)

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HORTICULTURAL REVIEWS

Volume 28

Horticultural Reviews is sponsored by: American Society for Horticultural Science

Editorial Board, Volum.e 28 Carole E. Bassett Steve van Nocker Rodomiro Ortiz

HORTICULTURAL REVIEWS Volume 28

edited by

Jules Janick Purdue University

John Wiley &- Sons, Inc. NEW YORK / CHICHESTER / WEINHEIM / BRISBANE / SINGAPORE / TORONTO

This book is printed on acid-free paper. § Copyright © 2003 by John Wiley & Sons, New York. All rights reserved. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4744. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, (212) 850-6011, fax (212) 850-6008, E-Mail: [email protected]. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold with the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional person should be sought. Library of Congress Cataloging-in-Publication Data: ISBN: 0-471-21542-2 ISSN: 0163-7851

10 9 8 76 5 4 3 2 1

Contents Contributors Dedication: M. Allen Stevens

viii xi

Fredrick A. Bliss 1. Plant Nomenclature and Taxonomy:

An Horticultural and Agronomic Perspective

1

David M. Spooner, Wilbert L. A. Hetterscheid, Ronald G. van den Berg, and Willem Brandenburg I. II.

III. IV. V. VI. VII. VIII. IX. X.

Introduction Species Concepts in Wild Plants Classification Philosophies in Wild and Cultivated Plants Brief History of Nomenclature and Codes Fundamental Differences in the Classification and Nomenclature of Cultivated and Wild Plants A Comparison of the ICBN and ICNCP Possible New Codes Cultivated Plant Nomenclature and the Law Cultivar Epithets and Trademarks Recommendations for a Universally Stable Crop Nomenclature Through Changes and Use of the ICNCP References Literature Cited

2 11 20 29 33 39 45 48 49

50 51 53

2. Grafting of Herbaceous Vegetable

and Ornamental Crops

61

Jung-Myung Lee and Masayuki Oda I. II.

Introduction Grafting Technology

63 65 v

CONTENTS

vi

III. IV. V. VI.

Physiology of Grafting Crop Examples Grafting for Crop Improvement Conclusion and Prospects Literature Cited

78 84 109 115 116

3. Health Promoting Phytochemicals in Vegetables Mosbah M. Kushad, John Masiunas, Kathy Eastman, Wilhelmina Kalt, and Mary A. L. Smith

125

Introduction Major Classes of Phytochemicals in Vegetable Phytochemicals Content and Health Benefits of the Four Major Vegetable Groups Conclusions and Future Research Needs Literature Cited

126 129

I. II. III. IV.

150 165 166

4. Detection and Elimination of Viruses and

Phytoplasmas from Pome and Stone Fruit Trees

187

Margit Laimer I. II. III. IV. V. VI. VII.

Introduction Pathogens Pathogen Detection Elimination of Viruses Elimination of Phytoplasmas Indexing, Mass Propagation, and Germplasm Conservation Conclusions Literature Cited

5. Pear Fruit Volatiles Francesca Rapparini and Stefano Predieri I. II. III. IV. V.

Introduction Analysis of Chemical Composition Biogenesis Factors Affecting Qualitative and Quantitative Emission of Pear Volatiles Volatile Compounds' Influence on Quality

189 191 198 205 218 219 221 224

237 239 241 279 289 303

vii

CONTENTS

VI.

Summary and Conclusions Literature Cited

6. The Physiology of Flowering in Strawberry

Rebecca 1. Darnell, Daniel J. Cantliffe, Daniel S. Kirschbaum, and Craig K. Chandler I. II. III. IV. V. VI.

Introduction Floral Morphology Environmental Effects on Reproductive Growth Floral Induction Models Genetics of Floral Induction Conclusions Literature Cited

306 308 325

326 326 327 333 342 344 345

7. Flower and Fruit Thinning of Peach

and other Prunus

351

Ross E. Byers, Guglielmo Costa, and Giannina Vizzotto I. II. III. IV. V.

Introduction Reproductive Physiology Abscission Thinning Practices Future Prospects Literature Cited

352 355 362 365 383 384

8. The Reproductive Biology of the Lychee

393

Raphael A. Stern and Shmuel Gazit I. II. III. IV. V. VI.

Introduction Flowering Pollination The Fertilization Process and Initial Fruit Set Fruit Development and Abscission Concluding Remarks Literature Cited

394 397 422 428 433 443 444

Subject Index

454

Cumulative Subject Index

456

Cumulative Contributor Index

478

Contributors Fredrick A. Bliss, Seminis, Vegetable Seeds, Woodland, CA, 95695 Willem Brandenburg, Plant Research International, PO Box 16, 6700 AA Wageningen, The Netherlands Ross E. Byers, Department of Horticulture, Virginia Polytechic Institute and State University, Alson H. Smith Jr. Agricultural Research and Extension Center, 595 Laurel Grove Road, Winchester, VA, 22602 Daniel J. Cantliffe, Horticultural Sciences, University of Florida, P.O. Box 110690, Gainesville, FL, 32611 Craig K. Chandler, Gulf Coast Research and Education Center, University of Florida, 13138 Lewis Gallagher Road, Dover, FL, 33527 Guglielmo Costa, Dipartmento di Colture Arboree, University of Bologna, Via Fanin 50, Bologna, 40126, Italy Rebecca L. Darnell, Horticultural Sciences, University of Florida, P.O. Box 110690, Gainesville, FL, 32611 Kathy Eastman, Center for Economic Entomology, Illinois Natural History Survey, University of Illinois, 172 Natural Resources Building, 607 East Peabody Drive, Champaign, IL, 61820 Shmuel Gazit, The Kennedy-Leigh Center for Horticultural Research, The Hebrew University of Jerusalem, Faculty of Agriculture, PO Box 12, Rehovot, 76100, Israel Wilbert L. A. Hetterscheid, VKC/NDS, Linnaeuslaan 2a, 1431 JV Aalsmeer, The Netherlands Wilhelmina Kalt, Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Center, Kentville, Nova Scotia, B4N lJ5, Canada Daniel S. Kirshbaum, Horticultural Sciences, University of Florida, P.O. Box 110690, Gainesville, FL, 32611 Mosbah M. Kushad, Department of Natural Resources and Environmental Sciences, University of Illinois, 279 Madigan Laboratory, 1201 West Gregory Drive, Champaign, IL, 61801 Margit Laimer, Plant Biotechnology Unit, Institute of Applied Microbiology, University of Agricultural Sciences, Nussdorfer Lande 11, 1190, Vienna, Austria, [email protected] Jung-Myung Lee, Kyung Hee University, Department of Horticulture, Suwon, 449-701, Korea John Musiunas, Department of Natural Resources and Environmental Sciences, University of Illinois, 279 Madigan Laboratory, 1201 West Gregory Drive, Champaign, IL, 61801 Masayuki Oda, Osaka Prefecture University, Graduate School of Agriculture and Biological Science, Sakai, Osaka, 599-8531, Japan Stefano Predieri, Istituto di Biometeorologia-Firenze, Sezione di Bologna, Via Gobetti 101,40129 Bologna, Italy viii

CONTRIBUTORS

ix

Francesca Rapparini, Istituto di Biometeorologia-Firenze, Sezione di Bologna, Via Gobetti 101, 40129 Bologna, Italy Mary A. L. Smith, Department of Natural Resources and Environmental Sciences, University of Illinois, 1021 Plant Sciences Laboratory, 1201 South Dorner Drive, Champaign, IL, 61801 David M. Spooner, USDA, Agricultural Research Service, Vegetable Research Unit, Department of Horticulture, University of Wisconsin, 1575 Linden Drive, Madison, WI, 53706-1590 Raphael A. Stern, MIGAL, Galilee Technology Center, PO Box 90000, Rosh Pina, 12100, Israel Ronald G. van den Berg, Biosystematics Group, Department of Plant Sciences, Wageningen University, PO Box 8010,6700 ED Wageningen, The Netherlands Giannina Vizzotto, Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Udine University, Via delle Scienze 208, Udine, 33100, Italy

M. Allen Stevens

Dedication: M. Allen Stevens Allen Stevens' distinguished career in horticulture covers over 30 years in agricultural research. He spent nearly a decade each at the University of California, Davis, the Campbell Soup Company, and Petoseed Co., now Seminis Vegetable Seeds. He once remarked to me that after about ten years, he was ready for new challenges. The changes in venue served him well, because he met each new opportunity with enthusiasm and his accomplishments attest to his success. At UC Davis where he held the title of Geneticist and Professor, he and coworkers published over 100 scientific articles and book chapters. They received well-deserved recognition through the National Canners Association Award, 1968 and 1977; the Asgrow Award, 1971 and 1978; the National Food Processors Association Award, 1980; and the Homer C. Thompson Award, 1983. While at the university, he was advisor to graduate students in plant breeding and genetics as a major professor and member of numerous graduate degree committees. Allen has especially enjoyed working with young breeders, to whom he emphasized the importance of the art of plant breeding and the "eye" for selecting those rare recombinants with exceptional promise as well as application of sound scientific principles. He understood the practical nature of the seed trade and realized that broad knowledge of horticulture and close personal contact with growers, processors, and consumers were essential to success but often under-appreciated. It was enjoyable and a great learning experience to evaluate field trials with him and see the fine points that others often overlooked. His contributions as a plant breeder spanned both public and private sectors and include development of the outstanding processing tomato cultivar, 'UC82,' which was grown worldwide, and 'UC204,' 'Alta,' 'Lassen,' and 'Shasta,' which were leaders in California. A westerner at heart, Allen is really at home west of the Rockies. He was born and raised in southern Utah; graduated from Utah State University, receiving the BS degree (Agronomy) in 1957, and the MS degree (Soil Fertility) in 1961. Following his first degree, he served as a pilot in the U.S. Army. Allen worked as an Extension Agent in Oregon, then studied with the late William A. (Tex) Frazier at Oregon State University, where he received his doctorate in horticulture. His graduate studies xi

xii

DEDICATION: M. ALLEN STEVENS

about the chemistry and genetics of flavor in snap beans set the theme for many of his subsequent contributions to breeding vegetable crops for greater productivity and improved nutritional and organoleptic traits. Fittingly, he pursued these topics as a tomato breeder in the Department of Vegetable Crops at UC Davis and the Campbell Soup Company, and continued to give support as a research administrator. Although the value of breeding for better quality and improved nutritive content in vegetables and fruits has long been recognized among researchers, it has been a difficult concept to incorporate into commercial varieties, often because of limited interest from consumers. Times may be changing, however, as the value of improved nutritional content is more widely recognized. If so, his pioneering work may be rediscovered. No one better understands the importance of diverse germplasm for a successful breeding program than Allen. He was particularly interested in and gave support for conservation and use of plant genetic resources. He served as a member of the National Plant Genetic Resources Board and Board of Directors of the Genetic Resources Communications System in the United States. As Chair of the University of California Tomato Genetic Resources Task Force, he played a major role in establishing an endowment fund for support of the Charles M. Rick Tomato Genetic Resource Center located at UC Davis. In recognition of that and other important contributions to the University of California and California agriculture, Allen received the Award of Distinction, from the College of Agricultural and Environmental Sciences at UC Davis in 1995. His plant breeding, research, administration and service activities are known around the world. He was a visiting scientist at the Hebrew University in Rehovot, Israel. For the Asian Vegetable Research and Development Center located in Taiwan, he served as USAID Scientific Liaison Officer and a member of the Board of Trustees. He had global responsibilities for R&D at the Campbell Soup Company and Seminis Vegetable Seeds and served on the Executive Committee of the International Food Biotechnology Council. He was consultant to the FAO of the United Nations and to USAID on vegetable production and improvement. Allen has been a visionary and inspiring leader in professional organizations that represent their respective fields of influence in agriculture. He has participated in most facets of the American Society for Horticultural Science at one time or another, where he served as Vice President of both the Research and Industry Divisions, as President in 1993, and as Chairman of the Board of Directors when he organized the strategic planning initiative. That provided an important blueprint for the Society to better serve an ever-changing membership. He is an elected

DEDICATION: M. ALLEN STEVENS

xiii

Fellow of the American Society for Horticultural Science and the American Association for the Advancement of Science. I first became acquainted with Allen by reading about his research on flavor components of snap beans, which he conducted as a graduate student at Oregon State University in the early 1960s. Later, I referred to that information periodically as I looked for new opportunities for bean improvement. The 'Bush Blue Lakes' beans that came from the OSU program are still remembered as standards for high quality. It has been my pleasure to work with him on numerous committees and task forces dealing with topics covering germplasm resources, intellectual property rights, and education of plant breeding students, as well as many activities within the American Society for Horticultural Science. In 1998 as Vice President for Research and Development, he asked me to join Seminis Vegetable Seeds, where I had the opportunity to work with him daily. I, like many others, had the good fortune to draw on his great breadth of knowledge about practical vegetable breeding and the commercial seed business. Since his retirement from the position of Vice President of Research & Development at Seminis, he and his wife Hermese have considerably more time for extensive travel-now for enjoyment, rather than mainly for business. They have the opportunity to see more of their children and grandchildren and to revisit family and friends and the land he knew as a youngster in Utah. In addition to being an accomplished breeder, scientist, and administrator, Allen is a superb amateur photographer, displaying professional skill and standards of quality and accomplishment similar to those he displayed as a professional horticulturist. Don't be surprised if you see some of his beautiful photographs in the popular press. Fredrick A. Bliss Seminis Vegetable Seeds Woodland, CA 95695

1

Plant Nomenclature and Taxonomy An Horticultural and Agronomic Perspective David M. Spooner* U.S. Department of Agriculture Agricultural Research Service Vegetable Crops Research Unit Department of Horticulture University of Wisconsin 1575 Linden Drive Madison Wisconsin 53706-1590 Wilbert L. A. Hetterscheid VKC/NDS Linnaeuslaan 2a 1431 JV Aalsmeer The Netherlands

Ronald G. van den Berg Biosystematics Group Department of Plant Sciences Wageningen University PO Box 8010 6700 ED Wageningen The Netherlands Willem A. Brandenburg Plant Research International PO Box 16 6700 AA, Wageningen The Netherlands

I. INTRODUCTION A. Taxonomy and Systematics B. Wild and Cultivated Plants II. SPECIES CONCEPTS IN WILD PLANTS A. Morphological Species Concepts B. Interbreeding Species Concepts C. Ecological Species Concepts D. Cladistic Species Concepts E. Eclectic Species Concepts F. Nominalistic Species Concepts

*The authors thank Paul Berry, Philip Cantino, Vicki Funk, Charles Heiser, Jules Janick, Thomas Lammers, and Jeffrey Strachan for review of parts or all of our paper.

Horticultural Reviews, Volume 28, Edited by Jules Janick ISBN 0-471-21542-2 © 2003 John Wiley & Sons, Inc. 1

2

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

III. CLASSIFICATION PHILOSOPHIES IN WILD AND CULTIVATED PLANTS A. Wild Plants B. Cultivated Plants IV. BRIEF HISTORY OF NOMENCLATURE AND CODES V. FUNDAMENTAL DIFFERENCES IN THE CLASSIFICATION AND NOMENCLATURE OF CULTIVATED AND WILD PLANTS A. Ambiguity of the Term Variety B. Culton Versus Taxon C. Open Versus Closed Classifications VI. A COMPARISON OF THE ICBN AND ICNCP A. Nomenclatural Types and Standards B. Denomination Classes and the Reuse of Epithets C. Botanical Hybrid (Species) Names D. The Species Category in Cultivated Plant Taxonomy (Cultonomy) E. The (Notho-)Genus Category in Cultivated Plant Taxonomy (Cultonomy) F. Ties Between the ICBN and ICNCP VII. POSSIBLE NEW CODES A. Biocode B. PhyloCode VIII. CULTIVATED PLANT NOMENCLATURE AND THE LAW IX. CULTIVAR EPITHETS AND TRADEMARKS X. RECOMMENDATIONS FOR A UNIVERSALLY STABLE CROP NOMENCLATURE THROUGH CHANGES AND USE OF THE ICNCP REFERENCES LITERATURE CITED

I. INTRODUCTION Now the whole world had one language and a common speech. Then they said, "Come, let us build ourselves a city, with a tower that reaches to the heavens, so that we may make a name for ourselves and not be scattered over the face of the whole earth." The Lord said, "Come, let us go down and confuse their language so they will not understand each other." So the Lord scattered them from there over all the earth, and they stopped building the city. That is why it was called Babel becaOuse there the Lord confused the language of the whole world. (Genesis 11 :1, 3, 4, 6-9; New International Bible)

Communication of taxonomists to agronomists and horticulturists can be hindered by specialized terminology that aids concise and effective communication of complex ideas among taxonomists, but may seem intractable and pedantic to agriculturalists. Our goals in this review are to provide agronomists and horticulturists basic conceptual tools of taxonomy: (1) to help understand the taxonomic classification in wild and cultivated plants; (2) to question whether the concept underlying this

1. PLANT NOMENCLATURE AND TAXONOMY

3

taxonomy is appropriate; (3) to help understand why new data may require changes in nomenclature; and (4) place the taxonomy of crops in the context of legal requirements that depend on a taxonomic name. Different taxonomic concepts of wild plants and cultivated plants are reviewed because both classes are used in breeding and germplasm evaluation. The goals and practices of two codes of plant nomenclature, the International Code of Botanical Nomenclature (ICBN) and the International Code of Nomenclature for Cultivated Plants (ICNCP) are compared, the former (Greuter et al. 2000) used primarily for wild plants, and the latter (Trehane et al. 1995) used exclusively for cultivated plants. The plethora of specialized terms used in this review is presented as a glossary in Table 1.1. Table 1.1

Glossary of terms used highlighted in bold italic in the text.

artificial classification. Classification that may be based on any special-purpose criteria that users view as relevant to group plants, not based on evolutionary relationships (see natural classification). basionym. The original name of a taxon, which may be changed in rank, say from variety to species. For example, when Solanum jamesii Bitter var. brachistotrichium Bitter was recognized as a species, its name became Solanum brachistotrichium (Bitter) Rydb., but the basionym remains Solanum jamesii Bitter var. brachistotrichium Bitter. biological species concept. The concept of a species as a population or group of populations that freely interbreed but are reproductively isolated from other populations. biosystematics. A term that originally referred to the use of breeding programs (by biosystematists) to infer evolutionary relationships among organisms; the term later became broadened to refer to a wide variety of experimental data gathering programs. cenospecies. Assemblages of related ecospecies that when crossed produce highly to completely sterile hybrids. cladogram. A branching phylogenetic tree of individuals or taxa, rooted on an outgroup(s) produced by a method that minimizes evolutionary changes (by parsimony, maximum likelihood, or other methods) of characters believed to be homologous among a group of organisms. cladistic species concepts. A philosophy and set of methods that use cladistic criteria to determine the limits of species. closed classification system. Hierarchical system where the categories at every rank are totally filled up by the sum of the categories at the next lower rank. comparium. A group of related cenospecies that cannot be crossed with one another. ecological species concepts. A philosophy that ecological factors are primary in forming and maintaining a species. (continues)

4

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

Table 1.1

(continued)

compilospecies. Genetically aggressive, highly polymorphic species, often of complex hybrid origin, often containing more than one ploidy level, often very weedy, and obscuring other species boundaries. conservation. In order to avoid disadvantageous changes in the nomenclature of families, genera, and species entailed by the strict application of nomenclatural rules, and especially of the principle of priority, names may be retained as legitimate even though initially they may have been illegitimate, by petitioning in the journal Taxon, and later vote at the International Botanical Congress. Conservation operates only within the taxa in which they have been voted upon, and is not operative if the taxon is classified in another group. convariety (convar). A group of cultivars. These can be roughly comparable to cultivar groups, but convarieties, unlike cultivar groups, do not necessarily contain named varieties, and convarieties are members of traditional "Linnaean" ranks. The ICNCP replaced this term with the term cultivar-group, and convarieties should not be used in modern cultivated plant taxonomy. crop. The total of all cultivars/cultivar-groups that constitute an agricultural, horticultural, or silvicultural product; examples: potato, cabbage, or tulips. cytodemes. Groups of plants characterized by having a constant chromosome number, with all accessions of the same cytodeme being fully interfertile, while those of different cytodemes are essentially cross-sterile. cultigen. A taxon with only cultivated representatives; example: Triticum aestivum, the species name encompassing all hexaploid wheat varieties. cultivar. A systematic group of cultivated plants that is clearly distinct, uniform, and stable in its characteristics and which, when propagated by appropriate means, retains these characteristics. cultivar-group. A group of properly named cultivars, based on one or more criteria. cultivated plant. One whose origin or selection is primarily due to the intentional activities of mankind. Such a plant may arise either by deliberate or, in cultivation, accidental hybridization, or by selection from existing cultivated stock, or may be a selection from minor variants within a wild population and maintained as a recognizable entity solely by deliberate and continuous propagation (Trehane et al. 1995). culton. A systematic group of cultivated plants; there are two types of culta: the cultivar and the cultivar-group. cultonomy. Cultonomy is the entire body of principles, philosophies, and methodologies leading to classifications of cultivated plants into culta, and following the rules of ICNCP. dendrogram. A branching diagrammatic representation of a set of individuals or taxa, constructed from overall similarity of a set of characters among organisms, which generally is not provided any phylogenetic interpretation. denomination class. Agreed upon systematic group (often a genus) within which a cultivar epithet may only be used once.

1. PLANT NOMENCLATURE AND TAXONOMY

Table 1.1

5

(continued)

eclectic species concepts. A philosophy that species are defined, formed, and maintained by a variety of biological factors, including morphological, interbreeding, ecological, and phylogenetic factors. ecospecies. An assemblage of ecotypes and are separated by incomplete sterility barriers. ecotype. All members of a species fitted to survive in a particular environment; different ecotypes within species have no interbreeding barriers. epithet. Part of the full name of a species; a complete species name consists of the name of the genus to which the species belongs, plus the specific epithet, plus the author of the species. form. The lowest rank in the taxonomic hierarchy (below variety), meant to convey minor variants in nature. gene pool classification. A classification of cultivated plants focused on the crossability of species to an individual crop plant, with gene pool 1 being the crop and those species easily crossable to it, gene pool 2 being species crossable to the crop with some difficulty, and gene pool 3 being species crossable to the crop with extreme difficulty. homologous. Characters that arise by common descent. ICBN. International Code of Botanical Nomenclature (latest version is Greuter et al. 2000). ICNCP. International Code of Nomenclature of Cultivated Plants (latest version is Trehane et al. 1995). ingroup. A putatively monophyletic group that is the prime subject of a cladistic analysis. indigen. Wild taxa in their natural habitat and distribution area. interbreeding species concepts. A philosophy and set of methods that define species almost entirely on the ability of species to exchange genes naturally or artificially, as assessed by artificial crossing programs, studies of mechanisms to facilitate gene flow, and biological isolating mechanisms. landrace. Cultivar that originated as a product of (the first stages of) mass selection (and not as a product of modern plant breeding), generally confined to a certain region. lumper. Refers to a taxonomist who focuses more on similarities than differences, discounting the importance of minor variation among individuals, and tending to recognize fewer taxa (see splitter). maximum likelihood. A set of methods used to construct cladograms based on certain evolutionary models of character state changes (compare to parsimony). monophyletic group. A group that includes an ancestral species and all of its descendants. morphological species concepts. A philosophy and set of methods that define species entirely on morphological or anatomical characters. natural classification. Classification based on the evolutionary relationships between the entities to be classified. (continues)

6

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

Table 1.1

(continued)

nomenclatural types. Objects (usually a herbarium sheet) to which a name of a taxon is permanently attached. When a species is described, a type specimen is designated that serves as a reference point for others to compare their concept of names. nominalistic species concepts. A philosophy that questions the very existence of species, and believes that individuals or interbreeding populations are the only population system with any objective reality. nothotaxon. A taxon of hybrid origin (as notho species, nothogenus). open classification. Nonhierarchical system of flexible groups that do not automatically need to be grouped together in larger groups, nor subdivided in smaller groups. orthologous. Genetic characters that are homologous from a speciation event, that is, identical by descent. outgroup. Any group used in a cladistic analysis that is not included in the taxon under study, which is used to root a phylogenetic tree. ordination plot. A phenetic analysis (as principal components analyses, principal coordinates analyses, multidimensional scaling analyses), showing overall similarity of individuals or taxa on two- or three-dimensional plots. paralogous. Characters that have arisen as a result of gene duplication. paraphyletic group. A nonmonophyletic group containing some, but not all representatives of a taxon; said another way, an incomplete group of descendants from one common ancestor with one or more descendants missing. parsimony. A set of methods that assumes that the simplest solution is the most likely one. It is used to construct cladograms, and assumes that minimizing the number of character state changes on a tree is the best approximation of phylogenetic history. plesiomorphy. An ancestral character, not viewed as useful in cladistic analyses for defining monophyletic groups. pluralist species view. The idea that species are formed and maintained by a variety of criteria including morphological, geographical, biological, and ecological criteria. polyphyletic group. A nonmonophyletic group where the common ancestor is placed in another taxon; in other words, a group in which the members do not ultimately derive from one common ancestor, where the descendants of one or more other groups are included. priority. A principle in the ICBN stating that the earliest validly published name is the proper name assigned to a species. sister group. The most closely related monophyletic outgroup to the ingroup. splitter. Refers to a taxonomist who focuses more on small differences among taxa, emphasizing minor variation among individuals, and who tends to recognize more taxa (see lumper). standard. A specimen, seed sample, or illustration kept and maintained in a conserved place to illustrate the diagnostic characteristics of a cultivar (used in the ICNCP).

1. PLANT NOMENCLATURE AND TAXONOMY

Table 1.1

7

(continued)

symplesiomorphy. A set of shared primitive characters, viewed as useless in cladistic analyses for defining monophyletic groups. synapomorphy. A set of shared derived characters, viewed as useful in cladistic analyses for defining monophyletic groups; said another way, characters shared by two or more taxa as a result of their immediate common ancestry. taxon. A systematic group of plants in a hierarchical system. total evidence analysis. A philosophy that cladistic analyses should be constructed with many separate sets of data. type (nomenclatural type). That element to which the name of a taxon is permanently attached, whether as a correct name or as a synonym (used in the ICBN). variety. A "botanical" variety is a rank in the taxonomic hierarchy below the rank of species and subspecies and above the rank of form (form/variety/subspecies/species). Another meaning, as used in legal texts is synonymous with cultivar (see Section V.A., Ambiguity of the Term Variety).

A. Taxonomy and Systematics A plant's name is the key to its literature. Van Steenis (1957)

One of the greatest assets of a sound classification is its predictive value. Mayr (1969)

Taxonomy is the theory and practice of describing, naming, and classifying organisms (Lincoln, Boxshall, and Clark 1998). Systematics is a related term, sometimes used synonymously, but involves a broader discipline of discovering phylogenetic relationships through modern experimental methods using comparative anatomy, cytogenetics, ecology, morphology, molecular data, or other data (Stuessy 1990). It also could be more generally defined as the science of developing methods and philosophies for the systematic grouping of organisms. Whatever term one chooses (we use taxonomy here for simplicity), taxonomists are basically involved with: (1) determining what is a species (or their subdivisions, as subspecies), (2) distinguishing these species from others through keys and descriptions and geographic boundaries and mapping their distributions, (3) investigating their interrelationships, and (4) determining proper names of species and higher order ranks (as

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D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

genera or families) using international rules of nomenclature. In addition, some taxonomists investigate processes of evolution that lead to the existing pattern of species and their interrelationships. There are many rationales for biological taxonomy, including the need (1) to understand the world about us and to conceptualize and order this through classifications; (2) to have classifications for identification and communication; (3) for a convenient information retrieval system; (4) to use stable names that maintain continuity of the literature; (5) to construct a predictive classification; and (6) to construct a useful framework to understand phylogenetic relationships. Taxonomy has special use for conservators including (7) to provide a useful reference system for biodiversity conservation; (8) to aid gene bank managers to rationally organize collections; (9) to aid germplasm collectors to plan expeditions based on gaps in a genebank (Warburton 1967; Mayr 1982; Stuessy 1990; Judd et al. 1999; Woodland 2000). For agriculturists and horticulturists, stability of names and prediction are major rationales, but the prediction rationale is controversial, as we shall discuss. The need for stable names is only amplified with crop plants where the frequency and need for information retrieval from the literature, through the convenient label of a species name, is much greater than for other plants. One reason for using Latin names or scientific names or Linnaean binomials for plants is to avoid the potential Babel of different common names for the same entity (synonyms). As we discuss, however, many factors are in conflict with this extremely practical goal of stability, leading to different scientific names for the same plant. What factors conflict with a stable nomenclature? 1. Different classification philosophies have fundamental differences

in primacy put on morphological, crossability, phylogenetic, ecological, molecular, or other data that may provide different names to taxa. 2. Powerful new technologies (molecular systematics and computer algorithms to analyze these data) are revising knowledge on species limits and species interrelationships. 3. Taxonomists are far from agreement between the often competing goals of stability of names and potentially improved predictivity of new classifications. 4. Revised phylogenetic data and emerging classification philosophies threaten to overturn long-held traditional classifications. Most users of taxonomy intuitively accept the putative predictive component of classifications (Mayr 1969). Claims of the predictive value of classifications can be found in Michener (1963), Rollins (1965), War-

1. PLANT NOMENCLATURE AND TAXONOMY

9

burton (1967), Sokal (1985), and Stuessy (1990). For example, Warburton (1967) states: [Prediction] means that one can describe a trait as characteristic of all members of a taxon before it has been verified for all. It also means that if organisms have been classified together as a taxon because they have all been found to share certain traits, they will later be found to share other traits as well.

For plant breeders, prediction would mean that germplasm could be chosen or avoided based on past positive or negative evaluations. Germplasm evaluations organized with species or higher ranks are common in the literature, for example, species-specific statements of breeding value of wild potato germplasm are found in Ross (1986), Hawkes (1990), and Ruiz de Galerreta et al. (1998). Clearly, not all accessions of a species share traits, but lacking prior evaluation data, taxonomy provides a useful guide to make inferences on unevaluated germplasm based on present knowledge. While differences in classification philosophies provide fascinating debate among taxonomists and advances the field, the remaining biological community largely focuses on stability of names for purely practical considerations. Some see a failure of the stability goal as the failure of taxonomists to fulfill their service role. Many taxonomists, however, focus on improved phylogenetic classifications as primary. B. Wild and Cultivated Plants Botanists have generally neglected cultivated varieties as beneath their notice.

Darwin (1868) Most modern taxonomists do next to nothing with cultivated plants; many deliberately avoid studying or even collecting them.

Anderson (1952) Almost one-third [of Conley K. McMullen's Flowering Plants of the Galapagos, 1999] covers cultivated species. That seems to place a rather excessive emphasis on the least interesting plants, but undoubtedly tourists will appreciate the information.

Ulloa Ulloa (2001)

Cultivated plants have various definitions, but all focus on the activities of humans. De Wet (1981) simply considered a cultivated plant one that

10

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

is adapted to the man-made habitat. Schwanitz (1967) defined cultivated plants as: the result of evolution processes, that were going on in prehistoric and historic times and are still going on nowadays, both under direct and indirect influence of mankind.

Trehane et al. (1995) defined a cultivated plant as: one whose origin or selection is due to the activities of mankind. Such plants may arise either by deliberate or chance hybridization or by further selection from existing cultivated stock or they may be selected from a wild population and maintained as an entity by continuous cultivation.

Frequently, domestication is regarded as just a special form of evolution as it happens unconsciously and the same mechanisms of selection are at work (Hanelt 1986; Van Raamsdonk 1993; Van Raamsdonk and van der Maesen 1996; Zohary 1984). Specialized terms (Table 1.1) are used to refer to cultivated plants that are important for effective communication and classification. A cultigen is defined as taxon with only cultivated representatives, such as Triticum aestivum L., the species name encompassing all hexaploid wheats. The term taxon for this definition is controversial, however, as we will discuss. This term is contrasted with an indigen, that is, a wild taxon in its natural habitat and distribution area, that is, a noncultivated plant. A culton is a systematic group of cultivated plants, and the ICNCP recognizes two types of culta: the cultivar and the cultivar-group. A cultivar is a nomenclatural term referring to the most basal taxonomic unit of cultivated plants. A cultivar-group refers to an assemblage of similar named cultivars. A landrace is a cultivar that originated as a product of (the first stages of) mass selection (and not as a product of modern plant breeding), mostly confined to a certain region. It also had been referred to as an indigenous cultivar or a primitive cultivar. Schwanitz (1967) outlined some special features of cultivated plants that can make for rapid divergence from their progenitors: (1) increase of number of desired plant parts; (2) increase of size of desired plant parts by allometric growth; (3) loss of undesired morphological or chemical traits; and (4) loss of defense mechanisms. All of these traits may arise rapidly, make the cultivated plant quite different from its progenitor(s), obscuring the connection between them. In addition, the study of relationships of crops to progenitors can be hindered by hybridization with weeds producing "crop-weed complexes."

1. PLANT NOMENCLATURE AND TAXONOMY

11

Crop-weed complexes have long been a subject of extensive systematic study and reviews (De Wet and Harlan 1975; Hanelt 1986; Harlan 1965, 1975; Pickersgill1971, 1981, 1986; Van Raamsdonk and Van der Maesen 1996). Most of these studies point out the complex interaction among weeds, domesticates, and their wild relatives. Extensive hybridization makes the classification of crop-weed complexes especially difficult. Pickersgill (1981, 1986) suggested that weedy progenitors evolve to cultivated plants, but cultivated plants can also evolve back to weeds. Modern cultivars are produced by extensive artificial hybridization, and pedigree records are often incomplete or unavailable for proprietary reasons. For example, separate modern cultivars of potato have in total incorporated germplasm from 16 wild species in the pedigrees (Ross 1986; Plaisted and Hoopes 1989). Despite these separate pedigrees, all are classified as the single species Solanum tuberosum 1. II. SPECIES CONCEPTS IN WILD PLANTS The discussion of species concepts has become a cottage industry. Not only does the number of pages and full-length books devoted to the topic continue to grow, but new concepts of species proliferate at an extraordinary rate.

Rieseberg and Burke (2001)

Species have a central place in taxonomy as they form the basic units of biological classification (Davis and Heywood 1963; Greuter et al. 2000), but there is no consensus on how to define species, and likely never will be. Why? New species concepts have changed considerably with the development of new data and theory, and are likely to continue to change. Each development has led to new species concepts, and Mayden (1997) lists a total of 22. Many concepts, however, are minor variations of others, and some are rarely applied. The following are six major classes of species concepts. A. Morphological Species Concepts

Morphological species concepts define species entirely on morphological or anatomical characters. Because of their utility, they are frequently applied, especially historically when taxonomists worked in large herbaria and the form of the plant was the major data set available. This can operate effectively by a method referred to as sheet shuffling, whereby a collection of herbarium specimens is initially sorted into species based on a subjective impression of overall form. This can be followed by

12

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

microscopic examination to gain additional data to modify species delimitation. Cronquist (1978) defined this practical application of the morphological species concept as the taxonomic morphological species concept: Species are the smallest groups that are consistently and persistently distinct, and distinguishable by ordinary means.

The characters leading to this subjective judgment are often unclear, sometimes even to the taxonomist applying them. Typically, characters of special value are weighted, for example more weight is given to reproductive than to vegetative characters. A potential problem with this is that because the methods of the taxonomist are not always evident, preference for one of several conflicting taxonomic treatments are often made based on a taxonomist's reputation, rather than on the inherent qualities of a classification. The advent of computers allowed the practical application of multivariate techniques to taxonomic data. In· practice, morphological, anatomical, chemical, or any character type was appropriate for analyses. The basic idea was that many characters were overlooked in defining species, and that species were best defined by an objective and equal treatment of all characters, reproductive and vegetative (Sneath and SokaI1962). An added claim was that these methods opened up these classifications to scrutiny, as data and analytical techniques were open for evaluation by all and not hidden and inscrutable impressions of experts. In practice, a taxonomist scores quantitative or qualitative characters and enters them on rectangular data matrices (data entry cells with characters versus individuals). Various algorithms then transform this matrix into a triangular similarity (or dissimilarity) matrix of individuals by individuals. Different individual data reduction techniques then convert a similarity matrix to a graphical display of phenetic trees (phenograms or dendrograms), ordination plots (as principal components analyses, principal coordinates analyses, or multidimensional scaling analyses). Decisions are made on species limits based on clustering of individuals, but there is no universally accepted objective criterion to determine the degree of clustering to define species or higher taxonomic levels. Sokal and Crovello (1970) defined this phenetic morphological species concept as "dense regions of hyperdimentional space" (referring to clustering of individuals in ordination analyses). This concept can provide misleading results of species boundaries in certain crops however where only a few genes have remarkable mor-

1. PLANT NOMENCLATURE AND TAXONOMY

13

phological effects as in the case of Brassica oleracea where the same species has been selected for forms as divergent as broccoli, brussels sprouts, cabbage, cauliflower, kale, and kohlrabi. B. Interbreeding Species Concepts

The interbreeding species concepts focus almost entirely on the ability of species to exchange genes naturally or artificially, as assessed by artificial crossing programs, studies of mechanisms to facilitate gene flow, and biological isolating mechanisms. Mayr (1942) advanced the biological species concept as "Species are groups of interbreeding natural populations that are reproductively isolated from other such groups." This concept matches that held in the minds of the general public and is intuitively appealing, but many practical and theoretical problems were raised. Procedurally, it is almost impossible to apply to a group of any size because replicated pair-wise crosses are needed in most interspecific combinations to be confidently interpreted (Sokal and Crovello 1970). As well, data from greenhouse situations are not always applicable to the field, and varying degrees of crossing success are not easily interpreted. Also, the concept is inapplicable to species reproducing apomictically. The lifetime of crossing studies by Rick (1963, 1979) in tomato is a notable application, but this depth of study is exceptional and rarely has been applied to other groups. Such crossing studies were common in the 1940s to 1960s and the term biosystematics originally referred to the use of breeding programs (by biosystematists) to infer evolutionary relationships among organisms. The term later became broadened to refer to a wide variety of experimental data gathering programs. Because of the broad definition of the term the need for this term has lessened (Stuessy 1990). The difficulty to define differing degrees of intercrossability led to qualifier terms. Harlan and de Wet (1963), working in grasses, recognized compilospecies as genetically aggressive, highly polymorphic species, often of complex hybrid origin, often containing more than one ploidy level, often very weedy, and obscuring other species boundaries. They suggested that such species were typical progenitors of crops. Grant (1981) referred to semispecies as populations of plants on the way to becoming species but yet without sufficient reproductive isolation, and used Lotsy's (1925) term synganleon to refer to a broadly sympatric set of semispecies. Some biosystematic terms were placed in a hierarchy of relationships (Clausen et al. 1945; Clausen 1951; Grant, 1981), from ecotype (lowest)

14

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

to ecospecies, cenospecies, and comparium. An ecotype consists of all members of a species that are fitted to survive in a particular environment, and different ecotypes within species have no interbreeding barriers. An ecospecies is an assemblage of ecotypes and are separated by incomplete sterility barriers. Cenospecies are assemblages of related ecospecies that when crossed produce highly to completely sterile hybrids. A comparium is a group of related cenospecies that cannot be crossed with one another. Patterson (1985) advanced a variant of the biological species concept termed the recognition species concept. Mayr's (1942) biological species concept suggested that biological isolating mechanisms were an accidental by-product of genetic reconstruction during speciation. Patterson, on the other hand, suggested that specific forces were responsible for such reconstruction, and viewed biological isolating mechanisms as an active, positive force in speciation. His concept stimulated the search for adaptations that assist the process of meiosis and fertilization, but he realized that isolation and recognition are just two components of the same process. C. Ecological Species Concepts

Van Valen (1976) was confounded by the perplexing array of variation in oaks. Oaks have broadly sympatric sets of very similar species, often hybridizing among each other, which he termed multispecies. These were similar to the compilospecies of Harlan and de Wet (1963, described earlier). He noted that despite many hybrids, species maintained their integrity in specific habitats. For example, Quercus bicolor Willd. (swamp white oak) was broadly sympatric with Quercus macrocarpa Michx. (burr oak) in the Great Lakes and Ohio River basins, and they frequently hybridized. The former, however, grew in wet bottomlands, streamsides and swamps, and the latter in mesic habitats of rich woods and fertile slopes. Van Valen stated, The control of evolution is largely by ecology and the constraints of individual development. He outlined the ecological species concept as: A species is a lineage (or a closely related set of lineages) which occupies an adaptive zone minimally different from that of any other lineage in its range and which evolves separately from other lineages outside its range.

He contended that reproductive isolation of allopatric populations is of minor evolutionary importance, and that ecological factors are more closely related to genetic differences than reproductive isolation.

1. PLANT NOMENCLATURE AND TAXONOMY

15

D. Cladistic Species Concepts

The most recent, conceptually difficult, and terminology-laden set of species concepts are grouped here under cladistic species concepts. They arose out of the ideas of Hennig (1950, 1966) who used phylogenetic history as the sole criterion for grouping taxa, irrespective of morphology, interbreeding behavior, or ecological considerations, except as they may be used to help reconstruct phylogenetic history. He never used cladistics to help define species, but his concepts have been applied this way as will be discussed. Basic cladistic terms are briefly explained; the reader is directed to Wiley et al. (1991) for more detailed explanations. Cladistics refers both to a set of methods for inferring phylogeny and a philosophy of systematics in which only monophyletic groups are accepted. Not everyone who uses cladistic methods, however, accepts a cladistic philosophy of classification, and some do not consider cladistics to be appropriate to recognize species. A monophyletic group encompasses an ancestor and all of its descendants, as determined by a cladistic analysis that produces phylogenetic branching trees (cladogram). The basic procedure to construct cladograms is to try to begin with a putatively monophyletic group, referred to as an ingroup, such as "species A" or "tuber-bearing solanums," or "the sunflower family." Evolutionary relationships within the ingroup are determined by the use of an outgroup(s) that are analyzed for the same characters and are used to construct the tree. A sister group is the most closely related monophyletic outgroup to the ingroup, and further outgroups also can be used for a multiple outgroup analysis (Maddison et al. 1984). (See Fig. 1.1.) Characters are scored for all ingroup and outgroup taxa, just as in phenetic studies. Any character type can potentially be used, including

Sister group Second outgroup ~ E

Fig. 1.1.

Ingroup

~

Terms relative to cladograms.

D

cl

16

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

morphological or molecular characters (as DNA base pairs or restriction endonuclease sites). Most analyses score these characters qualitatively, as presence or absence (0-1), or as a range of discrete character states (0-1-2-n). Great care is taken to score only homologous characters arising from common ancestry, avoiding characters that may look similar but actually arise in parallel from different ancestors. Orthologous characters are homologous by a speciation event, meaning that they trace their ancestry to a common progenitor, and are taken as the only useful type of homologous character. Molecular taxonomists are searching for single-copy nuclear genes for phylogeny construction, and doing everything possible to avoid paralogous characters that have arisen from gene duplication. Such duplicated genes can evolve separately in the same lineage, may falsely appear to be homologous, but can provide misleading phylogenetic information. Cladograms are then constructed from these data by various methods, but a common method is to use the parsimony criterion, that invokes the minimum number of evolutionary changes to construct the tree. Other techniques also are used, such as maximum likelihood (Felsenstein 1981; Swofford et al. 1996) that searches for trees that may be longer but that represent character changes based on certain evolutionary models. The tree is rooted based on characters of the outgroup(s), and in this method monophyletic groups are supported only by synapomorphies (shared derived characters) relative to the plesiomorphies (ancestral or primitive characters) of the outgroup(s). Cladograms may look like phenetic trees (dendrograms), but phenetic analyses are based on overall similarity and dendrograms are constructed by an average of all characters, not individual characters on each branch as in cladograms. Pheneticists infer only overall similarity of organisms from their phenograms, not phylogeny, and most cladists interpret cladograms phylogenetically. Monophyletic groups are then determined from the cladogram that trace to a single internode (all of which are supported by synapomorphies). Cladists avoid recognizing all nonmonophyletic groups, includingparaphyletic groups (groups containing some, but not all descendants of the most recent common ancestor), and polyphyletic groups (groups where the common ancestor is placed in another taxon). Paraphyletic species have been recognized, however, as described in Figure 1.2. There is a wide diversity of opinion on application and interpretation of these concepts, providing further problems. For example, phylogenetic results of the same organisms obtained from different data sources are frequently in conflict (Wendel and Doyle 1998). Some advocate analyzing data separately to discover datasets providing misleading results,

17

1. PLANT NOMENCLATURE AND TAXONOMY

Monophyletic

Paraphyletic

Fig. 1.2.

Polyphyletic

Cladistic relationships relative to cladograrns.

while others advocate combining all data into a single matrix for a total evidence analysis (e.g., Eernisse and Kluge 1993). Cladistic results also can be affected by poor choice of outgroups, by analysis of unrecognized nonorthologous characters, by different choice of cladistic algorithms to construct trees, by insufficient ingroup or outgroup sampling, and by different methods to handle missing data. There is also debate among cladists whether cladograms truly reflect recency of common ancestry (process cladists), or whether they need to be theory neutral and only show patterns decoupled from assumptions of ancestry (pattern cladists or transformed cladists) (Ereshefsky 2001). Perhaps the greatest source of debate is the use of cladistics at all at the species level. This is because cladistic procedures assume divergent taxa, yet individuals within species often (generally) hybridize, leading some to consider cladistics to be an inappropriate method to define species (Templeton 1989). Another complication is the differences in opinion in cladistic species concepts. The most strict interpretation of the cladistic species concept was advanced by the process cladists Mishler and Brandon (1987) as the autapomorphic species concept: A species is the least inclusive taxon recognized in a classification, into which organisms are grouped because of evidence of monophyly ... that is ranked as a species because it is the smallest "important" lineage deemed worthy of formal recognition.

18

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

Outgroup

T1

Plesiospecies AIA2A3A4AS A6 A 7

Apospecies B 1 B 2 B 3 B 4 Bs

--::--------;---:--~:---;----=---:-------~:----=---=----:--~

Fig. 1.3. Apospecies and plesiospecies as depicted by Olmstead (1995). Under this evolutionary model, a set of populations is shown at initial time To when a speciation event occurs, as depicted by the thick horizontal line designating a synapomorphy forming the species. At initial time To, the new apospecies leaves a remnant set of populations that are now paraphyletic (plesiospecies). Later (at time T 1 ) extinction of populations leads to monophyly of both species. Bold lines designate populations surviving to time T 1 • This shows the theoretical need to withdraw the strict criterion for monophyly in cladistic species concepts.

The criterion for "important lineage" necessary to define a species can vary from ecological, reproductive, or developmental criteria. Recently, Rieseberg and Brouillet (1994) and Olmstead (1995) have argued that geographically localized models of speciation typically produce a monophyletic daughter species and remnant paraphyletic progenitor species, and argue that a strict concept for monophyly fails for many species. Olmstead (1995) termed the former apospecies and the latter plesiospecies. He traced a hypothetical set of populations over time To (initial species divergence) and T 1 (later time with full development of apospecies) that showed the necessity of recognition of paraphyletic species if apospecies are to be recognized at all (Fig. 1.3). Cracraft (1989) has a tendency to lean in the direction of pattern cladistics and advanced the phylogenetic species concept as: A species is an irreducible (basal) cluster of organisms, diagnosably distinct from other such clusters, and within which there is a parental pattern of ancestry and descent.

1. PLANT NOMENCLATURE AND TAXONOMY

19

This definition emphasizes smallest diagnosable units by a practical set of diagnostic characters, as discovered by cladistic procedures, but makes no inference that these are monophyletic. E. Eclectic Species Concepts

The former species concepts highlight single processes as definitive for species. Eclectic species concepts, in contrast, take a pluralistic view that species are formed and maintained by a variety of criteria. For example, Doyden and Slobobchikoff (1974) constructed a flow chart detailing a variety of morphological, geographical, biological, and ecological criteria to define species. Mayr (1982) modified his biological species concept to include an ecological component: A species is a reproductive community of populations that occupies a specific niche in nature. Stuessy (1990) concluded his discussion of species concepts with a basic agreement with Crum (1985): Although subjectivity is involved with decision making, a species is only as good as the knowledge and insights used in its delimitation. Certain methodologies help. So do good sense and good judgment based on meaningful experiences, and the more the better.

Templeton (1989) outlined the cohesion species concept as: The most inclusive group of organisms having the potential for phenotypic cohesion through intrinsic cohesion mechanisms through genetic and or demographic exchangeability.

He attempted to define specific mechanisms that drive the evolutionary process to speciation. He considered that his concept attempts to utilize the strengths of [biological, evolutionary, and recognition species concepts] while avoiding their weaknesses with respect to the goal of defining species in a way that is compatible with a mechanistic population genetics framework.

Ereshefsky (2000) outlined several classes of species concepts, and advanced a pluralist species view that no single correct definition of species exists and that a number of alternative concepts may be legitimate. F. Nominalistic Species Concepts

Some question the very existence of species, and believe that individuals or interbreeding populations are the only population system with any

20

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

objective reality. This concept arose out of the philosophy of nominalism, arguing that only individuals are real and that classes of any kind (as species, genera, or families) are artificial constructs. For example, Burma (1954) stated: species are highly abstract fictions. Levin (2000) likewise argued that only the local population is the unit of evolution, and species are artificial. Some evidence supported nominalistic concepts. Ehrlich and Raven (1969) documented many cases of reduced gene flow in both plants and animals that would preclude any cohesive force to maintain species. They contended: Selection alone is both the primary cohesive and disruptive force in evolution ... for sexual organisms it is the local interbreeding population and not the species that is clearly the evolutionary unit of importance.

Rieseberg and Burke (2001) countered this view, arguing that prior studies grossly underestimated levels of gene flow, and that only very low rates of gene flow are actually needed for the diffusion of strongly advantageous alleles needed to maintain species integrity. III. CLASSIFICATION PHILOSOPHIES IN WILD AND CULTIVATED PLANTS

A. Wild Plants The previous section reviewed a variety of types of data and analytical and philosophical methods used to define species, and similar criteria are used to group species into higher ranks (as genera, families, and orders). The early classifications were based on intuitive interpretations of morphological data, and in many cases, they defined groups that have continued to be maintained. For example, the grass family, sunflower family, and many other traditional taxa are clearly natural as determined by molecular data. The intuitive, interbreeding, phenetic, cladistic, and eclectic classification philosophies mentioned in the previous section for species also are used to group species within genera, and all but the interbreeding classification philosophies have been used to classify above the genus level (reviewed in Stuessy 1990; Judd et al. 1999). Many botanists today examine cladistic relationships, but major disagreement rests on how to translate cladistic results into a classification. Some argue (e.g., Stuessy 1990), that cladistic data are only one component of phylogenetic relationships, other components being chronistic (time of divergence of

21

1. PLANT NOMENCLATURE AND TAXONOMY

clades), patristic (amount of character divergence within lineages), and phenetic (overall similarity). There are no algorithms to incorporate all of these data types into a classification, however, unlike classifications based on phenetics or cladistics, and intuitive judgments are still used by many to construct these eclectic classifications. B. Cultivated Plants The inconsistencies and lack of agreement of taxonomists dealing with the same materials are remarkable, to say the least, and are even more striking when the treatment of differing crops are compared.

Harlan and de Wet (1971)

The major goals of taxonomy reviewed suggest that for agronomists and horticulturists, stability and predictivity would be very important. The philosophies and practices to define wild species and to group them into genera and higher-level ranks are wide and diverse. The question remains-what would happen if different taxonomists were to work on the same group of plants and produced different classifications? Would one be "better," and by what criteria could we judge one classification to be "better"? These questions can be explored by comparing such different classifications, and we give examples from tomato, potato, Brassica, lettuce, Prunus, and wheat. One of the major reasons for discrepancies among taxonomic treatments is that the taxonomy of plants is often complicated by the occurrence of outcrossing, selfing, apomixis, clonal propagation, or polyploidization, producing different variation patterns that can be difficult to subdivide into easily recognizable units. The taxonomy of cultivated plants has the extra complication of the influence of the domestication process on variation patterns, with domestication having major and rapid effects on morphological characters used for classifications. 1. Tomato. Spooner, Anderson, and Jansen (1993) examined outgroup relationships of tomato (many recognize as the genus Lycopersicon Mill.) to potato and other members of the Solanaceae L. (Fig. 1.4). The results convincingly showed tomato to be firmly internested in the genus Solanum L. Based on these results, Spooner et al. (1993) followed a cladistic classification to assign tomato to the genus Solanum, matching the original treatment of Linnaeus (1753), and a minority of other taxonomists who foresaw this generic relationship based on morphological data. Subsequent molecular studies unequivocally supported the cladistic placement of tomato in Solanum (Olmstead and Palmer 1992,1997;

22

D. SPOONER, W. HETTERSCHEID, R, VAN DEN BERG, AND W. BRANDENBURG

, - - - - - - - - Capsicum

Outgroups . . . . - - - - - - - - - Datura ] Cyphomandra (now Solanum) Solanum pseudocapsicum

'--__...;----- s. quitoense

j Other Solanum

' - - - - - - - S. macrocarpon L-

S. nigrum

Lycopersicon esculentum ]

L. chmielewskii

Tomato

L. peruvianum S.lycopersicoides ] Tomato outgroups in S. sitiens sect. Lycopersicoides S. ochranthum and sect. Juglandifolium S. agrimonifolium ] S. phureja S. verrucosum

Potato

S. albornozii

S. bulbocastanum

S. brevidens

]

S. etuberosum

Solanum sect. Etuberosum

S. fernandezianum

S. suaveolens ] S. muricatum L.-

S. taeniotrichum

Solanum sect. Basarthrum

S. appendiculatum ] , . . - - - - - S. dulcamara Other Solanum LS. jasminoides

Fig. 1.4. One of two-most parsimonious cladograms (as a phylogram) of chloroplast DNA restriction site data examining wild tomatoes (here labeled Lycopersicon), their sister groups (Solanum sect. Lycopersicoides, sect. juglandifolium) , wild potatoes (Solanum sect. Petota), and further outgroups in Solanum sect. Etuberosum, sect. Basarthrum, and other Solanum (modified from Spooner et al., 1993).

Bohs and Olmstead 1997, 1999; Peralta and Spooner 2001). These unequivocal cladistic results are stimulating many taxonomists to place tomato in Solanum, but many agronomists and horticulturists have not accepted the name (but see Van der Heuvel et al. 2001). Most users of Lycopersicon clearly base their reluctance entirely on a desire to maintain nomenclatural stability rather than adherence to any particular classification philosophy. Ingroup relationships within tomato have varied greatly. Muller (1940), Luckwill (1943), and Child (1990) treated tomato based on tax-

23

1. PLANT NOMENCLATURE AND TAXONOMY

Luckwill (1943)

Rick (1919)

Child (1990)

GBSSI sequence

Subgenus Eulycopersicon Esculentum complex Series Lycopersicon Group 1 L. esculentum (8) L. esculentum (2) S. lycopersicum S. lycopersicum L. pimpine11ifolium - - - L. pimpinellifolium - - - S o pimpinellifolium--- S. pimpine11ifolium L. cheesmaniae S. cheesmaniae S. cheesmaniae L. pennellii ~ Series Neolycopersicon Group 2 L. hirsutum -~ ' - - - - S. pennel1ii S. chmielewskii L. chmielewskii "-.\_ Series Eriopersicon S. perovianum N L. parvinor~m ~ S. habrochaites S. neorickii Subgenus Eriopersicon Peruvianum complex S. chmielewskii Group 3 L. perovianum (5)~-4+- L. chiJense S. chilense S. pennel1ii L. pissisi- ? L. perovianum S. peroVianum S. habrochaites L. cheesmaniae (2) S. neorickii S. neorickii S. chilense L. hirsutum (2) S. perovianum S L. glandulosum

Fig. 1.5. A comparison of taxonomic treatments of wild tomatoes from Luckwill (1943; a taxonomic morphological species concept), Rick et al. (1979; a biological species concept), Child (1990; taxonomic morphological species concept), and a possible cladistic interpretation of a GBSSI DNA sequence cladogram of Perlata and Spooner (2001; Fig. 1.6).

onomic morphological species concepts. The treatments of Rick (1963, 1979) and Rick, Laterrot, and Philouze (1990) grouped the species completely differently based on interbreeding concepts (Fig. 1.5). Peralta and Spooner (2001) produced a phylogeny of tomato based on DNA sequences of the single-copy GBSSI (waxy) gene (Fig. 1.6 on page 24). It could be interpreted to distinguish three groups. One of the species, the highly polymorphic Solanum (Lycopersicon) peruvianum 1. would be placed into two groups, one consisting of populations from northern Peru, and another of populations from central to southern Peru (Fig. 1.5). A phenetic morphological study by Peralta and Spooner (in press) supported all species, including a north and south Solanum peruvianum species. A taxonomic monograph of tomato is in preparation by Iris Peralta, Sandra Knapp, and David Spooner. 2. Potato. Ingroup relationships within potato have differed even more than within tomato. Harlan and de Wet (1971) advanced the gene pool concept (a variant on the biological species concept) based on their frustration with traditional taxonomy to provide consistent answers to relationships of crops and their wild relatives. They initially tried to use taxonomic treatments of crops to give insight into materials to use in their breeding programs. They noted, however, such great discordance between taxonomic treatments of potato, maize, wheat, and sorghum that they rejected traditional treatments and constructed the gene pool classification. They compared the taxonomic treatments of potato of

24

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

Solanum lycopersicum Solanum pimpinellifolium Solanum cheesmaniae 70(3) ......---- Solanum peruvianum north 1 - - - - - Solanum chmiewelskii 100 Solanum neorickii Ingroup 1 - - - - - Solanum peruvianum south 1 - - - - - - Solanum chilense \ - - - - - Solanum habrochaites Solanum pennellii 64(2) 100 Solanum j uglandifolium Solanum ochranthum 100 Solanum lycopersicoides 86 (3) Solanum sitiens L..--~":"'Solanum tuberosum Outgroup 97 Solanum bulbocastanum Solanum jamesii 100 Solanum etuberosum Solanum palustre Solanum muricatum 24 (l)

...----+--

L--

L..--

1--

Fig. 1.6. Abstracted results ofa GBSSI (waxy) gene phylogeny of wild tomatoes and outgroups (Peralta and Spooner 2001).

Bukasov (1933) and Bukasov and Kameraz (1959) to Hawkes (1963), and noted that Hawkes (1963) recognized about one-half as many species, and grouped these species very differently into series. Their classification starts from the crop itself. Crossability is represented in a graph with three genepools, with the primary genepool 1 being the crop and wild species easily crossable to it, and the second and third being the rest of the plant kingdom, according to degree of crossability to the crop (genepool 2 crossable with some difficulty, genepool 3 crossable with great difficulty). Genepool1 is based on the biological species concept and is then to be subdivided in two subspecies, one with spontaneous populations, the other containing the cultivated "races" (their "race" not being equivalent with cultivar of cultivated plant classification). This system is a very special purpose classification and not an alternative to any form of taxonomy in general. Why? Every primary genepool chosen results in a separate classification based entirely on the choice of each crop used for comparison. This could lead to as many dif-

1. PLANT NOMENCLATURE AND TAXONOMY

25

ferent classifications of plants as there are primary genepools for comparison. This is unacceptable, since genepools 2 and 3 contain close and less close relatives of the primary genepool "species" and do in fact thus represent the entire rest of plant kingdom (genepool 41). The relationships of the genepools with the rest of the plant kingdom remain unresolved. Another objection is that the category of subspecies is misused for convenience to contain either wild plants or cultivated material. This last option creates an unfortunate hybrid between the taxonomy of wild plants (the category itself) and of cultivated plants (the actual content of the category). Similar problems exist in the biological species concept. The system proposed by Harlan and De Wet has already led to many "infraspecific" classifications of crops, using Latin binomials for cultivated plant groups that as we argue should be avoided. Spooner and van den Berg (1992) followed up on the Harlan and de Wet (1971) comparison of potato with an examination of later taxonomic treatments of Bukasov (1978) and Hawkes (1990), and added Correll (1962) and Gorbatenko (1989) (Fig. 1.7). All four of these authors apparently applied a taxonomic morphological species concept, but Hawkes (1990) also took intercrossability data into account. The treatments differ in the number of series recognized, the number of species in each series, and the different affiliation of species to these series. Likewise, Spooner and van den Berg (1992) also compared the nearsimultaneous independent publication of the potatoes of Bolivia by Hawkes and Hjerting (1989) and Ochoa (1990). Their treatments differed in the number of species recognized, their affiliation to series, the taxonomic rank used to recognize divisions of species (botanical varieties or subspecies), and in their hypotheses on which of these species are of hybrid origin and whether they form introgressant populations with other species. 3. Lettuce. The influence of domestication on taxonomic classification is shown very clearly in cases where through the domestication process the morphology of the plants are changed, and species are described based on these morphological characters. In the case of lettuce, the obvious differences between cultivated and wild material led to the distinction of the species Lactuca sativa 1. as separate from its presumed wild ancestor L. serriola 1. Cultivated material lacks the prickles on the midrib of the lower side of the leaves, and shows prominent heads. De Vries and van Raamsdonk (1994) reiterated this separation in a numerical morphological analysis. However, it is highly questionable whether botanical species should be recognized using characters that are clearly the results of human selection. An alternative classification would be to consider wild and

26

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG Correll 1962 Bukasov 1978

]uglandifolia (Rydb.) H.wk.. 1944 MoreUiformia H.wk., 1956 Bulbocastana (Rydb.) H.wk.~ 1'/44 Pinnatisecta (RyJb.) H.wk.. 1944 Trifida Correll 1950 Cardiophylla Bul< .. CA,....1I1952 Polyadenia Buk.exCorre1l1952

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Fig. 1.7. A chronological history of hypotheses of placement of wild potato species into series by Correll (1962), Bukasov (1978), Gorbatenko (1989), and Hawkes (1990). The nonitalicized series names of Bukasov were treated as series but not validly published. Solid lines connecting series indicate the maintenance or transfer of species between series. Dotted lines indicate that an author did not treat these series. The numbers in circles are the number of species accepted for the series. (From Spooner and van den Berg 1992).

1. PLANT NOMENCLATURE AND TAXONOMY

27

cultivated lettuce as the same species, that would, unfortunately, have to be called 1. sativa, this being the earlier name. Different nomenclatural solutions can be proposed. The name Lactuca serriola could be considered a synonym of L. sativa. Alternatively, Frietema de Vries (1996) recognized two subspecies: subsp. sativa encompassing the groups of cultivated lettuce and subsp. serriola (L.) Frietema de Vries for the wild material. 4. Brassica. The taxonomy of cabbage

(Brassica L. spp.) is extremely complex. Even the delimitation of genera in this part of the family Brassicaceae can be questioned. Raphanus L. and Sinapis L., the genera including radish and mustards, are closely related to the genus Brassica. Viable hybrids have been produced from Brassica x Raphanus crosses (described in the nothogenera x Brassicoraphanus Sagerent or x Raphanobrassica Karpechenko, depending from which taxon the female parent originated). Within the genus Brassica, a number of species form a closely-knit network. Cytogenetic studies ofD (1935) showed that three primary species (B. oleracea L., B. rapa L., and B. juncea (L.) Czern.) formed amphidiploid species, resulting in the "triangle of D." Molecular work confirmed these findings and established the male and female contributions of each primary species, using chloroplast DNA restriction site data (Palmer et al. 1983) and nuclear restriction site polymorphism data (Song and Osborn 1992). Brassica oleracea displays an extreme variability, where through different directional selection a large number of crops was produced, each targeting a different organ of the plant (leaves surrounding the terminal bud: cabbage and kale; enlarged axillary buds: Brussels sprouts; inflorescence: cauliflower and broccoli; swollen bulblike stem: kohlrabi). All of these variants were classified in botanical varieties, which in modern cultonomic terms would be cultivar-groups. A number of wild taxa associated with Brassica oleracea have been described but their exact number and delimitation have remained a matter of debate. Harberd (1972) proposed the concept of cytodemes: groups characterized by having a constant chromosome number, all accessions of the same cytodeme being fully interfertile while those of different cytodemes are essentially cross-sterile. He listed 11 species as members of the Brassica oleracea (2n=18) cytodeme that nearly all have been regarded as subspecies of Brassica oleracea at some time. Snogerup (1979,1980) recognized only seven wild taxa, reducing some species to a B. rupestris-incana complex, which itself contains eight species names. Hanelt (2001) listed ten species in a Brassica oleracea group, and followed Helm (1963) for the infraspecific classification of the cultivated forms within B. oleracea, using subspecies, convarieties and varieties.

28

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

5. Prunus. Cherries, plums, peaches, apricots, and almonds all are classified as species within the genus Prunus 1., besides many other wild species and species that are in use as ornamentals. Rehder (1960) discussed 77 cultivated species out of the nearly 200 species. Other sources mentioned up to 430 species within the genus (Royal Horticultural Society, 1992). A major difference is found in the infrageneric classification. This treatment subdivides Prunus into a number of subgenera and these in sections. Plums (Prunus domestica 1.) and apricots (P. armeniaca 1.) are accommodated in different sections of subgenus Prunophora (Neck.) Focke ex Engl. and Prantl. Almond (Prunus dulcis [Mill.] D. A. Webb) and peach (P. persica Batsch) are placed together in subgen. Amygdalus (1.) Benth. and Hook. f., and sour and sweet cherries (P. cerasus 1. and P. avium 1.) in subgen. Cerasus Pers. The flora of the USSR (Shishkin and Yuzepczuk 1971) and Hanelt (2001), however, recognized several genera within a subfamily Prunoideae Focke. Not only is the taxonomic level of many groups changed from subgenus to genus, but also the affinities between the different crops is interpreted differently, with sour and sweet cherries still together in one genus Cerasus, but almonds and peaches now placed in the separate genera Amygdalus 1. and Persica Mill., respectively. 6. Wheat. Hybridization and polyploidization have played an important role in the origin of bread wheat (Zohary and Feldman 1962). At the diploid level crosses between the wild species Triticum boeoticum Boiss. (wild einkorn) and a species from a different grass genus, Aegilops speltoides Tausch, produced tetraploid material (wild and cultivated emmer). The process was repeated on the tetraploid level with another species from the genus Aegilops 1., A. squarrosa, resulting in hexaploid Triticum tauschii (Casson) (= T. aestivum auct. Mult. non 1.) with a genome formula AABBDD reflecting the contributions of at least three species with different genomes. The apparent crossability of Aegilops and Triticum 1. led Stebbins (1956) to propose the unification of these two genera, which was supported by Bowden (1959). MacKey (1981) suggested that one might even lump the whole tribe Triticeae Dumort. based on the criterion of crossability. He favored a separation of the genera because of the discontinuity developing in this complex, with Aegilops evolving towards weediness, and Triticum, under the influence of human selection, following a completely different trend in ear construction. The number of species within the genus Triticum varies dramatically among treatments. Thellung (1918) recognized only three wheat species (T. monococcum 1., T. turgidum 1., and T. aestivum). MacKey (1966,

1. PLANT NOMENCLATURE AND TAXONOMY

29

1968) recognized six biological species within the genus Triticum, and MacKey (1981) criticized the detailed hierarchical subdivision of the genus by Dorofeev and Korovina (1979), who used the categories subgenera, sections, species, subspecies, convarieties, subconvarieties, varieties, and forms. Dorofeev and Korovina (1979) recognized 27 different species, subdivided in no less than 1031 varieties. Hanelt (2001) listed eight species divided into various subspecies. These comparisons illustrate that the very wide differences in taxonomic interpretations of the same group persist, continuing to present agronomists and horticulturists with a confusing decision as to which one to use. Different taxonomists continue to provide alternative taxonomic treatments of the same group of organisms, as was so effectively shown by Harlan and de Wet (1971). These treatments are sometimes vastly different based on different classification philosophies. A purpose of this chapter is to explain these different philosophies and show why the treatments of crops differ, and why names are being changed. Adherents to different philosophies will strongly argue that their classification is "better" in the sense of being more predictive. However, we are aware of no objective test of this claim of predictivity in any crop plant, and such tests are needed to test predictivity of alternative classifications. There is no taxonomic consensus emerging on classification philosophies, and different treatments of both wild and cultivated plants are likely to persist.

IV. BRIEF HISTORY OF NOMENCLATURE AND CODES

One of the most remarkable features of the human species is our ability to use a highly developed system of speech to communicate. There are unique words to identify unique objects but also many words to identify collections of items. These collective terms are the result of our innate need to classify objects into groups, and groups of groups. Such collective terms represent not only the group itself but also the way in which humans have decided to classify the objects. Needless to say, different cultures may use different criteria to group a set of objects, resulting in different classifications for that same set. Interestingly, the very term classification dates back to the Greek botanist Theophrastus in the third century BeE. If terms are not made distinct between different groups in classifications, confusion will eventually arise. For biological classifications that have universal applicability, a standardized system of nomenclature would eventually be necessary. Scholarly texts by early botanists (e.g. herbalists) identified groups of plants by using long Latin (the academic

30

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

language at the time) phrases enumerating characters of those plants. This cumbersome method cried out for a simplification. The first successful attempt to standardize names to classify all known plants was from Linnaeus (1753). His classification was based on similarities and differences among groups of plants. Those showing "relevant" similarities were grouped together. Linnaeus' most fundamental object in his classification was the species. He used the species in its oldest definition, being an immutable entity (the typological species, an outdated morphological species concept that allows little to no variation within species). The next higher level of grouping of species was the genus. Linnaeus used a combination oftwo Latin terms (one for the genus and one for the specific epithet) to singularly identify and name a species. Thus binary nomenclature was born, which we still use today (Stafleu 1971). A full species name consists of three elements: (1) a genus name, (2) a species epithet, and (3) a taxonomic author (e.g., Solanum tuberosum L.). Sometimes two authors follow a plant name, as in the wild potato name Solanum brachistotrichium (Bitter) Rydb. Friedrich Bitter described the plant as a variety, and Per Alex Rydberg transferred it to the species level. This originally published name is the basionym, or the original name later transferred in rank (here from variety to species). This use of two authors is not meant to serve as a credit device, but rather as a very useful way to trace the nomenclatural history of names. Brummitt and Powell (1992) provide a reference of taxonomic author abbreviations that is listed in many standard instructions to authors of scientific journals. This is useful because of many variants on abbreviations that introduce confusion in citations. Heiser and Janick (2000) point out that authors of many crops are improperly listed in standard references of names (e.g., Liberty Hyde Bailey Hortorium 1976), and may be unnecessary. New references (e.g., Wiersma and Leon 1999; Hanelt 2001) are properly listing species names of crops to help alleviate this problem, and bolster Heiser and Janick's argument to eliminate taxonomic authors in journals. Standard ranks in the taxonomic hierarchy from lowest to highest are form, variety, subspecies, species, series, section, genus, tribe, family, order, class, division, and kingdom. If more ranks are needed, a potentially infinite number can be created for all ranks by using qualifier terms such as sub or super, for example to create subgenus or supergenus. Families have standard endings of -aceae (e.g., Rosaceae), orders -ales (Rosales). Eight economically important angiosperm families have alternative family names of long usage not ending in -aceae that are maintained to keep links to the older literature. These are: Apiaceae

1. PLANT NOMENCLATURE AND TAXONOMY

31

(Umbelliferae), Arecaceae (Palmae), Asteraceae (Compositae), Brassicaceae (Cruciferae), Clusiaceae (Guttiferae), Lamiaceae (Labiatae), Fabaceae (Leguminosae), and Poaceae (Gramineae). Most ranks currently used were developed in the eighteenth century, and are mental constructs that are not comparable across all plants. There are no objective criteria or set of characters to indicate what taxonomic level is a genus, family, and order. As such, families or any rank are not comparable regarding age or diversity. Put in a phylogenetic context, traditional ranks are not necessarily equivalent in that they do not designate sister clades. Ranks only have meaning in a relative (not absolute) sense in that a genus is less inclusive than a family, and a family is less inclusive than an order (Stevens 1998). Because there are no universally accepted definitions of what constitutes a genus, species, or other rank, they are interpreted differently by different taxonomists. Lumpers are taxonomists who focus more on similarities than differences, discount the importance of minor variation among individuals, and tend to recognize fewer taxa. Splitters, on the other hand, focus on small differences among individuals and recognize more taxa. Both of these terms have negative connotations as they refer to extremes of taxonomic interpretation, but in reality they are relative terms and are best applied when independent taxonomists treat the same group in different ways. Botanists agreed that nomenclature should be as stable as possible and not change drastically with new classifications. De Candolle (1867) devised a set of nomenclatural rules, which finally led to the first edition of the Regles de la Nomenclature Botanique, later editions being published as the ICBN. The ICBN is amended every six years, based on votes at the International Botanical Congress. The last Congress was held in St. Louis, Missouri, in 1999, resulting in the latest edition of the ICBN (Greuter et al. 2000). The ICBN has six main principles. The first three enter into our discussion of how plants get their names, and how new names may be assigned to species that previously had another name.

Principle I. Botanical nomenclature is independent of zoological and bacteriological [and viral] nomenclature. As discussed in Section VILA., however, some suggest a unified code (BioCode) should be sought. Principle II. The application of names of taxonomic groups is determined by means of nomenclatural types. A nomenclatural type is that element (usually a herbarium sheet) to which the name of a taxon is permanently attached. What this means is that

32

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

when a species is described, a type specimen needs to be designated that serves as a reference point for others to compare to their concept of names. Principle III. The nomenclature of a taxonomic group is based upon priority of publication. This means that the earliest validly published name is the proper name assigned to a taxon. In order to provide an "out" to the strict application of these rules for well-known and frequently used plant names (as economic plants), the ICBN provides a way to avoid name changes based on principles II and III called conservation of names of families, genera, and species. This is done by petitioning for a name to be conserved (nomina conservanda). Anyone can petition to have names conserved by a published proposal in the journal Taxon. This proposal is voted on at the general assembly of the International Botanical Congress, and if a majority accepts the proposal the name is added to a list of conserved names in the ICBN that are valid despite having violated rules of the Code. For example, it was discovered that the proper name for the cultivated tomato within the genus Lycopersicon was not the well-known name Lycopersicon esculentum Mill., but rather 1. lycopersicum (L.) H. Karst. A petition was made and later voted on by a majority at a Congress to conserve the name L. esculentum. Conservation is valid however, only within the genus Lycopersicon. If a later taxonomist decided that tomato was better classified in the genus Solanum, the conservation has no validity outside of the genus Lycopersicon. This point is frequently misunderstood by those who think that conservation is meant to preserve classifications (e.g., Merrick 2000). Conservation is only a nomenclatural device that operates within the taxa in which they have been voted upon, and is not operative if the taxon is classified in another group. What does it take to validly publish a new species or other taxon name? Not much. There is no license needed, nor do you need to have an academic degree, or be a botanist, or to be part of any professional organization. Anyone can validly publish a new taxon if they: (1) provide a description or diagnosis (a short statement of how the new taxon differs from something similar) in Latin; (2) designate a type specimen (generally a single herbarium specimen affixing the name to the new taxon); (3) follow rules outlined in the ICBN such as using the proper form of a name regarding spelling and not using names that have been validly published before; (4) effectively publish the new name. Effective publication typically is through peer-reviewed scientific taxonomy journals, but it is stipulated in the ICBN only as "distribution of printed matter (through sale, exchange, or gift) to the general public or at least to botanical institutions with libraries accessible to botanists generally."

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33

Botanists keep track of most new taxon names through indexing services such as the Gray Card Index (Gray Herbarium, Harvard University, Cambridge, Massachusetts) or the Index Kewensis (Royal Botanic Gardens, Kew, UK), or the International Plant Name Index (http://www.ipnLorg/), maintained by these two organizations and the Australian National Herbarium. We've described the basics of nomenclatural principles and rules inherent in the ICBN. This system is commonly referred to as "Linnaean nomenclature" or "traditional nomenclature." The term Linnaean nomenclature is actually a misnomer, however, because some key features of the ICBN postdate Linnaeus, such as the principle of priority, the reliance on type specimens, or even the use of families. Therefore we use the term traditional nomenclature here. Although many early botanical texts dealt with cultivated plants, their role in the development of plant nomenclature was very limited (Hetterscheid et al. 1996). Focus was directed mainly toward plants in the wild, untouched by humans. Cultivated forms were to get "fancy-names" (as Rosa 'Splendor') but these names would have to be linked to botanical Latin names of the species, subspecies, or botanical varieties from which the cultivated plants were derived. As such, the nomenclature of cultivated plants became a mere appendix of the nomenclature of wild plants. Cultivated plants have many differences from wild plants, however, and the linking of their nomenclature to the ICBN became untenable. In 1952, a proposal was published (Lanjouw et al. 1952) for an independent set of nomenclature rules for cultivated plants, and in 1953 resulted in the first edition of the International Code of Nomenclature for Cultivated Plants (ICNCP; Stearn 1953). Other changes to the Code are discussed in three separate Proceedings of the International Symposia on Taxonomy of Cultivated Plants: (1) Acta Horticulturae 182(1), 1985; (2) Acta Horticulturae 413(2), 1994; (3) Taxonomy of Cultivated Plants, Proceedings of 3rd International Symposium, Royal Botanic Gardens, Kew, 1999. The reason for divorcing both codes was hardly fundamental but more of a practical nature. Most botanists dealing with the ICBN did not want to discuss cultivated plant nomenclature at length, and were glad to dispose of these rules to a different nomenclatural body. V. FUNDAMENTAL DIFFERENCES IN THE CLASSIFICATION AND NOMENCLATURE OF CULTIVATED AND WILD PLANTS

In the interest of nomenclatural stability it may at first glance seem wise to standardize the nomenclature of biological objects once and for all. This would not allow, however, for changes to accommodate new

34

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

theories of evolution and classification. For example, Linnaeus's very useful development of binomial nomenclature predated modern ideas of evolution espoused by Darwin (1859). If traditional nomenclature was "frozen" since the time of Linnaeus we would be burdened with an archaic system with little biological relevance. Hence, classification rules change along with new data and needs (every six years for the ICBN, irregularly for the ICNCP). We outline major emerging new proposals for classification changes for the ICNCP.

A. Ambiguity of the Term Variety The term variety has caused much confusion. One meaning, as used by the ICBN (the "botanical variety"), is a particular rank in the taxonomic hierarchy below the rank of species and subspecies and above the rank of form (form/variety/subspecies/species). Another meaning, as used in the ICNCP (the "cultivated variety" or "cultivar"), refers to cultivated variants originating through human influence. Regarding the use of variety in the ICBN, species variation has been subdivided through infraspecific classifications. The relationships of the infraspecific categories allowed in the ICBN are strictly hierarchical, and as such they are differentiated by their degree of uniqueness: subspecies within a species should differ less among themselves than separate species, varieties should differ less among themselves than subspecies, and forms less than varieties. In practice, however, different taxonomists treat variation patterns differently. For example, sometimes a species is subdivided in subspecies and these in varieties, but in other cases a species is subdivided directly into varieties and the subspecies rank is not used at all (Hamilton and Reichard 1992). Some taxonomists feel that recognizing subspecies indicates a geographical component, with subspecies being mostly allopatric, while varieties may be sympatric. This is not a formal or universally held distinction between these ranks, however. The term becomes especially confusing with the wish to assign cultivated plants to the species from which they originate, resulting in the application of the term to cultivated plants in a form that appears as a botanical variety. The use of the rank variety for cultivated plants goes back to Linnaeus (1753). In many cases Linnaeus started his treatment of a species with the wild plant, mentioning cultivated varieties at the end (Wijnands 1986). Linnaeus clearly considered varieties as minor variants due to the influence of climate or soil, or in the case of cultivated varieties, of human influence. He later stated that the grouping of cultivated plants should be the task of beginners in botany, while qual-

1. PLANT NOMENCLATURE AND TAXONOMY

35

ified botanists should study species and higher taxonomic levels (Linnaeus 1764). Many later workers on the taxonomy of cultivated plants continued the practice of applying variety names for cultivated plants, burdening nomenclature with formal names with all the inherent problems of typification and priority that these entail. In these systems (e.g. Helm, 1957, 1963) the varieties are often grouped in artificial higher categories like convariety (or convar). Convarieties can be roughly comparable to cultivar groups, but convarieties, unlike cultivar groups, do not necessarily contain named varieties, and convarieties are members of traditional "Linnaean" ranks. The ICNCP replaced this term with the term cultivar-group, and convarieties should not be used in modern cultivated plant taxonomy (Trehane et al., 1995). Some modern influential works, however (e.g., Hanelt 2001), ignore rules of the ICNCP and continue to use the term convar (convariety). The term cultivated variety (cultivar) in the ICNCP, in contrast, is used in a very different way. The botanical variety has its fixed position in the taxonomic hierarchy. The cultivated variety stands outside this hierarchy because it could have resulted from many different processes as selection or a complex series of interspecific hybridizations, making it impossible to assign it a position in the hierarchy. Because of this, the nomenclature of the cultivated variety follows the ICNCP, dispensing with Latin epithets used in hierarchical ranks in the ICBN. Presently, however, names originally published as botanical varieties still refer to cultivated material. These entities can be reclassified as cultivars, or if a botanical variety was described to encompass many cultivated morphotypes (as is the case in the classification of Brassica oleracea) they can be reclassified as cultivar-groups (van den Berg 1999). Thus, the botanical variety Brassica oleracea var. gemmifera can be reclassified as Brassica oleracea Gemmifera Group, encompassing the many cultivars of brussels sprouts. However, the term variety for cultivar is still in wide use in legal documents all over the world. ICNCP deals with this in stating that the term variety as used in such texts is fully equivalent to cultivar (ICNCP, art. 2, note 2; art. 2.4). Legally, variety can have additional definitions. For example, the U.S. Plant Variety Protection Act (PVPA) uses the term "cultivar" in a manner similar to the botanical "variety," but with the additional stipulation that "development" must take place from wild stock, as through breeding or genetic engineering. That is, discovery of unique variants alone does not make a cultivar eligible for protection under the PVPA. In addition, PVPA protection of varieties is granted with the additional requirements that it is "new," "distinct" from other cultivars, "uniform," and "stable."

36

D. SPOONER, W. HETTERSCHEID, R. VAN DEN BERG, AND W. BRANDENBURG

The botanical rank form has also been used extensively to describe minor variants of cultivated plants. Its use in the classification of wild plants is generally discouraged because the entities that could be described as forms are usually such minor morphological variations that it is arguable whether their distinction is useful. For cultivated plants these forms may easily be reclassified as cultivars. The same goes for the many informal and often ill-defined terms like strain, sport, type, and so on. If any such entity is worthy of recognition and description, it will be best to employ the general term cultivar for all of these. B. Culton Versus Taxon

A fundamental difference between the ICNCP and ICBN is their respective approach toward classification. Groups of plants used in the ICBN to classify and name are collectively designated as taxa (singular: taxon). The ICNCP uses the terms cultivar and cultivar-group for cultivated plants. Although it claims that they are taxa, these terms do not fit the definition of taxa for several reasons. This may become clear by the definition in the ICNCP of the term cultivated plant: A cultivated plant is one whose origin or selection is primarily due to the intentional activities of mankind. Such a plant may arise by deliberate or, in cultivation, accidental hybridization, or by selection from existing cultivated stock, or may be a selection from minor variants within a wild population and maintained as a recognizable entity solely by deliberate and continuous propagation.

A key point is the influence of humans on the origin of cultivated plants, disrupting natural evolutionary and environmental factors and constraints. Plants in the wild are subject to natural selection, whereas cultivated plants are subject to conscious or unconscious human selection. Hetterscheid, van den Berg, and Brandenburg (1996) have argued that classifications of cultivated plants and wild plants have different goals. Whereas wild plants are classified in a system that seeks to clarify evolutionary relationships, cultivated plants are (or should be) classified according to special purpose user-defined criteria, with stability of names as primary, requiring a totally different classification philosophy. Practitioners of the taxonomy of cultivated plants have not yet completely accepted this (Hetterscheid and van den Berg 1996). Since the term taxon is used as a basis for evolutionary classifications, it seems illogical to use the same term for very different kinds of classifications. The most important consequence of this is the substitution of the concept of "culton" for "taxon" for systematic groups of cultivated plants

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(Hetterscheid and Brandenburg 1995a, 1995b), but this term has not yet been included in ICNCP rules to the full extent. The definition reads: A culton is a systematic group of cultivated plants based on one or more user-criteria. A culton must have a name according to the rules of the International Code of Nomenclature for Cultivated Plants.

This definition emphasizes the essential role of human activity, in using the term "user-criteria" as the sole basis for the creation of systematic groups of cultivated plants (culta). This does not preclude studies of the origin of cultivated plants from existing natural populations. The point here is to divorce the nomenclature of cultivated plants from closed classifications that imply relationships, because artificial selection and hybrid origins often render this system nonsensical and nomenclaturally unstable. C. Open Versus Closed Classifications

Classifying plants involves putting sets of individual plants in boxes, where the boxes are the ranks in the taxonomic hierarchy (e.g., species, genus, family, order). On the basis of classification criteria, a number of individuals are put in a box. This system of boxing has one important principle: every box belongs in a higher, more inclusive (larger) box and, vice versa, every box contains one or more boxes itself, with the largest box being "life." In classification terms, this equates to: one or more species add up to form a genus, one or more genera add up to form a family, all the way through the taxonomic hierarchy. When we supplant the term box with taxon we have described the classification system of the ICBN and which is called a closed classification system. The ICBN says that there are an infinite number of levels (ranks) that can be constructed and named. Some ranks are specifically mentioned (e.g., the ranks called subspecies, species, genus, family, order) but their number may be increased infinitely. This is the nature of the hierarchy of levels typical in traditional nomenclature. Another mechanism typical for closed classifications is that when the individuals in a certain box (taxon) are going to be put in smaller boxes, all those individuals must be in smaller boxes and not one may be left on its own in the larger box. For that one leftover, a separate box has to be created and even named. The content of the boxes (taxa) is determined for a particular group of individuals by a taxonomist studying that group. Currently, evolutionary relationship is the primary criterion for grouping plants in taxa.

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Such classifications are called natural classifications because they describe relationships that taxonomists think are "real" in nature (see "Culton Versus Taxon"). Typical for such classifications is the use of as many characters as possible to gain a solid evolutionary basis. This frequently is contrasted to artificial classifications that are based on nonevolutionary special-purpose criteria. Whereas taxonomists of wild plants follow this closed classification system, taxonomists of cultivated plants have good reason not to use it (Hetterscheid, van den Berg, and Brandenburg 1996). Cultivated plants are created according to the whim of individuals. They arise by considerable hybridization and rapid selection for extreme forms. Classifying cultivated plants is directed by needs of individual groups of users, completely unlike the reason for classifying wild plants. Whereas one group may want to classify certain cultivated plants based on resistance against pests, another group may want to classify those same plants based on ornamental value. As such, the classification criteria for a certain group of cultivated plants may vary considerably, leading to the need of several coexisting special purpose classifications. Typically such classifications use few criteria and are sometimes called "artificial" classifications. In the philosophy of open classification (Brandenburg, Oost, and van de Vooren 1982; Brandenburg, 1986) special purpose classifications are allowed. In this system, the only boxes created are those needed for utility to users. That is, cultivated plants not possessing the characters of interest are left out of that particular classification. This is quite logical, because if we would include this last group, it would have to be based on not having a number of characters, which is contrary to the goal of the classification in the first place. For instance, in a classification of a crop the attribute of leaf shape may have led to the recognition of a box called the Laced-leaf Group and a box called the Dentate-leaf Group. The mere existence of these two boxes does not mean that all plants of that crop not having laced or dentate leaves automatically define a third box because this would have to be defined as "plants not having dentate or laced leaves," which is quite the opposite of the original classification intent. That particular "left-over" group would contain a very heterogeneous assemblage of cultivars, which is diametrically opposite the whole idea of user-criteria driven classification (for examples see Hetterscheid and van den Berg 1996; Hoffman 1996; Hetterscheid et al. 1999). Another simplifying attribute of open classifications compared to closed ones is the avoidance of complex hierarchies and hierarchy names. Currently, classifying cultivated plants in an open classification system only requires two categories, the cultivar and the cultivar-group

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(van den Berg 1999). Fewer names means greater stability of names (Hetterscheid 1999) and this is further aided by the fact that the ICNCP provides substantially fewer nomenclatural mechanisms compared to the ICBN. VI. A COMPARISON OF THE ICBN AND ICNCP The essential differences between codes of nomenclature for wild plants (ICBN) and cultivated plants (ICNCP) are shown in Table 1.2. A. Nomenclatural Types and Standards In the ICBN it is mandatory to designate a conserved specimen (the type specimen) to which a new species name is permanently linked. This specimen is an exclusively nomenclatural device and is designated during a new species description. Type specimens are important in revisionary studies when a taxonomist reconsiders species boundaries. For

Table 1.2

Major differences between the ICBN and the ICNCP.

International Code of Botanical Nomenclature (ICBN)

International Code of Nomenclature for Cultivated Plants (ICNCP)

Nomenclature rules for taxa (groups proposed on the basis of evolutionary classification criteria)

Nomenclature rules for culta (man-made entities)

Exclusively devised for objects classified in a closed classification system

Exclusively devised for objects classified in an open classification system

A potentially infinite number of categories

A limited number of categories, presently the cultivar and cultivar-group

Categories are not defined

The cultivar is defined

No basal rank

The cultivar is the basal unit and cannot be subdivided

Names are fixed to types

A cultivar's name and circumscription are fixed to standards

Basic binomial consists of a genus name plus a species epithet

Basic binomial consists of a (notho-)genus name plus a cultivar epithet

No nomenclature devises apart from the ranked categories

The denomination class as an extra nomenclature device

Reuse of names forbidden (homonymy)

Reuse of names allowed in certain cases

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example, if a taxonomist considers two species to really be only one, type specimens determine the proper application of competing names. In such a case two or more names compete for the final choice of a name, and the ICBN provides rules to determine what is the proper name and what is the synonymized name. Generally the older name must be used (the principle of priority). In other cases, even in the absence of combining two species, existing names may be found to be incorrect because nomenclatural rules were never applied properly from the beginning. In the ICNCP, however, cultivars are essentially not subject to revision. They are static units and once they are defined by a set of characters they are immutable, with fixed boundaries. To accomplish this, the ICNCP presents an alternative mechanism to fix both a cultivar name and its description (ICNCP Art. 32) at the same time with nomenclatural standards that are analogous to type specimens in the ICBN. These standards are specimens or collections of specimens and/or documentation designated by the author, and are deposited in public herbaria. Thus a standard is not exclusively a nomenclatural tool but also an immutable cultivar-defining device. B. Denomination Classes and the Reuse of Epithets A denomination class is a nomenclatural device found only in the ICNCP. It is defined (ICNCP Arts. 6.1, 17.2) as a taxon, or a designated subdivision of a taxon, or a particular cultivar-group, within which cultivar epithets must be unique. The botanical genus is the most often and widely used denomination class, but it can be any taxon as described below. A cultivar epithet must only exist once in every genus because the very nature of cultivars often defies assignment to botanical species, whereas at the level of genus (or hybrid genus) cultivars can be assigned. That is to say that attributing a cultivar to a genus is a relatively simple task and seems to work fine, whereas assigning it to a species is difficult or impossible because there are so many interspecific hybrids (sometimes of multiple hybrid origins) and sometimes companies keep pedigrees a secret. Therefore, a species epithet is not a mandatory part of a full cultivar name, but a (notho)genus is. Thus it would not be allowed to have two ornamental fig cultivars named 'Beauty' because within the genus Ficus only one such name would be allowed. The situation Ficus elastica 'Beauty' and Ficus altissima 'Beauty' would thus not be allowed because both could be Ficus 'Beauty,' and Ficus is a denomination class (the species is not mandatory for nomenclature of the ICNCP).

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In the ICBN, however, a species epithet must never be used twice in the same genus. This would create two homonyms (the same name for different entities). In contrast, Article 26 of the ICNCP allows reuse of cultivar epithets in two cases. The first case allows reuse of epithets only among, but not within, a denomination class as explained with Ficus (ICNCP Art. 26.1). The second case allows reuse of epithets within the same genus, species or other denomination class, subject to various conditions (ICNCP Art. 26.2). For various reasons (mostly in crops with numerous cultivars and long and ongoing breeding histories) a genus may be subdivided in smaller denomination classes. In this case, a particular cultivar epithet may be used in everyone of the designated denomination classes in that one genus. Thus that particular cultivar epithet is allowed to exist more than once in combination with a particular genus name. A well-known example is the three denomination classes within the Brassicaceae: (1) Brassica campestris + B. juncea + B. napus + B. nigra + B. rapa + Sinapsis, (2) B. oleracea, (3) the rest of Brassica. The same cultivar epithet could be applied in all three denomination classes, indicating different cultivars, the names of which would be indistinguishable if they would be combined only with the genus name. Another example is the situation in beets where two denomination classes are present within the species Beta vulgaris, making the use of the cultivar-group names necessary. Often these denomination classes are in fact perfectly useful cultivar-groups even though they may still bear cumbersome ICBN based names (ICNCP appendix IV provides a list of denomination classes at other levels than botanical genus). Sometimes one or more botanical species are used as denomination classes (e.g., in tobacco the species Nicotiana rustica and N. tabacum together form one denomination class). Quite often genera are taken together to form a denomination class or a mixture of genera and species (e.g., in the melon family, a denomination class is established for the genus Citrullus + the genus Cucurbita + the species Cucumis melo). Such supra-generic or mixed denomination classes are often established because the botanically established limits between the taxa in the class are narrow and the breeding history of their cultivars easily transcends these limits. Such a breeding history makes it difficult to impossible to establish the taxon to which one could assign such cultivars. This classification uncertainty would be especially vulnerable to the destabilizing effects when cultivars are shifted from one taxon to another, with a later discovery that a cultivar epithet already exists in that other taxon, thus leading to a mandatory change of the epithet. In short,

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denomination classes enhance cultivar name stability by creating a nomenclatural framework that is less sensitive to taxonomic change. Despite this mechanism influencing the extent of reuse of cultivar epithets, reuse may also occur within a denomination class. The conditions for this are laid down in Article 26.2 of the ICNCP. It allows reuse of an epithet when the original cultivar bearing that epithet is no longer in existence. This decision is to be taken with great caution.

c.

Botanical Hybrid (Species) Names

The ICBN contains a fairly extensive section solely describing special rules for the naming of hybrids, called the Hybrid Appendix. A nothotaxon is a taxon of hybrid origin and designated by the prefix notho- (as nothospecies, nothogenus). The tie between the ICNCP and the Hybrid Appendix of the ICBN is clear from ICBN Art. 28.1 Notes 1 and 3 that read: Note 1: Hybrids, including those arising in cultivation, may receive names as provided in App. I (App. I is the Hybrid Appendix). Note 3: Nothing precludes the use, for cultivated plants, of names published in accordance with the requirements of the botanical Code.

These unfortunate notes still remain in the latest edition of the ICBN (Greuter et a1. 2000), as does the unaltered Hybrid Appendix. A proposal to deconstruct the Hybrid Appendix and omit Notes 1 and 3 did not make the voting floor of the latest International Botanical Congress in St. Louis in 1999. Why do we consider the tie of the ICNCB to the Hybrid Appendix of the ICBN unfortunate? Hybrid nomenclature as described in the ICBN Hybrid Appendix is a typical set of rules based on a concept of cultivars as taxa rather than culta. It contains rules that we consider superfluous for the nomenclature and classification of cultivated plants because of the complexities of determining their relationships. They complicate matters considerably. The ICNCP specifically discourages the use of the Hybrid Appendix in circumstances of cultivated plant breeding (Recommendation 16A). Use of the Hybrid Appendix from the ICBN for describing cultivars also will increase nomenclatural instability in cultivar names because these are taxa, not tied to the ICNCP, and they have less stability as names than those in the ICNCP where stable names are part of this code's rules. Under current nomenclatural rules, when a breeder creates a new cultivar using hybridization techniques, they may use either the ICNCP or the ICBN to create a name for it. When they use the ICBN, especially

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the Hybrid Appendix, the result is a so-called Latin hybrid binomen (a genus name and specific epithet with a multiplication (x) between the genus name and hybrid species epithet, for example Begonia xsvalbardensis. The multiplication sign indicates that plants with such a name are part of the progeny of a hybridization event. When they use the TCNCP, the result will be a cultivar name (e.g., Begonia 'Darling'). Under this system, two names designate the same cultivar. Since the cultivar category is especially founded to accommodate the results of all sorts of breeding techniques (including hybridization under nonnatural circumstances, TCNCP Art. 2), we consider it logical that only the TCNCP be used to name cultivars of hybrid origin, and to reserve the Hybrid Appendix for the naming of hybrids originating under natural conditions and the formation of hybrid generic names (so called notho-genera). This automatically implies that Notes 1 and 3 of TCBN Art. 28 can be deleted in future editions of the Code. D. The Species Category in Cultivated Plant Taxonomy (Cultonomy)

The use of species epithets in the full name of a cultivar would be taxonomically superfluous because the combination of a genus name (or other denomination class) and a cultivar epithet to form the full cultivar name is sufficient to create a unique name. This is essentially the same mechanism that creates unique species names as seen in the TCBN. Nomenclatural rules for cultivated plants must establish that such a combination must be unique and then provide means to maintain that uniqueness. The development of cultivars is a process where species and generic boundaries either combine germplasm of different taxa or involve processes of selection where determining origins is very difficult. Therefore, the species category cannot be used a priori in a universal way as part of the full cultivar name. The species category may be used when a cultivar is known to be directly selected from stock to which the species binomen is still applicable (e.g., selection of introduced ornamentals from a natural population). On the other hand, most successful ornamental crops have pedigrees of extensive hybridization that have blurred species boundaries almost entirely (e.g., Gerbera, Lilium, Dianthus, Chrysanthemum). Most agricultural crops have pedigrees of complex cultigenetic gene pools (e.g., Brassica, Beta, Zeal. The use of species epithets in a full cultivar name also introduces nomenclatural instability because the species epithets are subject to change by taxonomic revisions of species boundaries. With the use of only the genus name as part of the full name of a cultivar, possibilities

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of name changes are reduced. This is often not yet fully recognized in statutory circumstances, where outdated classifications of crops at the level of botanical species and infraspecific levels are still in use (see Hetterscheid et al. 1999 for such a case in Allium and its "cultonomic" solution). Where people may object that species names are useful as classificatory devices, the ICNCP provides a better alternative with the cultivar-group. That is the primary category for grouping cultivars without having to resort to an ICBN category. E. The (Notho-)Genus Category in Cultivated Plant Taxonomy (Cultonomy)

The botanical genus name is the only really necessary ICBN-based item in cultivated plant nomenclature and taxonomy. The generic identity of cultivars is usually still apparent even after a prolonged history of breeding. Therefore, a cultivar classification may be covered by a genus name as an umbrella. The combination of genus name and cultivar name becomes the necessary and sufficient basis for all cultivated plant nomenclature. The rules of the ICNCP are essentially based on this concept. The matter may seem to be complicated by breeding efforts that transcend the limits of established plant genera. In such cases the ICBN Hybrid Appendix provides a relatively simple mechanism to create artificial genera (nothogenera) and their names to expand the possibilities of the genus level as a nomenclatural device for cultivated plants. There is a possibility that entirely unnatural nothogeneric names (only containing culta) should be lifted from the ICBN and transferred to the ICNCP to act as purely nomenclatural devices. Examples are many artificial orchid nothogenera, representatives of which will never be found in nature but only in cultivation. Thus the genus as a nomenclatural device remains as the sole useful category common to both the ICBN and ICNCP, although for different purposes. F. Ties Between the ICBN and ICNCP

At present not all ties between the ICBN and ICNCP are severed, nor do they have to be. The remaining ties are: The use of the term taxon in the ICNCP (hopefully to be severed). The reasons for deleting the ICBN term taxon from the ICNCP were discussed earlier. The word culton must replace taxon. 2. The use of botanical hybrid (species) names in the ICNCP (hopefully to be severed). 3. The use of the species category in the ICNCP (to be limited). 1.

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4. The use of the (notho-)genus category in the ICNCP (to be main-

tained). VII. POSSIBLE NEW CODES Two vigorously debated proposals for changes in the ICBN have emerged that if adopted could affect the ICNCP as well. One of these, termed the BioCode, concerns a debate about producing a unified code for the nomenclature of all life. The other, termed the PhyloCode, has arisen out of new phylogenetic discoveries and cladistic classification theory. A. BioCode

At present there are five nomenclature codes for organisms. The first two of these are for plants and have been discussed in this chapter: 1. International Code of Botanical Nomenclature (ICBN; Greuter et al. 2000); 2. International Code of Nomenclature for Cultivated Plants (ICNCP; Trehane et al. 1995);

3. International Code of Zoological Nomenclature (ICZN; Ride et al. 1999); 4. International Code of Nomenclature of Bacteria: Bacteriological Code (BC; Lapage et al. 1992);

5. International Code of Virus Classification and Nomenclature (VC; Van Regenmortel et al. 2000).

All of these codes have different rules, creating a degree of confusion in nomenclature across disciplines. During the last decade, zoologists and botanists have discussed the possible merits of a unified code for the nomenclature of the above first four codes (not viruses). This initiative, called the BioCode (Greuter et al. 1996, 1997), has not yet been adopted, but may again be proposed at the next International Botanical Congress. The potential advantages would be for biologists working with a wide range of organisms, as ecologists and conservationists, to have a unified code of nomenclature. Others, however, argue that the BioCode would increase nomenclatural instability and generate confusion by the use of new rules and name changes (Brummitt 1996). The BioCode would not cater to the classification and nomenclature of cultivated plants in a direct sense. The discrepancy between taxa and culta will prohibit this. The BioCode would contribute only partly to the stability of names of cultivated plants inasmuch as a part of their names may still be dependent on that code (notably at the plant genus level).

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For our purposes, Art. 35 of the fourth draft of the BioCode (Greuter et al. 1997) would be relevant. It stipulates the separate status of cultivars and their nomenclature and that the ICNCP would remain independent from the BioCode: 35.1. Distinguishable groups of cultivated plants and fungi, whose origin

or selection is primarily due to the intentional actions of mankind (e.g., cultivars and cultivar-groups), are not covered by this Code, but are denominated under the provisions of the International Code of Nomenclature for Cultivated Plants.

B. PhyloCode

Knowledge of phylogenetic relationships of all life is being changed very rapidly by the explosion of new data arising from molecular systematics, and the development of computers and computer algorithms able to handle these data. Many long-accepted taxa, from species to division, are being shown to be paraphyletic or polyphyletic. New knowledge of life's kingdoms arose from DNA sequence data from the small subunit of ribosomal RNA (SSU rRNA). These phylogenies showed three "domains" (Archaea, Bacteria, Eukarya) with subordinate kingdoms within them, and showed lateral transfer of genomes through endosymbiotic events (Doolittle 1999). A recently deduced amino acid sequence dataset (from DNA sequence data) of four protein genes (a-tubulin, ~-tubulin, actin, and elongation factor 1-alpha) tested relationships within the Eukarya domain. The results showed a number of striking differences to the SSU rRNA phylogeny. New relationships among angiosperms were demonstrated by DNA sequences of chloroplast and ribosomal genes (APG 1998; Chase et al. 2000; Savolainen et al. 2000; and Soltis et al. 2000). Many of these changes are summarized in the Tree of Life Project (Maddison and Maddison 1998), a Web-based searchable database useful for locating phylogenetic information about a particular group of organisms. It is intended eventually to treat all groups of organisms, and is organized by a nested set of phylogenetic trees (cladograms). Traditional nomenclature as outlined in the ICBN encounters problems when naming these newly discovered clades. The problem relates to the reliance on a system of ranked hierarchical categories (the closed classification system described earlier) to try to name clades. Under the ICBN, every species is by definition part of a genus, all genera are part of families, all the way up the taxonomic hierarchy, and every rank needs to be named. Traditional rank-based nomenclature and PhyloCode

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nomenclature both attempt to have a name apply to only one taxonomic group and for each group to have only one name. In the ICBN, priority dictates that the correct name is (generally) the earliest name provided within a rank. As a consequence, if a clade is reclassified at a different rank, its name is usually changed. Under the PhyloCode, however, priority operates independent of rank; the earliest phylogenetically defined (Fig. 1.8) name for a clade has priority over later-defined names for the same clade. Problems in ICBN-based nomenclature to name clades are manifested in various ways as a result of this rank-based nomenclatural system. For example, it is difficult to name clades, one at a time as they are discovered, without having to change the names of other clades. It becomes difficult to name the clades one wants to name without giving formal names to groups of uncertain cladistic support (Cantino 2000). De Queiroz and Gauthier (1992, 1994) proposed that a new nomenclatural system was needed to name clades, in which the use of ranked categories such as the genus and family is not mandatory, and these categories (if used) have no bearing on names. In their system, called phylogenetic nomenclature, names are to be applied to clades by anyone of three criteria: (1) node-based clade names; (2) stem-based clade names; and (3) apomorphy-based clade names (Fig. 1.8). Cantino and de Queiroz (2000) wrote a draft version of the PhyloCode that is advanced for discussion and modification. Like the BioCode, it is constructed to be applicable to all organisms, not just plants. Approachable discussions of the PhyloCode and its ramifications can be found in Milius (1999), Withgott (2000), and Cantino (2001). Node based

Stem based

Apomorphy based

Fig. 1.8. Three classes of phylogenetic definitions (modified and redrawn from De Queiroz and Gauthier [1994]). These three trees represent a cladogram rooted on an outgroup, A and B can represent species or entire clades, and the horizontal bar under Apomorphy based represents a shared derived character (synapomorphy) delimiting a clade. Those advocating the PhyloCode propose to name clades based only on cladistic relationships and to remove the role of ranks in the application of names. Node-based names are defined as a clade stemming from the most recent common ancestor of species A and B. Stem-based clades comprise all organisms that share a more recent common ancestor with species A than with species B. An apomorphy-based name is defined as the clade stemming from the first species to possess a particular trait.

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Needless to say, a proposal for such radical changes in nomenclature has attracted its critics (e.g., Brummitt 1996). The PhyloCode produces a more logical system for naming clades, which avoids the rank-related problems of the ICBN. However, it would drastically change a nomenclatural system that has served as a standard reference system since its inception in 1753, and critics claim that it could disrupt ties of names of all ranks, even species binomials, to the biological literature. De Queiroz (1996) and Cantino (2000) addressed these concerns. They claim that (1) many familiar names will continue to be used as they are now because these names already refer to clades (although not by explicit definition); (2) the PhyloCode does not have to supplant other codes but rather can coexist with it; (3) the abandonment of species binomials does not have to be so drastic in that species names can maintain a form that resembles existing binomials but with some qualifying label to distinguish them as PhyloCode names. We cannot predict the future of the PhyloCode, but it addresses critical concerns about nomenclature and is sure to be discussed and experimented with for some time. VIII. CULTIVATED PLANT NOMENCLATURE AND THE LAW

National and international laws and treaties governing trade, intellectual property, breeding, and germplasm exchange of cultivars outline the economic importance of cultivated plants. Most of those using these laws are passive users of existing nomenclature of cultivars in the sense that they do not describe new names. In such laws cultivar and crop names are merely used to define the objects of the laws. Such laws may be influenced, knowingly or unknowingly, by changes in plant taxonomy. In order to keep such changes to a minimum, systems are in existence that stabilize cultivar and crop names by declaring certain names or classifications impervious to nomenclatural changes. A prominent example is the List of Stabilized Plant names as issued by the International Seed Testing Association (ISTA, see Websites). This list stabilizes names of economically important crops and may be incorporated in national or international laws. The 1STA nomenclature committee maintains the list. Plant names have greater impact in the International Union for the Protection of New Varieties of Plants (UPOV) convention. This international treaty was developed under the aegis of UPOV (see Websites), based in Geneva. The convention lays down a system of legal protection of newly bred cultivars of plants. Breeders in countries that have signed reciprocal agreements to this convention may acquire legal ownership

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of the cultivars they have developed. Breeders are given the opportunity to release cultivars under restricted circumstances and receive a profit on their investments. All the above has no direct effect on naming cultivars. However, the same convention issues a document called the "UPOV Recommendations on Variety Denominations." This document contains some imprecise recommendations on how cultivar names (the word variety in English-written legal texts is equivalent to the international term Gultivar) could be established. The main purpose of this document is to ensure that, as far as possible, protected cultivars are given the same name in all member states. The recommendations are clearly related to articles in the ICNCP but they are unfortunately not as precise, and provide much opportunity for diverse interpretations. The result is that nomenclature of cultivars differs from one member state to the other. This situation automatically leads to the establishment of cultivar epithets under legal ("statutory") circumstances that would not be allowed using the ICNCP. However, since the ICNCP has no legal status, such deviating epithets are nonetheless to be accepted (ICNCP Principle 7). IX. CULTIVAR EPITHETS AND TRADEMARKS

Trademarks used in the trade of cultivated plants are an increasing source of nomenclatural confusion. Some breeders try to get ownership of the name of a cultivar as well as ownership of the cultivar itself. The UPOV convention and the U.S. Patent Act (see Websites) expressly prohibit this. A cultivar epithet may not be a protected trademark at the same time. This is a very logical consequence of the main purpose of a cultivar epithet: a label to be used worldwide to designate a particular cultivar in communication. A trademark may not be used worldwide and basically only at the discretion of the trademark owner. Obviously those two purposes are entirely different. The trademark serves to identify the products of a certain grower or company and may be used to enhance the focus of the public to the quality of the products of that particular grower or company. It is a typical commercial trading tool. However, all the individual products sold under the use of a trademark must still have generically usable individual names for purposes of communication and reference, and this is where the cultivar epithet comes in. Unfortunately, practice is less strict. Currently, trademarks for the sale of cultivars are often made to look like cultivar epithets, which is quite often illegal but usually goes unnoticed. The actual cultivar epithet is suppressed entirely, or printed in small letters. Usually cultivar epithets in such cases are constructed to be unspeakable "words" or

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numerals, thus focusing attention of the public to the commercially well-sounding trademark. For example, in 1988, UK Plant Breeders' Rights Grant No. 3743 was issued for a rose with the cultivar epithet 'Korlanum.' The cultivar is marketed under the trade designations Surrey, Sommerwind, and Vente D'lhe in different countries (ICNCP Art. 11, Ex. 2). The ICNCP contains an article that enhances clarity in this matter (Art. 17.7) by stating that a cultivar epithet should be identified in a full name by demarcation marks (single quotation marks). X. RECOMMENDATIONS FOR A UNIVERSALLY STABLE CROP NOMENCLATURE THROUGH CHANGES AND USE OF THE ICNCP

One may be daunted by all the apparent conflicts between the taxonomy of wild and cultivated plants, and if the important goal of stability of names is attainable at all. The following discussion focuses on recommendations for cultivated plant taxonomy. We support the sole use of the ICNCP for classification of cultivated plants. Attempts to merge the two have been tried ever since Linnaeus, notably at the infraspecific level. Hetterscheid and van den Berg (1996) introduced the term cultonamy for the taxonomy of cultivated plants. Although not totally accepted, here are the essential characters of the system that will enhance crop nomenclature stability: 1. Switching of emphasis in cultivated plant taxonomy from a plant-

centered focus to a human-centered focus. 2. Acceptance of the culton as the fundamental systematic concept for

the taxonomy of cultivated plants and simultaneous rejection of the taxon for that role. 3. Acceptance of the open classification philosophy for culta and simultaneously rejecting the use of closed systems of classification with extensive hierarchies. 4. Acceptance of the primacy of the ICNCP for nomenclatural purposes in cultivated plant taxonomy and using the ICBN only in a secondary capacity in governing names of taxa to which a classification of a crop may be linked. 5. Reducing the Hybrid Appendix of the ICBN so as to exclude rules solely based on phenomena exclusively apparent in cultivated plants. The Hybrid Appendix thus needs to be brought back to serve only nomenclatural purposes for wild plants. Consequentially, hybrids of cultivated origin must not be named according to

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Hybrid Appendix rules (no further creation of hybrid species names for such plants) but exclusively using the ICNCP and its categories. 6. Avoidance, where possible, of using ICBN-based species epithets in full names of cultivars and in cultonomic classifications. 7. Creating interest groups of users of crops that are in need of classification and evaluate their input in terms of characters to be used in the classification proposal. 8. Promoting the use of ICNCP-based nomenclature in national and international law to obtain a worldwide standardization and stabilization of cultivar nomenclature for all circumstances where cultivar names are part of legal documents.

REFERENCES Books Brummitt (1992). A list of vascular plant families and genera with taxonomic authors and family affiliations. Brummitt and Powell (1992). A standardized list of taxonomic author abbreviations for plants. Many abbreviation styles have been in use, creating confusion that this book helps to eliminate. Hanelt (2001). Provides taxonomy, updated scientific names, common names, economic use, and distribution of economically important plants. Lincoln et al. (1998). Provides definitions to many of the nomenclatural and taxonomic concepts discussed. Mabberley (1997). Provides generic and family names, family affiliations of genera, uses, and distribution of angiosperms. Smartt and Simmonds (1995). Provides useful compilations of data on crops compiled by individual experts, including cytotaxonomy, early and recent history of the crop, and evolutionary data. Wiersema and Leon (1999). Provides updated scientific names, common names, economic use, and distribution of economically important plants.

Websites American Association of Botanic Gardens and Arboreta (AABGA). www.aabga.org/. Supplies information about North American Botanic Gardens and arboreta. American Society of Plant Taxonomists (ASPT). www.sysbot.org/. Promotes research and teaching in taxonomy, systematics and phylogeny of plants. The site also lists taxonomic expertise of society members. BioCode (draft 1997). http://www.rom.on.ca/biodiversity/biocode/biocode1997.html. Provides the entire text of the latest BioCode proposal and background information. Biosys. http://www.biosis.org/. Provides access to information relevant to the life sciences in general. Community Plant Variety Office (European Plant Breeders' Rights) (CPVO). http://www .cpvo.eu.int/.

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Food and Agricultural Organization of the United Nations (FAa). www.fao.org/. Provides basic information on crop plants statistics. This organization has a Commission on Registration and Nomenclature, coordinating and guiding the International Cultivar Registration Authorities (ICRAs). There are ICRAs for genera of horticultural interest. An updated list will be found on this Website. Gardenweb. www.gardenweb.com/. Provides horticultural information and provides a glossary of horticultural and botanical terms. Germplasm Resources Info Network (GRIN). www.ars-grin.gov/npgs/tax/. Provides data on U.S. germplasm holdings and coordinates various nomenclatural lists, such as the ISTA and AOSA nomenclatural lists, a list of noxious weeds, and the list of rare and endangered plants under the CITES rules. Index Herbariorum. http://www.nybg.org/bsci/ih/ih.html. Index of all public herbaria in the world. International Association of Plant Taxonomy (IAPT). www.botanik.univie.ac.at/iapt/. Contains information dealing with botanical nomenclature, its publications (Taxon and Regnum Vegetabile), and IAPT publishes the International Code of Botanical Nomenclature and International Code for the Nomenclature of Cultivated Plants. International Cultivar Registration Authorities (ICRAs). http://www.ishs.org/sci/icra.htm. Provides a list of all ICRAs and several query possibilities, and information on how to become an ICRA, and about the nomenclatural codes. Internet Directory of Botany. www.botany.net/IDB/. Contains important information on botanical museums, botanic gardens and arboreta in the world, and various botanical associations. International Plant Genetic Resources Institute (IPGRI). www.ipgri.cgiar.org/. Provides information about the IPGRI which is an international institute that deals with plant genetic resources of all important crop plant species, sets up networks of cooperating Plant Genetic Resources Centers, and publishes at regular intervals international descriptor lists for crop plant species in order to facilitate international exchange of seeds and information. International Plant Names Index (IPNI). http://www.ipni.org/. Provides three important nomenclature databases on one site. Provides information as to where and when plant names were first published. International Seed Testing Association (ISTA). www.seedtest.org/.Primarily responsible for establishing standards for international seed testing, 1STA also publishes the 1STA List of Stabilized Plant Names International Society for Horticultural Sciences (ISHS). http://www.ishs.org/. Promotes and encourages research in all branches of horticulture. Journey into the World of Phylogenetic Systematics. http://www.ucmp.berkeley.edu/ clad/clad4.html. Provides information for those who want to know more about cladistics. Multilingual Multiscript Plant Name Database. http://gmr.landfood.unimelb.edu.au/Plantnames/. Lists many common names of plants in many languages and scripts in a database. Also extensive links to Web information on selected groups of plants. Plantscope. www.plantscope.nll. Lists all ornamental plants in the Dutch trade. Much attention to correct nomenclature, descriptions and images, and classifications according to cultonomic principles (cultivar-groups). Plant Variety Protection Office Webpage, United States Department of Agriculture. http://www.ams.usda.gov/science/PVPO/pvp.htm. Explains the history and administration of the U.S. Plant Variety Protection Act. Phylocode. http://www.ohiou.edu/phylocode/. Dedicated to the PhyloCode initiative.

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Phylogenetic Systematics (cladistics). http://members.tripod.de/GBechly/glossary.htm. Provides terms used in cladistics, personal opinion, and species definitions. Plant Patents. http://www.plantpatent.com/. Catalogs information about U.S. Plant Patents by Vincent Gioia (plant patent expert). Plant Patents (official site). http://www.uspto.gov/web/offices/pac/plant/. U.S. Plant Patents official governmental site. Royal Horticultural Society (RHS). www.rhs.org.uk/. Provides information on many aspects of horticulture; one of its well-known aspects is the plant finder providing updated information on plants. TROPICOS. http://mobot.mobot.org/W3T/Search/vast.html. Maintained by the Missouri Botanical Garden with current information on plant names. Union for the Protection of New Varieties of Plants (UPOV). http://www.upov.int/. Explains how the Union works.

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Pickersgill, B. 1986. Evolution of hierarchical variation patterns under domestication and their taxonomic treatment. p. 191-209. In: B. T. Styles (ed.), Infraspecific classification of wild and cultivated plants. Clarendon Press, Oxford. Plaisted, R 1., R W. and Hoopes. 1989. The past record and future prospects for the use of exotic potato germplasm. Am. Potato J. 66:603-627. Rehder, A. 1960. Manual of cultivated trees and shrubs. 2nd ed. Macmillan Publishing Co., New York. Rick, C. M. 1963. Barriers to interbreeding in Lycopersicon peruvianum. Evolution 17:216-232. Rick, C. M. 1979. Biosystematic studies in Lycopersicon and closely related species in Solanum. p. 667-678 + 1 pI. In: J. G. Hawkes, R N. Lester, and A. D. Skelding (eds.), The biology and taxonomy of the Solanaceae. Linnean Soc. London Symp. Ser 7. and Academic Press, New York. Rick, C. M., H. Laterrot, and J. Philouze. 1990. A revised key for the Lycopersicon species. Tomato Genet. Coop. Rep. 40:31. Ride, W. D. L., H. G. Cogger, C. Dupuis, O. Kraus, A. Minelli, F. C. Thompson, and P. K. Tubbs. 1999. International code of zoological nomenclature. 4th ed. Int. Trust for Zoological Nomenclature, Natural History Museum, London. Rieseberg, L. H., and 1. Brouillet. 1994. Are many plant species paraphyletic? Taxon 43:21-32. Rieseberg,1. H., and J. M. Burke. 2001. The biological reality of species: Gene flow, selection, and collective evolution. Taxon 50:47-67. Rollins, R C. 1965. On the basis of biological classification. Taxon 14:1-6. Ross, H. 1986. Potato breeding: Problems and perspectives. Adv. Plant Breed. Suppl. 13. Verlag, Paul Parey, Berlin. Royal Horticultural Society. 1992. Dictionary of gardening, London. Ruiz de Galerreta, J. I., A. Carrasco, A. Salazar, I. Barrena, E. Iturritxa, R Marquinez, F. J. Legorburu, and E. Ritter. 1998. Wild Solanum species as resistance sources against different pathogens of potato. Potato Res. 41:57-68. Savolainen, V., M. F. Fay, D. C. Albach, A. Backlund, M. van der Bank, K. M. Cameron, S. A. Johnson, M. D. Lledo, J.-c. Pintaud, M. Powell, M. C. Sheahan, D. E. Soltis, P. S. Soltis, P. Weston, W. M. Whitten, K. J. Wurdack, and M. W. Chase. 2000. Phylogeny of eudicots: A nearly complete familial analysis based on rbcL gene sequences. Kew Bul. 55:257-309. Schwanitz, F. 1967. Die Evolution der Kulturpflanzen. Bayerischer Landwirtschaftsverlag, Munich. Shishkin, B. K., and S. V. Yuzepczuk. 1971. Flora of the U.S.S.R 10:509-604. Smartt, J., and N. W. Simmonds. 1995. Evolution of crop plants. Longman Scientific and Technical, Essex, England. Sneath, P. H. A., and R R Sokal. 1962. Numerical taxonomy: The principles and practice of numerical classification. W. H. Freeman and Company, New York. Snogerup, S. 1979. Experimental and cytological studies of the Brassica oleracea group. Webbia 34(1):357-362. Snogerup, S. 1980. The wild forms of the Brasica oleracea group (2n=18) and their possible relations to the cultivated ones. p. 121-132. In: S. Tsunoda, K. Hinata and C. Gomez-Campo (eds.) Brassica crops and wild allies, biology and breeding. Japan Scientific Societies Press, Tokyo. Sokal, R. R. 1985. The continuing search for order. Am. Nat. 126:729-749. Sokal, R R, and T. J. Crovello. 1970. The biological species concept: a critical evaluation. Am. Nat. 104:127-153.

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Soltis, D. K, P. S. Soltis, M. W. Chase, M. K Mort, D. C. Albach, M. Zanis, V. Savolainen, W. H. Hanh, S. B. Hoot, M. F. Fay, M. Axtell, S. M. Swensen, L. M. Prince, W. J. Kress, K. C. Nixon, and J. S. Farris. 2000. Angiosperm phylogeny inferred from 18S rDNA, rbcL, and atpB sequences. Bot. J. Linnean Soc. 133:381-461. Song, K. M. and T. C. Osborn. 1992. Polyphyletic origins of Brassica napus: New evidence based on organelle and nuclear RFLP analyses. Genome 35:992-1001. Song, K. M., T. C. Osborn, and P. H. Williams. 1988. Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs). Theor. Appl. Genet. 75:784-794. Spooner, D. M., G. J. Anderson, and R. K. Jansen. 1993. Chloroplast DNA evidence for interrelationships oftomatoes, potatoes, and pepinos (Solanaceae). Am. J. Bot. 80:676-688. Spooner, D. M., and R. G. van den Berg. 1992. An analysis of recent taxonomic concepts in wild potatoes. Genet. Res. Crop. Evol. 39:23-37. Stafleu, F. A. 1971. Linnaeus and the Linnaeans. Regnum Veg. 79:1-386. Stearn, W. T. (ed.). 1953. International code of nomenclature for cultivated plants. Int. Assoc. Plant Taxon. Utrecht. Stebbins, G. L. 1956. Taxonomy and the evolution of genera, with special reference to the family Gramineae. Evolution 10:235-245. Stevens, P. F. 1998. What kind of classification should the practicing taxonomist use to be saved? p. 295-319. In: J. Drandsfield, M. J. K Coode, and D. A. Simpson (eds.), Plant diversity in Malesia III: Proc. 3rd Int. Flora Malesiana Symposium 1995. Royal Botanic Gardens, Kew. Stuessy, T. F. 1990. Plant taxonomy: The systematic evaluation of comparative data. Columbia Univ. Press, New York. Swofford, D. L., G. L. Olsen, P. J. Waddell, and D. M. Hillis. 1996. Phylogenetic inference. p. 407-514. In: D. M. Hillis, C. Moritz, and B. K. Mable (eds.), Phylogenetic systematics, 2nd ed. Sinauer Associates, Sunderland, MA. Templeton, A. R. 1989. The meaning of species and speciation: a genetic perspective. p. 3-27. In: D. Otte and J. A. Endler (eds.), Speciation and its consequences. Sinauer Associates, Inc., Sunderland, MA. Thellung, A. 1918. Neuere Wege und Ziele der Botanischen Systematik, erlautert am Beispiele unserer Getreidearten. Naturw. Wochenschr., Neue Folge 17(33):470. Trehane, P., C. D. Brickell, B. R. Baum, W. L. A. Hetterscheid, A. C. Leslie, J. Mcneill, S. A. Spongberg, and F. Vrugtman. 1995. Int. code of nomenclature of cultivated plants. Regnum Veg. 133:1-175. U, N. 1935. Genomic analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Japan. J. Bot. 7: 389-452. Ulloa Ulloa, C. 2001. Book review of Flowering plants of the Galapagos, by C. K. McCullen, 1999. Comstock Publishing/Cornell Univ. Press, Ithaca, New York. Syst. Bot. 26: 696-697. Van den Berg, R. G. 1999. Cultivar-group classification. p. 135-143. In: S. Andrews, A. C. Leslie, and C. Alexander (eds.), Taxonomy of cultivated plants: Third international symposium, Royal Botanic Gardens, UK. Van der Heuvel, K. J. P. P., D. W. M. Barendse, and G. J. Wullems. 2001. Effects of giberellie acid on cell division in anthers of the giberellin deficient gib-l mutant of potato. Plant BioI. 3:124-131. Van Raamsdonk, L. W. D. 1993. Wild and cultivated plants: The parallellism between evolution and domestication. Evol. Trends Plants 7:73-84. Van Raamsdonk, L. W. D., and L. J. G. van der Maesen. 1996. Crop-weed complexes: The complex relationship between crop plants and their wild relatives. Acta Bot. Neerl. 45: 135-155.

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2

Grafting of Herbaceous Vegetable and Ornamental Crops Jung-Myung Lee Kyung Hee University Department of Horticulture Suwon, 449-701, Korea Masayuki Oda Osaka Prefecture University Graduate School of Agriculture and Biological Science Sakai, Osaka, 599-8531, Japan

1. INTRODUCTION II. GRAFTING TECHNOLOGY A. Techniques 1. Conventional Manual Grafting 2. Comparison of Grafting Methods Used in Korea and Japan 3. Tools, Clips, and Grafting Aids 4. Monitoring Grafting Success 5. Acclimatization 6. Micrografting B. Robots III. PHYSIOLOGY OF GRAFTING A. Graft Union B. Graft Compatibility C. Translocation D. Growth Effects 1. Vigor 2. Physiological Disorders 3. Stress Tolerance

Horticultural Reviews, Volume 28, Edited by Jules Janick ISBN 0-471-21542-2 © 2003 John Wiley & Sons, Inc. 61

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IV. CROP EXAMPLES A. Watermelon 1. Current Status 2. Rootstocks 3. Grafting Methods 4. Cultural Practices 5. Problems Associated with Grafted Plants B. Cucumber 1. Current Status 2. Rootstocks 3. Grafting Methods 4. Cultural Practices 5. Problems Associated with Grafted Plants C. Melons 1. Current Status 2. Rootstocks 3. Grafting Methods 4. Cultural Practices 5. Problems Associated with Grafted Plants D. Tomato 1. Current Status 2. Rootstocks 3. Grafting Methods 4. Cultural Practices 5. Problems Associated with Grafted Plants E. Eggplant 1. Current Status 2. Rootstocks 3. Grafting Methods 4. Cultural Practices F. Pepper 1. Current Status 2. Rootstocks 3. Grafting Methods 4. Cultural Practices G. Other Solanaceous Crops H. Crucifers I. Cactus 1. Current Status 2. Rootstocks 3. Grafting Methods 4. Problems Associated with Grafted Plants V. GRAFTING FOR CROP IMPROVEMENT A. Flower and Tuber Induction B. Graft Chimeras and Graft-Induced Mutants C. Chimeral Engineering VI. CONCLUSION AND PROSPECTS LITERATURE CITED

J. LEE AND M. ODA

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I. INTRODUCTION

Graftage involves the joining together of plant parts by means of tissue regeneration, in which the resulting combination of parts achieves physical union and grows as a single plant (Janick 1986). The origins of grafting for fruit crops trace to antiquity. Grafting was known to the Chinese at least since 1560 BCE and is discussed in the agricultural writings of Aristotle (384-322 BCE) and Theophrastus (371-287 BCE). Grafting of grape is referred to in the Hebrew and Christian bible (Hartmann et al. 1997). Grafting is most studied in fruit and nut crops since the technique is commonly used as a means of vegetative propagation. Typically, buds or stems of a desirable clone (the scion) are inserted in a rootstock either produced by seed or by vegetative propagation so that the cambium tissues align and form a graft union. Grafting is also used for cultivar change, repair, or invigoration of older established trees. The use of graftage overcomes the difficult-to-root problems of many fruits such as apple, which makes propagation by cuttings impractical. Furthermore the interaction of stock and scion may affect growth and productivity (Beakbane and Rogers 1956; Rom and Carlson 1987). Small trees are created by the use of dwarfing rootstock. Disease resistance and hardiness can be achieved by the creation of plants composed of more than one genetic component. Grafting has also been used as a means to study the transmission of signals affecting vernalization and photoperiod (Suge 1992) as well as the transmission of virus into indicator plants, and to eliminate viruses (Section II.A6). Grafting of herbaceous vegetable crops is also an old practice. Grafting in cucurbits was briefly described in a seventeenth century book written by Hong (1643-1715) in Korea. He described methods of producing large gourd fruit by approach grafting two plants and then thinning to a single shoot after the union. Two of the grafted plants were approach grafted once again and with shoot thinning produced one vigorous shoot with four root systems. By allowing only one or two fruits per vine, extra-large gourd fruit were produced that could be used as large storage container for rice (Hong 1710; PSNCK 1982). A similar grafting method had been described in an ancient Chinese book dating to the fifth century (Anon. 530-545). Vegetable grafting, however, does not appear to have been a common practice until the twentieth century in Asia. Detailed information on watermelon grafting appeared in scientific journals in the 1920s (Ashita

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1927). Scions of watermelon (Citrullus lanatus) were grafted to rootstocks of bottle gourd (Lagenaria siceraria) to overcome the yield decline problems associated with successive cropping mostly due to soil-borne diseases in both Korea and Japan. Grafting had been practiced on small scale among farmers in Sungju, Kimhae, Haman areas in Korea from the early 1950s. The grafting was done with large seedlings and the efficiency of grafting was less than 50 percent so that a worker could produce only 150 seedlings per day (Ashita 1930, 1934). Wet or moistened straw or paper was used to hold the grafted union in a desirable position. This compares to daily production per worker of 800 to 1200 grafted seedlings at the present time using grafting clips or tubes and over 10,000 plants per day with robots. The development and utilization of plastic films in agriculture in the 1960s triggered a rapid increase in seedling production under plastics and led to the production and distribution of grafted seedlings in Korea and Japan. Scientific investigations of new rootstocks became very active in the late 1960s (Kim 1984). In 1959, scions of eggplant (Solanum melongena) were grafted in large scale onto rootstocks of Solanum integrifolium (' Akanasu', the scarlet eggplant) to avoid the injury caused by soil-borne diseases such as verticillium wilt, fusarium wilt, bacterial wilt, and nematodes. Cucumbers (Cucumis sativus) were also extensively grafted well before 1959 in Japan (Fuji and Itagi 1962) to reduce the damage caused by soil-borne diseases and to promote scion vigor. In the 1960s grafting was introduced as a commercial practice in Japan and Korea for cucumber and tomato (Lycopersicon esculentum). By 1990, the percentage of grafted plants for the production of fruit bearing vegetables (eggplants, cucumber, tomato, and various melons) reached 59 percent of the area in Japan and 81 percent in Korea (Lee 1994). At present, virtually all the cucurbits for greenhouse cultivation are being grafted in Korea and Japan. The cultivated area of grafted vegetables in Japan and Korea in 2000 is presented in Table 2.1. Despite the wide use of vegetable grafting in Asia, information on this technology has been generally unavailable to English-speaking audiences. However, at the present time greenhouse growers in the Netherlands favor grafted tomato seedlings if they intend to harvest more than six clusters per plants. Rootstocks for vegetables have been widely advertised in vegetable seed catalogs throughout the world including many European countries. There are many Korean and Japanese books on vegetable grafting that would be useful to expand this unique technique to various parts of the world. The purpose of this chapter is to review progress in grafting technology for vegetable and ornamental production achieved in Japan and Korea.

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Table 2.1. Cultivation area of some vegetables in 2000 and percentage of grafted plants in Japan and Korea (MAFF/Japan, 2000 or MAF/Korea, 2000). Korea

Japan Field + Tunnel

Crop

Total area (ha)

Watermelon Cucumber Melonso Tomato Eggplant Pepper b

14,017 10,160 6,142 6,459 11,815 2,684

Grafted (%)

92 55 0 8 43

_c

Greenhouse Total area (ha) 3,683 5,440 8,258 7,141 1,785 1,468

Grafted (%)

98 96 42 48 94

Field + Tunnel

Greenhouse Grafted

(%)

Total area (ha)

90 42 83 0 0 0

21,299 5,964 9,365 4,752 413 5,085

98 95 95 5 2 5

Total area (ha)

Grafted

13,200 1,728 1,047 258 650 75,574

(%)

°Include field and greenhouse melons in Japan and melons and oriental melons in Korea. bMostly sweet peppers in Japan and green hot peppers in Korea. CData not available. Source: Statistical data from Ministry of Agriculture, Forestry (and Fisheries).

II. GRAFTING TECHNOLOGY A. Techniques Graftage is a process that involves: (1) the choice of stock and scion species, (2) creation of a graft union by physical manipulation, (3) healing of the union, and (4) acclimation of the compound plant. 1. Conventional Manual Grafting. There are a number of methods

applicable for conventional herbaceous grafting. Some of the most frequently used methods are listed by crop in Table 2.2 and are diagrammed in Fig. 2.1. Conventional grafting is carried on by growers or by commercial plug seedling nurseries (Fig. 2.2).

Hole Insertion Grafting (HIG). Grafting methods vary with the kind of crops being grafted, preferences and experience of the growers, and the kind of grafting machines or robots available. For watermelons, holeinsertion hypocotyl grafting (Fig. 2.1A) is favored by many farmers in many areas because of the smaller seedling size of watermelon as compared to the size of the rootstock, which is usually squash or bottle gourd. Watermelon seeds are sown seven to eight days after the sowing

J. LEE AND M. ODA

66 Table 2.2.

Major grafting methods in selected vegetables. Grafting method used (%) Tongue approach

Hole insertion

Splice

Korea Japan

60 9

35 84

1 0

Melon

Korea Japan

70 62

10 37

Cucumber

Korea Japan

35 86

Tomato

Korea Japan

Eggplant

Korea Japan

Location

Crop Watermelon

Cleft

Pin

OtherO

0 7

0 0

4 0

15 0

0 0

0 0

5 1

1 13

60 0

0 0

0 0

4 1

1 59

25 25

0 0

0 14

70 0

4 2

15

0 23

0 0

10 76

70 0

5 1

(lOther grafting includes modified insertion grafting, double splice grafting, and combined grafting. Source: Lee (2000)

A

B

F

G

c

D

H

E

J

Fig. 2.1. Various grafting methods commonly used for vegetables. (A) Hole insertion grafting; (B) Modified hole insertion grafting; (C) Tongue approach grafting; (D) Splice grafting; (E) Double splice grafting; (F) Epicotyl insertion grafting; (G) Cleft grafting; (H, I) Pin grafting; (J) Splice grafting for solanaceous vegetables.

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Fig. 2.2. Women grafting tomato and pepper in a commercial plug seedlings nursery in Korea (photographed February 2001).

of gourd seeds (rootstock) or three to four days after sowing squash rootstock seeds. Grafting is made seven to eight days after the sowing of watermelon seeds. Both the scion and rootstock should be uniform and strong enough to take the grafting operation. The true leaf including the growing point should be carefully and thoroughly removed and a hole is made with a bamboo or plastic gimlet or drill at a slant angle to the longitudinal direction. The hypocotyl portion of the watermelon is prepared by slant cutting to have a tapered end for easy insertion. Care should be given to avoid the insertion into the stem pith since this greatly interferes with formation of a rapid union and facilitates later protrusion of watermelon adventitious roots into the soil after downward elongation through the pith cavity of the rootstock. Some growers insert young watermelon seedlings (usually somewhat etiolated seedlings with cotyledons still in folded position) into the hypocotyl (Fig.2.1B). For tomato and eggplant, rootstock seed is sown five to ten days before the scion, and grafting is made 20 to 25 days after sowing scion seed. Rootstock seedlings having 2.5 to 3.0 true leaves are decapitated five to ten mm above the first node and a hole is made at a slant angle with a

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bamboo stick. The scion, with two true leaves, is cut 10 mm below the cotyledonary node and, after removing the cotyledons, a slant cut is made on both sides of the scion hypocotyls to have a long, gradually tapering wedge. After inserting the scion into the hole in the rootstock, the grafted plant is placed in a high humidity conditioning room, chamber, or tunnel for subsequent healing. HIG requires a higher skill level as compared to tongue approach grafting (TAG) and a suitable conditioning chamber or facility. However, HIG is very popular among many large commercial growers because it does not require additional labor needed for clipping, transplanting, cutting, and clip removal. In addition, healthy and stronger seedlings can be obtained because more of the vascular bundles can be connected as compared to TAG (Oda 1994). Depending on the grower, the rootstocks for grafting are cut near the ground level for efficient grafting operation and the seedlings, after the grafting has been made, are inserted into rooting medium or substrate in plug cell (40 to 50 cells per tray) for rooting and subsequent growing. Special attention should be given to provide optimum conditions for rapid union, that is, high humidity, high temperature, and hopefully adequate light (natural or artificial). Kits for easy-to-build healing or conditioning chambers are commercially available in Japan and Korea at reasonable prices.

Tongue Approach Grafting (TAG). Tongue approach grafting (Fig. 2.1e) is usually favored by less experienced farmers and those who do not have a greenhouse with good microclimate control system. Even though this method needs more space and labor as compared to other methods, a higher rate of seedling survival is possible even for beginners. Furthermore, no special facilities and machines are needed for this grafting technique. Since the grafting operation would be much more efficient with both scion and rootstock seedlings having similar height, the seeds of scion (usually watermelons, cucumbers, and melons) are sown five to seven days earlier than the rootstock seeds. The growing point of the rootstocks should be carefully removed before grafting to reduce the unnecessary loss of nutrient for the bud growth and to promote the rapid union of graft interface. Occasionally one cotyledon may also be removed when removing the growing point to ensure complete removal of the growing point and to avoid overcrowding in limited space on the greenhouse bench. The grafting cut for rootstock should be made in a downward direction and the scion cut in an upward direction at an angle, usually 30 to 40 degrees to the perpendicular axis, and deep enough to allow the fusion of as many vascular bundles as possible. After the graft is com-

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pleted, specially designed clips are placed to fix the graft position. Grafted plants are then planted together in a nine to 12 em diameter pot. The grafted plants are partially shaded for one or two days before placing them under normal greenhouse growing conditions. The lower hypocotyl of the scion of several test plants is cut to examine the degree of graft-take ten to 12 days after grafting, and, based on the response, the remaining plants can be handled as described in the following. The root and lower hypocotyl of the scion are removed from the grafted plant by simply cutting off at the desired position, preferably at the closer position to the grafted position held by the clip. The clips are usually removed at later stages, shortly before transplanting. An experienced person can graft about 1500 plants per day, but grafting machines and robots specifically designed for this kind of grafting are also available at varying prices (see Section ILB). TAG is the oldest and perhaps the most convenient grafting method for herbaceous plants (Hong 1710). The method can be used for basically any kind of plants such as cucurbits, solanaceous plants, and many other types. Grafting is performed with very young seedlings and preferably at the hypocotyl portion of the rootstock and scion of cucurbitaceous plants and at the lower epicotyl portion in solanaceous crops. In spite of the simple and easy grafting operation and higher rate of survival, this method is not extensively used by commercial seedling growers mainly because of the (1) labor required for grafting, (2) labor needed for cutting the rootstock again, (3) needs for removal of clips after union, (4) larger space needed for growing grafted plants as compared to other methods, (5) and the frequent rooting from the scion after transplanting if the seedlings are transplanted too deep (Lee 1994).

Splice Crafting (SC). Splice grafting (Fig. 2.1D, E, J) is very popular among experienced growers and commercial plug seedling nurseries. Splice grafting can be done by hand, machine, or robot and can be applied to most vegetables. The major advantage is the production of strong and healthy grafted seedlings since all the vascular bundles of the scion are fused with those of rootstock and the graft union is strong enough to take all the rough post-graft handling. Intact or excised (rootremoved) rootstock seedlings may be used depending on the growers' preference (Lee et al. 2000). For the cucurbit rootstocks, one cotyledon and the growing point are removed for grafting. After placing the scion on the rootstock (Fig. 2 .1D, E), ordinary grafting clips as in the tongue approach grafting are used to fix the grafted position tightly together. For solanaceous crops, grafting is usually made at lower epicotyl and fixed (Fig. 2.1J) with ordinary clips, elastic tube-shaped clip with side slit, or

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ceramic pins (see Pin Grafting) developed specifically for this type of grafting.

Cleft Grafting (CG). Cleft grafting (Fig. 2.1G) in herbaceous plants may be somewhat different from those of woody plants (Hartmann et al. 1997). Usually a portion of the stem is cut longitudinally. The rootstock seedlings are decapitated and a longitudinal cut is made in a downward direction, 1 to 1.5 cm long and %depth of the stem diameter. The scion is pruned to have one to three true leaves and the lower stem is cut to slant angle to make a tapered wedge. After placing the scion into the split made on the rootstock, a clip is placed to hold in position until the union. Various types of grafting clips, differing in material, size, shape, and others, have been developed for grafting (Fig. 2.3). Pin Grafting (PG). Pin grafting (Fig. 2.1H, I) is basically the same as splice grafting. However, instead of placing grafting clips to hold the grafted position, specially designed pins are used to hold the grafted position in place. The ceramic pin developed by Takii Seed Company

Fig. 2.3. Various grafting aids. (A) Ordinary grafting clips; (B) Elastic tube with side slit; (C) Elastic tubes for grafting robots; (D) Ceramic pins developed by Takii Seed Co.; (E) Soft and elastic tubes with side slit being used in The Netherlands for tomato and pepper.

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in Japan is about 15 mm long and 0.5 mm in diagonal width of the hexagonal cross section. The pins are made of natural ceramic so it can be left on the plant without any problem. The price of ceramic pin is fairly high so that alternative methods are being sought. Experimental results revealed that bamboo pins, rectangular in cross-sectional shape, could successfully replace the expensive ceramic pins at much lower price. 2. Comparison of Grafting Methods Used in Korea and Japan. Grafting methods most favored by growers are quite different in Korea and Japan for watermelons, indicating the significance of growers' experience and individual preference (Table 2.2). For example, splice grafting is very popular among growers in Korea (60 percent) as compared to Japan (32 percent). Tongue approach grafting is still very popular among growers in Korea while this method is seldom used in Japan. Preferences in grafting methods can be expected to quickly undergo changes as new grafting methods and/or grafting machines including grafting aids are developed. For watermelons, two major grafting methods are commonly practiced for both manual and machine grafting, hole insertion grafting, and splice grafting. However, other types of grafting can also be safely used for watermelons as well as for other crops.

3. Tools, Clips, and Grafting Aids. A number of grafting tools to perform grafting and clips to hold the graft union together have been developed by various agricultural companies (Oda 1999). Unfortunately, however, most of them have not been widely used by the growers. Simple grafting aids, such as grafting clips, tubes, tapes, and pins have been selectively but widely used for grafting (Fig. 2.3). The ordinary grafting clips consisting of a round spring made out of plastic (Fig. 2.3A), have been most extensively used for tongue approach grafting in cucurbits and other crops. The clips, although slightly different in size and shape depending on manufacturers, are inexpensive, easy to operate and handle for various stem sizes, and can be used many times. Various other clips, especially elastic tube-shaped clips with or without attachment for supporting pole for the grafted seedlings (Fig. 2.3B, C, D), are also widely used by many commercial growers for manual grafting as well as for machine or robot grafting. Much smaller elastic slit-tubes are being used in The Netherlands (Fig. 2.3E) for tomato and pepper grafting. A ceramic pin is a very handy and efficient aid to fix the grafted interface, and highly suitable for machine or robot grafting. It can be used manually, with a simple pencil-shaped device, or with machine or robots. Adhesive tape or glue is another means of holding the grafted counterparts in place (Morita 1988; Oda and Nakajima 1992).

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Specially designed knives and gimlets for grafting (Fig. 2.1. A, B, F) have been manufactured and are used by growers in different parts of the world. A special knife with a self-feeding connection of skimmed milk to inactivate some potent viruses has been developed in The Netherlands and in Korea. A handheld grafting device constructed with changeable stainless steel, single-edge razor blades, makes it possible to simultaneously create a uniform wedge and a receptacle in the stem of Phaseolus vulgaris (White 1979). Rapid changes have been taking place recently and it is evident that marked progress will be made on these devices with the improvement of grafting technology and introduction of new and efficient grafting robots (Section II.B). 4. Monitoring Grafting Success. Grafting success can be judged subjec-

tively by observations of experienced growers and researchers. A transverse cut across the graft union is still the major way of judging in most nurseries. Recently, objective determination of grafts take have been developed for mass production of grafted seedlings using measurement of electrical resistance across the graft interface, by thermal imaging of leaf temperature, assessment of hydraulic connections by displacement transducer, and electrical wave transmission. In tomato grafts, electrical resistance for the first two to three days increased rapidly in step with the formation and thickening of the isolation layer (Yang, Zhang, and Low 1992). In the next three to four days electrical resistance decreased steadily, as the isolation layer ruptured and disappeared during callus proliferation interdigitation. Then, resistance began to drop to the level of the intact stem, which seemed to indicate that symplastic connection and vascular unification had been completed. In Amaranthus tricolor/Lycospersicon esculentum grafts, resistance increased steadily with the establishment of an isolation layer, which remained unruptured. Thermal cameras with an image processor have been used to evaluate the quality of graft take based on leaf temperature. With successful grafting, water moved smoothly from the root to leaves of the scion, decreasing temperature due to transpiration. Leaf temperatures of successfully grafted plants were 2° to 3°e lower than poor grafts. Seedlings with thicker leaves are better for grafting because thicker leaves can maintain higher moisture content facilitating faster graft union. A displacement transducer that assessed the functional hydraulic connection between rootstock and scion was used to evaluate TAG. The thickness of scion and rootstock leaves was measured under repeated water stress and the change in leaf thickness was associated with histological observations. The major hydraulic connection within the graft

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS

73

union of tomato became functional over about 48 hours from the fifth day after grafting, consistent with the appearance of wound-xylem bridges at this time (Turquois and Malone 1996). Measurement of electrical wave transmission from scion to rootstock across the grafting interface may be another technique to evaluate grafting. Transmission was related to histological changes during the development of the graft union (Lu et al. 1996). 5. Acclimatization. Proper acclimatization is critical for grafted plants to survive. Acclimatization involves healing of the cut surface and hardening for field or greenhouse survival (Oda 1999). Acclimatization may be achieved simply by enclosing the rootstock and scion in a black plastic bag (to avoid heat buildup) until the union is formed (Denna 1962). Growers usually achieve acclimatization by use of plastic tunnels (Fig. 2.4). In many commercial nurseries, the grafted plants, usually in cell trays of 50 to 72 cells, are placed on a greenhouse bench and the trays are sealed with a single layer of semitransparent 0.01 mm high-density polyethylene film to reduce the moisture loss and kept sealed for five to seven days without additional irrigation. Partial shading may be needed during the daytime to avoid excessive heat buildup. For tomato, a combination of high humidity and weak light, slightly higher than the light compensation point, prevents wiltingof grafted tomato scions and promotes healing of the cut surfaces of grafts. Films reducing thermal radiation on acclimatization tunnels depressed the rise of leaf temperature and increased the favorable range of light intensity

/

A

Fig. 2.4.

Plastic film

.

' '""

Plastic tunnels for acclimatization of grafted seedlings.

B

74

J. LEE AND M. aDA

for graft healing (Nobuoka, Oda, and Sasaki 1996). Under these high light intensity and high humidity conditions, healing of the graft union is accelerated by air movement (Nobuoka, Oda, and Sasaki 1997). Several types of acclimatization chambers have been developed and widely used by commercial plug seedling growers in Japan and Korea (Lee, Bang, and Ham 1998; Kawai et al. 1996; Oda 1999). 6. Micrografting. Micrografting refers to in vitro grafting using meristematic tissues. It is often carried out in tree crops such as citrus to eliminate a virus from infected plants, because virus particles do not exist in the apical meristem. Micrografting has been used in herbaceous plants to evaluate the physiology of grafting and determine the chemical basis of cell-to-cell contacts. Explants derived from the internodes of tomato, Nicandra physaloides, and Datura stramonium were grafted in vitro (Jeffree et al. 1987). Internode pectins from one species reduced the ability of homografts or other species to form vascular connections between rootstock and scion. Using C14-sucrose, it was determined that sucrose translocation across the graft interface started four days after grafting and increased later (Schoning and Kollmann 1995). Translocation appeared to occur via wound phloem, since at this time the first complete wound-phloem bridge traversed the graft interface. Apical segments ranging from about 200 /-lm down to 50 /-lm in depth were successfully grafted back to their parent plants, and apices developed completely normally. Complete shoots could be regenerated from an apical segment less than 1/1000th mm 3 containing about 600 cells, which was smaller than any graft previously recorded (Gulline and Walker 1957).

B. Robots The first robot developed was the "Cutting-off Cotyledon Grafting" (CCG) system developed by lAM BRAIN (Suzuki et al. 1995) to graft cucurbit vegetables (Table 2.3, Fig. 2.5). The robot took into account variation of seedling shape, location of cutting and gripping, cutting, and attachment. Seedlings were cut at the point of attachment of the cotyledon to the hypocotyl at an angle of 10° for the scion and 30° for the rootstock. The prototype grafting robot was constructed in 1987 and the second in 1989. It took three seconds to make a grafted plant with 95 percent survival rate (Onoda, Kobayashi, and Suzuki 1992). The demonstration model robot was deemed practical and the results were transferred to an agricultural machinery company that developed machines for the market (Kobayashi et al., 1996). A prototype

Table 2.3.

Grafting machines and robots developed in Japan and Korea.

Grafting machine

'I

Ql

Company (Country)

Crop(s)

Efficiency

Others

CCG

lAM BRAIN (Japan)

Cucumber

500/hr

Cutting-off cotyledon grafting (CCG).

G 892

lAM BRAIN (Japan)

Cucumber

1200/hr

First prototype grafting robot (1987).

Super Angel G-710

Nasmix Co. (Japan)

Cucurbits

600-800/hr

Super Idol 31 silica rubber split tube for fixing. Same as above.

Super Angel G-720

Nasmix Co. (Japan)

Solanaceous

600-800/hr

Grafting Robot AG 1000

Yanma (Japan)

Solanaceous

1000/hr

TGRa Grafting Robot

Technical Grafting Res. Institute (Japan)

Solanaceous and Cucurbits

800/hr

Instant glue is used and all 128 seedlings in a tray can be grafted at once.

Pin-grafting Robot

Takii (Japan)

Solanaceous

NAb

Ceramic pin is used. Plug-in method without other grafting clips or glue.

Plug-in Grafting Robot (-machine)

Osaka Prefecture Univ. (Japan)

Solanaceous and Cucurbits

NAb

Grafting Robot/Korea (Plug-in type)

Kyungpuk Univ. (Korea)

Solanaceous and Cucurbits

900/hr

Not yet commercialized.

Pin-grafting Robot

RDA (Korea)

Solanaceous

1200/hr

Ceramic pin is used.

Tongue Approach Grafting robot

SungKyunKwan Univ. (Korea)

Cucurbits

900-1200/hr

Ordinary clip is used.

Semi-automatic Grafting Machine

Yupoong (Korea)

Cucurbits

400/hr

One operator is needed per machine.

°Technical Grafting Research Institute. bData not available. Source: Kurata (1994); Lee et al. (1998); Hwang, Kim, and Ko (1999)

J. LEE AND M. ODA

76

D

E

Fig. 2.5. Grafting robots. (A) Grafting Robot AG 1000 developed by Yanma, Japan; (B) Grafting robot developed by Iseki, Japan; (C) Full-automatic pin grafting machine developed by Rural Development Administration, Korea; (D) Full-automatic grafting machine for cucurbits developed by Kyungpook National Univ., Korea; (E) Yupoong (tongue approach) grafting machine developed by Yupoong Co., Korea.

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS

77

semiautomatic grafting system was also developed by Hwang, Kim, and Ko (2000) in Korea. Several grafting robots have been manufactured by the Rural Development Administration (RDA), Korea (Kang 2000) and will be distributed to the commercial plug seedling growers at relatively reasonable prices. Since 1987, grafting robots for plugs have been developed for tomato grown in plugs. The robot manipulates grafting plate holders to grasp scions and rootstocks. The plants are held in V-shaped hollows on a "hollow plate" with spongy rubber mounted on a "drive plate" on the inner side that firmly grasps the axes of scions and rootstocks. The hollow plate and the drive plate are fixed with holders. The scion axis is cut parallel to the low side of the grafting plate and the rootstock axis is cut parallel to the upper side. The grafting plate holding the scions is placed on the plate holding the rootstock (Oda et al. 1994). The scion and rootstock were attached with adhesive (alkyI2-cyanoacrylate) and hardener (polyalkyleneglycol derivatives). The adhesive grafting was successful in eggplant, cucumber, and grapevine (Morita 1988). Chinese cabbage could also be grafted on turnip at the two-leaf stage using the adhesive and hardener (Oda and Nakajima 1992). The grafting machine constructed for this technique was released by a commercial company. Growth and yield of tomato (Oda et al. 1995) and eggplant (Oda et al. 1997) grafted with this robot were equivalent to conventional manual grafts. However this robot, which produces a horizontal cut was applicable to solanaceous crops but not to cucurbits. The low cucurbit survival of horizontally-cut grafts at the hypocotyl was ascribed to the loss of the cotyledons from the rootstock and the smaller number of vascular bundles coming into contact with the cut surface of the scion and rootstock. Survival was increased when the angle formed by the expanding direction of the scion and rootstock cotyledon was 90° instead of 0° (Oda et al., 1994). Survival and growth of cucumber scions were enhanced when the difference in the diameter of the horizontally cut scion and rootstock hypocotyls was minimized (Oda, Tsuji, and Sasaki 1993). Low survival of cucumber plants by horizontal-cut grafting was attributed to low pressure in the spliced cut surfaces and loss of cotyledons on the rootstock (Oda et al. 2001). The length of the hypocotyls of figleaf gourd (Cucurbita ficifolia) used as a rootstock could be controlled by a seed treatment of uniconazole and a spray of gibberellic acid. Various other prototype grafting robots were also described by Kurata (1994). Three grafting robots have been developed in Korea, two in 1998

78

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LEE AND M. ODA

and one in 1999 (Table 2.3), and one was commercialized in 2001. The pin-grafting robot developed by Rural Development Administration for solanaceous crops can graft 1200 seedlings per hour. The simple and economic grafting machine was developed by Yupoong and has been very popular in Korea. This machine, priced about $400 (US), has been exported for more than ten years to many Asian countries and some European countries. This machine can graft up to 600 seedlings per hour by tongue approach grafting, mostly in cucurbitaceous crop. However, an experienced operator is needed to run this machine effectively and efficiently. III. PHYSIOLOGY OF GRAFTING

Numerous publications related to grafting are concerned with physiological aspects such as flowering, ion transport, and organic substances (Table 2.4). Publications concerned with disease, rootstock, and fruit quality are also listed. Grafting induces significant changes in almost all aspects of growth and development. Some of these physiological processes or changes are further explained. A. Graft Union

The graft union in woody crops is based on growth and proliferation of the cambium layer. It is formed from the intermingling and interlocking of callus tissue produced by the stock and scion cambium in response to wounding. Under the influence of the existing cambium, the callus tissue differentiates new cambium tissue and this redifferentiates xylem and phloem to form a living, growing connection between stock and scion (Janick 1986). The sequence of structural events in compatible grafts of herbaceous plants has been reviewed by Andrews and Marquez (1993). Basically, ruptured cells of the graft interface collapse and form a necrotic layer that disappears during subsequent events. Living cells from both stock and scion then extend in the necrotic zone. A callus bridge of interlacing parenchyma cells forms by cell division, rupturing and invading the necrotic layer. During these events the tensile strength of the graft increases due to physical cohesion between stock and scion. New vascular cambium is differentiated from parenchyma cells and secondary xylem and phloem are produced by the reconstituted cambium providing a vascular connection between rootstock and scion. In some herbaceous plants, especially in cucurbits, the stem (both the hypocotyl and the epicotyl) has a central pit or cavity and usually six vascular bundles

Table 2.4.

Research reports and some references on various aspects of vegetable grafting.

Category Disease and Stress Resistance Bacterial wilt Fusarium resistance

Nematode Verticillium wilt Viral wilt Others Grafting Chimeras & genetic change

Graft incompatibility Grafting machines & aids Grafting methods Grafting robots Growth regulators Micrografting Virus detection

Reference Tikoo, Mathaii, and Kishan 1979; Monma et a1. 1997; Matsuzoe et a1. 1993a Harrison and Burgess 1962; Gindrat, Ducrot, and Caccia 1977; Kuniyasu and Yamakawa 1983; Alam et a1. 1994; Chung, Yoon, and Choi 1997 Ali et a1. 1992; Matsuzoe et a1. 1993b; Mian, Ali, and Akhter 1995 Gindrat, Ducat, and Caccia 1976; Kuniyasu and Yamakawa 1983 Alexander 1963,1971; Iwasaki and Inaba 1990; Park et a1. 1999 Derbyshire and Green 1961; Spender and Weichhold 1964; Tachibana 1982; Matsubara 1989; Liao and Lin 1996; Shoji 1989 Winkler 1908,1910; Mirso 1954; Yagishita et a1. 1990; Lindsay et a1. 1995; Goffreda et a1. 1990; Taller et a1. 1998 Jeffree and Yeoman 1983; Szteyn 1959; Parkinson, Jeffree, and Yeoman 1987; Oda, Nagaoka, and Tsuji 1992; Andrews and Marquez 1993; Oh 2000; Ko 1999 Kobayashi et a1. 1996; White 1979; Yagishita and Hirata 1987; Morita 1988; Kang 2000 Ashita 1930, 1934; Demla 1962; Shackleton 1965; Harnett 1974; Yamakawa 1982; Oda 1994; Honami 1977; Lee 2000 Kurata 1994; Oda et a1. 1997; Kang 2000 Lockhart 1957; Suge 1984; Tachibana 1988; Andrews and Marquez 1993; Lu et a1. 1996 Schoning and Kollmann 1995; Natali and Cavallini 1989; Wang and Kollmann 1996 Miller 1958 (continues)

'J

(.0

Q:)

o

Table 2.4.

(continued)

Category Physiology Flowering & Others

Mineral transport Organic substances Rootstock Resources Breeding

Germplasm Interspecific hybrids Yield and Quality Growth

Quality Yield Other Problems Cultural practices Graft-induced disease Quality reduction

Reference

Kher et al. 1953; Lam and Cordner 1955; Kruzilin and Svedskaja 1959; Lardizabel and Thompson 1988,1990; Andrews and Marquez 1993 Brown, Chaney, and Ambler 1971; Tachibana 1982, 1988; Masuda and Gomi 1984; Ikeda, Okitsu, and Arai 1986; Zaiter, Coyne, and Clark 1987; Ahn et al. 1999; Ruiz and Romero 1999 Ikenaga et al. 1990; Hirata and Yagishita 1986; Dawson 1942; DeStiger 1961; Yagishita et al. 1990; Tiedemann and Carstens-Behrens 1994; Golecki et al. 1998 Kuniyasu and Yamakawa 1983; Matsuo, Ichiuchi, and Kohyama 1985; Yazawa et al. 1980; Zijlstra, Groot, and Jansen 1994; Sakata 2000, Takii 1995 Limberk 1951; Oda et al. 1992; Robinson and Decker-Walters 1998; Decker-Walters 1998; Heo 2000 Yazawa et al. 1980; Kuniyasu and Yamakawa 1983, Andrews and Marquez 1993; Parkinson, Jeffree, and Yeoman 1987; Oda et al. 1992; Oh 2000 Madec 1963; Abelhaffez, Harssema, and Verkerk 1975; Tachibana 1988; Lee et al. 1998; Lee, Bang, and Ham 1999a Hong 1710; Yamamoto et al. 1989; Matsuzoe et al. 1996; Oda et al. 1996; AVRDC 2000 White 1963; Coggins and Lesley 1968; Natali and Cavallini 1989; Ruiz et al. 1997; Asao et al. 1999; Lee et al. 1999b Tachibana 1982; Oda et al. 1996; Lee et al. 1999a Kuwata et al. 1981; Kim 1984; Uematsu et al. 1992; Kim and Lee 2000 Matsuda and Honda 1981; Matsuzoe et al. 1996; Lee et al. 1998; Lee et al. 1999b

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS

81

in definite position (ada, Tsuji, and Sasaki 1993). Complete union of these six vascular bundles is often impossible depending on the grafting methods and research is focused to achieve fast and complete union of vascular connection. B. Graft Compatibility

In general, grafting compatibility is related to taxonomic affinity but there are significant exceptions. Graft incompatibility, reviewed by Andrews and Marquez (1993), is differentiated from graft failure that often results from environmental factors or lack of skill of the grafter. Graft incompatibility under conditions when grafting could be expected to be successful includes failure to unite into a strong union, failure of the grafted plant to grow in a healthy manner, or premature death following grafting. Physiological incompatibility may be due to lack of cellular recognition, wounding responses, growth regulators, or incompatibility toxins. Ko (1999) studied the graft compatibility in cucurbits and concluded that most of the tested cucurbits scions could be grafted on several rootstock species with only a few exceptions (Table 2.8), However, incompatibility could be changed depending on the grafting methods and growing environments. Similar reports have been pointed out by Lee (1989,1994).

c.

Translocation

Uptake, as well as the translocation of various substances such as ions, photosynthates, plant hormones, alkaloids, and viruses, can be influenced by rootstocks or by grafting. Since one of the basic purposes for grafting is the utilization of the vigorous root systems of the rootstocks, grafted plants usually show increased uptake of water and minerals as compared to the self-rooted plants. However, the ion concentration in xylem sap from grafted plants may be actually lower than the ones from self-rooted plant (Masuda and Gomi 1984), due to the increased absorption of water and possible dilution in the xylem sap. Grafting influences the absorption and translocation of ions, that is, phosphorus, nitrogen, magnesium, and calcium (Gluscenko and Drobkov 1952; Masuda and Gomi 1984, Ikeda, Okitsu, and Arai 1986; Kim and Lee 1989). Absorption and translocation of some microelements such as iron and boron is also influenced by rootstocks (Brown, Chaney, and Ambler 1971; Zaiter, Coyne, and Clark 1987; Gomi and Masuda 1981). One of the most striking effects of the rootstock is the absorption of ions by grafted plants or by rootstocks at much lower temperatures

82

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(Tachibana 1982; Masuda and Gomi 1984; Ahn et al. 1999). This enhanced uptake of minerals appeared to be closely associated with the activity of enzymes responsible for absorption (Ahn et al. 1999). Some well-known alkaloids, for example nicotine, are synthesized in the root and move upward through the xylem. Thus, nicotine can accumulate in tomato grafted onto tobacco rootstocks (Dawson 1942). However, it is also known that the alkaloid synthesized and translocated from the roots could be effectively destroyed or metabolized by the scion (Poda-Cikalenko 1960). Solanum aculeatissimum contains the steroid saponins aculeatiside A and aculeatiside B at high levels in the root, whereas they are lacking in all organs of tomato. When S. aculeatissimum was grafted onto tomato, a small amount of steroid saponins was found in the leaves and the stem of S. aculeatissimum but not in the roots of the tomato. However, when tomato was grafted onto S. aculeatissimum, steroid saponins appeared only in the roots of S. aculeatissimum. The steroid saponins seemed to be synthesized mainly in the roots of S. aculeatissimum (Ikenaga et al. 1990). Cytokinins are major plant hormones, principally synthesized in the root near the root tip region. Cytokinins move upward exerting significant influence on plant growth. Plants with vigorous root systems produce more cytokinins and the yield increase induced by a vigorous rootstock is closely associated with the amount of cytokinins in the ascending xylem sap (Kato and Lou 1989). The cytokinin composition varies greatly with the kind of cucurbitaceous crops. For example, only zeatin and dihydrozeatin are found in cucumbers whereas large quantities of isopentenyladenine and isopentenyl adenosine are found in squash and gourd (Table 2.5). A small portion of scion stem, approximately 15 to 20 em in length, can effectively modify the composition of cytokinins in the ascending xylem sap, indicating the rapid and efficient changes of cytokinin composition in plant tissue (Lee et al. 1999b, 2001). Among the cucurbits tested, only Sicyos angulatus did not show any measurable amount of cytokinins in the xylem sap. Stem growth of Sicyos angulatus and any scions grafted onto Sicyos angulatus was different than those grafted onto other vigorous rootstocks. The translocation of some floral stimuli has been reported in Sicyoscucumber graft combinations and in Perilla fructescens, a typical shortday plant (Takahashi, Saito, and Siuge 1982; Suge 1984). The transmission of virus from rootstock to scion and vice versa has been widely reported in tomato (Alexander 1963; Alexander 1971) as well as in many cucurbits. Some of the noxious seed-borne viruses in rootstock seeds such as strains of tobacco mosaic virus, could be a disaster for the watermelons

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS

83

Table 2.5. Cytokinin composition in xylem sap collected from intact and grafted plants of cucumber, squash, and figleaf gourd plants.

Cytokinin content (ng/mL sap) Zeatin

Zeatin riboside

Dihydro-zeatin riboside

Isopentenyl adenine

Total

Cucumber (Cucumis sativus)

0.76

4.55

0.80

Trace

6.11

Squash A (Cucurbita moschata)

Trace

3.67

0.43

3.63

7.73

Squash B (Cucurbita maxima)

Trace

4.06

0.57

1.84

6.47

Figleaf gourd (Cucurbita fidfolia)

12.20

Crop (Scion/rootstock)

Trace

4.54

1.48

6.18

Cucumber/Cucumber

0.55

5.58

0.96

Trace

7.07

Cucumber/Squash A

1.65

4.29

0.20

Trace

6.14

Cucumber/Squash B

Trace

5.36

0.19

Trace

5.55

1.49

5.08

0.65

Trace

7.22

Cucumber/Figleaf gourd

or other commonly grafted plants, unless the seeds are treated with dryheat to inactivate the virus (Kim and Lee 2000). D. Growth Effects 1. Vigor. Rootstocks affect the growth and yield of scion plants and vegetable crops grafting is often performed to provide vigor. Cucumber plants grafted on pumpkin rootstocks grown in sand culture were higher in dry matter than self-rooted cucumber (Shimada and Moritani 1977). Tomato grafted to disease resistant rootstocks 'K', 'KV', 'KVF', and 'KN' were more vigorous and produced higher yields than self-rooted plants (White 1963). Some rootstocks may also depress growth and yield of scion plants. Growth was reduced in tomato with rootstocks of Datura patula (Kramer 1957), Solanum sodomaeum, and S. auriculatum (Shackleton 1965), and eggplant (Abdelhaffez, Harssema, and Verkerk 1975). Some tomato rootstocks invigorated eggplant (Topoleski and Janick 1963). 2. Physiological Disorders. Fruit physiological disorders often appear after grafting depending on the rootstock (Chung 1995b; Matsuda and Honda 1981). Abnormal fruit fermentation occurs when 'Prince' melon

J.

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LEE AND M. ODA

(Cucumis melo) is grafted onto squash (Cucurbita spp.) or oriental melons are grafted onto vigorous interspecific squash (Chung 1995b). Other abnormalities include reduction in soluble solids, persistent green color in the suture stripe, and appearance of nonviral yellow mottle symptoms in leaves (Ryu, Choi, and Lee 1973; Lee 1989; Lee, Bang, and Ham 1998). 3. Stress Tolerance. Tolerance to temperatures, drought, flooding, and salt stress may be influenced by the rootstock (Matsubara 1989). The increased performance at low soil temperatures with certain rootstocks in cucurbits is one of the main benefits of grafting. The rootstock' Shintozwa No l' (Cucurbita maxima x Cucurbita moschata) has excellent resistance to low soil moisture for watermelon. IV. CROP EXAMPLES

Grafting as a commercial practice in the cultivation of herbaceous plants principally occurs in crop species of the Cucurbitaceae (watermelon, cucumber, and various melons) and the Solanaceae (vegetable fruit crops such as tomato, eggplant, and bell pepper). Grafting is also a common practice in the Cactaceae in species such as Gymnocalicium mihanovichii ('Ruby Ball') to produce a number of bizarre ornamental forms that have been widely accepted throughout the world. Grafting is also practiced in a number of crops in a limited way for virus identification (Kim and Lee 2000), for crop improvement (Coggins and Lesley 1968; Poinsettia Growers Association 1995; Son 2000), and for research investigations. Common and scientific names of vegetables and ornamental cacti are listed in Table 2.6.

Table 2.6.

Common and scientific names of crops used in text.

Common name CucurbitaceaeO African horned cucumber Bitter gourd, bitter melon, bitter cucumber Bottle gourd Bur (=star) cucumber Cucumber Figleaf gourd

Scientific name

Common usage

Cucumis metuliferus

Food

Momordica chaentia Lagenaria siceraria

Food, ornamental Food, rootstock, container Rootstock, food Food Food, rootstock

Sicyos angulatus Cucumis sativus Cucurbita ficifolia

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS Melon, muskmelon, net melon, cantaloupe Oriental melon Oriental pickling melon, pickling melon Pumpkin, winter squash Sponge gourd Squash Vegetable marrow, summer squash Watermelon Wax gourd, White gourd

Brassicaceae Cabbage Chinese cabbage Kale Kohlrabi Mustard Radish Rapeseed Turnip Cactaceae Ruby Ball (Bimoran)

Sanchui

Solanaceae Apple of Peru Datura Eggplant Fox face Mahorka Nightshade Plate brush Potato Scarlet eggplant Scented tobacco Thorn apple Tobacco Tomato

85

Cucumis melo Cucumis melo var. makuwa

Food Food, medicine

Cucumis melo var. cocomon Cucurbita maxima Luffa cylindrica Cucurbita moschata

Food Food, ornamental, rootstock Food, industrial Food, rootstock

Cucurbita pepo Citrullus lanatus Benincasa hispida

Food Food Food, rootstock

Brassica oleracea var. capitata B. campestris spp. pekinensis B. oleracea var. acephala B. campestris var. gongylodes B. juncea Raphanus sativus B.napus B. campestris ssp. Rapa

Food Food Food, Food Food, Food, Food, Food

Gymnocalycium mihanovichii var. friedrichii Chamaecereus silvestris

Ornamental, medicinal, and food Ornamental

Nicandra physoloides Datura patula Solanum melongena S. mammosum

Medicine Medicine Food, medicine Decoration, ornamental Tobacco Medicine Food Food Food, medicine Food, ornamental Tobacco Medicine Tobacco, ornamental Food Food, spice, medicine

Nicotiana rustica S. nigrum S. tOlvum S. tuberosum S. integrifolium S. laciniatum N. affinis D. stramonium N. tabacum Lycopersicon esculentum S. indicum

fodder spice animal feed oil

aNumerous cucurbitaceous crops were described elsewhere by Decker-Walters (1998), Robinson and Decker-Walters (1998).

86

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LEE AND M. ODA

A. Watermelon 1. Current Status. Watermelon was the first crop used for vegetable grafting, but detailed scientific information was only available from the late 1920s. Watermelons were first grafted to squash (Cucurbita moschata) rootstocks to control soil-borne diseases caused by successive cropping. Most of the problems associated with successive cropping such as yield decreases, fusarium wilt, and wax or bloom development on the fruit rind were ameliorated with the use of rootstocks (Ashita 1927). Tongue approach grafting and cleft grafting were the two major methods for watermelon grafting during the earlier stages and the advantage was rapidly recognized by the farmers (Ashita 1930, 1934). The use of plastic films triggered a revolution in horticulture and was widely adopted, especially in temperate countries with short growing seasons. Remarkable yield increases, usually more than 200 percent under protected structures as compared to open field, attracted strong interest by many growers. As a result, the area of protected cultivation sharply increased in many Asian countries such as Japan, Korea, and China and the demand for grafted seedlings markedly increased despite their high cost. Heo (2000) tested the response of various Citrullus germplasm to some major watermelon diseases (Table 2.7) and found out that three lines of Citrullus Janatus, 'Calhoun Gray', 'Mudeungsan M13', and PI 560901, showed strong resistance to Fusarium races 0 and 1, whereas others showed only partial resistance. Three of four resistant lines of hybrids between C. citroides x C. Janatus exhibited strong resistance to Fusarium, indicating the high potential for breeding resistant lines for new cultivars or rootstocks. This is a very significant finding since fusarium disease (Fusarium oxysporum f. sp. lagenariae) is spreading among gourd rootstocks in major watermelon producing areas and causing severe crop losses, especially later in the growing season. However, there are other advantages such as low temperature tolerance, salt tolerance, wet-soil tolerance that cannot be expected in watermelon rootstocks (Heo 2000). 2. Rootstocks. The original purpose of watermelon grafting in Korea and Japan was to avoid the yield decline caused by successive cropping because growers did not possess enough land to apply proper crop rotations. Squash was the first rootstock for watermelon but the rationale for grafting has been steadily extended with the introduction of new rootstocks and the development of new cultural techniques for the management of microenvironments in protected areas.

87

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS Table 2.7.

Disease reaction of major Citrullus germplasm and their hybrids.

Genotype

Citrullus lanatus Calhoun Gray Charleston Gray Dixilee Fairfax Grif 12335 Grif 12336-3 Mudeungsan M1 Mudeungsan M13 PI 185636 PI 203551-2 PI 518611-1 PI 560006 PI 560901

Anthracnose

Gummy stem blight

Colletotrich um orbiculare

DipJodia bryoniae

SO HR HR HR MR MR S S MR MR MR HR S

Fusariumwilt

Fusarium oxysporum f. sp. niveum Race 0

Race 1

S S S S S S S S S S S S S

HR HR MR

HR S HR HR SR MR MR HR MR MR SR S HR

HR S Segregate b S S MR MR Segregate MR S Segregate HR HR

SR HR MR MR HR SR

S MR HR HR SR MR SR S HR

Citrullus citroides PI 189225 PI 271769 PI 271775-1 PI 271779 PI 296341 PI 299379 PI 326515-2 PI 492261 PI 482299-1 PI 482299-2 PI 482299-3 PI 482322 PI 482342

S S S SR S S S HR HR S S

C. citroides x C. lanatus hybrids PI 271769 x Calhoun Gray S PI 271769 x Charleston Gray HR PI 296341 x Calhoun Gray S PI 296341 x Charleston Gray HR C. citroides hybrid PI 271769 x PI 296341

S

HR HR MR SR

S HR MR MR MR S MR MR SR MR S

S

HR

HR

S S

HR HR

HR HR

S

HR

MR

S

HR

HR

fJHR, Highly resistant; MR, Moderately resistant; SR, Slightly resistant; and S, Susceptible. bSegregation of resistant and susceptible plants. Source: Heo (2000)

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Rootstocks for cucurbits are presented in Table 2.8. Bottle gourd is the most popular rootstock for watermelons followed by interspecific hybrid squash, Cucurbita maxima x C. moschata. Since most of the seedlings and young plants were grown early in the spring in frames, tunnels, or in greenhouses, low temperature tolerance along with disease tolerance, have been key functions of a good rootstock. The vigorous root system of the rootstock was also regarded as highly important in the early days since watermelon seedlings grafted on the vigorous root systems of gourd and squash could absorb water and nutrient more efficiently than the self-rooted seedlings. However, the quality of the fruit in grafted plants could be markedly reduced as compared to the self-rooted seedlings unless special care was given to the grafted seedlings. Decrease in sugar or soluble solids content, appearance of yellowish bands in the red flesh, off flavor, insipid taste, and incidence of internal breakdown due possibly to the reduced absorption of calcium in relation to nitrogen, appeared to be some of the commonly observed problems associated with the use of grafted seedlings (Ryu, Choi, and Lee 1973). Graft compatibility should be carefully considered when researching suitable rootstock for specific crops. All rootstocks do not exhibit similar degrees of graft compatibility with scions. 'Shintozwa', an interspecific hybrid squash between Cucurbita maxima and C. moschata, shows good compatibility with all scions whereas figleaf gourd is compatible only with cucumbers. Since the purpose of grafting is to take the advantage of the strong vigor and resistance to various diseases and stresses, it is important to understand the overall response of cucurbitaceous plants, both the rootstocks and scions, to biological environmental stresses (Table 2.8). 'Shintozwa' and figleaf gourd rootstock have the best salt tolerance while the bur cucumber (Sicyos angulatus) has the best nematode tolerance. Other secondary problems associated with grafting such as higher incidence of late blight, anthracnose, gummy stem blight, cucumber green mottle mosaic virus (CGMMV) may cause problems in growing of grafted seedlings (Kim and Lee 2000). 3. Grafting Methods. For small growers, the grafting method is based on the skill of the operator and availability of affordable grafting machines. Tongue approach grafting, the oldest method, is still the most widely used grafting method among small growers but for large growers, laborsaving is critical. For example, the grafting time required for 100 grafts is 71.7 minutes for tongue approach grafting as compared to 60.5 minutes for hole insertion grafting and 46 minutes for pin grafting, a savings of 15.6 percent and 35.8 percent, respectively (Lee et al. 2000). The

Table 2.8.

Response of cucurbits to biological and environmental stresses.

Fusarium Rootstock and scion

~

Nematode

Ia

II

III

IV

Rootstockb Shintozwa Hongtozwa Figleaf gourd Bottle gourd Wax gourd Bur cucumber AH cucumbere

HW HR MR MR HR HR HR

HR HR SR HR MR HR HR

HR HR MR HR HR HR HR

HR SR S S HR HR HR

Scion Watermelon Cucumber Oriental melon

S HR HR

SR SR HR

HR HR S

HR HR HR

M.

M.

incognita

halpa

Low temp tolerance

S S S S S S S

S S S S SR HR MR

HR MR HR SR SR SR SR

HR MR HR MR SR SR '?

HR S S

SR S S

S HR S

SR SR S

Graft compatibility

High salt tolerance

Watermelon

Cucumber

HC SC IC HC HC HC HR

HCd HC HC HC HC MC HC

Oriental melon HC HC IC IC HC HC

aI, Fusarium oxysporum f. sp. niveum; II, F. oxysporum f. sp. cucumerinum; III, F. oxysporum f. sp. melonis; and IV, F. oxysporum f. sp. lagenariae. bShintozwa (Cucurbita maxima xC. moschata), Hongtozwa (G. moshchta), figleaf gourd (G. ficifolia), bottle gourd (Lagenaria siceraria), wax gourd (Benincasa hispida), bur cucumber (Sicyos angulatus), and AH cucumber (Cucumis metuliferus), respectively. cHR, highly resistant; MR, moderately resistant; SR, slightly resistant; and S, susceptible. dHC, highly compatible; MC, moderately compatible; SC, slightly compatible; and IC, incompatible. eAH: African horned cucumber. Source: Ko (1999)

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]. LEE AND M. aDA

distribution of efficient grafting machines or grafting robots will have a profound influence on the preferences for grafting method. 4. Cultural Practices. Yield increases obtained by using rootstocks can

be substantial, but more frequently it is not yield increase that most of the growers seek. Rather, it is a matter of crop or no crop, because once a field begins to show symptoms of fusarium wilt or some other noxious soil-borne disease, harvest of high-quality fruit can never be expected. There are considerable differences in resistance of rootstocks and scions to various pathogens indicating the need for rootstock selection depending on the situation. 'Shintozwa', bur cucumber, and African horned cucumber are highly resistant to all race of fusarium, while wax gourd is highly resistant to three races and moderately resistant to one race (Table 2.8). In addition to disease resistance, low temperature tolerance of rootstocks is important in many cucurbits. In order to meet the year-round demand for fresh vegetables, most cucurbits are frequently sown during the cool season and grown under unfavorable microclimatic conditions in the winter greenhouse. Since watermelons favor high temperature for optimum growth, inadequate temperature conditions in winter greenhouses greatly inhibits fast and normal growth of seedlings. However, when the seedlings are grafted onto bottle gourd or hybrid squash (Cucurbita maxima x Cucurbita moschata), normal and fast seedling growth can be expected even at somewhat lower or inadequate temperature conditions, thus reducing the energy needed for heating. Resistance to other stress conditions includes high salt and nematodes (Table 2.8).

Because most of the rootstocks have much more vigorous root systems and wider distribution throughout the soil, grafted seedlings also absorb water and nutrients much more efficiently than the self-rooted seedlings. Apparently less fertilizer is needed to maintain normal growth and less frequent irrigation should be practiced. It is routinely recommended to reduce the amount of fertilizers for grafted plants as compared to the selfrooted plants by 30 percent or more depending on the kind of rootstocks used. With grafted watermelon, care should be taken to avoid high soil moisture condition, especially late in the growing season, to avoid the fruit quality deterioration due to high soil moisture and higher uptake of nitrogen fertilizers. 5. Problems Associated with Grafted Plants. In addition to the labor and skill required for grafting and postgraft management, growers may encounter unexpected problems associated with the use of grafted

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seedlings as compared to self-rooted seedlings. These include the incidence of unexpected diseases such as virus infection in rootstock seeds, unsatisfactory resistance or the occurrence of secondary disease such as anthracnose in bottle gourd rootstock, poorly fused vascular bundles in grafted unions, changes of graft-compatibility with the growing season, environmental stresses, and inferior quality of fruit harvested from the grafted plants (Lee 1994; Lee et al. 1998). Furthermore, there are additional costs and investments that must be considered such as the cost of rootstock seeds, the expense needed for the grafting operation, and the capital input for the purchase of machines or robots. B. Cucumber 1. Current Status. Cucumbers usually do not show such an extensive

spread of soil-borne disease as compared to watermelons and melons especially when grown outdoors during the summer. Cucumber cultivars developed for winter greenhouses are more susceptible to various diseases and unfavorable environmental conditions than those developed for outdoor growing. Thus, only about 50 percent of the fieldgrown cucumbers were grafted in Korea and Japan. However, cucumbers grown in greenhouses and under protected structures, including those hydroponically grown, are very commonly grafted because disease resistance is essential for unfavorable microenvironments in winter greenhouses and during the extended harvesting period later on. Other reasons favoring grafting include low temperature tolerance, yield increase, ease of management, higher fruit quality, and extension ofharvest period in hydroponics system (Asao et al. 1999). Cucumber seedlings are larger than watermelons and thus, grafting methods, as well as the purpose of grafting, are different from those of watermelons (Table 2.2). 2. Rootstocks. Squash, figleaf gourd, and star cucumber are commonly used as rootstocks for cucumber, but figleaf gourd is by far the most popular (Table 2.9). Figleaf gourds possess good to excellent resistance to fusarium wilt (Ko 1999), good tolerance to low temperature conditions (Tachibana 1982, 1986, 1988; Ahn et al. 1999), and good ability of absorbing water and nutrients from the soil or in hydroponics system (Masuda and Gomi 1984; Shimada and Moritani 1977) or from nutrient solution even at low temperature conditions (Tachibana 1986, 1989). Interspecific squash rootstocks such as 'Shintozwa' are more tolerant of high temperature conditions and are commonly used for cucumber grown during summer conditions. Rootstocks can be used to change fruit quality. Cucumber grafted to certain strains of squash (butternut types)

CD N

Table 2.9.

Rootstocks for cucurbitaceous crops and some related characteristics.

Scion/Rootstock Watermelon Bottle gourd (Lagenaria siceraria) Squash (Cucurbita moschata) Interspecific hybrid squash (Cucurbita maxima x C. moschata)

Pumpkins (Cucurbita pepo) Wintermelon (Benincasa hispida) Watermelon (Citrullus lanatus) African horned cucumber (Cucumis metuliferus) Cucumbers Figleaf gourd (Cucurbita tidfolia) Squash (Cucurbita moschata)

Cultivar

Major characteristics

Possible disadvantage

FR Dantos, Partner, Renshi, FR Combi, TanTan Chinkyo, No.8, Keumkang

Vigorous root system, resistant to fusarium and low temperature Vigorous root system, resistant to fusarium and low temperature Vigorous root system, resistant to fusarium and low temperature, excellent vigor and high temperature tolerance Vigorous root system, resistant to fusarium and low temperature Good disease resistance

New fusarium race, susceptible anthracnose Poor fruit shape and quality Reduced fertigation required; reduced quality

Kanggang, Res. #1 Tuffness, Kyohgoh, NHRI-1

Fusarium tolerance, but not resistance Excellent fusarium resistance and good nematode tolerance

Not enough vigor and disease resistance Medium to poor graft compatibility

Heukjong, Black Seeded Figleaf gourd Butternut. Unyong #1, Super Unyong

Good low temperature tolerance and disease resistance Good fusarium tolerance and bloomless fruit skin

Fruit quality may be reduced Affected by Phytophthora

Shintozwa, Shintozwa #1, Shintozwa #2, Chulgap Keumsakwa, Unyong, Super Unyong Lion, Best, Donga

Poor fruit shape and quality Incompatibility

Interspecific hybrid squash (Cucurbita maxima x C. moschata) Bur cucumber (5icyos angulatus)

Shintozwa, Keumtozwa, Ferro RZ, 64-05 RZ, Gangryuk Shinwha, Chulgap Andong

African horned cucumber (Cucumis metuliferus)

NHRI-1

Melons-Oriental Melons Squash (Cucurbita moschata) Interspecific hybrid squash (Cucurbita maxima x C. moschata)

Baekkukzwa, No.8, Keumkang, Hongtozwa Shintozwa, Shintozwa #1, Shintozwa #2

Pumpkin (Cucurbita pepo)

Keumsakwa, Unyong, Super Unyong

Melon (Cucumis melo)

Rootstock #1, Kangyoung, Keonkak,Keumgang NHRI-1

African horned cucumber (Cucumis metuliferus)

Good fusarium and low temperature tolerance

Slight quality reduction expected

Good fusarium tolerance, low and high soil moisture tolerance and nematode tolerance Excellent fusarium resistance and good nematode tolerance

Reduced yield

Good fusarium and low temperature tolerance Good fusarium resistance, low and high soil temperature tolerance, and high soil moisture tolerance Good fusarium resistance, low and high soil temperature tolerance, and high soil moisture tolerance Fusarium tolerance and good fruit quality Good fusarium tolerance, low and high soil moisture tolerance and nematode tolerance

Phytophthora infection

Weak temperature tolerance

Phytophthora infection, poor fruit quality Phytophthora infection

Phytophthora problem Weak temperature tolerance

Source: Asao et al. (1999); Chung (1995a, 1995b); Igarashi, Kanno, and Kawaide (1987); Gomi and Masuda (1981); Kang, Choi, and Lee 1992; Kim and Lee (1989); Ko (1999); Matsuo, Ichiuchi, and Kohyama (1985); Lee (1989, 1994, 2000); Lee at al. (1998); Oda (1999); Oh (2000); Tachibana (1981) co w

94

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produced fruit with shiny bloomless skin as compared to those from ungrafted ones or from figleaf gourd rootstock (Choi et al. 1985; Yamamoto et al. 1989; Yamakawa 1982). The shiny bloomless fruits are favored by the Japanese, but not by the Koreans. Bur cucumber has been used to overcome the problems of fruit quality reduction caused by grafting onto more vigorous rootstocks such as 'Shintozwa' (Lee 1992). Cucumber grafted onto bur cucumber also exhibits some degree of fusarium resistance and marked resistance to rootknot nematode (Ko 1999). However, bur cucumber has not been used extensively because the fruit yield increase is less than expected. Furthermore, the uneven seedling size at the time of grafting due to the poor and slow seed germination makes grafting difficult and inefficient (Lee, Bang, and Ham 1999a). Cucumbers grafted onto bottle gourd grow well under greenhouse conditions but show incompatibility-like symptoms in the field (Oh 2000). Cucumbers can also be safely grafted onto wax gourd and African horned cucumber (Table 2.9), but further studies are needed before these rootstocks can be recommended. Rootstocks show striking differences in the absorption of water and nutrients from the soil or from the nutrient solution. In addition to the nutrient, it was also found that the total amount of cytokinins and their composition was quite different depending on the rootstock and the scion, leading to significant variation in the cytokinin composition in the xylem sap (Tachibana 1988; Lee et al. 1999c). 3. Grafting Methods. Tongue approach grafting and splice grafting are extensively used for cucumbers. The length of hypocotyl of figleaf gourd, the most popular rootstock for cucumber, frequently remains very short even after a considerable period after seed germination especially when the seedlings are grown under full sunlight at relatively low temperature conditions. Since rootstock seedlings with somewhat longer and strong hypocotyls are favored for some grafting, the seeds of figleaf gourd are soaked in gibberellin solution before sowing to promote hypocotyl elongation for easy grafting (Oda 1994). However, when excised rootstock seedlings are used for grafting, gibberellin treatment to the rootstock seeds should be minimized since the GA treatment tended to decrease rooting. 4. Cultural Practices. Cucumbers are frequently grafted only for low tem-

perature tolerance for off-season growing. However, grafting is routinely practiced for nutrient solution culture systems for extended fruit harvesting (Asao et al. 1999). Seedlings grafted by tongue approach graft-

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS

95

ing should be handled very carefully to minimize the possible separation of grafted interface during the handling and planting. Grafted seedlings should be planted shallow enough to prevent the rooting from the lower hypocotyl portion of the cucumber; adventitious rooting from the scion allows the grafted seedlings to become easily infected by soilborne diseases (Lee 1989). When grafted by the tongue approach method, removal of one cotyledon with the growing point of the rootstock seedling may be practiced in order to provide more space for scion growth. Reduction of fertilizer is recommended when grafted onto vigorous rootstocks such as interspecific hybrid squash, but cucumbers are not as sensitive to high nutrient (or nitrogen) level as watermelons or melons. Bloom development on the fruit skin can be considerably influenced by the rootstocks (Yamamoto et al. 1989). There is a significant negative correlation between the bloom development and absorption of silica from the soil. Plants or lines with higher content of silica on the leaf surface exhibit strong resistance to certain diseases and it is believed that the bloom development on fruit skin is also closely associated with the fruit quality preferences. However, the relationship between bloom development and quality characteristics has not as yet been firmly established. Sex expression in cucumber was not significantly influenced by grafting itself or by rootstocks (Lee 1994). Table 2.10 shows the soluble solids and sugar contents in cucumber grafted onto figleaf gourd and bur cucumber (Lee et al. 1999b). Even though the highest yield was obtained with figleaf gourd rootstock, fruit quality was much lower than from ungrafted plants. Fruit harvested from the plants grafted onto bur cucumber did not show such decreases in soluble solids and sugar concentrations. Thus, it is clear that fruit quality can be maintained by selecting proper rootstocks as well as by cultural practices such as reduced application of nitrogen fertilizers. Table 2.10.

Quality of cucumber fruits as affected by rootstocks. Sugar concentration (mg·L-l)

Soluble solids Scion/rootstock

(%)

Fructose

Glucose

Total

Cucumber own-rooted

4.17 aO

0.90a

1.55 a

2.45 a

Cucumber! Sicyos angulatus

4.16 a

0.81 b

1.49 a

2.30 b

Cucumber! Cucurbita fidfolia

3.66 b

0.65 c

1.33 b

1.98 c

°Mean separation by Duncan's multiple range test at 5 percent. Source: Lee et al. (1 999a)

96

J. LEE AND M. aDA

5. Problems Associated with Grafted Plants. Quality deterioration by grafting has been recognized in several fruit characteristics such as flavor and fruit shape. Fruits from grafted plant tended to be slightly shorter than those from untreated plants. Fruit firmness may also be decreased by grafting and shelf life of the fruit can also be decreased (Lee 1989; Lee et al. 1999a). Many of these graft-associated problems could be minimized by careful selection of rootstocks for each growing season.

C. Melons 1. Current Status. There are various melons commonly grown in Asia. These include oriental melons (Cucumis melo var. makuwa) , rock melon (var. cantalupenis), netted melon (var. reticulata), winter melon (var. inodorus), snake melon (var. flexuosus), pocket melon (var. dudaim) , and many others (Filov 1960; Robinson and Decker-Walters 1998). Most of the melons grown in Korea are oriental melons and virtually all oriental melons are grafted. However, grafting to vigorous rootstocks may also induce undesirable physiological disorders such as vigorous vegetative growth, delay in fruit maturity and harvesting, uneven maturity, and internal breakdown of fruit caused by unbalanced uptake of nitrogen and calcium into the fruit. The incidence of preharvest internal decay or internal breakdown in oriental melons is greatly influenced by the kind of rootstocks (Chung 1995b).

2. Rootstocks. Oriental melons are commonly grafted to squash (mostly

Cucurbita moschata) whereas other melons are more often grafted to melon rootstocks with well-known resistance to various diseases. Interspecific hybrid squash (Cucurbita maxima x Cucurbita moschata) makes a good rootstock for oriental melons because of good compatibility, excellent vigor, disease resistance, and a vigorous root system. Other squash, mostly Cucurbita moschata, may also be used as potential rootstocks but graft incompatibility, which usually appears during the later stages of plant growth, should be carefully tested under various cultural and environmental conditions. Many growers would like to use ordinary squash with a somewhat less vigorous root system rather than the interspecific hybrids to obtain earlier harvest and better quality rather than high yield (Table 2.11). However, care should be taken to finish fruit harvesting early in the growing season since some of the widely used squash rootstocks tended to deteriorate much earlier than the interspecific hybrids at high temperatures. Rootstocks having good to excellent resistance to nematodes are badly needed in many successive growing areas through Korea. Limited suc-

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS

97

Table 2.11. Effects of rootstocks on soluble solids contents and percent fermented fruits in two cultivars of oriental melon (Cucumis melo var. makuwa Makino).

Cultivar

RootstockO

Fruit fresh weight (g/fruit)

Soluble solids

Fermented fruit

(%)

(%)

Keumssaraki Eunchun

Shintozwa Baekkukzwa Hongtozwa Non-grafted

439 387 375 283

ab ab ab b

8.0 7.7 8.3 8.1

a a a a

42.3 a 46.9 a 21.3 b 0.0 c

Chammat Eunchun

Shintozwa Baekkukzwa Hontozwa Non-grafted

412 396 367 329

a a ab b

7.8 7.9 8.0 8.0

a a a a

15.4 a 9.2 b 5.2 c 0.0 d

OSee Table 2.9 for scientific names.

bMean separation within a cultivar within a column by Duncan's multiple range test at 5 percent.

cess was obtained with 'Andong' bur cucumber rootstock. The use of alternate rootstocks for nematode control is being sought for the major oriental melon producing areas in Korea. At the present time, bur cucumber and African horned cucumber are considered to be promising (Igarashi, Kanno, and Kawaide 1987). However, uneven seed germination and smaller size of the rootstocks are considered to be the major problem that must be solved before use in commercial production fields. Nematodes have been one of the most serious problems in oriental melons in greenhouses in Korea (Ko 1999). 3. Grafting Methods. Tongue approach grafting has been widely used for melons and oriental melons, but splice grafting has been increasing rapidly due to the efficiency of grafting and convenient supply from commercial plug seedling growers. Hole insertion grafting is seldom used for melons in Korea. Since the size of oriental melons is much smaller than the rootstocks if sown at the same time, earlier sowing of scion (oriental melon) is essential for tongue approach grafting. The seeds of the rootstock are sown when the scion cotyledons are fully expanded (usually seven to eight days after scion sowing). The grafting is usually made eight to ten days after the sowing of rootstock seeds and the hypocotyl portion of the scion below the graft is cut about 12 days after grafting. Seedlings are then transplanted in the field about 15 days after the cutting of scion hypocotyl or 40 to 45 days after the sowing of oriental melon.

98

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4. Cultural Practices. All melons, and especially those grown in green-

houses, are expensive in Asia and the quality factors are quite sensitive to cultural practices and environmental conditions. Even slight reductions in quality could result in reduced earnings from the produce. Therefore, some experienced growers tend to grow melons without grafting or with grafting onto melon rootstock rather than rootstocks belonging to other genera or other species. Squash rootstocks (Cucurbita moschata) with medium vigor are preferred for oriental melons by many farmers because of the possible poor fruit quality from the plants grafted onto vigorous rootstocks such as interspecific hybrids. Fruit set can be more easily controlled by using less vigorous rootstocks. However, thinning may be required to reduce the fruit set per plant to prevent the sudden decline of vigor and incidence of sudden wilt of vines. Use of rootstocks having strong vigor is only recommended for the extended growing throughout the summer in the greenhouse. Andromonoecious melon rootstocks induced the formation of staminate flowers on gynomonoecious melons and pumpkin rootstocks had a similar but smaller effect (Mockaitis and Kivilaan 1964). 5. Problems Associated with Grafted Plants. Even though fusarium wilt can be perfectly controlled with the use of resistant rootstocks, there are other problems associated with the use of grafted seedlings depending on cultural and environmental conditions. Internal fruit decay (or premature fermentation) is one of the typical problems aggravated by the use of rootstocks. Both the melons and oriental melons are very sensitive to internal decay and the use of vigorous rootstock apparently increases the incidence in oriental melon (Table 2.11). It is possible that the symptoms could be caused by some other factors such as low temperature, reduced absorption of calcium, or cultivar susceptibility.

D. Tomato 1. Current Status. Even though tomato is one of the major vegetables in the world, production in Asia is rather limited as compared to western countries and its use has been also mostly limited to table use until recently. The recent increase in tomato production in East Asia is primarily confined to fresh market tomato and the production is usually under protected structures (Table 2.1). Numerous diseases affect solanaceous crops as compared to cucurbits and various rootstocks are available for one or more diseases (Harrison and Burgess 1962; Gindrat, Ducrot, and Caccia 1977; Alam et al. 1994). It is usually recommended to use grafted seedlings for long-term grow-

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS

99

ing under protected structures. Grafted plants are also highly recommended for nutrient solution culture systems, not only for the excellent disease resistance of the rootstock, but to exploit the vigorous root system to support scion growth and guarantee stable fruit harvest for an extended period. However, because of the difficulty of the grafting technique, due mostly to the smaller size of seedlings at the time of optimal grafting and the requirement for postgrafting conditioning, the commercial sales of grafted solanaceous seedlings have been popular only recently in Korea. The recent introduction of various grafting methods and techniques as well as suitable grafting tools and aids, and postgrafting conditioning facilities now make it possible to produce highquality grafted seedlings even at the small farmer level (Lee et al. 1999). Recent introduction of promising rootstock cultivars in vegetable seeds catalogs in Japan, Korea, and some European countries (Hungnong 2001; Known-you 1998; Rijk Zwaan 2000; Sakata 2000; Takii 1995) and efficient grafting machines and robots have attracted the attention of many commercial seedling growers on a much larger scale. 2. Rootstocks. Eggplants were commonly used as rootstocks for tomato in earlier years (Yamakawa 1982), but many other excellent rootstocks have been developed recently and are widely used for various purposes (Table 2.12). Tables 2.12 and 2.13 show the resistance of selected tomato rootstocks to various diseases. It is apparent that rootstocks have quite different characteristics or resistance to various fungal, bacterial, and viral diseases (Bravenboer 1962; Harrison and Burgess 1962; Tikoo, Mathai, and Kishan 1979; Gindrat et al. 1977; Monma et al. 1997; Matsuzoe, Okubo, and Fujieda 1993b). Eggplant is not much used now except for some specific purpose. In Taiwan, total fruit yield and sugar content in tomato fruit were significantly increased by grafting tomato scions onto an eggplant root system (Oda et al. 1996; AVRDC 2001). Decrease in total yield was reported in Japan by grafting tomatoes on eggplant, but marketable fruit yield was increased (Oda, Okada, and Saasaki 2000).

3. Grafting Methods. Unlike the cucurbitaceous vegetables that are grafted at the hypocotyl portion during the cotyledonary stages, tomato is grafted at the epicotyl portion because tomato seedlings do not have a cavity in the middle of stem and the hypocotyl diameter is much smaller than cucurbit seedlings. Until recently, tongue approach grafting has been extensively used but other grafting methods are frequently used depending upon growers' preference (Table 2.2). Tongue approach grafting is made by cutting and grafting in the middle of the first internode (Fig. 2.1).

]. LEE AND M. ODA

100

Table 2.12. Rootstocks for solanaceous crops (Lycopersicon, Solanum, Capsicum, and Datura) and their performances.

Rootstock

Scion

Performance

1. esculentum

Tomato

Modify boron absorption

1. esculentum

Tobacco

1. esculentum 1. hirsutum

Tomato Tomato

Solanum spp.

Tomato

Nicotine and alkaloid absorption affected High temperature tolerance Resistant to corky root disease Resistant to bacterial wilt and nematode Yield increase Growth and yield reduction Growth and yield reduction Resistant to water-logging Growth and yield reduction

S. S. S. S.

Reference Brown, Chaney, and Ambler 1971 Dawson 1942 Okimura et al. 1986 Harrison and Burgess 1962 Tikoo et al. 1979 Matsuzoe et al. 1993a Shackleton 1965 Shackleton 1965 Shackleton 1965 Abdelhaffez et al. 1975 Oda et al. 1996

sodomaeum auricularum laciniatum melongena

Tomato Tomato Tomato Tomato

S. integrifolium S. sisymbrifolium

Tomato Tomato

S. torvum

Tomato

S. toxicarium

Tomato

S. melongena 1. hirsutum x 1. esculentum 1. esculentum x 1. hirsutum

Eggplant Tomato

Sugar content increase Disease resistance, no effect on sugar content Disease resistance, no effect on sugar content Disease resistance, no effect on sugar content Multiple disease resistance Low fusarium infection

Tomato

Multiple disease resistance

Tomato

Eggplant

Resistant to corky root (K), Root knot nematode (N), Verticillium wilt (V), and Fusarium wilt (F) Yield increase Low and high temperature tolerance Resistance to tomato brown root rot Resistance to bacterial wilt

Eggplant

High temperature tolerance

Okimura et al. 1986

Sweet pepper (green) Green pepper

Compatible with Capsicum only Superior growth and yield

Beyries 1974 Yazawa et al. 1980

Tomato

Low yield

Kramer 1957

Tomato Tomato

S. torvum x S. sanitwongsei S. integrifolium x S. melongena Capsicum spp. C. annuum x C. chinensis Datura patula

Matsuzoe et al. 1996 Matsuzoe et al. 1996 Matsuzoe et al. 1996 Monma et al. 1997 Harrison and Burgess 1962 Gindrat et al. 1977

Bravendoer 1962 Okimura et al. 1986 Kuniyasu and Yamakawa 1983 Monma et al. 1997

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS Table 2.13.

101

ResistanceQof tomato rootstocks to various diseases. Fusarium wilt

Corky root

R-l

R-2

Verticillum wilt

Bacterial wilt

Nematodes

Long-term, forcing, and semiforcing HRb Tm-2a, Tm-2 Vulcan Tm-2a, Tm-2 HR Magnet Tm-2 S Seogun S Shinmate Tm-2a, Tm-2

MR MR MR MR

S HR S HR

HR HR S HR

S HR S HR

MR MR MR MR

HR S S

MR MR MR

S S S

HR S S

HR HR MR

MR MR S

S

MR

S

S

MR

S

Use Rootstock

Tunnels Joint BFNT-R LS-89 BF-Okitsu 101

TMVQ gene

Tm-2a, Tm-2 Tm-2a, Tm-2

QTobacco mosaic virus. bHR, Highly resistant; MR, Moderately resistant (tolerant); and S, Susceptible. Source: Sakata (2000)

After grafting, the plants are planted in the same pot with intact root systems of scion and rootstock. Ordinary grafting clips are suitable for this kind of grafting. The grafting is slow and additional labor is required to cut off the scion root, removing the grafting clip, and for careful handling of the grafted seedling. Hole insertion grafting is used by some growers, but less frequently (Fig. 2.1). Even though the grafting operation is somewhat slow and graft-take percentage may not be high enough, the clipping operation is not needed and graft union fusion is much stronger than the tongue approach grafting. Splice grafting is now most widely used by commercial seedling growers and experienced farmers. The grafting operation is simple and rapid, and fusion of the graft interface is strong enough to take rough handling during transport and transplanting. Ordinary grafting clips can be used to fix the grafted interface together. However, more efficient grafting aids, such as ceramic pins, silica-rubber clips, or plastic tubes have been developed and widely used for hand or machine grafting (Fig. 2.3). For splice grafting, the position and angle of the cut may be somewhat different depending upon the grower and grafting method. The grafting clips are usually flexible enough and may slide off naturally with the increase in stem diameter. The price of this kind of plastic or silica grafting clips may be expensive, but the clips can be used for several years. Grafting pins are the most popular grafting aid for tomato.

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Smaller and more convenient clips or tubes are being developed and used for tomato and pepper in The Netherlands (Fig. 2.3E). Cleft grafting (Fig. 2.1G) is also used by many growers in Japan, but is rapidly decreasing due to the high labor needed for grafting and somewhat lower success rate. 4. Cultural Practices. Tomato scions grafted onto vigorous rootstocks

show much higher yield than ungrafted ones when grown in clean soils (Matsuzoe et al. 1993a). 'Seokwang' tomato grafted onto 'Kagemusha' rootstock showed a 39.3 percent increase in the number of fruit set and a 54.4 percent increase in marketable fruit yield as well as a significant reduction in the percentage of abnormal fruit (Table 2.14). Mean fruit weight showed only slight increases as compared to the number of fruit or marketable fruit yield. This increase in fruit yield or in the number of fruit set may vary considerably with scion cultivar, growing season, environment, cultural practices, and microclimatic conditions. Careful selection of rootstock most suitable for the scion and growing environment is crucial for successful cultivation of grafted tomato. In The Netherlands, growers are using grafted tomatoes for long-term cultivation, for example, harvesting more than six clusters per plant. At the present time, 50 percent of tomato seedlings produced for sale in a company for greenhouse production were grafted (K. Marsman pers. commun.). Grafted tomatoes are also routinely used for hydroponic culture systems in The Netherlands. Table 2.14.

Fruit yield of 'Seokwang' tomatoes as affected by rootstock.

Fruit no. per plant

Fruit wt. (g/plant)

Fruit no. per plant

Fruit wt. (g/plant)

Abnormal and normal fruit wt. (kg/plant)

3.0 5.9 2.6 3.4 3.9 3.4 2.9 4.5 5.3

500 757 427 471 608 474 414 629 760

13.5 10.2 12.1 14.9 14.6 14.9 13.0 10.0 10.7

2,736 1,947 2,562 3,088 2,799 3,020 2,699 1,836 2,000

3.24 2.70 2.99 3.56 3.41 3.49 3.11 2.47 2.76

Abnormal fruit

Rootstock Shinmate Vulcan Joint Kagemusia Dr. K Helper BFNT-R BF Okitsu 'Seokwang' (Check)

Normal fruit

GMean separation by Duncan's multiple range test, 5 percent. Source: Chung (1995a)

Marketable yieldG 80.1 b 57.7 d 75.9 c 91.5 a 82.9 b 89.5 a 80.0 bc 55.4 d 59.3 d

(t/ha)

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103

5. Problems Associated with Grafted Plants. In order to effectively control the noxious soil-borne diseases in solanaceous crops by the use of resistant rootstocks, it is important to have a good understanding of the causal organism including the race situation followed by careful selection of suitable rootstocks. Ungrafted susceptible tomato plants or tomato plants grafted to susceptible rootstocks usually show very rapid spread of disease at the fruit maturation stage in the late spring or early summer, but not usually during the early stages. In tomato, the race of Fusarium in the soil and tobacco mosaic virus (TMV) resistance of scions should be carefully considered for rootstock selection (Table 2.13). Because of the high price of grafted seedlings (usually three times higher than ungrafted seedlings of the same cultivar), grafted seedlings should be used for a long-term cultivation in the greenhouse including nutrient solution culture system. The use of virusfree rootstock seeds should not be neglected since some seed-borne viruses such as tobamovirus can be easily and very rapidly transmitted to the scion and other nearby plants, thus eventually destroying most of the plants in a short period of time (Kim and Lee 2000). The presence of one virus-infected plant in a greenhouse could be disastrous for all other healthy plants because tobamovirus can be quickly transmitted to nearby plants by grafting knife, grafting aids, thinning and training, and other practices. The tobamovirus (TMV, CGMMV, and others) in seeds can be partially inactivated by soaking the seeds in 10 percent Na 3 PG 4 solution followed by extensive water washing or by NaGCI and methyl bromide treatment. Complete inactivation is, however, possible only by dry heat treatment of the seeds as in the case of cucumber green mottle mosaic virus in watermelons (Kim and Lee 2000). There are two kinds of resistance in TMV (TIll and Tm-2) in scion and the graft compatibility may change with the resistance composition in the scion and rootstocks (Table 2.13).

E. Eggplant 1. Current Status. Eggplants are truly an Asian vegetable with 94.1 percent of the world's cultivated area (1,312,794 hal. China is by far the leading producing country (636,053 hal followed by India (425,000 hal, Indonesia (43,000 hal, and Turkey (32,600 hal. Eggplants are also very much favored by the Japanese (13,000 hal who have developed many eggplant dishes. In Japan, about 43 percent of field-grown eggplants and virtually all the eggplants for protected cultivation are grafted to various rootstocks. In contrast, only about 5 percent of the greenhouse eggplants are grafted in Korea.

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2. Rootstocks. Eggplant has a strong and extensive root system and high disease resistance is the major requirement for good rootstocks (Kato and Lou 1989). Selection of suitable rootstock in Solanum species should be based on the population of soil-borne pathogens in the relevant production area (Table 2.12). 3. Grafting Methods. Essentially the same grafting methods used in tomato are being used for eggplant even though the proportion may be considerably different. Eggplants are mostly grafted 50 to 53 days after the sowing of the scion (four to five true leaf stage) or 60 days after the sowing of the rootstock (five to six true leaf stage). Seeds of the rootstock are sown about three to seven days earlier than the scion seeds. However, the seeds of Solanum torvum, even after gibberellin treatment to break dormancy, should be sown 20 to 30 days earlier than the scion because of slow germination. Kato and Lou (1989) evaluated three eggplant cultivars grafted onto four different rootstocks including a self-rooted control. The highest eggplant fruit yield and most vigorous growth were obtained with 'VF' tomato rootstock and this attributed to differences in the number of thick roots and in cytokinin production from the root. Growth promotion and yield increases with tomato rootstocks have been confirmed by Monma et al. (1997) and Oda et al. (1997). Disease resistance oftomato rootstocks have been evaluated by Shishido, Zhang, and Kumakura (1995). 4. Cultural Practices. Uneven germination of eggplant seeds could be a

serious problem for efficient grafting, especially for machine and robot grafting. Seeds of some rootstocks are treated with gibberellic acid to promote germination (Yamakawa 1982). This could be a serious problem for efficient grafting, especially for machines and robot grafting with rootremoved rootstock seedlings, since gibberellin treatment could result in reduced rooting ability from rootstock hypocotyl. Alternating temperatures are given to some eggplant seeds to promote early and uniform germination. Continuous search for new germplasm possessing outstanding disease resistance should be done to solve the problems associated with differential absorption and distribution of mineral elements, quality problems, infection from seed-borne diseases, and others. F. Pepper 1. Current Status. As compared to tomatoes and eggplants, grafting of

peppers has been only recently introduced among growers. Hot pepper is the major vegetable in the Republic of Korea and comprises more than

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105

20 percent of the total vegetable production area of approximately 360,000 ha. Even though most of the area is for production of dry peppers, 4500 ha is being used for fresh pepper production under protected

structure. Use of grafted pepper is mostly confined to those grown under protected structures to prevent the severe incidence of Phytophthora disease caused mostly by successive cropping in protected houses and wet soil environment. Even though only about 5 percent of the green pepper producing area is planted with grafted peppers (about 6 million plants), it is anticipated that the cultivation area of grafted peppers will rapidly increase (Kim 1999; Choi et al. 1985). 2. Rootstocks. Most of the rootstocks being used for the pepper had been inbred lines noted for their marked tolerance to the Phytophthora disease. Those lines possessing excellent tolerance to the Phytophthora disease are rather susceptible to virus diseases, most noticeably TMV. In contrast to most other Solanaceous vegetables, Capsicum peppers were reported to be graft-compatible only with Capsicum peppers (Beyries 1974; Yazawa et al. 1980; Kim 1999). However, interspecific hybrids possessing excellent tolerance to Phytophthora and TMV have been developed for rootstock use. Promising hybrids are being actively developed in Japan and Korea (Kim 1999; Sakata 2000).

3. Grafting Methods. Splice grafting is the major method used for pepper grafting. Ordinary clips, ceramic pins, and elastic tubes with side slit are commonly used to hold the grafted position. Since pepper needs high temperature and high humidity conditions as compared to other vegetables, the grafted plants are usually placed in conditioning chambers or sealed with high density PE film for the healing (Fig. 2.4). 4. Cultural Practices. In general, cultural practices for grafted peppers

are similar to ungrafted ones. However, somewhat wider spacing is recommended for grafted peppers since the plants grow well from the beginning and remain healthy for much longer period that self-rooted plants, especially under the protected environments. G. Other Solanaceous Crops

Since potato can be used as rootstock for tomato, eggplant, and some other solanaceous plants, both the potato and the tomato can be harvested from a single grafted plant at the same time. "Totato," tomato plants growing on potato roots, have been grown in greenhouses and considerable yield of both the tomato and potato has been reported

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(Ra et al. 1992). However, this technique has not been commercially used by the farmers because the limitation in selection of suitable scion cultivars and considerable delay in potato harvest. H. Crucifers

Many Brassica crops can be successfully grafted onto each other even though the grafting operation requires a very delicate technique and postgraft care. In the future, robots may make it possible to graft plants within the Brassicaceae. Grafting has been successful with: (1) intraspecific grafts in Brassica oleraceae (cabbage [scion]/kale [rootstock], kohlrabi/ kale, and cabbage/kohlrabi) and within B. campestris (Chinese cabbage/turnip); (2) interspecific grafts (Chinese cabbage/kale, Chinese cabbage/cabbage, Chinese cabbage/Takana [B. juncea] and Takana/turnip); and (3) intergeneric grafts (Chinese cabbage/Japanese radish [Raphanus sativus], and Japanese radish/cabbage) (ada et al. 1992). I. Cactus

1. Current Status. Grafted cactus is one of the major exported floricultural plants in Korea. Korea has been exporting about 80 percent of the world's demands of 10 million grafted cacti per year for the last five years. The grafted cacti are being exported to 23 countries with The Netherlands, United States, and Canada being the leading importing countries. Active growing of grafted cactus was begun in late 1960s by growing several imported cultivars from Japan. The introduction of several prominent cultivars by private breeders contributed a decisive role in promoting international export from the late 1980s. Many outstanding cultivars have been developed by private breeders and public as well as governmental research institutes and most of these newly bred cultivars were distributed to the growers (Table 2.15). 'Ruby Ball' or 'Bimoran' strains (Gymnocalicium mihanovichii var. friedrichii Werd. Hort.) are by far the leading cacti for export. 'Bimoran' could be variously classified based on the color and shape. Other types are also being developed to provide new cultivars for export. Plate 2.1 shows some of the cultivars recently introduced by the National Horticulture Research Institute, Rural Development Administration, Korea. Even though the color changes considerably depending on the microenvironmental conditions, the basic pigmentation pattern is very unique and attractive enough to maintain its top popularity in the world market. 2. Rootstocks. 'Samgakju' (Hylocereus trigonus) has been widely used as rootstocks for most cactus grafting. However, some other rootstocks

Table 2.15.

Cactus scion cultivars and their characteristics.

Genotype and cultivar

Skin color

Bulb shape

Rib no.

Spine color

Ruby Balls or Bimoran (Gymnocalicium mihanovichii var. friedrichii Werd. Hort) Morning 1 Dark red Flattened global 10-11 Dark brown Morning 3 Bright red Flattened global 9-10 Dark brown

Morning 5 Koyang 8 Seoul 1 Black Ruby Late Fall Yeonmin Shinseong Binghwa Cheongsil Black Pearl Rainbow

Dark red Bright red Scarlet red Dark red Yellow Mixed orange & red Pink Pink Yellowish green Brilliant black Red+white+purple

Global Global Global Global Flattened global Flattened global Flattened global Global Global Global Flattened global

8-9 8 7-8 10 9 9-10 8-9 8-9 8 8 8-9

Myungweal

Yellow+red

Flattened global

9

Dark brown Brown Brown Light brown Brown Brown Brown White Dark brown Black Brown White

Sunghong Red+yellow Flattened global 9 White Ojak Dark purple+pink Flattened global 8 Brown Saekdong Red+yellow Flat 7-8 White Sanchui (originated from Baekdan, Chamaecereus silvestris f. variegata hort.) Mangwool Light yellow Cylindrical 10-11 Yellow Cylindrical 11-12 Yellow Bosong Yellow 9-10 Light brown Somsom Green Cylindrical White Sojung (Notocactus scopa var. sojung) and ..... ~

Gold Bosang (Rebutia minuscula var. bosang)

Source: Son (2000); Jeong et al. (2001)

Other characteristics Very good vigor, firm flesh Excellent grafting ability, many bulblets formed Hardy plant, fewer bulblets Large bulb size Popular traditional cultivar Vigorous grower, soft flesh Good grafting compatibility, hard flesh Fast grower Excellent color maintained Excellent bulblet producer Vigorous grower, good bulblet yield Excellent shape, few bulblets per plant Selection of right bulblets required for propagation Noted for well balanced growth and uniformity Well adapted to casual soil cultivation Fast and vigorous grower Good for long distance shipping Red touch at the top Thrives well even during the summer Dark brown apical buds New type to be named later.

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108

possessing other characteristics such as low temperature tolerance, high temperature tolerance, and good rooting characteristics have been selected for potential rootstocks (Table 2.16). Both scion and rootstock should be virus-free for the best performance. The cacti rootstocks are vegetatively propagated using cuttings. 3. Grafting Methods. The only grafting method used is the top-loading method (Fig. 2.6). Since those colored cactus lines do not have chlorophyll for photosynthesis, they should be grafted on photosynthesizing rootstocks for normal growth. Grafting can be performed any time of the year, but is done commonly from March to October. The upper stem tip of the rootstocks, Mylocereus trigon us and others (Table 2.16), is cut horizontally to the axis and the scion, preferably 10 mm in diameter at grafting, is placed on the central portion of the decapitated rootstock (Fig. 2.6). When placing the scion on the top of the rootstock, vascular bundles of each rootstock and scion should be in as close contact as possible. After placing the scion on the rootstock, cotton thread or specially designed grafting clip is used to fix the counterparts in desired position. The grafted plants are placed in conditioning room (30°C, 85 percent relative humidity) and kept dry to facilitate the fast healing and increase graft-take percentage.

Table 2.16.

Common rootstocks for cactus grafting and their characteristics.

Name

Shape of cross section

Skin color

Spines Eight short spines

Characteristics Most popular for all kinds

Samgakju (Hylocereus trigon us)

Triangular (shrunk)

Green

Yongsinmok (Myrtillocactus geometrizans)

Star shape with four to six angles

Tan green

Sodegaura (Eriocereus jusbertii)

Star shape with four to five angles

Green

Short and strong

Grows well even under cool and wet conditions

Waryoung (Eriocereus tortuusus)

Triangular (shrunk)

Green

Long & strong

Can be easily propagated by cutting

Guimyungak (Cereus peru vian us)

Star shape with five to six angles

Green

10 short

Can be propagated by cuttings and seeds

Requires high temperature for best growth

spines

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS

............. Vascular bundle

109

.i .

.

Cut Diameter of scion: 10 mm.

o After placing the scion on the top of rootstock to match the vascular bundle, thread or clips are used to hold the position.

* Standard rootstock length: 9 cm long * Diameter of rootstock: 30 - 35 mm Fig. 2.6.

Grafting of 'Ruby Ball' cactus on the top of a triangular rootstock.

4. Problems Associated with Grafting Plants. Since both the scions and

rootstocks are commonly propagated by cuttings and easily affected by virus, both virus free and/or virus-resistant cacti should be used for grafting. Severe growth inhibition and low graft-take percentage result with virus infection. V. GRAFTING FOR CROP IMPROVEMENT A. Flower and Tuber Induction

Grafting has been used to induce flowering and to induce early flowering. For example, sweet potato (Ipomoea batatas) is routinely grafted to other nontuberous root forming Ipomoea species such as 1. ruba, 1. carnes, and 1. tiliaceae to induce flowering. In this case, the presence of leaves on the scion and rootstock have had a profound influence on the flowerinducing response. Flowering was induced only when the rootstocks had expanded leaves, thus suggesting that flower-inducing substances are synthesized in the rootstock leaves and translocated through the graft union to induce flowering in the scion (Kher et al. 1953; Lam and Cordner 1955). Grafting sweet potatoes onto Ipomoea carnea ssp. fistulosa

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increased flower numbers, percentage of capsule set and number of seeds in all four tested cultivars, whereas the responses to bioregulator treatment differed among cultivars (Lardizabal and Thompson 1988, 1990). Graftage of a late maturing cultivar of pea (Pisum sativum) onto an early-maturing stock increased earliness (Haupt 1958). Considerable amount of seeds could be produced from biennial plants in the first year by grafting biennial seedlings onto annuals or onto flowering plants of the same species (Kruzilin and Svedskaja 1959). Grafting is commonly made between inflorescences of cruciferous crops to save a plant of special interest from being permanently lost due to severe infection of certain disease or to injury. The graft-take percentages are very high if the inflorescence grafting is made with a young succulent inflorescence. Gregory (1956) grafted tuberization-induced potato scions onto noninduced stocks and showed decisively that the tuberizing stimulus can carry the signal from the induced to noninduced parts; furthermore, the stimulus moves in a polar manner toward the base of the plant. Madec (1963) provided further support for the concept of a specific tuberizing stimulus in that tomato scions grafted onto potato rootstocks do not lead to tuber formation but potato scions do (Madec 1963). B. Graft Chimeras and Graft-Induced Mutants One of the earlier mysteries in horticulture was the nature of certain unusual plant forms that developed with certain graft combinations (Tilney-Bassett 1986; Marcotrigiano and Gradziel 1997). Winkler (1908, 1910) was able to demonstrate that these bizarre plants were not graft hybrids but were in fact chimeras. He was able to synthesize a true graft chimera of tomato (Lycopersicon esculentum) on black nightshade (Solanum nigrum) and vice versa (Hartmann et al. 1997). In the procedure, the scion of young grafted plant was cut severely near to the callus area arising during the healing ofthe grafted interfaces (Fig. 2.7). Adventitious shoots from all these calli became chimeras of tomato and nightshade tissue growing together and not hybrids. Winkler gave such mixed shoots the name "chimera" after the mythological monster that was composed of lion's head, goat's body, and dragon's tail (Hartmann et al. 1997). In addition to Winkler's chimeras, graft chimeras were made by several other workers utilizing a variety of species (Table 2.17). Some of these were unstable. The graft chimeras between tomato and eggplant were continuously converting to the tomato core, and the same experience was reported with the chimera between tomato and S. dulcamara. Good illustrations for the contents shown in Table 2.17 could be seen in Tilney-Bassett (1986). Graft-induced mutations have been reported in some cultivars of poinsettias and these have been used in breeding. In the 1980s, a poinsettia

2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS

Nightshade bud Tomato

b~

\

111

Chimeral bud /---.:.

Callus"..

Fig. 2.7.

Synthesis of graft chimeras in solanaceous crops.

breeder was able to induce branching in thousands of nonbranching poinsettias using the grafting techniques developed by Gregor Gutbier who began exploring the idea of grafting as a means of developing better poinsettias (Poinsettia Growers Association 1995). Gutbier's grafting technique has been studied in several universities and some poinsettia breeders incorporated it into their program (Stimart 1983; Dole and Wilkins, 1992). The nature of this permanent inheritable change has not been clearly explained. Topoleski and Janick (1963) investigated the possibilities of graftinduced hybrids in eggplant to confirm a positive report by a reputable Japanese geneticist (Sinoto 1955). Graft-induced hybrids were not confirmed but tomato rootstocks invigorated eggplant in some environments. In peppers, however, positive results were reported on graftinduced mutants. Yagishita and Hirata (1987) reported that morphological changes or possibly transformed genes caused by grafting were transmitted to progenies through seed. Considering the stability of the phenotypic changes (Ohta and Chuong 1975) and the characteristics of the graftinduced variants in pepper, Taller et al. (1998) considered that some of the characteristics of the stock were introduced into the progeny obtained from self-pollinated seeds of the scion. These workers concluded that the novel characteristics appearing on grafted plants and self-pollinated

,.... ,.... N

Table 2.17.

Examples of experimentally synthesized periclinal chimeras,c' Chimeral arrangement b

Components of chimeras

Source

Ll

L2

L3

Reference

Adventitious shoot from greenhouse grafting Spontaneous rearrangements of apical layers of existing chimera Regenerated shoots from tissue culture of chimeral leaf tissue

T T T G

G T G T

G G T T

Marcotrigiano and Gouin 1984 Marcotrigiano 1986

G

G

T

Marcotrigiano 1986

Nicotiana glauca (G) Solanum ariculare (S)

Adventitious shoot from greenhouse grafting

G

S

S

Marcotrigiano 2001

Solanum nigrum (N)S. tuberosum (T)

Mixed protoplast culture

T

N

N

Binding et al. 1987

Lycopersicon pennellii (P) - 1. esculentum (E)

Adventitious shoot from greenhouse grafting

P P

E P

E E

Goffreda et al. 1990

Nicotiana tabacum (N) Solanum iaciniatum (S)

Adventitious shoot from greenhouse grafting

N

N

S

Kaddoura and Mantell 1991 SS

Lycopersicon peruvianum (P) - 1. esculentum (E)

Adventitious shoot from greenhouse grafting

P P P

Nicotiana tabacum (T) - N. glauca (G)

N

E E P

E P E

Szymkowiak and Sussex 1992

Lycopersicon esculentum (wild-type+) and L. esculentum lateral suppressor (is)

Adventitious shoot from greenhouse grafting

Is

+

+

Szymkowiak and Sussex 1993

Nicotiana tabacum (T) N. glauca (G)

Forcing adventitious shoots from disbudded chimeras

G

T

G

Tian and Marcotrigiano 1993

Brassica campestris (C) B. oleraceae (0)

Regenerated shoots from in vitro grafting

C C C

o

o

o o o

Noguchi and Hirata 1994

aFor a table of pre-1978 synthetic solanaceous chimeras, see Tilney-Bassell (1986). bLl, Outmost layer; L2, Second outmost layer; and L3, Third outmost layer. Source: From Marcotrigiano and Gradziel (1997)

I-' I-' W

C

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J. LEE AND M. aDA

progenies were due to graft-induced mutations, based on RAPD markers in the rootstock, scion, and graft-induced variants. This appears to be the only analysis available on graft-induced permanent changes. Further research is needed on this phenomenon. C. Chimeral Engineering

While mutagenesis can induce nuclear chimeras and cell fusion can create heteroplastidic chimeras, direct synthesis of nuclear chimeras from known components is less common. The classical graft chimera technique has been successful in the Solanaceae (Tilney-Bassett 1986) and in the Brassicacea (Hirata et al. 1990). Noguchi, Hirata, and Yagishita (1992) developed a modified technique that capitalizes on the enhanced regeneration of a tissue-culture system with the proven methods of grafting. When seedling hypocotyls were grafted and the graft unions were cultured on a regeneration medium, a significant portion of the shoots formed chimeras. Theoretically, the most efficient method of synthesizing a chimera would be to generate a mosaic meristem in vitro by growing mixed callus cultures or cell suspensions and then transferring to shoot-inducing media to generate chimeras. This technique however has yielded few useful results (Marcotrigiano and Gouin 1984). There has been only one report of the intentional synthesis of chimeras following protoplast mixing. Several studies indicate that derivatives of the internal apical cell layers play a very significant role in developmental processes. Periclinal graft chimeras composed of Lycopersicon esculentum and 1. peruvianum (Table 2.17) were used to determine that carpel number and meristem girth were determined largely by the genotypes ofL3 (Marcotrigiano and Gradziel, 1997). A graft chimera was made with lateral suppressor (LS) in the L1 and wild~type cells in L2 and L3 (Szymkowiak and Sussex 1993). Even though LS plants do not form petals, the chimera made normal petals covered with an Is epidermis, indicating that the internal cell layers provided the cues necessary for the initiation of petal primordium. In a set of periclinal chimeras between Nicotinana glauca and N. tabacum, the number and position of axillary buds was greatly influenced by the genotype of the L3 (Tian and Marcotrigiano 1993). The major limitation to synthetic periclinal chimeras is the fact that the plant must be vegetatively propagated to maintain the chimeral arrangement. Although this could appear limiting, many fruits and ornamentals are vegetatively propagated and many chimeras that have originated spontaneously are extremely valuable. The best example may be the pink poinsettia.

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VI. CONCLUSION AND PROSPECTS

Cultivation of grafted vegetables has been successfully practiced in many Asian countries for several decades and is increasing in Europe. Many multinational seed companies are eager to develop and distribute rootstock seeds through their commercial seed catalogs. Watermelon and tomato are the two major vegetables for grafting and worldwide distribution. Grafting is routinely practiced in other vegetables (cucumbers, melons, oriental melons, greenhouse squash, eggplant, and capsicum peppers) and cactus. Introduction of excellent rootstocks possessing multiple disease resistance and efficient grafting machines including grafting robots will greatly encourage the extended use of grafted vegetables over the world. There are many problems commonly associated with grafting and cultivating grafted seedlings (Lee 1994). These include the additional cost for rootstock seeds, labor required for the grafting and raising grafted seedlings, lack of experience and technique for grafting and cultivation of grafted plants, and incidence of possible physiological disorders associated with grafting. However, there are enormous benefits from using grafted seedlings. These include increase in income by high yield and off-season growing, lower input of fertilizers and irrigation water due to the wide root systems of the rootstocks, considerable saving in agrochemicals due to high resistance of the rootstocks, extension of the harvest period, efficient maintenance of popular cultivars against diseases and other physiological disorders, no need for long-term crop rotations, overcoming problems due to saline soils, reduced expense needed for soil fumigation, ease of producing organically grown vegetables, and reduced use of agrochemicals. Partial or full utilization of these benefits will depend on various factors such as farm. size and degree of mechanization, cultivation practices such as crop rotation and transplanting, technology level, understanding the full benefits and risks of grafted seedlings, and the uses of protected cultivation and hydroponics. Use of grafted seedlings is strongly recommended for hydroponics culture of tomato, pepper, eggplant, and cucumber. Growers can now purchase grafted seedlings of any specific combination from many commercial plug seedling growers rather than doing the tedious grafting themselves. In this case, the grower should make an order for their seedlings in advance. This is especially true in Japan, Korea, The Netherlands, and perhaps in many other countries. With the invention of more efficient grafting robots and acclimatization facilities, the price of grafted seedlings could be considerably reduced in the future to meet grower expectations.

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Shoji, T. 1989. Respiratory responses of detached roots to lower temperatures in cucumber and figleaf gourd grown at 20° root temperature. J. Japan. Soc. Hart. Sci. 58:333-337. Son, B. G. 2000. The culture of cacti and succulents. Kyonggi Provincial RDA. Korea. Spender, B., and R. Weichhold. 1964. Tomato corky root and the possibilities of its control (in German). Deut. Gartenb. 11:96-99. Stimart, P. 1983. Promotion and inhibition of branching in poinsettia in grafts between se1£branching and non-branching cultivars. J. Am. Soc. Hort. Sci. 108:419-422. Suge, H. 1984. Nature of floral stimulus in Perilla as studied by grafting. I. Method of evaluation and the movement of floral stimulus as affected by N6-benzylaminopurine. Japan. Soc. Crop Sci. 53:423-429. Suge, H. 1992. Nature of floral stimulus in Perilla as studied by grafting. II. Role of endogenous gibberellins and relation of floral stimulus in floral organ. Japan. J. Crop Sci. 61:107-111. Suzuki, M., K. Kobayashi, K. Inoku, K. Miura, and K. Hirata. 1995. Development of grafting robot for cucurbitaceous vegetables (in Japanese). J. Japan. Soc. Agr. Machin. 57(2):67-75. Szteyn, K. 1959. Trials to overcome the incompatibility of crosses between Lycopersicon esculentum and Lycopersicon glandulosum by repeated grafting. Euphytica 8:145-150. Szymkowiak, E. J., and I. M. Sussex. 1992. The internal meristem layer (L3) determines floral meristem size and carpel number in tomato periclinal chimeras. Plant Cell 4:1089-1100. Szymkowiak, E. J., and I. M. Sussex. 1993. Effects of lateral suppressors on petal initiation in tomato. Plant J. 4:1-7. Tachibana, S. 1982. Comparison of root temperature on the growth and mineral nutrition of cucumber cultivars and figleaf gourd. J. Japan. Soc. Hart. Sci. 51:299-308. Tachibana, S. 1986. Effect of root temperature on lipid and its fatty acid composition in cucumber and figleaf gourd roots. J. Japan. Soc. Hart. Sci. 55:187-193. Tachibana, S. 1988. Cytokinin concentrations in roots and root xylem exudate of cucumber and figleaf gourd as affected by root temperature. J. Japan. Soc. Hort. Sci. 56: 417-425. Tachibana, S. 1989. Respiratory response of detached roots to lower temperature in cucumber and figleaf gourd grown at 20°C root temperature. J. Japan. Soc. Hart. Sci. 58: 333-337. Takahashi, H., T. Saito, and H. Siuge. 1982. Intergeneric translocation of floral stimulus across graft union in monoecious Cucurbitaceae with special reference to the sex expression of flowers. Plant and Cell Physiol. 23:1-9. Takii. 1995. Vegetable Seed Catalog. Japan. Taller, J., Y. Hirata, N. Yagishita, M. Kita, and S. Ogata. 1998. Graft-induced genetic changes and the inheritance of several characteristics in pepper (Capsicum annuum 1.). Theor. Appl. Genet. 97:705-713. Tian, H. c., and M. Marcotrigiano. 1993. Origin and development of adventitious shoot meristems initiated on plant chimeras. Develop. BioI. 155:259-269. Tiedemann, R, and U. Carstens-Behrens. 1994. Influence of grafting on the phloem protein patterns in Cucurbitaceae. I. Additional phloem proteins in Cucumis sativus grafted on two Cucurbita species. J. Plant Physiol. 143:259-260. Tikoo, S. K., P. J. Mathai, and R Kishan. 1979. Successful graft culture of tomato in bacterial wilt sick soil. Curro Sci. 48:259-260. Tilney-Bassett, R A. E. 1986. Plant chimeras. Edward Arnold, London. Topoleski,1. D., and J. Janick. 1963. A study of graft induced alternations in eggplant. Proc. Am. Soc. Hart. Sci. 83:559-570.

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Turquois, N., and M. Malone. 1996. Non-destructive assessment of developing hydraulic connections in the graft union of tomato. J. Expt. Bot. 47:701-707. Uematsu, S., K. Hirata, T. Shiraishi, T. Ooizumi, K. Sekiyama, H. Ishikura, and Y. Edagawa. 1992. Monosporascus root rot of bottle gourd stock of watermelon caused by Monosporascus cannonballus. Ann. Phytopath. Soc. Japan. 58:354-359. Wang, Y. Q., and R. Kollmann. 1996. Vascular differentiation in the graft union of in vitro grafts with different compatibility. Structural and functional aspects. J. Plant Physiol. 147:521-533. White, M. C. 1979. A hand-held grafting device for making uniform wedge cuts. Agron. J. 71:141-144. White, R. A. J. 1963. Grafted greenhouse tomatoes give heavier crops. N. Z. J. Agr. 106:247-248. Winkler, H. 1908. Solanum tubingense, ein echter Pfropfbastarde zwischen Tomate und Nachtschatten, Ber. Dtsch. Bot. Ges. 26a:595-608 (cited from Tilney-Bassett 1986). Winkler, H. 1910. Uber die Nachkommenschaft der Solanum-Pfropfbastarde und die Chromosomen-Zahlen ihrer Keimzellen. Z. Bot. 2:1-38. (cited from Tilney-Bassett 1986). Yagishita, N., and Y. Hirata. 1987. Graft-induced changes in fruit shape in Capsicum annuum L. 1, Genetic analysis by crossing. Euphytica 36:809-814. Yagishita, N., Y. Hirata, H. Mizukami, H. Ohashi, and K. Yamashita. 1990. Genetic nature of low capsaicin content in the variants strains induced by grafting in Capsicum annuum L. Euphytica 46:249-252. Yamakawa, K. 1982. Grafting (in Japanese). p. 141-153. In: S. Nishi (ed.), Handbook ofvegetable production. Yokendo, Tokyo. Yamamoto, Y., and M. Hayashi, T. Kanamaru, T. Watanabe, S. Mametsuka, and Y. Tanaka. 1989. Studies on bloom on the surface of cucumber fruits. 2. Relation between the degree of bloom occurrence and contents of mineral elements (in Japanese with English summary). Bul. Fukuoka Agr. Res. Cent. B9:1-6. Yang, S., G. Zhang, and C. Low. 1992. Electrical resistance as a measure of graft union. J. Plant Physiol. 141:98-114. Yazawa, S., T. Kenmi, N. Uemura, K. Adachi, and S. Takashima. 1980. Use of interspecific hybrids of Capsicum as rootstock for green pepper growing (in Japanese with English summary). Sci. Rept. Kyoto Pref. Univ. Agr. 32:25-29. Zaiter, H. Z., D. P. Coyne, and R. B. Clark. 1987. Temperature, grafting method, and rootstock influence on iron-deficiency chlorosis of bean. J. Am. Soc. Hort. Sci. 112:1023-1026. Zijlstra, S., S. P. C. Groot, and J. Jansen. 1994. Genotypic variation of rootstocks for growth and production in cucumber; possibilities for improving the root system by plant breeding. Scientia Hort. 56:185-196.

3 Health Promoting Phytochemicals in Vegetables Mosbah M. Kushad, John Masiunas, and Mary A. L. Smith Department of Natural Resources and Environmental Sciences University of Illinois 279 Madigan Laboratory 1201 West Gregory Drive Champaign, Illinois 61801 Wilhelmina Kalt Agriculture and Agri-Food Canada Atlantic Food and Horticultural Research Center Kentville, Nova Scotia, B4N 1J5 Canada Kathy Eastman Center for Economic Entomology Illinois Natural History Survey University of Illinois 172 Natural Resources Building 607 East Peabody Drive Champaign, Illinois 61820 I. INTRODUCTION II. MAJOR CLASSES OF PHYTOCHEMICALS IN VEGETABLE A. Organosulfur Compounds 1. Glucosinolates 2. Thiosulfides B. Polyphenolics 1. Phenolics/Flavonoids 2. Terpenoids

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D. Dietary Fiber E. Selenium

F. Folates III. PHYTOCHEMICALS CONTENT AND HEALTH BENEFITS OF THE FOUR MAJOR VEGETABLE GROUPS A. Crucifers 1. Phylochemical Content 2. Health Benefits B. Alliums 1. Phylochemical Content 2. Health Benefits C. Solanaceous Vegetables 1. Phylochemical Content 2. Health Benefits D. Other Vegetables IV. CONCLUSIONS AND FUTURE RESEARCH NEEDS LITERATURE CITED

I. INTRODUCTION

Medical researchers have made great strides in elucidating the causes and modifiers of many of the major chronic diseases affecting humans worldwide. For most of these diseases, diet has been identified as one of the main contributing factors. McGinnis and Foege (1993) ranked poor diet and lack of exercise second only to smoking as a leading cause of morbidity and mortality. Cancer and cardiovascular diseases are the two leading causes of death in the United States. The American Cancer Society estimates that more than 1.2 million cases of all types of cancers will be diagnosed this year, of which 552,200 are expected to die within 12 months. Mortality rate from cardiovascular disease in the United States accounts for more than 150,000 deaths per year. In addition to loss of human life, the economic consequences from these diseases is enormous. The National Institute of Health estimates the overall annual cost of cancer alone is about $102 billion in the United States. Cancer is often used to describe uncontrolled formation of neoplastic malignant cell tumors (metastasis). Metastasis is formed as a result of impairment of the mechanisms that regulate cell growth and differentiation leading to progressively unregulated cell growth (Friedberg 1986). The stages of cancer development include initiation, promotion, and progression. The interaction of a carcinogen with a normal cell results in an irreversible change in the cell's genetic material, which may then be promoted to form metastasis. The promotion step involves replica-

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tion of the altered DNA and hence proliferation of the affected cell (Williams and Weisburger 1991). However, depending on the nature of the carcinogen and its dose, the cell mayor may not become malignant. Possibility of intervention exists at each stage of the carcinogenic process through modulation of the metabolism of carcinogens, thereby preventing the formation of electrophilic intermediates that are capable of inducing mutagenesis. The induction of cancer in human and animal models has been shown to be regulated by chemical, physical, and genetic factors. However, major cancers in humans are caused by environmental agents such as smoking and chemical toxicants. Agents that cause cancer have been classified, based on their chemical and biological properties, into DNAreactive (genotoxic) and epigenetic carcinogens. Genotoxic carcinogens include carcinogens that function as electrophilic reactants that interact directly with DNA causing damage to the cell's genetic material. Epigenetic carcinogens are carcinogens that inhibit specific pathways involved in the expression of certain genetic components, causing unregulated cell growth. Some chemical toxicants can be classified as both epigenetic and genotoxic (Williams and Weisburger 1991). The American Cancer Society suggests that more than two-thirds of cancer deaths in the United States are avoidable and that a third of cancer deaths can be prevented by proper diet (Ferguson 1999). The role of diet in the etiology of certain types of cancer such as colorectal cancer, which is the second leading cause of cancer deaths in the United States (Potter et al. 1993), is gaining significant attention. Numerous epidemiological studies conducted in the United States and in other countries in the last two decades have concluded that of the food groups evaluated, high vegetable intake yielded the most consistent association with decreased risk of colorectal neoplasia (Trock et al. 1990; Neugut et al. 1993; Potter et al. 1993). Evidence supporting high vegetable intake and decreased risk of colorectal cancer include results from tests on adenomatous polyps which are the precursors to colorectal cancer (Peipins and Sandler 1994; Witte et al. 1996). Witte et al. (1996) reported significantly lower incidence of colorectal polyps in men and women ages between 50 and 74 years who consumed higher rates of vegetables including crucifers, garlic, and tofu. Interestingly, the study concluded that vegetables have more beneficial effects against colorectal polyps than fruits or fiber from grains. Similarly, Steinmetz et al. (1994) conducted a cohort study aimed at 41,837 women aged 55 to 69 years who completed a food frequency questionnaire consisting of 127 items and were monitored for cancer for five years through the Iowa State Health Agency. They found a 20 to 40 percent reduction in risk of colon cancer in populations with

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higher vegetable consumption, especially garlic and dietary fiber. This association appears to be stronger for cancer of the distal colon than the proximal colon. Other studies have also estimated lower risk of colon cancer, ranging from three- to eightfold, due to high vegetable and fruit intake (Hu et al. 1991; Iscovich et al. 1992; Zaridize et al. 1993). However, the most significant conclusion was made by Doll and Peto (1981) who estimated that more than 90 percent of colon cancer in the Untied States may be avoidable through alterations in the diet. Breast cancer is another serious disease that has been linked to dietary factors. Lee et al. (1991) reported that Asian women who consumed a traditional diet high in soy products had lower incidence of breast cancer. This finding was confirmed by Wu et al. (1996) who reported a correlation between tofu intake and a reduced rate of breast cancer in a population-based case-control study of Chinese American, Japanese American, and Filipino American women. Adjustment for migration rate showed that the second generation, but not the first, lost this protection (Ziegler et al. 1993). Later studies have identified the isoflavonoids genistein and daidzein as the active ingredients in soybean and other leguminous vegetables that induced the protection against breast and other forms of cancer (Goldwyn et al. 2000). Citing more than 200 scientific studies, Steinmetz and Potter (1991) and Block et al. (1992c) concluded that five to eight servings of fruits and vegetables per day would reduce cancer of the colon, lung, stomach, bladder, pancreas, and breast. Certain vegetables have been shown to protect against specific types of cancer. For example, the crucifers (Brassicaceae), including broccoli, brussels sprouts, kale, and cabbage have been shown to protect against lung and chemically induced cancers (Verhoeven et al. 1996). The alliurns (Liliaceae), including garlic, chive, and onion have been shown to protect against stomach cancer (Dorant et al. 1996; Gao et al. 1999), the solanaceous vegetables (Solanaceae), including tomatoes and peppers, have been shown to protect against esophageal, gastric, and prostate cancers (Cook-Muzaffari et al. 1979; Buiatti et al. 1989; Giovannucci et al. 1995), and the chenopods (Chenopodiaceae), including spinach and chard have been shown to inhibit DNA synthesis in proliferating human gastric adenocarcinoma cells (He et al. 1999). A high vegetable diet has also been associated with lower risk of cardiovascular disease in humans and animals. Gaziano et al. (1995) conducted a 43 item food-frequency questionnaire for five years to assess cardiovascular disease in 1273 Massachusetts residents whose ages were greater than 65 years old. They found that residents in the upper quartile of high-carotene vegetable intake had 46 percent lower risk of death from cardiovascular disease than those in the lowest quartile. In another

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study of 5133 Finnish adults for 14 years, Knekt et al. (1994) found a 34 percent reduction in mortality due to cardiovascular disease among those who consumed vegetables rich in vitamin E and C. More recently, Liu et al. (2001) monitored the influence of vegetable intake on the incidence of cardiovascular disease among 15,220 male physicians without a history of heart disease, stroke, or cancer. The participants were administered a food frequency questionnaire at the onset of the study and at two, four, and six years of follow-up, that consisted of eight vegetable items including broccoli, brussels sprouts, carrots, spinach, lettuce, squash, and tomatoes. Questions were also asked about frequency of intake of a specific portion size. Results showed that participants who consumed more than two servings of vegetables per day had 25 percent less cardiovascular disease than those who consumed less than one serving per day. The effect of vegetable intake on cardiovascular disease in this study appears to be continuous and without an apparent threshold, which suggests that the higher the vegetable intake the lower the risk of cardiovascular disease. Based on overwhelming evidence, the American Heart Association (2000) has concluded that a diet high in vegetables and fruits may reduce the risk of cardiovascular disease in humans. In addition to reducing cancer and cardiovascular disease, a diet high in vegetables has also been linked to reducing rheumatoid arthritis, anemia, diabetes especially among women, macular degeneration, and gastric ulcer (Zhang et al. 1989; Abdel-Salam et al. 1997; Linos et al. 1999; Ford and Mokdad 2001; Ohnishi and Ohnishi 2001). The exact mechanisms by which vegetable consumption reduces human diseases have not yet been fully understood, however, the general consensus among dieticians, physicians, and nutritionists is that phytochemicals in vegetables are responsible for mitigating some of these diseases. II. MAJOR CLASSES OF PHYTOCHEMICALS IN VEGETABLE

Phytochemicals, phytonutrients, and phytonutriceuticals have been used interchangeably to describe chemical compounds derived from plants that have health-promoting properties. In this review we have chosen phytochemicals to describe nonnutrient chemicals found in vegetables that have biological activity against chronic diseases. Nutrientchemicals such as carbohydrates, amino acids, and proteins will not be covered in this review, even though they have indirect effect on chronic diseases. Furthermore, phytochemicals that have deleterious effects on

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human health, such as steroidal alkamines in solanaceous vegetables (Friedman and McDonald 1997; Chen and Miller 2001), will not be discussed. Most phytochemicals are found in relatively small quantities in vegetable crops. However, when consumed in sufficient quantities, phytochemicals contribute significantly toward protecting living cells against chronic diseases. Harborne (1999) classified phytochemicals based on their chemical structures into four major classes: terpenoids, phenolic metabolites, alkaloids, and nitrogen-containing compounds. Beecher (1999) classified major phytochemicals based on their biological activities into ten different classes including: (1) carotenoids (a and ~ carotene, ~-cryptoxanthin, lutein, lycopene, and zeaxanthin), (2) glucosinolates (sulforaphane, indole-3 carbanol), (3) inositol phosphates (phytate, inositol tetra and penta-phosphate), (4) cyclic phenolics (chlorogenic acid, ellagic acid, and coumarins), (5) phytoestrogens (isoflavones, daidzein, genistein, and lignans), (6) phytosterols (campesterol, ~ sitosterol, and stigmasterol), (7) phenols (flavonoids), (8) protease inhibitors, (9) saponins, and (10) sulfides and thiols. In this review we have grouped the major phytochemicals in vegetables into six classes based on their structural similarities and the type of vegetables in which they are found. Ao Organosulfur Compounds

Vegetable tissues contain many organosulfur compounds, however, most of them have no biological activity against chronic diseases. The two major organosulfur compounds in vegetables that have been found to have biological activity against important human diseases are glucosinolates and thiosulfites. 10 Glucosinolateso Glucosinolates or ~-D-thioglucosidesare found in 15 families of dicotyledonous plants (Rohdman 1991). Using molecular and morphological markers, Rohdman et al. (1996) identified two separate lineages of glucosinolate producing plants. One is the order Capparales, which includes the Brassicaceae and the order Euphorbiales, which includes the genus Drypetes. The biosynthesis of glucosinolates has been the subject of several comprehensive reviews (Underhill et al. 1973; Rosa et al. 1997; Fahey et al. 2001). Briefly, the glucosinolate molecule comprises a skeletal ~ thioglucose moiety, a sulfonated oxime moiety (glucone), and an aglucone R-group that defines the structure of each glucosinolate. The

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biosynthetic origin of glucosinolates is derived from the carbon skeleton of a number of commonly known amino acids (Ettlinger and Kjoer 1968; Underhill et al. 1973; Kjoer 1976). In crucifers, for example, the dominant glucosinolates are derived from methionine, phenylalanine, tryptophan, valine, and tyrosine (Bjerg et al. 1987), while in the genus Cappareseae, glucosinolates are derived from alanine (Olsen and Sorensen 1981). About 120 glucosinolates have been identified in plants thus far, but only about 20 have been detected in vegetable crops (Fahey et al. 2001). Based on their R-group structure, glucosinolates have been classified into aliphatic, aromatic, and indolyl. Several studies have suggested that intact glucosinolates have no biological activity against cancer, however, the breakdown products have been shown to stimulate mixed-function oxidases involved in detoxification of carcinogens. Loft et al. (1992) reported that rats fed a diet containing intact glucosinolates from broccoli had no effect on the metabolism of the carcinogens antipyrine and metronidazole, however when intact glucosinolates were hydrolyzed by myrosinase (thioglucosideglucohydrolase; EC 3.2.3.1) prior to feeding, detoxification of the two carcinogens was enhanced by 67 percent and 200 percent, respectively, suggesting that glucosinolate breakdown products and not intact glucosinolates are biologically active against chemical toxicants. The myrosinase enzyme has been the subject of several investigations. Multiple myrosinase isozymes of differing physiochemical properties have been identified from a number of crucifer species and organs within the same plant. At least three myrosinase subfamilies (A, B, and c) have been identified in crucifer plants thus far (Phelan and Vaughan 1980; Lenman et al. 1993; Machlin et al. 1993; Falk et al. 1995). A fourth subfamily of myrosinases has been identified in Arabidopsis thaliana (Xue et al. 1995). Lenman et al. (1993) reported that different plant organs express different myrosinase genes. For example, myrosinase genes of the subfamily A were expressed in developing seeds, while myrosinase genes of the subfamily B were expressed in cotyledons, young leaves, and other fully-developed organs (Lenman et al. 1993; Erikson et al. 2000). Analysis of two eDNA clones encoding myrosinase from radish (Raphanus sativus) roots showed that the enzyme is of the subfamily B and it is localized mostly in the epidermis and vascular cambium, but very little is present in the parenchyma tissue of the vascular cambium where there are no glucosinolates present (Hara et al. 2000). Plant myrosinase isozymes that have been identified thus far have been characterized as glycoproteins with varying carbohydrate content ranging from 10 to 20 percent of the total molecular mass, mostly as

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L-fucose and L-mannose (Bones and Slupphaug 1989). Myrosinase gene expression and enzyme activity were detected only in tissue that accumulated glucosinolates, however, no correlation was found between the rate ofmyrosinase activity and glucosinolate content (Bones 1990). The evolutionary significance of the expression of multiple isoforms of myrosinase is unknown. However, it is possible that each isoform is associated with the breakdown of a specific group or an individual glucosinolate. This may also explain why different isoforms are expressed in different tissues. As it will be discussed later in Section IILA on crucifers, different vegetables and different organs produce different types and amounts of glucosinolates. In mature tissue, intact glucosinolates and myrosinase are localized in separate organelles. Myrosinase is primarily localized in myrosin cells. These cells were first discovered by Heinricher (1984) as idioblasts and later characterized as myrosin cells (Sharma 1971). There is little information on the localization of glucosinolates in plant tissue. Yiu et al. (1984) suggested that they are sequestered in the vacuole. However, Kelly et al. (1998) examined the localization of sinigrin and myrosinase in Brassica juncea using polyclonal antibodies and electron microscopy and found that during early stages of seedling development (4 h) they are co-localized in aleuronelike protein storage cells. However, at the later stages of seedling development (100 h), the aleuronelike protein storage cells degraded, fused to form the vacuole, lost their myrosinase activity, but retained their sinigrin content. They also found that only myrosinase and no glucosinolates are present in myrosin cells. When tissues of crucifer plants are cut or chewed, myrosinase is released from the myrosin cells and depending on the reaction conditions, it converts glucosinolates into either isothiocyanates or nitriles. However, when these tissues are cooked or steamed, the enzyme is completely inactivated. It has been suggested that myrosinase activity produced by the stomach microflora is capable of breaking down glucosinolates into active products (Campbell et al. 1995). While intact glucosinolates have no biological activity against chronic diseases, certain myrosinase catalyzed-breakdown products of glucosinolates have been shown to reduce chemically induced carcinogenesis. However, not all glucosinolate breakdown products have anticancer activity. The myrosinase-catalyzed breakdown of glucosinolates into active products is pH dependent. The hydrolysis of glucosinolates by myrosinase proceeds via the formation of unstable aglycones, which at neutral pH rearrange to form isothiocyanates, oxazolidinethiones, or indole-3-carbinol. Under acidic conditions, nitriles are the dominant product. The myrosinase mediated breakdown of glucosinolates is also

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affected by ferrous ions, thiol compounds, and epithiospecifier protein. In the presence of ferrous ions, the relative amount of nitriles to isothiocyanates increases, regardless of pH (Uda et al. 1986) and studies with 2-hydroxy-3-butenyl glucosinolates have shown that both ferrous ions and epithiospecifier protein are required for the formation of nitriles (VanEtten and Wolff 1972). Similarly, thiol compounds, such as Lcysteine and glutathione, in the presence of ferrous ions lead to the breakdown of glucosinolates into nitriles (Uda et al. 1986). Sulforaphane (4-methylsulfinylbutyl isothiocyanate) and indole-3carbinol are the two most widely studied glucosinolate breakdown products exhibiting anticarcinogenic properties. Sulforaphane is the breakdown product of the aliphatic glucosinolate glucoraphanin, while indole-3-carbinol is the breakdown product of the indolyl glucosinolate glucobrassicin. Other breakdown products of glucosinolates that have been shown to protect human and animal cells against carcinogenesis are phenethyl isothiocyanate, 1-cyano-2-hydroxy-3-butene (crambene), and breakdown products from sinigrin and glucoiberin that have yet to be characterized (Smith et al. 1998; Bonnensen et al. 2001; Niedoborski et al. 2001). Studies on laboratory animals and human subjects have shown that induction of detoxification enzymes (Phase II enzymes) and inhibition of activation enzymes (Phase I enzymes) are two major mechanisms of cancer prevention by glucosinolate breakdown products (McDanell et al. 1989). Although the exact mechanism has not been fully characterized, it has been suggested that Phase II enzymes are capable of conjugating with activated carcinogens and converting them into inactive water soluble compounds that can be easily cleared by the kidney (Bradlow et al. 1999). Isothiocyanates such as sulforaphane, indole-3-carbinol, and phenethylisothiocyanates (from nasturtiin) have been shown to be potent inducers of the Phase II enzymes glutathione-S-transferase, quinone reductase, NADPH reductase, and glucouronyl transferase (Zhang and Talalay 1994; Tawfiq et al. 1995). Kore et al. (1993) reported that indole-3-carbinol induced quinone reductase and glutathione-Stransferase in rats, and Zhang et al. (1992) reported that sulforaphane induced quinone reductase in hpa1c1c7 cells. The increase in the Phase II enzyme, glutathione-S-transferase, in rats fed indole-3-carbinol was observed two days after treatment with indole-3-carbinol and persisted for up to 28 days (Wortelboer et al. 1992). Tawfiq et al. (1995) tested the influence of intact glucosinolates and myrosinase hydrolyzed glucosinolates and the nature of the glucosinolate side-chain on the induction of the Phase II enzyme quinone reductase in murine hepatoma (Hepa1c1c7). They found that intact glucosinolates (except progoitrin

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and glucosinalbin) had no effect on quinone reductase activity, while myrosinase hydrolyzed glucosinolates induced quinone reductase activity at a concentration as low as 1.8 11M. Tawfiq et al. (1995) also found that the rate of quinone reductase induction by isothiocyanates was dependent on the nature of the glucosinolate side chain. For example, glucosinolate breakdown products from glucoiberin had the highest increase in quinone reductase activity (76%), followed by gluconasturtiin (46%), and sinigrin (31 %). Another mechanism for the glucosinolate breakdown products involves inhibition of enzymes involved in the induction of cancer. Chemical carcinogens generally require metabolic activation in order to be able to bind to DNA and contribute to cancer development. Phase I enzymes are responsible for in vivo and in vitro metabolic activation of most carcinogens in human and animal cells. Studies have shown that, under certain conditions, the products of Phase I enzymes serve as substrates for Phase II enzymes, which convert them into electrophilic carcinogens that can easily be excreted through the urine (Talalay and Zhang 1996; Kassahun et al. 1997). There is ample evidence from animal studies that shows that some glucosinolate breakdown products inhibit the catalytic activity of Phase I enzymes (cytochrome P450 enzymes). Cytochrome P450 enzymes are a battery of Phase I enzymes that have been shown to metabolically activate chemical carcinogens such as nitrosamines and aflatoxins (Vondracek et al. 2001). Maheo et al. (1997) reported that treatment ofhepatocytes from rat and human livers with sulforaphane caused significant reduction in the expression and activity of cytochrome P450 3A4 and lA1. Barcelo et al. (1996) reported that sulforaphane inhibited cytochrome P450 2E1. Wang et al. (1997) established that glucosinolate breakdown products from glucoiberine, sinigrin, progoitrin, and benzyl glucosinolates at concentrations as low as 1 J.1M reduced the transcription of cytochrome P450 lAl in human Hep G2 cells caused by the chemical carcinogen ~-naphthoflavone. The glucosinolate breakdown products' influence on Phase I and Phase II have been classified into two groups, mono- and bifunctional inducers based on their effect on Phase I and Phase II enzymes. Monofunctional inducers such as sulforaphane and ~-phenylethylisothio cyanates are glucosinolate breakdown products that induce Phase II enzymes and either have no effect or inhibit Phase I enzymes, while bifunctional inducers are glucosinolates breakdown products that induce both Phase I and Phase II enzymes. Bifunctional inducers include indole-3-carbinol, its parent glucosinolate glucobrassicin, and neoglucobrassicin (Spanins et al. 1982; Bradfield and Bjeldanes 1984).

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In addition to modulating Phase I and Phase II enzymes, glucosinolates and their breakdown products have also been shown to have a direct effect on cancer development by suppressing the promotion phase of cancer cell formation (Wattenberg 1992; Thorling 1993), and reducing tumor invasion and metastasis (Meng et al. 2000b). Recent evidence also suggests that isothiocyanates may regulate cancer cell development by promoting apoptosis. Apoptosis, or programmed cell death, is a genetically encoded active cell destruction. It serves as a counterpart to mitosis by maintaining adequate cell number and eliminating damaged cells that are no longer needed by the organism. Gamet-Payrastre et al. (2000) reported that sulforaphane induced cell cycle arrest followed by apoptosis in an actively proliferating HT29 cell line established from human colon cancer. Similarly, indole-3-carbinol has also been shown to promote apoptosis in rodent cell lines (Telang et al. 1997). Glucosinolate breakdown products have also been shown to prevent and/or suppress estrogen-dependent cancers, such as cervical and breast, in both animal and human cells (Jin et al. 1999; Bell et al. 2000; Meng et al. 2000a). The mode of action may be by blocking the estrogen receptor function (Chang et al. 1999). 2. Thiosulfides. Thiosulfides are organosulfur compounds found in alliurns including garlic, onions, leeks, chive, and green onions. Fenwick and Hanley (1985a,b) and Lancaster and Shaw (1989) reported that S(2-propenyl)-L-cysteine sulfoxide) (alliin), y-glutamylcysteine, and Smethyl-L-cysteine sulfoxide (methiin) are the major thiosulfides in intact tissues of alliums. Other minor thiosulfides include S-propenyl-Lcysteine sulfoxide and a more recently discovered thiosulfide, S-ethylL-cysteine, commonly known as ethiin (Rubec et al. 1999). To our knowledge, none of the thiosulfides found in alliums have been detected in other vegetables, except methiin which was detected in cabbage leaves (Thomas and Parkin 1994). The pathways for biosynthesis of thiosulfides in alIiurns have not been very clearly defined because of their diversity and instability. For example, alliin, the most abundant thiosulfide in intact garlic, is highly unstable. When tissue is cut, chewed, or dehydrated, cytosolic alliin is rapidly lysed by the vacuolar enzyme alliinase or alliin lyase (EC 4.4.1.4) into a highly unstable diallyl thiosulfinate transient intermediate called allicin. Allicin then converts into several lipid-soluble alkyl alkanethiosulfinates, including allyl sulfide, diallyl disulfide, diallyl trisulfide, allylmethyl disulfide, ajoene, and 2- and 3-vinyl dithiins (Egen-Schwind et al. 1992; Kubec et al. 2000). These lipid soluble compounds form the

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odiferous and aromatic characteristic of garlic. Water soluble forms of allyl sulfides have been detected in aged extracts from garlic (Lawson 1993). Fermentation of intact garlic tissue results in the conversion ofyglutamyl-S-allyl cysteine into the water soluble S-allyl cysteine and allymercaptocsyteine (Lawson 1993). These compounds, however, are not detected in intact tissue or during cooking. The thiosulfides detected in onions are S-propenyl-L-cysteine sulfoxide, S-methyl-L-cysteine sulfoxide, and S-propyl-L-cysteine sulfoxide (Edwards et al. 1994; Thomas and Parkin 1994; Yoo and Pike 1998). Several surveys of the thiosulfide content of alliums have been conducted in the last decade (Block et al. 1992a; Breu 1996; Calvey et al. 1997). The type and amount of the thiosulfides varied considerably, depending on the level of alliinase activity and the nature of the substrate. Krest et al. (2000) examined alliinase activity in 17 species within the Liliaceae. They found high alliinase activity in allium and rhizerideum and low activity in melanocrommyum. Alliinase activity was also found to correspond with the level of its substrate (Krest et al. 2000). Consumption of alliums has been found to retard growth of several types of cancers. It has generally been accepted that the in vivo and in vitro antiproliferative effect of allium vegetables results from the breakdown of alliin into active organosulfur compounds (Sundaram and Milner 1993, 1996; Siegers et al. 1999). Knowles and Milner (2000) listed seven allyl sulfides (allicin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, ajoene, S-allyl cysteine, and S-allylmercaptocysteine) that have been reported to inhibit human tumor growth including prostate, colon, skin, breast, lung, lymphoma, erythroleukemia, and lymphocyte tumors. They proposed that the antiproliferative effects of the organosulfur compounds occur through common modes of action. Sundaram and Milner (1993) suggested that the allyl moiety of the organosulfur compounds is important in mediating their antiproliferating effect. This was supported by the finding that diallyl disulfide caused approximately 90 percent inhibition of human colon cancer growth, while its saturated analog dipropyl disulfide had no effect. However, there are significant differences in the ability of allyl sulfides to inhibit tumor cell growth. Knowles and Milner (2001) reported that diallyl disulfides were twice as effective as S-allylmercaptocysteine in inhibiting human colon tumor growth. In addition, studies from Milner's laboratory (Sundaram and Milner 1993; Sakamoto et al. 1997) found that the rate of tumor growth inhibition was also dependent on the number of sulfur atoms in the allyl sulfide molecule and that lipid soluble allyl sulfides are more effective than the water soluble allyl sulfides. For example, diallyl trisulfide was

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reported to be at least 2.5 times more effective in controlling lung tumor growth than diallyl disulfide. Sakamoto et al. (1997) and Sigounas et al. (1997) reported that 0.25 mmol/L S-allymercaptocysteine completely inhibited human erythroleukemia proliferation, whereas 5 mmollL Sallylcysteine had no effect. The lipid soluble diallyl sulfide, disulfide, and trisulfide were reported to be more effective in suppressing canine tumor cell proliferation than an equimolar concentration of the watersoluble S-allylcysteine, S-ethylcysteine or S-propylcysteine (Sundaram and Milner 1993). Knowles and Milner (2000) proposed a scheme for the antiproliferative effect of organosulfur compounds that included modification of at least eight major cellular mechanisms among them reducing cholesterol synthesis, inhibiting cellular adhesion, altering the release of growth factors, and enhancing apoptosis. Examination of the mechanism of cancer prevention by organosulfur compounds indicate that they function as blocking agents. Blocking agents, as defined by Wattenberg (1997), act during the initiation stage of carcinogenesis by either inhibiting the activation of procarcinogens, trapping reactive species, or by enhancing Phase II detoxification enzymes. Studies have shown that DNA adducts induced in animal cells during exposure to chemical carcinogens, such as dimethylbene[a]anthracene and N-methylnitrosourea, are suppressed by a S-allylcysteine supplement (Lin et al. 1994; Amagase et al. 1996). Similar to glucosinolates, several allyl sulfides have also been shown to enhance activities of Phase II detoxification enzymes, such as glutathione S-transferase and quinone reductase. Guyonnet et al. (1999) examined the effect of diallyl sulfide, diallyl disulfide, dipropyl sulfide, and dipropyl disulfide on intestinal, kidney, and liver Phase II enzymes including quinone reductase, glutathione S-transferase, glucouronosyltransferase, and microsomal epoxide hydrolase and found that the four compounds induced all Phase II enzymes in all four organs, except quinone reductase which was induced only by diallyl disulfide. Similarly, Hatono et al. (1996) reported a 41 percent increase in glutathione S-transferase in rat liver 12 h after a single oral administration of 3.5 mmollkg body weight of S-allylcysteine and the increase lasted for 72 h after the treatment. In addition to enhancing the activity of Phase II detoxification enzymes, allyl sulfides also inhibit Phase I enzymes, such as cytochrome P450 2E1, which have been shown to activate chemical carcinogens, and promote apoptosis (Brady et al. 1991; Kwak et al. 1994; Surh et al. 1995; Reicks and Crankshaw 1996; Seiss et al. 1997). Ajoene, an organosulfur compound from garlic, induced apoptosis in human leukemic cells but not in healthy blood cells, by stimulating free-radical peroxide production, and the effect was reversed by antioxidants (Dirsch et al. 1998).

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Another important health benefit of allyl sulfides is their ability to protect humans and laboratory animals against cardiovascular diseases. Cardiovascular diseases are controlled by a multitude of factors including high serum cholesterol, high levels oflow-density lipoproteins, high blood pressure, and platelet aggregation. Liu and Yeh (2000) examined the effect of 11 water and lipid soluble organosulfur compounds from garlic on the incorporation of [2- 14 C]-acetate into cholesterol in rat hepatocyte cells. They found that seven of the allyl sulfides tested inhibited cholesterol synthesis by as much as 55 percent and that higher concentrations (1-4 mmol/L) of diallyl disulfide, diallyl trisulfide, and dipropyl disulfide resulted in complete inhibition of cholesterol synthesis. Oxidation of low-density lipoprotein molecules has also been suggested as a key factor in the development of cardiovascular diseases (Luc and Fruchart 1991). Ide and Lau (1997) reported that S-allylcyteine and other thiosulfides prevented low density lipoprotein oxidation in endothelial cells, caused by reactive oxygen species. There is considerable evidence linking blood platelets in the development of cardiovascular diseases. Platelets are blood cells that are essential for blood clotting during tissue damage. The inner most layer of artery walls consists of the endothelial cells. These cells are aligned with the blood stream flow and have a nonstick surface. Mechanical or chemical disruption of the continuity of the endothelial cell layer causes platelet activation. Platelet activation has also been observed in smokers and patients suffering from hypertension. Platelets attach themselves to the damaged vessel walls and then to themselves causing a blood clot in order to repair the damage. During atherosclerosis, circulating platelets precipitate on the endothelial cell layer causing plaque, which thickens and/or blocks the arteries, leading to a heart attack. Blood clots, also known as thrombus, may move along the blood stream from the coronary arteries into the brain causing a stroke. Several substances, including aspirin (acetylsalicylic acid), warfarin, and organosulfur compounds from garlic and onion, have been suggested to reduce platelet aggregation, thereby lowering the risk of a heart attack and/or stroke (Patrono 2001; Rahman 2001). Lawson et al. (1992) demonstrated that diallyl disulfide, diallyl trisulfide, and ajoene, from garlic, have in vitro antiplatelet activity. Similarly, Morimitsu et al. (1992) reported that organosulfur compounds found in onions, mostly cepaenes, also have antiplatelet activity. Inhibition of platelet aggregation can be achieved by either inhibition of membrane receptors or by interception of signaling pathways (Geiger 2001). At least three mechanisms have been proposed for this inhibition. They include interfering with ADP-induced platelet aggregation, block-

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ing the glycoprotein lIb/IlIa receptor, and inhibiting cyclooxygenase, which is involved in arachidonic acid induced platelet aggregation (Geiger 2001). Organosulfur compounds extracted from onions and garlic have been shown to inhibit cyclooxygenase activity and arachidonic acid metabolism in animal tissue by as much as 75 percent (Srivastava 1984; Srivastava and Justesen 1989; Wagner et al. 1990) and ADP-induced human platelet aggregation (Chen et al. 2000). In addition to inhibiting the two leading causes of death in humans, cancer and cardiovascular diseases, thiosulfides have also been shown to stimulate the immune system, by activating T cell proliferation (Lau et al. 1991; Feng et al. 1994) and reducing blood glucose level in diabetics by stimulating insulin secretion by the pancreas (Sheela et al. 1995; Augusti and Sheela 1996).

B. Polyphenolics Polyphenolics constitute the largest ubiquitous group of phytochemicals in vegetables. They are partially responsible for the sensory (astringency and bitterness) as well as nutritional quality of plant tissue. There are nearly 8000 structurally known polyphenolics in the plant kingdom, mostly in herbs, vegetables, and fruits ranging in size from simple phenolics to complex and highly polymerized compounds with molecular weights of more than 30 kda (Harborne 1994). Because of the complexity of the structure and functions of polyphenolics, this review will be limited to those compounds that are present in substantial amounts in vegetables and that have been shown to have phytochemical activity. Polyphenolics generally arise from two main biosynthetic pathways: the shikimic acid pathway and the acetate pathway (Harborne 1989). Harborne (1989) divided polyphenolics according to their basic chemical structure into at least ten different classes. Phenolics (mostly flavonoids) and terpenoids represent the most important classes of polyphenolics in vegetables. Scalbert and Williamson (2000) estimated that the total human intake of phenolics is about 1 g/day, which consists of two-third flavonoids and one-third phenolic acids. 1. PhenolicslFlavonoids. Phenolics including flavonoids, simple phenolic acids (e.g., gallic acid and resorcinol), and phenylpropanoid derivatives of hydrocinamic acids (caffeic, coumaric, and ferulic) are among the most important phytochemicals in plants. In this review we limit our discussion to flavonoids, since they are the largest group of phenolics present in vegetables. Flavonoids are among the most widely distributed and most abundant plant phenolics. Harborne (1993) estimated that there are more than

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4000 secondary plant metabolites in the flavonoid family. They consist

of a diphenylpropane common structure which contains two aromatic rings linked through a three carbons oxygenated heterocyclic-structure (Harborne and Mabry 1982). Flavonoids are further classified into anthocyanins, flavonols, flavones, and isoflavonoids. The first products of the flavonoid biosynthetic pathway are the chalcones. Chalcones are synthesized from p-coumaryl-CoA and 3 malonyl-CoA and catalyzed by chalcone synthase. Isomerization of chalcones by chalcone isomerase produces naringenin and liquiritigenin, which serve as substrates for flavonoid biosynthesis. The health benefit of flavonoids is linked primarily to their antioxidant potential. Induction of virtually all human diseases occur as a result of processes that generate compounds that have highly reactive oxidative properties (free radicals) or exposure to chemical toxicants that affect specific molecular targets such as cellular DNA or low density lipoproteins. In animal models, flavonoids regenerate vitamin C, which in turn regenerates vitamin E (Cossins et al. 1998). However, despite the number of in vitro studies that have been published thus far, there is little information from human feeding studies that elucidate the antioxidant mechanisms taking place in the human body (Robards and Antolovich 1997). To overcome the potential hazard from oxidative damage in the body, consumption of a diet rich in the antioxidant phenolics, including flavonoids, are considered the first line of defense against highly reactive toxicants. The average daily intake of flavonoids in the United States is estimated to be about 1 g/day, and among Japanese women flavonoids are the main source of antioxidants in the diet (Arai et al. 2000).

Anthocyanins. Anthocyanins exist mainly as glycosides and acylglycosides of anthocyanidins, usually as C3 mono-, bi-, and/or trisides (Herrmann 1976). Anthocyanins give vegetable leaves and fruits their purple and/or red color appearance, such as in purple cabbage, purple broccoli, eggplant, purple potato, purple sweet potato, rhubarb, red radish, and red onion (Goda et al. 1997; Cai et al. 1998; Noda et al. 1998; Rumpunen and Henriksen 1999; Fossen and Andersen 2000; Cao et al. 2001). Anthocyanins have also been shown to protect mammalian cell lipoproteins from damage by free radicals. For example, nasunin, an anthocyanin from eggplant, has been shown to inhibit brain cell lipid peroxidation caused by oxygen and hydroxyl free radicals (Noda et al. 1998) and to reduce intestinal absorption of cholesterol (Kayamori and Igarashi 1994). Flavonols. Flavonols include quercetin, kaempferol, fisetin, and myricetin. Flavonols exist primarily in the glycosylated form with a

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hydroxyl group at C3 position (Harborne 1993). Quercetin is the most important flavonoid in vegetables. It has been detected in onions and to a lesser extent in tomato and bean (Crozier et al. 1997). Kaempferol, myricetin, and fisetin have been detected in onion, lettuce, endive, and horseradish (Eloesser and Herrmann 1975; DuPont et al. 2000; Miean and Mohamed 2001). The effect of flavonols on carcinogenesis and cardiovascular diseases in human and laboratory animals has been studied by several investigators. Several mechanisms have been proposed for the mode of action of these compounds including inhibition of estrogen binding in mammalian cells (Baker et al. 1998), induction of the Phase II enzyme quinone reductase in murine hepatoma cells (Hou et al. 2001), antioxidant protection from oxygen radicals (Lio et al. 1993; Bohm et al. 1998), and induction of apoptosis (Kuntz et al. 1999; Sakagami et al. 2000).

Using data from the National German Food Consumption Survey, Bohm et al. (1998) estimated that flavonols intake among the German population was about 11.5 mg/day, mostly from vegetable, tea, red wine, and fruit (Bohm et al. 1998). Earlier studies have suggested that only free flavonols, without the sugar molecule (aglycones), are absorbed through the human gut. However, in a more recent study, Hollman and Katan (1998) showed that the glycosidic form (natural form within the plant cell) are more readily absorbed than the aglycone form.

Flavones. Similar to flavonols, flavones including apigenin and luteolin also exist as O-glycosides with a hydrogen atom in the C;, position, but at lower concentrations. Flavones have been detected in conjugated form in celery (Crozier et al. 1997), tomato, eggplant, garlic, and onion (Paganga et al. 1999; Miean and Mohamed 2001). Miean and Mohamed (2001) surveyed 62 fruits and vegetables and found only celery, among vegetables, that contains both apigenin and luteolin. The mechanism of action of flavones on chronic diseases is similar to that of other flavonoids. They were proposed to function primarily as antioxidants by conserving a-tocopherol content of low density lipoproteins and membrane lipids in the reduced state (de Whalley et al. 1990). Isoflavonoids. In contrast to flavonoids, isoflavonoids including daidzein and genistein have limited distribution in plants. They exist mainly in legumes including soybean, chickpea, and lentil (Halbrock 1981). Smaller quantities have also been detected in other vegetables, such as broccoli, asparagus, alfalfa sprouts, okra, and mushroom (HornRoss et al. 2000; Liggins et al. 2000; Yu et al. 2000). Liggins et al. (2000) assayed 114 vegetables and vegetable preparations, common in the European diet, for daidzein and genistein and found that 48 contained no

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detectable levels, 41 contained less that 0.1 mg/kg dry weight, and 25 contained more than 0.1 mg/g dry weight. It is estimated that the average total daily intake of isoflavonoids in the United States is about 154 J.lg, mostly from soybean and peas (deKleijn et al. 2001), while in Japan the average daily intake is about 47 mg/day (Arai et al. 2000). Isoflavonoids in the Japanese diet were reported to exceed that of carotenoids, vitamin E, and nearly half the total intake of vitamin C (Arai et al. 2000). Isoflavonoids are isomeric to other flavonoids and their metabolites have structures similar to mammalian steroids (Merken and Beecher 2000). Much of the research on the health benefits of isoflavonoids has been done on daidzein and genistein. Recently, Goldwyn et al. (2000) comprehensively reviewed the health benefit of isoflavonoids. Special emphasis was given to the phytoestrogenic response of genistein. Genistein was reported to have an estrogenlike activity at low concentration and antiestrogenic activity at high concentration, as well as the highest affinity for binding to the estrogen receptors (Brandi 1997). As a phytoestrogen, genistein is believed to block estrogen perception by actively competing for binding sites and/or by blocking estrogen synthesis (Bingham et al. 1998). Laboratory studies have confirmed the role of early exposure to estrogen in reducing hormone-dependent and chemically induced cancers in animal models (Grubbs et al. 1985), however there are significant health risks associated with the use of estrogen. Consumption ofisoflavonoid-rich foods have been reported to induce estrogenlike activity. Lee et al. (1991) found that Asian women who consumed a diet rich in soy products had relatively low risk of breast cancer. In another study, Ziegler et al. (1993) conducted a multigeneration breast cancer incidence in Asian American women. They found a significant increase in breast cancer in the second generation compared to the first, which they attributed in part to a reduced intake of soy-based foods among the second generation. Further evaluation of soy-based foods have confirmed that the main antibreast cancer ingredient is genistein (Lamartiniere 2000). Daidzein has very low estrogen activity, however it is further metabolized in the large intestine into equol. Equol is a potent phytoestrogen and an antioxidant, but the rate of conversion varies among individuals, which could affect the health benefits of this compound (Wiseman 1999). 2. Terpenoids. Terpenoids represent one of the most important groups of phytochemicals in vegetables. Of particular interest are the roles of tocopherols and carotenoids in promoting human health. The antioxi-

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dant activity of tocopherols and carotenoids have attracted significant scientific attention, especially their in vitro and in vivo roles in reducing atherosclerosis by inhibiting low density lipoprotein oxidation (Diaz et al. 1997; Holvoet and Collen 1998).

Tocopherols. Generically referred to as vitamin E, tocopherols consist of eight naturally occurring fat-soluble tocols and tocotrienols (Fritsma 1983). They are made up of four tocol structures consisting of a saturated phytyl C16 side chain (a, ~, y, and 8-tocopherols) and four tocotrienols bearing three double bonds in the phytyl side chain (a, ~, y, and 8tocotrienol). The tocopherol name is derived from the Greek words tokos and pherein; together they mean "to-conceive-a-child" in reference to their health promoting properties. The most predominant and most active form of vitamin E is alpha-tocopherols (Thakur and Srivastava 1996). Vitamin E deficiency is believed to be associated with the pathogenesis of cardiovascular disease including low density lipoprotein oxidation, cytokine production, production of lipid mediators, platelet function, and smooth muscle cell proliferation, as well as interaction of the endothelium with immune and inflammatory cells (Azzi et al. 1995; Ozer et al. 1995). Gey and Puska (1989) reported that vitamin E is protective against nearly 80 cellular abnormalities including cardiovascular disease, cancer, sterility, muscular dystrophy, changes in the central nervous system, and anemia development (Srivastava and Goswami 1988; Jialal et al. 1990; Sokol 1990). The current recommended dietary allowance for vitamin E intake from natural foods is about 15 mg/day (National Research Council 1989). Carotenoids. Carotenoids are 40 carbon dimer molecules of symmetrically aligned polyisoprenes. There are more than 600 carotenoids found in nature, however only 40 are present in a typical human diet, and only 20 have been identified in animal blood and tissue (Agarwal and Rao 2000). The most abundant carotenoids in vegetables are a-carotene, ~ carotene, lycopene, lutein, zeaxanthin, and ~-cryptoxanthin. These carotenoids account for more than 90 percent of the carotenoids present in the human diet (Gerster 1997). Three of these carotenoids (a-carotene, ~-carotene, and ~-cryptoxanthin) can be converted into the provitamin A (retinol), while lycopene, lutein, and zeaxanthin have no vitamin A activity. In humans the level of carotenoids is regulated by their dietary intake, except retinol level which is regulated by the liver where it is stored (Collins 2001). Serum concentration of carotenoids is believed to be the most reliable marker for consumption of food rich in carotenoids. Krinsky (1993) examined human diet, serum, and tissue and found that their major

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carotenoid content included ~-carotene and lycopene. However, several studies have found significant interindividual variability in serum concentration and serum response to a single dose of ~-carotene (Dimitrov et al. 1988; Brown et al. 1989; Johnson and Russell 1992). Carotenoid concentration in the blood serum is also influenced by several dietary and genetic factors such as fiber, fat, efficiency of absorption, and rate of metabolism (Bowen et al. 1993; Castenmiller and West 1998). The biological roles of carotenoids have been classified into three categories: functions, actions, and associations. In plants and photosynthetic bacteria, carotenoids function as accessory pigments in photosynthesis and protect animal and plant tissues against photosensitization. However, in humans the only known function of carotenoids is linked to their vitamin A activity. Carotenoids have also been shown to have a variety of different actions including potential antioxidant activity and inhibition of mutagenesis and transformation. Carotenoids actions include possible inhibition of macular degeneration, cataracts, decreased risks of some cancers, and heart diseases (Olson 1999). People consuming diets rich in tomato and tomato based products, which are rich in the carotenoid lycopene, or with high levels of lycopene in their bodies, were found to be less likely to develop stomach and rectal cancers than those who consume lesser amounts of lycopene rich vegetables or have lower levels of lycopene in their system (Giovannucci 1999). The antioxidant activity of carotenoids differs among the different compounds. DeMascio et al. (1989) reported that the singlet oxygen quenching ability of lycopene is twice that of ~-carotene and ten times that of a-tocopherol. The bioavailability of ~-carotene from vegetables in the human diet is limited. Studies have estimated that ~-carotene bioavailability is 22 to 24 percent from broccoli, 19 to 34 percent from carrots, and 3 to 6 percent from leafy vegetables (van het Hof et al. 2000). However, according to the Life Sciences Research Office and the Federation of American Societies for Experimental Biology (LSRO/FASEB 1995), vegetable intake in the U.S. food supply contributes about 36 percent, of vitamin A, fruit intake contributes about 3 percent, and meats, fats, and other dairy and poultry products contributes the rest. Several factors have been shown to influence carotenoids bioavailability in humans, the most prominent of which is the food matrix. Studies have shown that combination of fatty foods with carotenoid-rich vegetables enhanced carotenoids uptake. Roels et al. (1958) reported a one- to fivefold increase in ~-carotene uptake in boys deficient in vitamin A, when their diet was supplemented with olive oil. More recent studies have shown that the bioavailability of lycopene from tomato has

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increased dramatically by heat treatment in the presence of oil (Stahl and Sies 1992; Gartner et al. 1997). For example, lycopene was found to be more bioavailable from tomato paste than from fresh tomato due to heat treatment and the presence of higher oil content in the paste (Stahl and Sies 1992). Carotenoid bioavailability is also influenced by the nature of the carotenoid. Lutein, which has no vitamin A activity, is five times more readily available in the human body than ~-carotene (van het Hof et al. 1999). C. Vitamin C

Vitamin C (ascorbic and dehydroascorbic acids) is an essential vitamin to humans and other mammals that lack the ability to synthesize this vitamin, because they are deficient in the enzyme L-gulonolactone oxidase, an enzyme involved in the biosynthesis of vitamin C via the glucuronic acid pathway (Woodall and Ames 1997). The biological function of vitamin C is based on its ability to donate electrons, which provide intra- and extracellular reducing power for a variety of biochemical reactions. Buettner (1993) reported that the reducing power of vitamin C is capable of neutralizing most of the physiologically relevant reactive oxygen and nitrogen species in the human body. In mammalian cells, vitamin C serves as a cofactor for reactions that require reduced iron and/or copper metalloenzymes (Englard and Seifter 1986; Halliwell and Whiteman 1997; Tsao 1997). Several enzymes involved in the biosynthesis of collagen, carnitine,and neurotransmitters require vitamin C as a cofactor (Burri and Jacob 1997; Tsao 1997). Vitamin C has been reported as an electron donor for eight enzymes involved in amino acid and hormone synthesis (Levine et al. 1996). It is involved in the synthesis and modulation of nervous system hormones by serving as a cofactor for dopamine ~-hydrolase, which is involved in the conversion of dopamine into norepinephrine and in modulating neurotransmitter receptors (Englard and Seifter 1986; Katsuki 1996). The substantially high cellular levels of vitamin C provide antioxidant protection in the eye against photosynthetically generated free radicals (Delamere 1996) and against plasma and low density lipoprotein oxidation (Frei et al. 1989; Jialal et al. 1990). Vitamin C also functions as a reducing agent for mixed-function oxidases involved in drug metabolism by inactivating a wide variety of xenobiotic substances (substances that are foreign to the cells such as drugs and carcinogens) and hormones (Tsao 1997). One of the most important indirect functions of vitamin C is its ability to regenerate other biologically important antioxidants, such as glutathione and vitamin E, into their reduced state (Jacob 1995;

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Halpner et al. 1998). The oxidized one- and two-electron forms of vitamin C are readily converted back into a reduced state by reductants, such as glutathione, NADH, and/or NADPH. A lack of vitamin C in the diet can cause the potentially fatal disease scurvy, which can be prevented with as little as 10 mg vitamin C per day, an amount easily obtained through consumption of vegetables (Levine 1986). Deficiency of vitamin C in human and animal models has also been linked to reduced absorption of amino acids and lower levels of intestinal brush-border membrane proteins and phospholipids (Dulloo et al. 1981); while higher intake of vitamin C has been linked to lower risk of cardiovascular disease and several types of cancers, enhanced cognitive function and memory, decreased risk of asthma, and protection from the common cold. Singh et al. (1995) reported a twofold decrease in coronary heart disease in patients with higher plasma vitamin C compared to the lower group. Several other studies have also confirmed the association between higher plasma vitamin C concentration and lower risk of cardiovasular disease, ranging in benefit from 26 to 60 percent (Gale et al. 1995; Nyyssonen et al. 1997; Simon et al. 1998). Vitamin C consumption has also been shown to reduce breast, cervical, colorectal, pancreatic, lung, and gastric cancers (Wassertheil-Smoller et al. 1981; Fontham et al. 1988; Freudenheim et al. 1990; Howe et al. 1990; Howe et al. 1992; O'Toole and Lomabard 1996). However, despite the many studies that suggest protective effects for vitamin C against chronic diseases, the data are not consistent or specific to indicate a vitamin C requirement by the human body. Nearly 90 percent of vitamin C in a typical human diet comes from vegetables and fruits, mainly from citrus, tomato, tomato-based products, and potato. The average dietary intake of vitamin C by adult men and women in the United States is estimated at about 98 mg/day, which is about 50 percent less than the amount recommended by the five daily servings of fruits and vegetables suggested by the U.S. Department of Agriculture and the National Cancer Institute. Nearly 25 percent of men and women in the United States consume less than 60 mg/day vitamin C (Bendich 1997). Intake of vitamin C, higher than the Recommended Daily Allowance of 200 mg/day, has not been shown to increase the plasma steady-state concentration of the vitamin or cause any health risk (Levine et al. 1996). However, concentrations greater than 3 g/day have been suspected of causing gastrointestinal disturbance, kidney stone formation in certain individuals, and vitamin Bl2 breakdown (Herbert et al. 1978; Sauberlich 1994).

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D. Dietary Fiber

The health benefits of a high fiber diet has been recognized since the fifth century Be, when Hippocrates, the father of modern medicine, recommended a diet high in fiber and low in refined foods. Dietary fiber, complex carbohydrates, and/or nonstarch polysaccharides are terms used interchangeably to refer to the residual portions of the plant cell, such as polysaccharides, lignin, and other substances, which are resistant to digestion by human gastrointestinal secretion (Pilch 1987). Dietary fiber is classified into water soluble and water insoluble fiber. Water soluble fiber includes pectins, and gums and nonwater soluble fiber includes lignin, cellulose, and hemicellulose (Anderson 1985). Anderson and Bridges (1988) evaluated dietary fiber content of several food groups. They found that vegetables contain about 32 percent dietary fiber, based on dry weight, which they estimated to be equivalent to the amount found in cereal products. Pectin constitutes nearly 30 percent of the dietary fiber content in vegetables (Marlett 1992). Soluble and insoluble fibers have different clinical effects. Anderson et al. (1990) reported that soluble fibers tend to delay gastric emptying by slowing food passage through the small intestine, while insoluble fibers tend to hasten food passage through the small intestine. There is also a strong relationship between dietary fiber intake and lower risk of certain chronic diseases, such as type II diabetes (diabetes at an advanced age), coronary heart disease, peptic ulcer, hypertension, some types of cancer, and appendicitis (Trowell 1978; Salmeron et al. 1997; Levi et al. 1999). Several mechanisms have been proposed for this inhibition, including improved insulin sensitivity and/or decreased insulin requirement (Harold et al. 1985), lower blood pressure (Rouse et al. 1983), and decreased total serum cholesterol and low-density lipoprotein concentrations (Anderson et al. 1990). Other effects of dietary fiber include increased stool-bulking, which dilutes chemical carcinogens, and increased production of the anticarcinogen butyric acid through fermentation of dietary fiber by the colon microflora. Le Marchand et al. (1997) conducted a population-based case-control study among different ethnic groups and genders in Hawaii to evaluate the role of dietary fiber from various commodities on the risk of colorectal cancer. Their findings suggested a strong inverse association between vegetable dietary fiber (soluble and insoluble) and colorectal cancer. In contrast, they reported that dietary fiber from cereals and fruits had lower association with colorectal cancer than vegetable fiber.

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E. Selenium

Interest in selenium as a phytochemical has increased dramatically in the last three decades as a result of several studies that demonstrated an increased risk of cancer with low selenium diet (Lane and Medina 1985; Ip 1986). Selenium is found in human hair, nails, and in the blood. Most selenium in animal and plant tissue is present as selenomethionine and selenocysteine. Animal tissues, however, are not capable of synthesizing selenomethionine, instead they obtain it from plant tissue. Selenium is not considered an essential element in plants. Estimates of the daily intake of selenium in the U.S. diet ranges from 60 to 216 Jlg/day (Fan and Kizer 1990). Plant-based selenomethionine accounts for nearly half of the daily requirement of selenium in humans, while the balance is obtained from water (as selenate and selenite), milk, fish, and animalbased products (Esaki et al. 1982). Vegetables, such as broccoli and garlic, grown on high selenium soil or fertilized with selenium have been shown to accumulate selenomethionine and selenocysteine (Ip and Lisk 1994; Finley 1998). Selenomethionine and selenocysteine are readily absorbed by human tissue (Swanson et al. 1991). Selenomethionine and selenocysteine function primarily through their association with certain proteins, recognized as selenoproteins (Stadtman 1991). Selenoproteins are proteins that contain selenium in stoichiometric amounts. The physiological function of selenomethionine is similar to that of methionine. Selenomethionine has been shown to substitute for methionine in these proteins, however selenocysteine does not substitute for cysteine (Sunde 1990). At least 14 selenoproteins have been identified in animal cells. Glutathione peroxidases, which protect polyunsaturated membrane lipids from oxidative degradation (Flohe 1988) and iodothyronine diodinases, which regulate thyroid hormone metabolism (Berry and Larsen 1992), are among the most important selenium-dependent proteins in human. Bosl et al. (1997) reported that any disruption of the selenoproteins synthesis in animal embryos can be lethal. Keshan disease, a disease of the heart, and Kashin-Beck disease, a disease of the cartilage, have been linked to selenium deficiency that occurs almost exclusively in children and adolescents (Keshan Disease Research Group 1979; Yang et al. 1988). These two diseases occur mostly in southeast Asia in areas with severe selenium deficiency (Ge et al. 1983). Cancer mortality correlation studies suggest an inverse association between serum-selenium levels, higher than those required for the full expression of selenoproteins, and cancer incidence. Data from a prospective study, which examined the association of selenium and ovarian can-

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cer risk, indicated that women in the higher group of serum-selenium content were four times less likely to develop ovarian cancer than women in the lower group (Helzlsouer et al. 1996). Similarly, Clark et al. (1996) conducted a selenium-enriched yeast supplement trial, involving 1312 patients (mostly men) who were prone to skin cell carcinoma. They found that men receiving 200 Jlg selenium per day were 50 percent less likely to develop or die from cancer than those receiving a placebo. Ip et al. (2000) found that selenium enriched garlic was twice as effective in reducing mammary cancer than selenium-enriched yeast. In another trial, Yoshizawa et al. (1999) reported a 33 percent reduction in prostate cancer in men receiving a 200 Jlg/day selenium supplement compared to the control. Selenium deficiency in human and animal models has also been linked to cardiovascular diseases (Overcast et al. 2001) and rheumatic arthritis (Knekt et al. 2000). Based on a few research findings that suggest a positive role of selenium in reducing chronic diseases, many dietary supplements of various strength have been introduced into the market. However, according to data from the Third National Health and Nutrition Examination Survey (Institute of Medicine 2000), selenium intake from food sources, including vegetables and fruits, in the United States is above the average daily requirement. F. Folates

Other important phytochemical compounds that have been linked to improved human health are the folates (folic acid and tetrahydrofolate). Folates are involved as cofactors in carbon transfer reactions in DNA biosynthesis and the methylation cycle (Scott et al. 2000). They are an absolute requirement for methionine, purine, and thymidylate synthesis. Tetrahydrofolate is also involved in the synthesis of Sadenosylmethionine, the universal methyl donor in all living cells, and in the control of glycine-to-serine conversion (Scott et al. 2000; Ames 2001). Ames (2001) reported that folate deficiency causes chromosome breakage as a result of extensive incorporation of uracil into the human DNA. Epidemiological studies have shown that folate deficiency contributes to an accumulation of homocysteine leading to neural-tube defects such as spina bifida in fetuses (Oakley et al. 1996). Folate deficiency is also linked to colon cancer risk, neurotoxicity, cognitive defects, and heart attacks (Mason 1994; Blount et al. 1997; Quinn et al. 1997). Ames (2001) estimated that 10 percent of the U.S. population is deficient in folate at a level that causes chromosome breakage, with higher risk among the

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poor. In 1992, the U.S. Public Health Service issued a recommendation that all women capable of childbearing consume 0.4 mg folic acid per day to reduce neural tube defect (Centers for Disease Control 1992). Similar recommendations have been issued by other countries, including Canada, England, and Australia. The U.S. Public Health Service identified three approaches to ensure adequate folate consumption among these women including higher fruit and vegetable consumption, daily supplements, and fortification of the U.S. food supply. Vegetables, such as broccoli, brussels sprouts, and potato were reported to contribute between 35 to 40 percent of the total intake of folate in the human diet (Scott et al. 2000). In 1992, the Food and Drug Administration proposed a folate fortification scheme for cereal grains and flour. III. PHYTOCHEMICALS CONTENT AND HEALTH BENEFITS OF THE FOUR MAJOR VEGETABLE GROUPS

In recent decades, health factors have become increasingly important in determining consumers' dietary preferences. The direct association between a diet high in vegetables and fruits and lower risk of chronic diseases has prompted many consumers to change their dietary habits, which may have contributed to the sharp increase in demand for vegetables and fruits during the last three decades. Table 3.1 lists family and common and scientific names of vegetables examined in this review. Many of the studies on diet and chronic diseases have focused mainly on intake of specific nutrients rather than whole foods (Ness and Powles 1997; Law and Morris 1998). Due to the chemical and physical complexity of vegetables, the effect of individual nutrients may differ from the effect of whole vegetables (Liu et al. 2001). The general belief among dieticians and health professionals is that the health benefit of vegetables should not be linked to only one compound or one type of vegetable, but rather a balanced diet that includes more than one type of vegetable is likely to provide better protection. In this section, we will highlight the phytochemicals composition and health benefits of the most commonly consumed vegetables in the United States, namely the crucifer, allium, solanaceous vegetables, as well as some other vegetables. A. Crucifers

Cultivation of a bygone ancient cabbage may have occurred as early as 8000 years ago along the northern coast of Europe. It was from this area that the wild ancestral cabbage was later introduced into the Mediterranean, eastern Europe, and Asia (Schery 1972; Heywood 1978; Snogerup

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Common and scientific names of plants referred to in the text.

Family

Common name

Scientific name

Apiaceae

Carrot Celery Parlsey

Daucus carota Apium graveolens Petroselinum crispum

Asteraceae

Lettuce Endive

Lactuca sativa Cichorium endivia

Brassicaceae

Arabidopsis Broccoli Brussels sprouts Cauliflower Cabbage Horseradish Kale Radish Rape Rutabaga Turnip, Chinese Cabbage

Arabidopsis thaliana Brassica oleracea Halica group Brassica oleracea Gemmifera group Brassica oleracea Botrytis group Brassica oleracea, Capitata group Armoracia rusticana Brassica oleracea, Acephala group Raphinus sativus Brassica napus Brassica napus, Napobrassia group Brassica rapa

Chenopodiaceae

Chard Spinach

Beta vulgaris Spinacia oleracea

Convolulaceae

Sweet potato

Ipomoea batatas

Cucurbitaceae

Cucumber Pumpkin Squash

Cucumis sativus Cucurbita argyrosperma Praecitrullus fistulosus

Liliaceae

Chive Garlic Leek Onion Rakkyo

Allium Allium Allium Allium Allium

Solanaceae

Eggplant Hot and Sweet pepper Potato Tomato

Solanum melongena Capsicum annuum Solanum tuberosum Lycopersicon esculentum

scoenoprasum sativum ampeloprasum cepa chinense

1980). Crucifer vegetables include broccoli, cabbage, brussels sprouts, cauliflower, and kale, which have evolved from the wild ancestral cabbage nearly 3000 years ago (Phillips and Rix 1993). Related crucifers such as chinese cabbage, turnip, radish, rutabaga, and rape have evolved from this ancestral cabbage in different parts of the world (Phillips and Rix 1993). It is believed that many crucifers may have been collected for medicinal purposes in northern Europe long before their domestication. Currently, crucifer vegetables are among the most popular vegetables in the

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United States diet and per capita consumption has risen steadily in the last three decades. The greatest rise in consumption was in broccoli, which increased by nearly 34 percent from 1990 to 1998. Worldwide, production of crucifers is in the top three vegetables after potato and tomato (FAO 1999). Cabbage production alone in 1998 totaled more than 47 million tonnes, of which 14 million tonnes were produced in China (FAO 1999). 1. Phytochemical Content

Crucifers provide the richest sources of the phytochemical glucosinolates in the human diet. They are also rich in vitamin E, tocopherols, vitamin C, fiber, and other phytochemicals. Of the nearly 120 glucosinolates that have been identified thus far, about 20 have been detected in crucifers, and of those, only three or four are present in significant amounts (Fahey et al. 2001). The most abundant glucosinolates are the aliphatic, followed by the indoles, and then the aromatic. The aliphatic glucosinolates include glucoraphanin, glucoerucin, progoitrin, epi-progoitrin, sinigrin, napoleiferin, gluconapin, and glucoalysin; the indoles include glucobrassicin and 4-hydroxyglucobrassicin, 4-methoxyglucobrassicin, and neo-glucobrassicin; and the aromatics include gluconasturtiin and sinalbin. Comparative studies of glucosinolate profiles indicate significant differences among individual crucifers (VanEtten et al. 1976; Carlson et al., 1981; Carlson, et al. 1985; Carlson et al. 1987a,b; Kushad et al. 1999; Ciska et al. 2000). In broccoli heads, the most significant glucosinolates are glucoraphanin, glucobrassicin, progoitrin, and gluconasturtiin (Carlson et al. 1987a; Goodrich et al. 1989; Kushad et al. 1999); in brussels sprouts, cabbage, cauliflower, collard, and kale the predominant glucosinolates are sinigrin, progoitrin, and glucobrassicin (VanEtten et al. 1976; Carlson et al. 1987a; Kushad et al. 1999); in turnip and rutabagas, the predominant glucosinolates are glucobrassicin, progoitrin, and gluconasturtiin (Carlson et al. 1981; Carlson et al. 1987b); and in radish, the predominant glucosinolates are glucoerucin, glucoraphanin, and glucobrassicin (Carlson et al. 1985; Ciska et al. 2000). Each of these vegetables also contain smaller amounts of other glucosinolates. There are also differences at different stages of development. Fahey et al. (1997) evaluated glucosinolate content of broccoli sprouts and found that they contain nearly 20- to 50-fold higher glucosinolates than tissue from mature plants. Differences in the amount of glucosinolates were also observed among accessions within each crucifer. For example, in 65 accessions of broccoli, glucoraphanin was the major glucosinolate and there was more than

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27-fold difference between the highest concentration in 'Brigadier' and the lowest concentration in 'Ev6-1.' Similar differences were also observed within other crucifers (Carlson et al. 1981; Goodrich et al. 1989). The bulk of the differences in the aliphatic glucosinolates is genetically regulated (Magrath and Mithen 1993; Magrath et al. 1994; Giamoustaris and Mithen 1996). Nonfunctional alleles on several loci are involved in this partitioning (Mithen et al. 1995; Giomoustaris and Mithen 1996). Differences in the indoles, in contrast to aliphatic glucosinolates, have been attributed to environmental factors. Growing season, soil sulfur, nitrogen fertility, plant spacing, and water stress have all been shown to increase indole glucosinolate levels in the edible portion of crucifers (Rosa et al. 1997). Glucosinolate levels are also affected by methods of food preparation. Goodrich et al. (1989) and Rosa and Heaney (1993) reported 40 to 80 percent leaching of glucosinolates from cabbage leaves, brussels sprouts, and broccoli heads into the cooking water. Leaching of glucosinolates was significantly lower for brussels sprouts than broccoli or cabbage, possibly due to the compactness of the sprout. Steam cooking also caused 40 percent loss of glucosinolates in broccoli and 20 percent loss in brussels sprouts (Goodrich et al. 1989). In contrast, microwave cooking had no significant effect on glucosinolate levels in broccoli and kale (M. M. Kushad, unpublished data). Crucifer vegetables are also rich in total antioxidants, with kale rated as the second highest among 22 vegetables tested (Cao et al. 1996). Brussels sprouts and broccoli were also ranked high in their antioxidant capacity containing significant amounts of vitamins C and E, and ~ carotene (Cao et al. 1996). Evaluation of (X- and ~-, (X-, and y-tocopherols, and vitamin C in broccoli, brussels sprouts, cabbage, cauliflower, and kale, that were also tested for glucosinolates, showed significant variations between and within these crucifers (Kurilich et al. 1999; Kushad et al. 1999). Kale had the highest amount of these vitamins, followed by broccoli, brussels sprouts, cabbage, and cauliflower. Vitamin C is the most abundant vitamin in all five crucifers tested (Kurilich et al. 1999). Analysis indicated that 79 percent of ~-carotene, 82 percent of (Xtocopherol, and 55 percent of vitamin C variability in broccoli were associated with genetic factors (Kurilich et al. 1999). Storage and processing also affect vitamins E and C, tocopherols, and carotenoids. Significant loss of vitamin C (up to 90 percent) occur in cold stored, frozen, blanched, and vacuum packaged compared to fresh broccoli, but only a slight decrease in ~-carotene (Kidmorse and Hansen 1999; Hussein et al. 2000). However, there are no published data on the effect of processing on tocopherols. Based on data from other tissue,

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tocopherols appear to be sensitive to high temperature, while cold temperature preserves tocopherols (Rudramma 2000; Sharma et al. 2000). Other antioxidants in crucifers include flavonoids. Hertog et al. (1992) evaluated methanol-extracted flavonoids from 28 vegetables and found that quercetin levels, in the edible part of most vegetables, were below 10 mg/kg, except in kale (110 mg/kg), broccoli (30 mg/kg), and onion (486 mg/kg). They also detected a smaller amount of quercetin in turnip 7.3 mg/ kg. In a similar study, Miean and Mohamed (2001) examined the flavonoid content of 62 vegetables and found that broccoli, cauliflower, cabbage, chinese cabbage, and a locally grown crucifer vegetable kailan (Brassica alboglabra) contained between 148 and 219 mg/kg flavonoids. Broccoli contained myricetin, quercetin, and luteolin; cauliflower contained myricetin and quercetin; kailan contained quercetin and apigenin; while cabbage contained only myricetin. Kale, broccoli, and turnip contained 211,72, and 48 mg/kg kaempferol, respectively (Hertog et al. 1992). Kaempferol had also been detected in cabbage leaves (Nielson et al. 1993), but Miean and Mohamed (2001) did not detect kaempferol in any of the tested crucifer vegetables. Human absorption and excretion offlavonoids from broccoli consumption was examined by Nielsen et al. (1997). Subjects fed a diet supplemented with broccoli for seven days had a small amount of kaempferol and no quercetin in their urine, which suggest that flavonoids are either poorly absorbed by the body and/or that they are absorbed and rapidly metabolized. Crucifers also contain significant amounts of dietary fiber. Dietary fiber content of cauliflower was estimated to be about 5 percent of the total fresh weight or about 50 percent of the total dry weight, consisting of about 40 percent nonstarch polysaccharides (Fermenia et al. 1999). Cellulose and lignin concentrations in brussels sprouts were estimated to be 36 percent and 14.5 percent, while in cauliflower they were estimated to be about 16 percent and 13 percent of the total dry matter, respectively (Rahn et al. 1999). Crucifers are capable of accumulating substantial amounts of selenium when grown on high-selenium soil. Banuelos and Meek (1989) reported that broccoli grown on selenium enriched soil accumulated sevenfold more selenium than cabbage, swiss chard, and collards. Broccoli plants grown outdoors on a sphagnum, peatmoss, and vermiculite medium and fertilized with sodium-selenate and selenite accumulated 278 mg/g dry weight selenium, in the edible florets, compared to the nonfertilized control, which accumulated only 0.13 mg/g dry weight (Finley et al. 2001). Broccoli sprouts grown hydroponically on high selenium also accumulated about 28 mg/kg dry weight selenium (Finley 1998). Cab-

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bage sprouts and fully developed heads also accumulated selenium and the accumulation was higher in the sprouts than in the mature heads (Bibak et al. 1999). In broccoli, selenium is stored as selenocysteine (Cai et al. 1995). Crucifers are also an excellent source of folate. Brussels sprouts and broccoli were ranked among the highest vegetable sources for folate, contributing about 110 to 135 and 70 to 90 ~g/100g, respectively (Scott et al. 2000; Konings et al. 2001). 2. Health Benefits. Most consumers in North America and Western Europe associate cruciferous vegetable consumption, especially broccoli, with health. Epidemiological data show that a diet rich in crucifers can reduce the risk from several types of cancers and that the risk can be significantly reduced by an intake of as little as 10 g per day (Graham 1983; Wattenberg 1993; Kohlmeier and Su 1997). The overwhelming evidence concerning the anticarcinogenic effect of phytochemicals in crucifers were from in vivo studies using animal models. In order to establish the relationship between whole vegetables and cancer prevention, Farnham et al. (2000) examined the diversity of induction of the Phase II detoxification enzyme quinone reductase, in murine hepatoma (hepa lclc7) cells, by 71 inbred and 5 hybrid lines of broccoli. They found that the rate of induction of quinone reductase in hepa lclc7 by the broccoli inbred lines ranged from 0 to 15,000 units and that the rate of induction was highly correlated (average r = 0.85, P = 0.0001) to the concentration of glucoraphanin in each broccoli inbred. These results suggest that there are significant differences in the health benefits among crucifers, which is important not only from a health point of view, but also as a marketing tool to promote a certain cultivar. In a recent study, Murray et al. (2001) reported that consumption of broccoli and brussels sprouts enhanced the breakdown of the suspected carcinogens heterocyclic aromatic amines in the human body. Heterocyclic aromatic amines are produced during grilling of meats. Broccoli sprouts have also been shown to increase Phase II enzymes and have afforded protection from chemically induced cancer in rodents (Fahey et al. 1997). Similarly, selenium-enriched broccoli has also been found to reduce colon cancer and mammary tumors in animal models (Finley et al. 2000, 2001). Despite evidence from these studies and several others not discussed in this article, that there may be an inverse association between cruciferous vegetable consumption and the risk of cancer, especially for colon, stomach, and rectal cancers, other studies have found no correlation and/or negative correlations. Verhoeven et al. (1996) conducted an

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exhaustive review of the epidemiological studies on crucifers and cancer risk and concluded that there was no statistically significant positive or negative trend. They found that out of the prospective cohort studies (based on answering sets of questions related to dietary habits), five reported protective effect (two lung, one colon, one stomach, and one all), one showed no benefit (prostate), and one showed an increased risk (prostate) from the consumption of one or more crucifers per day. In the same survey, case-controlled studies (studies based on feeding a crucifer diet to a set of selected human subjects) showed that of the 74 studies examined, 46 (62%) reported lower cancer risk to be associated with the consumption of at least one type of crucifer. The lower risk was most consistent for lung, colon, stomach, and rectal cancers, and less consistent for prostate and ovary cancers. However, based on one of the largest and most detailed reviews of diet and cancer, the World Cancer Research Fund concluded that a diet rich in crucifers is likely to protect humans against colon, rectum, and thyroid cancers, and when consumed with vegetables rich in other phytochemicals, can protect against cancers in other organs (World Cancer Research Fund 1997). B. Alliums

Alliums consists of nearly 500 species, but only a few have been domesticated for human consumption including onion, garlic, chives, leeks, and rakkyo. Alliums were probably first domesticated for their additive flavor to foods and for their health benefits. Alliums have been cited in the Egyptian Codex Ebers, a 35-century old manuscript as useful for the treatment of heat disorders, tumors, worms, bites, and other ailments (Rahman 2001). In recent decades, more epidemiological studies have associated allium consumption with decreased risk of chronic diseases than all other vegetables combined. However, despite an enormous volume of literature on the health benefits of alliums (Fenwick and Hanley 1985b), demand for these vegetables has remained relatively stable, especially among U.S. consumers. A noticeable increase, however, has occurred in the use of various allium preparations as dietary supplements. The following two sections will examine the phytochemical content and health benefits of raw and/or lightly processed alliums. 1. Phytochemical Content. The chemistry of alliums is complex and some of their components most likely have evolved for self-protection against attacking pests (Amagase et al. 2001). Alliums are rich in a wide variety of thiosulfides, which have been linked to reducing various chronic diseases. They are also rich in the flavonols quercetin and

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kaempferol, the cysteine-containing tripeptide glutathione, and selenium when grown on selenium-rich soil. Similar to glucosinolates in crucifers, the types and amounts of thiosulfides in alliums vary significantly. Typically, they contain 1 to 5 percent nonprotein sulfur compounds, on a dry weight basis (Kubec et al. 2000). Six isomers of thiosulfides have been identified in these vegetables thus far, namely alliin, isoalliin, propiin, methiin, and ethiin (Whitaker 1976; Lancaster and Boland 1990; Block 1992b; Kubec et al. 2000). Kubec et al. (2000) reported significant variability in the total thiosulfide (0.02 to 1.3% fresh weight) content and in the relative proportion of these compounds between and within alliums, even when grown under identical conditions. They found that total thiosulfide contents in green onion leaves, chive, and onion bulb were 0.2, 0.72, and 1.02 g/kg fresh weight, respectively. The type of thiosulfides in these vegetables were also variable. For example onion bulbs contained 34 percent methiin, 5 percent ethiin, 6 percent propiin, 5 percent alliin, and 49 percent isoalliin (Kubec et al. 2000), while garlic cloves contained about 92 percent alliin, 8 percent methiin, and trace amounts of ethiin, propiin, and isoalliin (Kubec et al. 1999). Although methiin, ethiin, propiin, alliin, and isoaliin, have no biological activity against chronic diseases, when alliums are crushed or chewed, these compounds are converted into active thiosulfides (allyl sulfides). Using supercritical fluid extraction and liquid chromatography in conjunction with atmospheric pressure chemical ionization massspectroscopy, Calvey et al. (1998) identified 11 allyl sulfides in garlic, including allicin and its allyl l-propenyl isomers as well as a smaller quantity of ajoene, and four classes of allyl sulfides in onions including bisulfine, zwiebelanes, as the major classes and small amounts of cepaenes, and lachrymatory factors. The second most important group of phytochemicals in alliums are flavonoids. Miean and Mohamed (2001) reported that onion leaves had the highest total flavonoid content among 62 different vegetables they tested and that total flavonoid content of onion leaves and garlic were about 2.7 and 1.0 g/kg dry weight, respectively (Miean and Mohamed 2001). In onion leaves, about 55 percent of the total flavonoids is quercetin, 31 percent kaempferol, and 14 percent luteolin. In onion bulb, more than 95 percent of the flavonoids is quercetin and only a trace amount ofkaempferol (Hertog et al. 1992). Quercetin in onion appeared mainly in the free-form as the aglycone (Herrmann 1976). White onion cultivars were reported to have significantly !ess quercetin than the red ones and most of the quercetin is present in the outer sca!es (Herrmann 1976). In garlic cloves, 72 percent of the tota! flavonoids is myricetin,

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23 percent apigenin, and 5 percent quercetin (Miean and Mohamed 2001). In chive, garlic chive, and leek the predominant flavonoid is kaempferol (Bilyk and Sapers 1985). Inhibition of cancer by selenium in animal models requires between 1 to 3 ppm (EI-Bayoumy 1991). However, most food crops contain very low concentrations of selenium, less than 0.01 ppm (Morris and Levander 1970). Ip and Lisk (1994) reported that 'Valencia' topset garlic (regular garlic) cloves contained 0.06 ppm selenium, while 'Stuttgart' onion bulbs contained 0.02 ppm. Selenium is taken up into the plant through the sulfur transport channels and the synthesis of the organic forms of selenium occurs through the sulfur assimilation pathway (Fenwick and Hanley 1985a). Based on this information, Lisk and coworkers (1992; Ip and Lisk 1994) showed that garlic plants fertilized with high selenium and low sulfur fertilizer accumulated between 110 and 150 ppm dry weight selenium, while onion plants accumulated up to 28 ppm. Ip and Lisk (1994) proposed that selenium-enriched garlic and onion provide an ideal system to deliver selenium efficiently and safely into the human body for cancer prevention. Allium vegetables have also been reported to contain a-tocopherol. In garlic and Japanese bunching onion (Allium fistulosum) leaves, atocopherol content was estimated at 1.23, and 14.5 mg/l00g fresh weight, respectively (Ching and Mohamed 2001). 2. Health Benefits. The health benefits of alliums, has been the subject of more than 3000 publications worldwide and nearly 1000 were published in the last decade alone (Amagase et al. 2001). In some of these studies, allium consumption has been linked to reducing cancer and cardiovascular diseases, stimulation of the immune system, detoxification of chemical toxicants in the body, restoration of physical strength, enhancement of resistance to various stresses, and antiaging effect (Amagase et al. 2001). Several epidemiological studies have examined the health benefits of alliums. A substantially lower incidence of gastrointestinal cancer was reported in several regions in China where garlic consumption can reach as high as 20 g/day compared to regions that have lower garlic consumption (Horwitz 1981). The cancer mortality rate among adult white males, in the Vidalia-onion producing region in Georgia, USA, is about one-half the rate in the rest of the state and one-third the national average (Riggan et al. 1983). In the Iowa Women's Health Study, the relative risk of colon cancer was 32 percent lower in women that consumed garlic more than once a week compared to those who did not consume garlic (Steinmetz et al. 1994). Ali and Thomson (1995) reported that subjects

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who ate 3 g of fresh garlic daily for 16 weeks had a 21 percent reduction in blood cholesterol. However, similar to epidemiological studies with other vegetables, the association between allium consumption and chronic diseases is inconclusive. Two case-control studies have examined the association between allium consumption and risk of breast cancer. In one study (Katsouyanni et al. 1986), no significant association was found between leek and onion consumption and breast cancer. In the second study (Levi et al. 1993), a significant decrease was observed with an increase in onion consumption, which persisted even after controlling for age, total energy intake, and education, while garlic had no effect in this study. In contrast, Dorant et al. (1995) did not observe any significant effect for onion, garlic, or garlic supplement on breast cancer. Fleischauer et al. (2000) analyzed the effect of raw and cooked garlic consumption on colorectal and stomach cancers in nearly 300 studies published between 1996 and 1999. They concluded from this analysis that there appears to be a positive association between raw and cooked garlic consumption and lower risk of colorectal cancers. However, they argued that there were inherent biases in many of these studies such as small sample size, improper dose estimation, and lack of accountability for the effect of other vegetables in the diet, since garlic is rarely consumed as a single food. Despite the conflicting findings on the health benefits of alliums, which may have occurred because of differences in preparations, testing, and/or experimental protocols, Rahman (2001) concluded that historical experience suggest a positive role for these vegetables in either protecting or preventing chronic diseases. He suggested a need for longterm controlled human studies using standardized preparations and protocols in order to determine the health benefits of these vegetables.

c.

Solanaceous Vegetables

Solanaceous vegetables are a diverse group, including potato, tomato, pepper, and eggplant. With the exception of eggplant, solanaceous vegetables are relative newcomers into the human diet. Tomato, sweet, and hot pepper are native to Central America, while eggplant is native to China and Southeast Asia (Toussaint-Samat 1992). Neither potato nor tomato were popular after their introduction into the Old World until about the middle of the eighteenth and early nineteenth century (Toussaint-Samat 1992). Today, they are the most popular items on nearly every country's cuisine. In 1999, world production of tomato, potato, eggplant, and pepper (hot and sweet) were 95.1, 29.4,

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21.2, and 18.1 million tonnes, respectively (FAO, 1999). Per capita consumption of tomato (fresh and process) in the United States has increased from 15.6 kg in 1980 to about 39 kg, while per capita consumption of potato has increased from 61 kg in 1993 to 72.4 kg in 1999. 1. Phytochemical Content. There are significant differences in the phytochemical content between solanaceous vegetables, and therefore each vegetable will be examined separately. Tomato. The major phytochemicals in tomato are the carotenoids, consisting of 60 to 64 percent lycopene, 10 to 12 percent phytoene, 7 to 9 percent neurosporene, and 10 to 15 percent carotenes (Clinton 1998). Based on a fresh weight basis, tomato (on average) contains about 35 mg/kg lycopene, with yellow cultivars containing about 5 mg/kg and red ones as high as 90 mg/kg (Scott and Hart 1995; Tonucci et al. 1995; Gerster 1997; Clinton 1998). Cherry types contain higher amounts of carotenoids than standard ones (Leonardi et al. 2000). Processed tomatoes (sauce, paste, juice, and ketchup) contain 2- to 40-fold higher lycopene than fresh tomatoes (Gerster 1997; Clinton 1998). Tomato contains significant amounts of a-, ~-, y-, 8-carotene ranging in concentrations from 0.6 to 2.0 mg/kg (Tonucci et al. 1995; Abushita et al. 2000; Leonardi et al. 2000), which ranks tomato as the fourth leading-contributor of provitamin A and vitamin A in the American diet (Arab and Steck 2000). The average daily intake of lycopene in the human diet is about 25 mg/day; nearly 85 percent is obtained from fresh and processed tomato products (Gerster 1997; Rao et al. 1998). Tomato fruits are an excellent source of ascorbic acid, about 200 mg/kg and are the major source of vitamin C next to citrus. Tomato contains small but significant amounts (1-2 mg/kg) of lutein, a-, ~-, and ytocopherols, and conjugated flavonoids (Abushita et al. 1997, 2000; Leonardi et al. 2000). In a study of 20 tomato cultivars, total flavonoids content ranged from 1.3 to 22.2 mg/kg with about 98 percent present in the skin (Stewart et al. 2000). Flavonoids in fresh tomato are present only in the conjugated form as quercetin and kaempferol (Crozier et al. 1997), but processed tomato products contain significant amounts of free flavonoids (Stewart et al. 2000). Flavonoids content is affected by cultivar and culture; for example, cherry tomatoes had higher conjugated flavonoids than standard varieties and field-grown fruits had higher flavonoids than greenhouse-grown (Stewart et al. 2000). Potato. In general, potato is perceived only as a source of carbohydrates,

but it is also an excellent source of essential amino acids. Potato contains a small amount of protein (less than 6 percent), but human feeding tri-

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als suggested that potato proteins are of a very high quality, possibly because they are rich in essential amino acids, such as lysine, and other metabolites, which may enhance protein utilization (McCay et al. 1987; Okeyo and Kushad 1995; Friedman 1996). In addition to high quality proteins, potato tubers accumulate significant amounts phytochemicals including phenolics, phytoalexins, and protease inhibitors. Potato contributes a small but significant amount of phytochemicals. Several yellow, red, and purple fleshed types with high phytochemical content have recently been introduced into the market. Total phenolics in potato tubers range in concentration from 0.5 to 1.7 g/kg (Reeve et al. 1969; Thomas and Joshi 1977). AI-Saikhan et al. (1995) reported significant differences in total phenolics among cultivars, with flesh color having no significant effect on total phenolics. Nearly 50 percent of the total phenolic compounds in potato are located in the peel and adjoining tissue, but decrease toward the center of the tuber (Hasegawa et al. 1966; AI-Saikhan et al. 1995), with chlorogenic acid representing about 90 percent of the total polyphenolic content (Friedman 1997). Potato tubers contain a moderate amount of vitamin C, in the range of about 10 to 140 mg/kg, depending on the cultivar and the growing season, but it declined rapidly (30 to 50 percent) during storage and cooking (Cieslik 1994; Okeyo and Kushad 1995; Hagg et al. 1998). Other antioxidants found in potato include 0.5 to 2.8 mg/kg a-tocopherol, 0.13 to 0.6 mg/kg lutein, and 1 mg/kg ~-carotene (Ong and Tee 1992; Packer 1994; Lachman et al. 2000). Cao et al. (1996) estimated the total antioxidant capacity of potato to be in the medium range among 22 commonly consumed vegetables. Potato also contributes a small amount of selenium (0.01 mg/kg) and folate (0.35 mg/kg) to the human diet (Djujic et al. 1995; Konings et al. 2001). Animal feeding studies have shown that nearly 90 percent of the starch in raw potato, known as resistant starch, escapes digestion in the small intestine. When resistant starch enters into the colon it is degraded by fermenting bacteria yielding short-chain fatty acids, which have been linked to lower cardiovascular diseases (Scheppach et al. 2001). However, cooking and frying for human consumption, degrade the bulk of resistant starch into digestible starch that cannot be converted by bacteria into short-chain fatty acids (Gormley and Walshe 1999). Resistant starch supplements from potato have shown mixed results on colon cancer in human (Heijen et al. 1998; Jenkins et al. 1998). Sweet and Hot Peppers. The oldest documented use of pepper in Mexico dates to 7000 BeE, making it one of the oldest cultivated plants in the

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world (Szallasi and Blumberg 1999). The major phytochemicals in hot peppers are capsaicinoids. More than 20 capsaicinoids, belonging to two groups, capsaicin and dihydrocapsaicin, have been identified in pepper. Capsaicin was discovered in 1846 and its structure, as an acid amide, was elucidated by Nelson (1919). Capsaicin constitutes about 70 percent of the pungent flavor in hot pepper, while its analogue dihydrocapsaicin represents 30 percent (Suzuki and Iwai 1984). The two groups differ in the presence or absence of double bonds in the fatty acid side-chain and within each group they differ in the length and branching point in the fatty acid side-chain (Krajewska and Powers 1988). Significant variations in the profile of capsaicinoids are found between and within pepper species, ranging from about 220 ppm (3,400 Scoville Heat Units, SHU) in Capsicum annum to 20,000 ppm (320,000 SHU) in Capsicum chinense (Thomas et al. 1998). Fresh pepper fruits are an excellent source of vitamin C, carotenoids, and flavonoids. Significant differences in vitamin C were observed between cultivars, but not between species. On average, fruits contain between 1 to 2 g/kg vitamin C, which is equivalent to 200 to 300 percent of the recommended daily allowance for adult men and women (Howard et al. 2000). The level of provitamin A carotenoids (a- and ~-carotene) is cultivar specific. Some cultivars of hot pepper have as much as 12 mg/kg total carotenoids, while others are below the detectable level (Howard et al. 1994, 2000). Major flavonoids in the peppers are quercetin and luteolin. They are present in conjugated form and their content varies among cultivars ranging from not detectable to 800 mg/kg (Lee et al. 1995).

Eggplant. The major phytochemical in eggplant is nasunin or delphinidin-3-(coumaroylrutinoside)-5-glucoside. Nasunin is part of the anthocyanin purple pigment found in the peel of eggplant, purple radish, red turnip, and red cabbage (Kayamori and Igarashi 1994; Noda et al. 1998). Matsuzoe et al. (1999) examined the profile of anthocyanins in several eggplant cultivars and found that nasunin represents between 70 to 90 percent of the total anthocyanins in the peel, however, the actual concentration of nasunin has not been reported. Nasunin is an antioxidant that effectively scavenges reactive oxygen species, such as hydrogen peroxide, hydroxyl and superoxide, as well as inhibits the formation of hydroxyl radicals, probably by chelating ferrous ions in the Fenton reaction (Noda et al. 1998). Kimura et al. (1999) reported that paraquatinduced oxidative stresses in rats, such as loss of body weight and increase in lung size, were suppressed by supplementing nasunin to the paraquat diet.

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Eggplant fruits also contain several other antioxidants including the carotenoids lycopene, lutein, and a-carotene, as well as the flavonoids myricetin and kaempferol (Ben-Amos and FishIer 1998; Miean and Mohamed 2001). Total antioxidant activity of eggplant was estimated to be about 190 ~mol Trolox equivalent per 40 gram serving size, which ranks it in the middle among 22 commonly consumed vegetables (Cao et al. 1996). 2. Health Benefits. The use of solanaceous vegetables in traditional medicine is ancient. Native American tribes used hot peppers to cure cramps, diarrhoea, gastric ulcers, rheumatism, as an appetite stimulant, and even for restoration of hair growth. Spanish explorers encouraged sailors to eat potato in order to avoid scurvy (Toussaint-Samat 1992; Szallasi 1995). Tomato, despite its relation to the toxic nightshades, such as Solanum nigrum, was regarded by Spanish explorers as a medicinal plant (Toussaint-Samat 1992). The popularity of solanaceous vegetables worldwide has made it the subjects of many clinical trials.

Tomato. In a number of epidemiological studies, dietary intake of tomatoes and tomato products have been found to be linked to lower risk of a variety of cancers. Results from the Health Professional Follow-up study concluded that, among various carotenoids in vegetables, only tomato lycopene was responsible for lower prostate cancer (Giovannucci et al. 1995). A cancer risk reduction of nearly 35 percent was observed when the test subjects consumed ten or more servings of tomato products per week and the effect was much stronger for subjects with more aggressive and advanced stages of cancer. In another study, a 50 percent reduction of all types of cancer was observed in a population of elderly Americans who included tomato in their diet (Colditz et al. 1985). Giovannucci (1999) reviewed 72 epidemiological studies on the relationship between tomato consumption and cancer. Among these studies, 57 reported an inverse association between tomato intake and the risk of cancer, especially prostate, lung, and stomach cancers, and 35 of these inverse associations were found to be statistically significant. Tomato consumption has also been linked to reduction of cardiovascular disease, possibly by reducing low density lipoprotein oxidation, inhibiting low-density lipoprotein synthesis, or enhancing low density lipoprotein degradation (Arab and Steck 2000). Other potential, but unproven, health benefits from tomato consumption include, delayed progression of Parkinson's disease, inhibition of cataract, and age related macular degeneration (Gerster 1997; Pollack et al. 1999; Agarwal

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and Rao 2000). The Society for Experimental Biology and Medicine (1998) conducted a multidisciplinary symposium on the role oflycopene and tomato products in disease prevention. The overall take-home message from this symposium was that tomato and tomato-based products promote health.

Potato. Few studies have been conducted on the health benefits of potato. In a hospital-based study, Hirose et al. (1995) listed potato as one of the vegetables that was correlated with a decreased trend in breast cancer among women. Potato carboxypepetidase inhibitors and proteinaceous protease inhibitors have been shown to serve as cancerchemopreventive agents by suppressing radiation and chemically induced malignant transformation in vitro and have strong anticarcinogenic activity in vivo (Blanco-Aparicio et al. 1998). Peppers. The health benefits of pepper are attributed primarily to capsaicin. Capsaicin is an ingredient in several commercial medicinal formulation for the treatment of muscle pains, toothaches, burning-mouth syndrome, gastric ulceration, painful diabetic neuropathy, postmastectomy pain syndrome, and osteo- and rheumatoid-arthritis. It is also prescribed for bladder hypersensitivity, vasomotor rhinitis, and hyperreflexia of spinal origin (Szallasi and Blumberg 1999). Eggplant. There are two studies on the health benefits of eggplant; one study showed that eggplant extract inhibited human fibrosarcoma HT180 cell invasiveness (Nagase et al. 1998); while the other study showed a significant decrease in blood levels of low-density lipoproteins and total cholesterol in human volunteers who were fed eggplant powder (Guimaraes et al. 2000). D. Other Vegetables

In addition to the three vegetable groups listed, others have also been reported to contain phytochemicals that may offer protection to humans against chronic diseases. With the exception of glucosinolates and thiosulfides, which are unique to the crucifers and alliums, the phytochemicals content of a number of other vegetables consists primarily of polyphenolics, vitamin C, fiber, selenium, and folate. The main difference is that each vegetable group contains a unique combination and amount of these phytochemicals, which distinguishes them from other groups and vegetables within their own group. For example, the Apiaceae is rich in flavonoids, carotenoids, vitamin C, and vitamin E. Celery and parsley for example are among the best vegetable sources for the flavonoid apigenin and vitamin E (Nielsen et al. 1999) and carrots have

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a unique combination of three flavonoids kaempferol, quercetin, and luteolin (DuPont et al. 2000; Ching and Mohamed 2001; Miean and Mohamed, 2001). The Asteraceae is rich in conjugated quercetin, flavonoids, and tocopherols. The Cucurbitaceae family is rich in vitamin C, carotenoids, and tocopherols. The Chenopodiaceae is an excellent source of folate (Scott et al. 2000; Konings et al. 2001). IV. CONCLUSIONS AND FUTURE RESEARCH NEEDS

Since the beginning of the twentieth century, contemporary health experts have been using scientific tools to document the relationship between human diet and health. Based on evidence gathered from these studies, there appears to be a general trend that suggests that phytochemicals in vegetables can protect the human body from several types of chronic diseases. However, despite the large number of studies that have proven the health benefits of a specific phytochemical, the overwhelming epidemiological evidence seems to suggest that the benefit cannot be attributed to one component or even to one vegetable, but rather, due to a synergism between phytochemicals. Because each vegetable group contains a unique combination ofphytochemicals that distinguish them from others, the U.S. Food and Drug Administration and Department of Agriculture have initiated the "five-vegetables-a-day program" in order to ensure that an individual's diet includes a combination of phytochemicals. The availability of a large selection of vegetables year-round have enabled consumers to include a variety of health promoting phytochemicals in their diet. However, consumers are not sufficiently informed about which combination of vegetables and/or phytochemicals to eat. Therefore, research on the health benefits of vegetables, from a horticultural perspective, needs to focus on two key areas in the near future. One area is to identify the genetic mechanisms that regulate the synthesis of key phytochemicals, such as the flavonoids, glucosinolates, and thiosulfides. The objective is to develop vegetable cultivars rich in a variety of phytochemicals in order to ensure that a mixture enters into the human diet. However, researchers should also be aware that by increasing certain phytochemicals in any vegetable, they may be compromising palatability, quality, or other nutritional components. For example, researchers who are attempting to increase glucosinolates in crucifers or thiosulfides in alliums need to be aware that some of these compounds have off-flavor characteristics. Another concern is related to the potential change in the balance of these compounds. For example, selenium and sulfur use the same transport channels into the plant and

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the same pathway to synthesize their organic forms. It is possible that by increasing selenium in the plant, sulfur uptake will be reduced, which may limit thiosulfide and glucosinolate synthesis. Another risk that needs to be considered when manipulating the components of foods is related to the potential harmful effects from higher levels of phytochemicals. For example, carotenoids are generally regarded as nontoxic, yet intervention trials with high ~-carotene supplement in smokers have been associated with increased lung cancer and heart disease. The second key area for future research is related to identifying optimum conditions for maintaining these compounds after harvest and processing. Studies have shown that the bioavailability of some of the phytochemicals increase dramatically after storage and processing, while others degrade rapidly (Harvard et al. 1999). For example, in spinach, folate content drops by nearly 50 percent after five days of storage at ambient temperature, however during cold storage nearly 90 percent was maintained (Scott et al. 2000). In contrast, cold temperature destroys vitamin C. Processed vegetables may have different effects from fresh vegetables, due to the increased or decreased availability of phytochemicals. For example, lycopene concentration in tomato paste is 30fold higher than in fresh tomato (Gerster 1997). Because of the potential health benefits and risks associated with enhancement of the phytochemicals content of vegetables, there is an obvious need to link horticultural research to the epidemiology of the modified products. LITERATURE CITED Abdel-Salam, O. M., ]. Szolcsanyi, and G. Mozsik. 1997. Capsaicin and the stomach. A review of experimental and clinical data.] Physiol Paris 91:151-171. Abushita, A. A., K A. Hebshi, H. G. Daood, and P. A. Biacs. 1997. Determination of antioxidant vitamins in tomato. Food Chern. 60:207-212. Abushita, A. A., H. G. Daood, and P. A. Biacs. 2000. Change in carotenoids and antioxidant vitamins in tomato as a function of varietal and technological factors. ]. Agr. Food Chern. 48:2075-2081. Agarwal, S., and A. V. Rao. 2000. Carotenoids and chronic diseases. Drug metab. Drug Int. 17:189-210. Ali, M., and M. Thomson. 1995. Consumption of a garlic clove a day could be beneficial in preventing thrombosis. Prostaglandins Leukot. Essen. Fatty acids 53:211-212. AI-Saikhan, M. S., L. R. Howard, and]. C. Miller. 1995. Antioxidant activity and total phenolics in different genotypes of potato (Solanum tuberosum, L.).]. Food Sci. 60:341-344. Amagase, K, K M. Schaffer, and]. A. Milner. 1996. Dietary component modify the ability of garlic to suppress 7,12-dimethylbenz[aJanthracene-induced mammary DNA adducts. J. Nutr. 126:817-824. Amagase, H., B. L. Petesch, H. Matsuura, S. Kasuga, and Y. Itakura. 2001. Intake of garlic and its bioactive components. ]. Nutr. 131:955S-962S.

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4

Detection and Elimination of Viruses and Phytoplasmas from Pome and Stone Fruit Trees Margit Laimer*

Plant Biotechnology Unit Institute of Applied Microbiology University of Agricultural Sciences Nussdorfer Lande 11, 1190 Vienna, Austria

1. INTRODUCTION II. PATHOGENS A. Filamentous Fruit Tree Viruses 1. Apple Chlorotic Leaf Spot Virus 2. Apple Stem Grooving Virus 3. Apple Stem Pitting Virus 4. Plum Pox Virus B. Isometric or Bacilliform Fruit Tree Viruses 1. Prunus Necrotic Ringspot Virus 2. Apple Mosaic Virus 3. Prune Dwarf Virus C. Nematode-transmitted Fruit Tree Viruses 1. Strawberry Latent Ringspot Virus 2. Raspberry Ringspot Virus 3. Tomato Ringspot Virus 4. Arabis Mosaic Virus

*Dedicated to Philipp Boxus, who apart from being an admirable scientist was an extraordinary person. This work has been supported by the EU-Project FAIR5 CT 3889 "Health certification of rosaceous species based on disease indexing of in vitro plants: Validation of diagnostics and diagnostic strategies." I thank our partners in the FAIR 3889 project Assunta Bertaccini, David Davies, Jean Kummert, Willi Jelkmann, and Thierry Candresse, and my coworkers in the Plant Biotechnology Unit of the lAM Wolfgang Arthofer, Veronika Hanzer, Sabine Strommer, Duarte Mendon~a, and Maria Heinrich.

Horticultural Reviews, Volume 28, Edited by Jules Janick ISBN 0-471-21542-2 © 2003 John Wiley & Sons, Inc. 187

188

III.

IV.

V. VI. VII.

M. LAIMER D. Phytoplasmas of Fruit Trees 1. Apple Proliferation 2. European Stone Fruit Yellows 3. Pear Decline PATHOGEN DETECTION A. Viruses 1. Indexing 2. Serology 3. Immuno-Tissue Printing (ITP) 4. Polymerase Chain Reaction (PCR) B. Phytoplasmas 1. Nested PCR and RFLP for the Detection of Phytoplasmas 2. IC-PCR for the Detection of Apple Proliferation Phytoplasma ELIMINATION OF VIRUSES A. Long-Term Treatments at Elevated Temperatures B. Meristem Culture C. Micrografting D. Use of Viricides E. In Vitro Thermotherapy F. Combinations of In Vitro Thermotherapy and Meristem Culture 1. Vienna Collection 2. Propagation of Malus Cultivars and Rootstocks In Vitro 3. Propagation of Prunus Cultivars and Rootstocks In Vitro G. Virus Distribution Throughout In Vitro Host Plants H. Thermotherapy Conditions I. Elimination Success ELIMINATION OF PHYTOPLASMAS INDEXING, MASS PROPAGATION, AND GERMPLASM CONSERVATION CONCLUSIONS LITERATURE CITED

ABBREVIATIONS ACLSV AP APHIS ApMV ArMV ASGV ASPV BA CP DAPI DAS-ELISA ESFY

Apple chlorotic leafspot virus Apple proliferation Animal and Plant Health Inspection Service Apple mosaic virus Arabis mosaic virus Apple stem grooving virus Apple stem pitting virus 6-benzylaminopurine Coat protein 4,6-diamidino-2-phenylindole Double antibody sandwich-enzyme linked immunosorbent assay European stone fruit yellows

4. DETECTION AND ELIMINATION OF VIRUSES AND PHYTOPLASMAS

ELISA GFLV IC-PCR ITP

IBA

kDa

LIV

Mab

2iP nt

NASBA PBS PCR PD PDV PNRSV PPV RFLP RpRSV RT-PCR SLRSV ssRNA TA TBRV ToRSV VC I.

189

Enzyme linked immunosorbent assay Grapevine fanleaf virus Immuno capture-polymerase chain reaction Immuno-tissue printing Indolebutyric acid Kilodalton Longevity of the infectivity of the sap in vitro Monoclonal antibody N 6 -(2-isopentyl)adenine Nucleotide Nucleic acid sequence-based amplification Phosphate buffered saline Polymerase chain reaction Pear decline Prune dwarf virus Prunus necrotic ringspot virus Plum pox virus Restriction fragment length polymorphism Raspberry ringspot virus Reverse transcription-polymerase chain reaction Strawberry latent ringspot virus Single strand RNA Temperature adaptation Tomato black ring virus Tomato ringspot virus Vienna Collection

INTRODUCTION

The great development of the fruit industry in the second half of the twentieth century relied on intensive breeding programs (Janick and Moore 1996) and the development of nursery activities for rapid and inexpensive production of plants to establish new orchards. The release of fruit plant cultivars to fruit plant growers takes several years. Diseases of unknown etiology must be checked by indexing methods currently in use. In the certification of fruit plants, there is an urgent need for the development of rapid, reliable, sensitive, and user-friendly methods for detection and identification of harmful organisms, and for an increased supply of healthy planting material. Worldwide sanitation programs include virus elimination treatments of elite plants in vivo (Waterworth 1993; Koizumi 1998; Mink et al. 1998), but in vitro methods offer valuable improvements, as will be discussed.

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Accurate global figures for crop losses due to viruses are not available, but it is generally accepted that losses due to viruses are second only to fungi (Matthews 1991). Virus symptoms vary according to their occurrence on the leaves (chlorotic alterations, deformations, enations, necrosis), fruits (alteration of shape, color, size, chemical composition), and wood (pits, different diameter between scion and rootstock, graft union necrosis). An excellent overview concerning the presence of these pathogens, in the Mediterranean Region, is presented by Di Terlizzi et al. (1998). In perennial crops, as fruit trees, lethal or crippling diseases can have serious consequences because of the time and land invested in such crops. In vegetatively reproduced plants, latent virus infections may be very widespread, reducing performance every year by a small amount. The extent to which yield is reduced will depend on many factors including cultivar of the host plant and strain of the virus present, the incidence and activity of any vectors, the time at which infection occurs, the nutritional state of the crop, the weather and the presence of other pathogens, and parasites. Viruses are known to be responsible for decreased rates of bud-take in nurseries (Rebandel et al. 1979; Zawadzka 1980).

In temperate fruit trees, pathogenic plant viruses cause considerable economic losses and are of major concern to worldwide phytosanitary agencies as no effective cure exists for already infected plants in the field. Detection and elimination procedures need to be improved to provide virus-free planting material to growers. In the case of pome fruits this will guarantee an improved health status of the orchards over a considerable period of time. It is necessary, however, to differentiate viruses according to their mode of transmission. In the case of temperate fruit trees, with the exception of plum pox virus, filamentous viruses have no known vectors and are only transmitted by vegetative propagation or by grafting of the plant material. Therefore plants, that are free of filamentous viruses, as established by the use of accurate, sensitive detection methods, remain healthy for several years. Plants infected with nematodetransmitted viruses (nepoviruses), with viruses transmitted by pollen (ilarviruses) or even by aphids (potyviruses) need continuous control after sanitation (epidemiological survey, eradication and/or control of the vectors). The same holds true for diseases caused by phytoplasmas transmitted by leafhoppers and other vectors. Information about some pathogenic agents considered by current phytosanitary regulations will be presented. Submitting pome and stone fruit tissue cultures to a combination of in vitro thermotherapy in combination with subsequent meristem prepa-

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ration allows the recovery of a higher number of healthy plants than by applying one method alone. This is particularly true in cases of single infections with certain viruses, where a high rate of success can be achieved (Knapp et al. 1995a; Gella and Errea 1998). However, the success rate decreases when cultures contain mixed infections or some viruses difficult to eliminate like the apple stem grooving virus (ASGV). By using the tissue printing method (Knapp et al. 1995b, 1995c) we were able to demonstrate that ASGV is present at a very low level, and can be found throughout the plant. The fact that even the youngest leaves and leaf primordia are infected stresses the importance of a precise meristem preparation (Knapp et al. 1997). The combination of disease elimination and disease indexing of in vitro plants, using reliable laboratory diagnostics, considerably reduces the efforts and contributes to savings of time, money and labor (da Camara Machado et al. 1998; Laimer da Camara Machado et al. 1999). Results described in the present document demonstrate the advantages of the use of in vitro thermotherapy combined with meristem preparation and PCR-based detection methods in sanitation programs. In this chapter, essential data on viruses and phytoplasmas are presented, detection methods are compared, different approaches for elimination and detection are evaluated, and 15 years experience of the Plant Biotechnology Unit of the Institute of Applied Microbiology (lAM) in the field of fruit tree pathogen elimination by tissue culture techniques are summarized.

II. PATHOGENS Rapid progress in plant pathology has resulted in the characterization of the causal agents of a number of viruses and phytoplasmas that affect fruit trees and their production (Nemeth 1986; Fridlund 1989; Desvignes 1999). This has led to the discovery that different diseases in different host plants are caused by the same viral pathogens, which requires extraordinary experience in identification (Di Terlizzi 1998a,b) and the development of more reliable diagnostic tools (Boscia and Myrta 1998; Pallas et al. 1998; www.boku.ac.at/iam/pbiotech/phytopath). When dealing with different pathogens in elimination programs, it is helpful to gather the most recent available information about their characteristics in order to specifically adapt the treatments. To present a few cases of major concern to European Phytosanitary Agencies, the following classification of morphological characteristics of the pathogens was chosen.

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A. Filamentous Fruit Tree Viruses

Many of the viruses affecting pome and stone fruit species have filamentous particles. These include apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV) and plum pox virus (PPV). 1. Apple Chlorotic Leaf Spot Virus. Apple chlorotic leaf spot virus

(ACLSV) has been distributed worldwide, but the mode of natural spread is unknown. In commercially grown apples it causes mainly latent infections, but is also known to cause the line pattern disease in Malus platycarpa, ring pattern mosaic of pear, dark green mottle of peach, false plum pox on apricot, cherry, and plum fruits (Lister 1970a; Desvignes and Boye 1988; Mink 1989; www.boku.ac.at/iam/pbiotech/phytopath/ v_aclsv.html). ACLSV, the type species of the genus Trichovirus (Martelli et al. 1994), has elongated, flexuous particles of 720 x 12 nm, encapsidating singlestranded, positive sense genomic RNA (Lister and Bar-Joseph 1981). The particles contain a single coat protein species with a molecular weight of about 22 kDa (Yoshikawa and Takahashi 1988). Nucleotide sequence and genomic organization of ACLSV is known (German et al. 1990; Sato et al. 1993). ACLSV was found to be unevenly distributed in affected trees (Gilmer et al. 1971, Fridlund 1973). Investigations on apple budwood and shoots in vitro showed that ACLSV was found to be unevenly distributed (Fridlund 1983; Knapp et al. 1995b). ACLSV in vitro shows little susceptibility toward temperatures of 55° to 60°C, but of all the viruses affecting Malus species, ACLSV can be relatively easily eliminated by short periods of heat treatment followed by shoot tip grafting (Campbell and Best 1964; Welsh and Nyland 1965; Campbell 1968; Cropley 1968a; Knapp et al. 1995b) and by apical meristem cultivation (Hansen and Lane 1985). 2. Apple Stem Grooving Virus. Apple stem grooving virus (ASGV), the type species of the genus Capillovirus (Bar-Joseph and Martelli, 1991), occurs worldwide latently in many commercial apple cultivars, but its natural mode of spread is unknown (Lister 1970b). 'Virginia Crab' was the first woody indicator available showing distinct symptoms including long stem grooves, graft union breakages, and shoot tip distortion (de Sequeira and Posnette 1969; Mink et al. 1971). Howell et al. (1996) selected three new and improved indicators, for example Malus micromalus, for the detection of ASGV being used at several virus certification programs.

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Cambial activity within the grooves apparently stops, and the furrows are filled mostly with parenchyma cells instead of normal elements of bark and wood (Plese et al. 1975). In large-scale, routine detection by enzyme linked immunosorbent assay (ELISA), ASGV requires special attention in sample selection, as it occurs at very low titers. Leaf tissue provides the most unreliable samples. According to the season, bark of young branches, cambium, or buds are the sample materials of choice (Fuchs et al. 1988). ASGV has elongated, flexuous particles of 600 to 700 x 12 nm, encapsidating a single-stranded, positive sense genomic RNA. The particles contain a single coat protein species with a molecular weight of about 27 kDa (Yoshikawa and Takahashi 1988). The entire genome has been sequenced (Yoshikawa et al. 1992) and consists of 6496 nt. In plant extracts the thermal inactivation occurs at 60° to 63°C and longevity of the infectivity of the sap in vitro (LIV) is 2 days. ASGV is one of the most recalcitrant viruses to be eliminated by any procedure, even when heat therapy and meristem dissection are applied (Campbell 1962; Campbell and Best 1964; Welsh and Nyland 1965; Cropley 1968a; Knapp et al. 1995a). 3. Apple Stem Pitting Virus. Apple stem pitting virus (ASPV), member of the genus Foveavirus (Martelli and Jelkmann 1998), is a quite stable virus and unevenly distributed in infected trees. Infection is usually latent in many cultivars and goes unnoticed, despite its high frequency. ASPV causes significant, though insidious losses in yield quality and quantity, often in complex with other latent viruses such as ACLSV and ASGV. Natural spread of ASPV seems to be through root grafts (Mink et al. 1971; Yanase et al. 1975; Yanase 1983; Nemeth 1986; Koganezawa and Yanase 1990; www.boku.ac.at/iam/pbiotech/phytopath/v_aspv .html). ASPV has filamentous particles of 800 x 12 to 15 nm, encapsidating a single-stranded, positive sense genomic RNA (Koganezawa and Yanase 1990). The particles contain a single coat protein species with a molecular weight of about 48 kDa. The nucleotide sequence of several strains of ASPV has been published (Jelkmann 1994; Yoshikawa et al. 2000; Komorowska and Malinowski 2001). 4. Plum Pox Virus. Plum pox virus (PPV) is a member of the genus

Potyvirus (Mayo and Martelli, 1993) and is the causal agent of the "Sharka" disease, which spreads rapidly in stone fruit orchards by aphid vectors. The disease results in heavy economic losses through a decrease in fruit yield and quality and ultimately in death of the infected trees

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(Kegler et al. 1964; Trifonov 1974; Minoiu 1978). PPV was classified by United States and European Union plant quarantine agencies as the most important pathogen in apricots, plums, and peaches (CABIIEPPO 1992) and is the only plant pathogen for which an Animal and Plant Health Inspection Service (APHIS) plan exists (Scorza 1991; www.boku .ac.at/iam/pbiotech/phytopath/v_ppv.html). PPV has filamentous particles of 764 x 20 nm (Kegler and Schade 1971), encapsidating a single-stranded, positive sense genomic RNA of 9741 nt and is present in serologically distinct strains (Kerlan and Dunez 1979; Quiot et al. 1995). The particles contain a single coat protein species with a molecular weight of about 36 to 37 kDa. The nucleotide sequences of four strains M, D, SC, and EI Amar have been published (Lain et al. 1989; Maiss et al. 1989,1995; Saenz et al. 2000; Teycheney et al. 1989). The 3' located CP region is highly conserved among the different strains, while the 3'NTR is strongly heterogeneous and therefore well suited for the distinction of the different strains (Wetzel et al. 1991). However, the NIb region can also be used for this purpose (Hammond et al. 1997). PPV is well known for its uneven distribution within woody host plants. Healthy branches can be found on otherwise heavily infected trees; in some cases healthy and infected buds even occur on the same branch (Trifonov 1969; Marenaud 1976; Morvan and Castelain 1976; Marenaud and Massonie 1977; Adams 1978; Knapp et al. 1996a). Casper and Meyer (1981) reported that even within the same leaf there may be infected and virus-free zones. Herbaceous hosts react very selectively according to PPV strains and isolates. Thermal inactivation occurs at 52° to 58°C, however LIV is three to four days. Heat treatment without shoot tip preparation is insufficient for PPV elimination (Knapp et al. 1996b, 1997) whereas the combination of heat treatment followed by meristem or shoot tip preparation or micrografting is highly successful (Mosella-Chancel et al. 1980; Knapp et al. 1997, 1998; da Camara Machado et al. 1998). Traditional breeding to find or to introduce resistance to PPV started in mid-twentieth century at Cacak (former Yugoslavia). At the same time, surveys of cultivars resistant or at least less affected by PPV were undertaken, but little hope exists to find a satisfactory solution by conventional plant breeding (Hartmann 1988; Cociu et al. 1997). If one considers the severity of the disease, the difficulty of controlling its spread, and the lack of resistant cultivars, the necessity of resistant cultivars is evident and a straightforward breeding strategy is required. Biotechnology offers completely new approaches through the isolation and transfer of "resistance" genes. Efforts to breed for resistance

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against this pathogen have been underway for several years. This involves sequences of the PPV CP in apricot and plum (Laimer da Camara Machado et al. 1992; Scorza et al. 1994; da Camara Machado and Laimer da Camara Machado 1995; da Camara Machado et al. 1995; Ravelonandro et al. 1997) and other regions of the viral genome for pathogen-mediated protection approach. B. Isometric or Bacilliform Fruit Tree Viruses

Prunus necrotic ringspot virus (PNRSV) and apple mosaic virus (ApMV) belong to subgroup III and prune dwarf virus (PDV) to subgroup IV of Ilarviruses (Hamilton 1991). 1. Prunus Necrotic Ringspot Virus. Many strains ofPNRSV exist and it is frequently present in mixed infections, for example with PDV, showing a synergy of symptoms (Fridlund 1965; Cropley 1968c; Fulton 1970a; Myrta et al. 2001) www.boku.ac.at/iam/pbiotech/phytopath/v_pnr .html. Mink et al. (1982) isolated three serotypes, CH-3, CH-9 and CH30 from cherries, which can be partly detected by Fultons polyclonal antiserum NRSV-G, or by Mab against PNRSV and ApMV (Halk et al. 1982), or by c-DNA probes to RNA3 (Crosslin et al. 1992). PNRSV consists of isometric particles of 23 nm diameter and larger bacilliform particles. PNRSV is rather unstable in plant juice with a thermal inactivation point between 55° to 62°C (Fulton 1981). As a multicomponent virus it consists of four positive sense ssRNA molecules. RNA3 is totally sequenced and the subgenomic RNA4 contains the cpgene (Hammond and Crosslin 1995). The virus invades all tissues of in vitro grown plantlets of Prunus domestica: epidermis, cortical parenchyma, and vascular tissue. Concentration of the virus is irregular in vascular tissue, but a slight decrease in concentration toward the apex was observed (E. Knapp et al. unpubl.).

2. Apple Mosaic Virus. There are several strains of ApMV and very susceptible apple cultivars show clear mosaic symptoms (Kristensen and Thomsen 1963; Dhingra 1972). ApMV infects birch, rose, and stone fruits causing line pattern symptoms (Fulton 1972; Gotlieb and Berbee 1973; www.boku.ac.at/iam/pbiotech/phytopath/v_apmv.html). ApMV is rather unstable and loses infectivity in crude plant sap within a few minutes and in buffer within a few hours. Thermal inactivation occurs at 54°C. ApMV also consists of four ssRNA molecules and all four are necessary to cause infection. The cp-gene has been sequenced (Alrefai et al. 1994; Pallas et al. 1995).

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3. Prune Dwarf Virus. There are several different strains of PDV and in mixed infections with other viruses it frequently shows synergistic effects on symptomatology. The most severe symptoms occur in combinations with nepoviruses like RpRSV, ArMV, or SLRSV (Marenaud and Sechet 1971). The degree of damage caused by PDV strongly depends on the Prunus species or cultivar and the viral isolate. In nurseries, bud take can be drastically reduced as can be yield in orchards (www.boku.ac.at/iam/ pbiotech/phytopath/v_pdv.html). PDV consists of isometric particles of 19 to 20 nm diameter, and bacilliform particles up to 73 nm in length (Fulton 1970b). PDV loses its infectivity in crude plant sap within 30 min. Thermal inactivation in plant sap occurs between 45° to 54°C (Waterworth and Fulton 1964). PDV consists of three positive-sense ssRNA molecules, but details about genome organization and expression are still lacking. The nucleotide sequence of RNA3 is available (Bachman et al. 1994).

c.

Nematode-Transmitted Fruit Tree Viruses

The nepoviruses arabis mosaic virus (ArMV), raspberry ringspot virus (RpRSV), strawberry latent ringspot virus (SLRSV), tomato ringspot virus (ToRSV), and tomato black ring virus (TBRV) infect strawberries and related small fruit crop species as well as many Prunus and apple species in temperate regions. Members of the genus Nepovirus have two positive-sense RNAs with capsids of a single polypeptide species forming icosahedral particles of 28 to 30 nm in diameter. Both RNAs are necessary for systemic infection (Murphy et al. 1995). Complete nucleotide sequences of RNA1 and RNA2 have been published for TBRV and ToRSV. For RpRSV and SLRSV the complete nucleotide sequence of RNA2 and for ArMV a large part of the RNA2 sequence are available (Blok et al. 1992; Kreiah et al. 1994; Loudes et al. 1995; Steinkellner et al. 1990). Virions of the genus Nepovirus are rather heat stable, their thermal inactivation point lying above 60°C. 1. Strawberry Latent Ringspot Virus. Strawberry latent ringspot virus (SLRSV) causes latent infections in peach cultivars, but infects also strawberry, raspberry, and different Prunus species. It is common in southern Europe (http://www.boku.ac.at/iam/pbiotech/phytopath/ v_slr.html). Damage is more serious, when SLRSV is present with other viruses, like the ilarviruses PNRSV or PDV. 2. Raspberry Ringspot Virus. Raspberry ringspot virus (RpRSV) has been detected on strawberry, raspberry, gooseberry, black currant, plum,

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and grape plants and is particularly involved in cherry diseases known as "Pfeffinger" in Switzerland, "Eckelrader" in Holland, and "European cherry rasp leaf" (www.boku.ac.at/iam/pbiotech/phytopath/v_rrv.htm!). 3. Tomato Ringspot Virus. In spite of its name, tomato ringspot virus (ToRSV) is known to be the causal agent of various diseases on many plant species, including apple union necrosis and decline, peach yellow bud mosaic, prunus stem pitting, and cherry leaf mottle (www.boku.ac .at/iam/pbiotech/phytopath/v_tor.htm!). 4. Arabis Mosaic Virus. Arabis mosaic virus (ArMV) is distributed on

numerous woody and herbaceous plants all over the world. Among fruit trees infections have been reported on cherry and peach (www.boku .ac.at/iam/pbiotech/phytopath/v_armv.html). D. Phytoplasmas of Fruit Trees

Phytoplasmas (first referred to as mycoplasmas or mycoplasma-like organisms) are associated with diseases of many perennial fruit crops in Europe, America, and Australia. They spread rapidly since they are transmitted by insects and by grafting. The poor taste and small size of fruits or the decline of infected trees makes these diseases an economically important threat for orchards. In Europe, currently, the most important fruit tree phytoplasmas are apple proliferation (AP), European stone fruit yellows (ESFY) and pear decline (PD), which are closely related (Jarausch et al. 1994, 2000; Lorenz et al. 1995; Lee et al. 1995; Schneider et al. 1995; Kison et al. 1997). Phytoplasmas are considered quarantine organisms of European and Mediterranean Plant Protection Organization (EPPO) (OEPP/EPPO 1986) and are included in the EPPO certification scheme for virus tested fruit trees (OEPP/EPPO 1991/1992). Phytoplasmas may be detected in fruit trees by several methods including DAPI tests, Southern blotting, grafting, ELISA (Kison et al. 1997; Desvignes 1999), and polymerase chain reaction (PCR) (Gibb et al. 1995, Lee et al. 1995, Lorenz et al. 1995, Schneider et al. 1995, Gundersen and Lee 1996, Smart et al. 1996, Kison et al. 1997). Since phytoplasmas are not completely biologically characterized, molecular tools based on restriction fragment length polymorphism (RFLP) analyses of 16S rDNA and/or the spacer region amplified by PCR and/or nested PCR are helpful in distinguishing different groups and subgroups (Lee et al. 1998; Ahrens et al. 1993; Heinrich et al. 2001). In fruit trees, it appears advisable to differentiate phytoplasmas according to host species (Seemuller et al. 1998).

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1. Apple Proliferation. AP first observed in Europe by end of the nineteenth century, was defined as a viral agent by Blattny et al. in 1963, and subsequently as a phytoplasma by Gianotti et al. (1968) and Petzold and Marwitz (1976, http://www.boku.ac.at/iam/pbiotech/phytopath/ v_ap.html). AP infects apples, where it causes typical symptoms of proliferated shoots and small fruits (Nemeth 1986) and is responsible for considerable crop losses (Gheorghiu 1976).

2. European Stone Fruit Yellows. ESFY has been proposed as the common name for phytoplasma-related diseases in European stone fruits (Kison et al. 1997). The presence of ESFY disease has been reported in France, Spain, Italy, Greece, Hungary, Romania, Switzerland, Germany, the former Yugoslavia, the UK, and Austria (Nemeth 1986; Morvan 1977; Davies and Adams 2000, Laimer da Camara Machado et al. 2001a) causing decline and death of apricot, Japanese plum, more rarely of peach (Llacer and Medina, 1988), almond, flowering cherry, and European plum (Seemiiller et al. 1998, www.boku.ac.at/iam/pbiotech/phytopath/ v_esfy.html).

3. Pear Decline. PD was described in the United States as a virus disease by Shalla et al. (1963), although symptoms were known since 1946 in pear orchards of the Pacific Coast. The disease is identical with the moria disease of pear known in Italy since 1934, in which the causal agent was believed to be a virus (Refatti et al. 1966). The phytoplasma nature of the pathogen was demonstrated by Hibino and Schneider in 1970. PD is a very serious disease. In California, between 1959 and 1962 more than 1.1 million diseased trees were recorded (Nichols et al. 1965), and in the pear growing areas of Trentino-Alto Adige (Italy) more than 50,000 trees were killed (Refatti, 1967, http://www.boku.ac.at/iam/ pbiotech/phytopath/v_pd.html). Elimination of phytoplasmas, classified in the kingdom of bacteria, has been attempted in vitro with some success by the use of antibiotics, such as tetracycline (Davies and Clark 1994) rather than by modified temperatures, which were used for in vivo treatments (Nemeth 1986). Many of the "virus diseases" easily eliminated by hot water dips or by heat treatment followed by shoot tip grafting were later discovered to be induced by phytoplasmas (W. E. Howell, pers. commun.). III. PATHOGEN DETECTION

Different methods for the detection of plant pathogens have been developed and are applied, depending on the pathogen to be detected, the time frame, equipment, and financial resources available. Internation-

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ally approved and recognized detection systems including serological and molecular laboratory assays and indicator hosts in greenhouse and field indexing are updated regularly by the ISHS International Working Group on Fruit Tree Viruses in their meetings (1992, 1998, 2001). Measures for pathogen elimination may depress the pathogen titer for a prolonged time period, which makes retesting necessary with a more sensitive, but also more expensive, detection method. The diagnostic concept should always be adapted to the situation, especially considering the ease of elimination of a particular pathogen, the complexity of the infectious status of the plants, and so on. Molecular diagnostic assays have the distinct advantage that they allow in a reliable and rapid manner an earlier selection of pathogen-free candidate plants and, thus, a reduction of effort.

A. Viruses Viruses can be detected by grafting or mechanical inoculations (field and greenhouse indexing), serology, electron mircoscopy, molecular hybridization, nucleic acid sequence-based amplification (NASBA), and PCR amplification. The ISHS Working Group on Virus Diseases of Fruit Trees has long recommended serological indexing (ELISA). Since the Bethesda, Maryland, meeting in 1997, major emphasis has been placed on the application ofPCR-based detection systems, due to their speed and increased sensitivity. In the framework of ED Project FAIR5 97/3889, improved protocols have been established for RNA preparation and for specific and broad-range detection procedures of fruit tree viruses (Laimer da Camara Machado et al. 2001a). 1. Indexing. Indexing is based on the ability of special indicator plants to develop typical disease symptoms after infection with a certain virus. The cultivar to be tested is grafted to the indicator or plant sap is transmitted mechanically. After a fixed incubation time, the presence of visible symptoms is determined. A general overview and further helpful links to approved indexing methods can be found online (www .boku.ac.at/iam/pbiotech/phytopath/det.html; wvvw.prosser.wsu.edul Faculty/howell2.htm). Even though indexing is the only technique demonstrating pathogenicity, there are also disadvantages such as the long time scale and space and trained personnel requirements.

2. Serology. The ELISA test was developed for the detection of plant viruses some decades ago by Clark and Adams (1977). Improvements include DAS-ELISA and Two-Step ELISA (Casper 1973,1983; Clark et al. 1978; Flegg and Clark 1979; Fuchs 1980; Adams et al. 1983). Serology is

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used routinely for large-scale testing of plants for the presence of many viruses (Boscia and Myrta 1998; Di Terlizzi et aL 1998a) because it is rapid, inexpensive, and convenient. In choosing the source of antisera to be used in a large-scale approach, it is advisable to compare different antisera for their affinity to the pathogen strain to be detected, to optimize different dilutions, incubation times, and particularly the tissue and season for testing. However, ELISA can only be applied for those viruses where specific antisera are available. It is further limited by the uneven patterns of distribution of certain pathogens in a tree, and by climatic influences that reduce the titer below the level of detection. 3. Immuno-Tissue- Printing (ITP). In sanitation programs after elimination treatments, some time may be required before virus replication again reaches a detectable threshold. The use of Immuno-Tissue Printing (Lin et aL 1990) allows the localization of viruses in tissues and therefore the improvement of elimination strategies in vitro. Immunotissue printing protocols for the localization of apple chlorotic leafspot virus (ACLSV), apple stem grooving virus (ASGV), and plum pox virus (PPV) in shoots of Prunus and Malus in vitro have been established for routine diagnosis in a virus elimination program (Knapp et al. 1995c). Stem discs of approximately 1 mm thickness are cut freehand from in vitro shoots using sharp razor blades. The freshly cut surface is pressed gently but firmly onto a nitrocellulose membrane. After removal of the discs, the membrane shows a detailed offprint of the structural organization of tissues. Membranes are developed by exposure to antibodies, BCIP (5-bromo4-chloro-3-indolyl-phosphate) and NBT (tetranitro blue tetrazolium chloride, Sigma Biochemicals and Reagents) solutions. Detection of viral antigen within a tissue or even within cells occurs by the precipitation of indigo-blue dye wherever antibodies are bound to viral antigen. 4. Polymerase Chain Reaction (PCR). PCR is a method for amplification of specific DNA regions, producing easily detectable amounts of DNA usually visualized by staining after agarose gel electrophoresis. In pathogen detection, PCR is 100- to 1000-fold more sensitive than ELISA (Candresse 2001). Therefore, it is specially suitable for the detection of infections in an initial stage, where the amount of pathogen in the plant is still low. While the DNA of phytoplasmas can be used directly as a PCR template, all known temperate fruit tree viruses contain RNA. For their detection, either an RNA purification or, in case of pathogens for which antisera are available, an immunocapture (IC) followed by a reverse

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transcription (RT) step is required to transcribe the viral RNA to a DNA that can be amplified by PCR (Nolasco et al. 1993; Nemchinov et al. 1995; Crossley et al. 1998; Jelkmann and Keirn-Konrad 1997). PCR for pathogen detection is carried out by using a range of general or specific primer pairs which represent short DNA sequences of the template DNA from the edges of the amplified or target region (Hadidi and Hammond 1989; Kinard et al. 1996; Spiegel et al. 1996; Marinho et al. 1998; Knapp et al. 1998; Nemchinov et al. 1998; Schwarz and Jelkmann 1998). Primers for several important fruit tree pathogens are recommended by the ISHS Working Group (2001). Some primers were also developed in the framework of FAIR 97/3889 (Table 4.1, http://www .boku.ac.at/iam/pbiotech/phytopath/det.html). Broad-range detection systems have become available allowing the simultaneous verification of presence or absence of even unrelated viruses by the use of broadrange versus specific primers (Kummert et al. 2000,2001; Candresse et al. 1998, 2000; Foissac et al. 2001; Rott and Jelkmann 2001). A comparison of print and spot-capture-PCR with heminested PCR and PCRELISA was presented by Cambra et al. (1998). B. Phytoplasmas

Phytoplasmas may be detected by orchard and nursery inspections due to typical morphological anomalies. ESFY can be reliably detected by indexing on GF 305 seedlings in the greenhouse with an incubation period of approximately four months (Desvignes 1999). In the sieve tubes of petioles, bark and roots, phytoplasmas can be detected by DAPI staining. PCR detection of phytoplasmas relies on the quality of the DNA template for amplification. Therefore, different protocols for DNA preparation are recommended. Plate Capture PCR or IC-PCR avoids an initial preparation step by use of antisera. 1. Nested peR and RFLP for the Detection ofPhytoplasmas. PCR detection of phytoplasmas is in many cases the only sensitive and rapid method currently available (Lee et al. 1995; Lorenz et al. 1995; Carraro et al. 1998; Jarausch et al. 1998; Veronesi et al. 2000; Laimer da Camara Machado et al. 2001b). However it requires DNA purification (Table 4.2) to allow subsequent amplification. Several general and specific primers, located in the 16S rDNA, intergenic spacer (IS), and 23S rDNA regions of phytoplasmas, have been described in the literature (Heinrich et al. 2001). A 1784 bp region corresponding to the 16S rRNA gene, 16/23S spacer region, and partial 23S rRNA gene region of an ESFY isolate from an infected field sample of

N

o N

Table 4.1.

Methods routinely applied for detection of viruses in the Plant Biotechnology Unit, lAM, BOKU, Vienna.

Pathogens

Methods to isolate viral RNA

Apple chlorotic leaf spot virus (ACLSV)

Apple stem grooving virus (ASGV)

Apple stem pitting virus (ASPV)

PCR product Primer sequences

[bp]

lmmuno capture RT-PCR and RT-PCR

A52: 5' CAG ACC CTT ATT GAA GTC GAA 3' (7213-7233 on ACLSV P863, German et al. 1990) A53: 5'GGC AAC CCT GGA ACA GA 3' (6875-6891) [Candresse et al. 1995]

358

Immuno capture RT-PCR and RT-PCR

ASGV4F: 5'GTT CAC TGA GGC AA AAG CTG GTC 3' (nt 3918-3940) ASGV4R: 5'CTT CCG TAC CTC TTC CAC AGC AG 3' (nt 4491-4469) [Kummert et al. 1998] ASGV5F 5' CCT GAA TTG AAA ACC TTT GCT GC 3' (nt 6019-6041) ASGV5R 3' C CTT GAC CTC CCA ATC CTC AGC AC 5' (nt 6339-6362) [Kummert et al. 2000]

573

ASPV1F: 5' CTT CAT GAA GTC GCA ACT TTG CAC CA 3'(nt 5691-5716, Jelkmann 1994) ASPV1R: 5' GCY TCY YTH CCA TTV AGA TCA TAC CT 3' (nt 6176-6151) [Kummert et a1. 1998]

485

RT-PCR

344

Apple mosaic virus (ApMV)

N

o

w

Immuno capture RT-PCR or RT-PCR

ApMV1: 5'- TGG ATT GGG TTG GTG GAG GAT-3' ApMV2: 5'- TAG AAC ATT CGT CGG TAT TTG-3' [Petrzik et al. 1997] ILAR1: 5' TTC TAG CAG GTC TTC ATe GA3' ILAR2: 5'CAA CCG AGA GGT TGG CA 3' [Candresse et al. 1998]

261 206

Prunus necrotic ringspot virus (PNRSV)

Immuno capture RT-PCR and RT-PCR

ILAR1: 5' TTC TAG CAG GTC TTC ATC GA3' ILAR2: 5'CAA CCG AGA GGT TGG CA 3' [Candresse et al. 1998]

Prunus necrotic ringspot virus (PNRSV)

Immuno capture RT-PCR and RT-PCR

PNRSV-F...C537: 5' ACG CGC AAA AGT GTC GAA ATC TAA A 3' * (nt 1600-1624, Guo et al. 1995, S78312) PNRSV-R. .. H83: 5' TGG TCC CAC TCA GAG CTC AAC AAA G 3' (nt 1176-1200, Guo et al. 1995, S78312) [MacKenzie et al. 1997]

430

Plum pox virus (PPV)

Immuno capture RT-PCR and RT-PCR

Pi (=PPV1): 5' ACC GAG ACC ACT ACA CTC CC 3' (nt 9515 - 9533, Maiss et al. 1989) P2 (=PPV2): 5'CAG ACT ACA GCC TCG CCA GA 3' (nt 9291-9311, Maiss et al. 1989) [Wetzel et al. 1991]

243

Prune dwarf virus (PDV)

Immuno capture RT-PCR and RT-PCR

PDV A5': 5' ATG GAT GGG ATG GAT AAA ATA GT 3'(1838-1860, Bachmann et al. 1994) PDV A3': 5'TAG TGC AGG TTA ACC AAA AGG AT 3'(1988-2010) [Parakh et al. 1995]

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Prunus armeniaca, found in Austria, has been sequenced. Based on this ESFY sequence, primers were designed for direct and nested PCR. PA2F/R are the primers for direct PCR, amplifying a product of 1187 bp between nt 482 and nt 1669. Nested NPA2F/R primers amplify a product ranging from nt 1182 to nt 1667 (Table 4.2). These primers work at high annealing temperatures, thus increasing specificity and decreasing the risk of false positives. They detect, with a high degree of reliability, different fruit tree phytoplasmas present in Europe, for example, AP, ESFY and PD (Heinrich et al. 2001). Polymorphism among fruit tree phytoplasma strains AP, PD, and ESFY was deduced on 19 different isolates from a micropropagated reference collection (Bertaccini et al. 2000) using PA2F/R as general primers, and RFLP analyses with three restriction enzymes, that is, TOll, Tsp509I and TaqI (Heinrich et al. 2001). 2. IC-PCR for the Detection of Apple Proliferation Phytoplasma. An antibody against the AP agent has become commercially available (Loi et al. 1998). Besides allowing the application of ITP, an immunofluorescent method for the detection of AP has also been developed (Loi, pers. commun.). An IC-PCR test method for AP was established, based on an IC step combined with PCR applying the newly developed primers. Compared to ELISA tests or tedious DNA extraction procedures and subsequent detection by PCR, this IC-PCR method had su-

Table 4.2. Methods routinely applied for detection of phytoplasmas in the Plant Biotechnology Unit, lAM, BOKU, Vienna.

Pathogens European stone fruit yellows (ESFY) Apple proliferation (AP) Pear decline (PD)

Methods for DNA isolation DNA isolation after Kobayashi et al. (1998)

DNA isolation after Bertheau et al. (1998)

Primer sequence Primers for direct PCR: PA2F: 5' - GCC CCG GCT AAC TAT GTG C - 3' PA2R: 5'- TTG GTG GGC CTA AAT GGA CTC - 3' (Heinrich et al. 2001) Primers for nested PCR: PAN2F-m: 5'- ATG ACC TGG GCT ACA AAC GTGA-3' PAN2R: 5'- GGT GGG CCT AAA TGG ACT CG - 3' (Heinrich et al. 2001)

PCR product [bpJ 1187

485

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205

perior performance due to its simplicity, sensitivity, and reproducibility and is therefore suitable for large-scale testing of apple material in vivo and in vitro (Heinrich et al. 2001). IV. ELIMINATION OF VIRUSES

As with all plant viruses, fruit tree viruses are host plant dependent nucleoproteins, which are often characterized by their sensitivity to heat, stability in plant sap, differential sensitivity to certain chemotherapeutants, and their molecular organization. Thermotherapy of virus infected plants has found a large application. However, the more fundamental research on the effect of increased temperatures on viral replication, translation, processing of proteins, and transport throughout the plant is rare and has become possible only through the availability of improved diagnostic tools. Different hypotheses concerning the possible mechanisms of virus elimination have been proposed. However, the main conclusion to be drawn from all the different experimental approaches is that elimination is an extremely pathogen/host dependent process and that no generalizations should be made. Kassanis (1954) concluded that increased temperatures create disadvantageous conditions for virus replication, nonreplacement of degraded pathogens, and an altered balance between synthesis and degradation causing the elimination of viruses. Nyland and Goheen (1969) countered that a balance between virus synthesis and degradation is only to be expected at sublethal temperatures for virus and host. They concluded that increased temperatures destroy essential chemical processes in the viral and the host plant life cycles, but that the plant has more possibilities for recovery (Mink et al. 1998). Empirical observations have indicated that the pathogen is not eliminated, but that the host tissue escapes through rapid growth (Campbell 1962). This was questioned by the fact that dormant buds may lose the virus during therapy (Welsh and Nyland 1965). There are still many issues that are poorly understood: why heat disturbs viral replication, which differential inhibitions of host and pathogen occur under increased stress conditions, and the effects temperature-induced metabolic disorders of the plants have on pathogens. Thermal inactivation of most viruses (leading to a loss of infectivity) requires higher temperatures than the plant can normally withstand (Kunkel 1936). Therefore, different techniques have been developed, including short-term treatments at elevated temperatures, like the treatment of budwood or cuttings for 10 min. in hot water at 70°C (Bawden 1964). Such a treatment is more effective against microorganisms than against viruses.

206

M. LAIMER

A. Long-Term Treatments at Elevated Temperatures

By the end of the 1950s, in vivo thermotherapy belonged to a standard package of measures against the spread of virus diseases in fruit trees. Shoot tips or buds are grafted onto healthy rootstocks and, according to the host-pathogen combination, treated for 20 or more days at 38°C. The variation applying the heat treatment before budding 2 to 5 mm shoot tips onto healthy rootstocks (Howell et al. 1995) has become the standard technique for obtaining virus-negative clones of fruit tree cultivars in many countries for decades (Mink et al. 1998). ApMV was the first pome fruit virus eliminated successfully (Posnette and Cropley 1956), followed by a series of stone fruit viruses (Nyland 1959, 1960, 1962). ACLSV was most easily eliminated, while ASGV was classified as recalcitrant (Campbell and Best 1964; Welsh and Nyland 1965; Campbell 1968; Cropley 1968a). The required duration of heat treatment may vary for a single pathogen in different host species (Cropley 1968b). For viruses with isometric particles, heat treatment is expected to inactivate viral pathogens, while for filamentous viruses only an inhibition of replication seems to occur (Nemeth 1986). Elimination of mixed infections was considered more difficult (Nyland 1960; Kornkamhaeng 1983). B. Meristem Culture

Initially heat treatment was done with potted plants; soon this was followed by in vitro meristem culture. Shoot tips were dissected and meristems were cultured under aseptic conditions (Ten Houten et al. 1968; Quak 1977). Meristems consist of actively dividing cytoplasm-rich cells without vacuoles. Intercellular inclusions of air and a direct spacial connection to the vascular bundles are missing. In many cases it appears that viruses are unable to enter the meristem, which may be due to morphological and metabolic aspects of apical meristems. There are at least three explanations: 1. The electronmicroscopy and fluorescent antibody technique devel-

oped by Mori and Hosokawa (1977) showed clearly a specific apical "immunity" for the plant viruses studied. This indicates an optimal explant size, 0.1 to 0.3 mm, if only the last cells are without virus and 0.5 mm to 1 or even 3 mm if the zone free from viruses is larger. 2. The vascular tissue ends below the meristematic dome, creating a spacial barrier, which inhibits the spread of the virus into newly formed tissues (Brants et al. 1962).

4. DETECTION AND ELIMINATION OF VIRUSES AND PHYTOPLASMAS

207

3. In metabolicly active meristems a competition for energy rich

organophosphates or nucleic acids leads to a disadvantage for viral replication (Hirth 1958). The presence of inhibitors such as phenolamines (Martin-Tanguy et al. 1978) or metabolic disorders due to cell injury during explant excision may be further explanations (Mellor and Stace-Smith 1977). None of these hypotheses is completely satisfying. However, they underline the generalization that smaller explants increase the chance for virus elimination. The application of tissue culture methods to the elimination of virus infections in plants began after the observation of Limasset and Cornuet (1949), that the virus titer in a systemically infected plant decreases as the shoot meristem is reached. Since the first success of Morel and Martin (1952), who succeeded in obtaining a virus-free dahlia from meristem tips removed from plants infected with mosaic and spotted wilt viruses, virus diseases have been eliminated from clones of a number of crop species (Quak 1977). However, this technique has not yet been generalized for trees (Boxus and Druart 1986). ACLSV and ASGV were eliminated from apple (Huth 1978; Hansen and Lane 1985; Huang and Millikan 1980), mosaic virus from fig (Muriithi et al. 1982), vein banding virus from gooseberry (Jones and Vine 1968), several viruses from Prunus sp. (Boxus and Quoirin 1974), GFLV from grapevine (Barlass et al. 1982; Legin et al. 1979), PNRSV from Prunus (Negueroles and Jones 1979), and chlorotic leafspot virus from apricot (Pena-Iglesias and Ayuso 1978). In most cases the rate of success for virus elimination is directly correlated to the size of the explants and the time length of the heat treatment. Navarro and Juarez (1977) obtained 100 percent success, when they picked apices of Citrus with a maximum of two leaf primordia, that is, 0.1 to 0.15 mm. Iri et al. (1982) suggested replacing heat therapy of grapevine, yielding only 27 percent virus elimination, by meristem-tip technique (0.2 to 0.3 mm) where 80 percent of regenerated plants were free of leafroll disease. Likewise, Mosella-Chancel et al. (1980) achieved 57 percent PNRSV elimination and 72 percent PPV elimination with 0.4 to 0.8 mm peach explants. Boxus and Druart (1986) found that results were more dependent on the virus strain than on the plant species. Gella and Errea (1998) confirmed these results in the elimination of ACLSV and ilarviruses from different Prunus species. Careful analysis of these data, makes it obvious that the assumption that virus elimination by tissue culture alone is due either to rapid growth or the inhibitory effects of phytohormones is untenable (Laimer

208

M. LAIMER

da Camara Machado 1996; Diekmann and Putter 1996). Various in vitro techniques, including the incorporation of chemicals to suppress or inactivate viruses, gamma radiation and somatic cell hybridization (Kartha 1984), somatic embryogenesis (Goussard and Wiid 1992), and fragmented apical culture (Barlass et al. 1982) have been attempted to eliminate viral infections from tissues or their regenerants. C. Micrografting Micrografting involves the excision of the extreme tip of the shoot, preferably a dome (0.1 to 0.5 mm), and eventually grafting it onto a rootstock. This is possible, both under in vivo and in vitro conditions (Jonard et al. 1983). With in vitro micrografting, the seedling to be used as a stock is decapitated, and the cotyledons are removed. In some cases, in vitro grown rootstocks rather than seedlings have been used, as in sweet cherry (Lacrotte 1981). The apex is placed on the decapitated surface in contact with the cambial zone. The apex then develops as a leafy shoot (Alskieff and Villemur 1978). Jonard (1986) has improved and refined this technique which has been used to eliminate viruses from a number of fruit tree species. Micrografting was 100 percent successful in eliminating viruses from Citrus and 60 percent successful in eliminating PPV from Prunus. By using this technique, disease-free citrus orchards were produced in the United States and Spain (Murashige et al. 1972; Navarro et al. 1983). Mosella-Chancel et al. (1980) achieved the elimination of PPV and PNRSV, after micrografting 0.4 mm long tips obtained from terminal shoots of peach on young decapitated plants of GF 305, a polyvalent indicator (Bernhard et al. 1969). Without thermotherapy this technique achieved an elimination of 72 percent of PPV (strain M) and 7 percent of PNRSV (strain G). D. Use of Viricides

Studies indicate the inhibition of viruses by the use of benzimidazoles and ribavirin (Virazole) in meristem-tip and protoplast culture of potato (Cassells and Long 1982; Cassells 1983). Some investigations were made also with woody species. Barlass et al. (1982) attempted to replace heat treatment by the use of virazole to eliminate grapevine fanleaf virus (GFLV) in meristem tip cultures. Hansen and Green (1983) were able to inhibit ACLSV in apple meristern tissue. A period of two months in the presence of 10 or 20 pM rib-

4. DETECTION AND ELIMINATION OF VIRUSES AND PHYTOPLASMAS

209

avirin was effective in eliminating ACLSV from all shoots. At these concentrations shoots showed no growth reduction, but only occasionally mild chlorosis. Phytotoxicity was evident in all shoots in the treatment with 80 11M, and to a lesser degree with 40 JlM ribavirin. Yamaga and Munakata (1991) reported the successful elimination of the difficult to eliminate ASGV by two subcultures of 80 days on 12.5 Jlg/ml ribavirin. James et al. (1997) developed a rapid RT-PCR detection assay for confirming the elimination of ASGV by chemotherapy. E. In Vitro Thermotherapy

Rapid progress in plant tissue culture-delivered protocols for a range of fruit tree species and cultivars has provided the basis for in vitro thermotherapy. From experiences gained by in vivo thermotherapy, protocols for long-term heat treatment of in vitro plantlets could be adapted. In vitro thermotherapy is carried out in growth chambers. Duration of treatment and temperature regimes are adapted to the host/pathogen combination, taking into consideration the heat sensitivity of the host plant. Galzy and Compan (1968) applied heat therapy for in vitro grown grape plantlets, eliminating grapevine fanleaf nepovirus after three months at 35°C. F. Combinations of In Vitro Thermotherapy and Meristem Culture In many host/pathogen combinations the rate of elimination is lower when applying heat treatment or meristem culture alone, thus a combination is advisable. After thermotherapy of micropropagated shoots, a meristem is dissected at a dimension of 0.2 to 0.5 mm and cultured on appropriate culture media (Fig. 4.1). This approach has some obvious advantages: 1.

2. 3. 4. 5.

thermotherapy can be initiated immediately after a tissue culture is established, which means six months versus two years of preparation of the plant material to be treated; in vitro shoots are poorly lignified and allow a better effect of the heat treatment on the pathogen; moderately increased temperatures induce increased cell division activity and allow the plant to escape the pathogen through rapid growth; in vitro thermotherapy requires shorter treatment periods and is independent of the seasons; time and space are saved and work under aseptic conditions also allows storage without the danger of reinfection;

210

M. LAIMER

~. " " ,

'

'

""',.,',I, """ ", , "', ',' ,PteOlarati, ,

"'.~,>

,",' ._ ,'_' _••• _. '0'"

," "

'

"

,

,

, ,,''. " ' ,'" ".,' ' ,

,

Figure 4.1.

Virus elimination by in vitro thermotherapy and meristem preparation.

6. the primary in vitro shoots represent a simplified model for host-

pathogen interactions; 7. the system is more flexible and more readily adapted to a range of

cultivars to be freed from pathogens; and

4. DETECTION AND ELIMINATION OF VIRUSES AND PHYTOPLASMAS

211

8. a larger amount of pathogen free plant material is available after treatment for distribution, since it can directly be followed by an in vitro propagation phase. Quak (1977) demonstrated that for viruses that are difficult to eliminate by meristem-tip culture, a combination of meristem tip culture with heat therapy increases the number of virus free plants, even with large explants. Naumann (1981) showed this beneficial influence on virus-infected raspberries. Nepovirus infections were cured efficiently from infected Daphne species by combining meristem preparation with heat treatment (Sweet et al. 1979). Postman and Hadidi (1995) reported the successful elimination of apple scar skin viroids by in vitro thermotherapy and meristem preparation, demonstrating the validity of this approach also for other pathogens. 1. Vienna Collection. At the lAM an in vitro collection of pome and stone

fruit species was established from material infected with viruses and phytoplasmas named the Vienna Collection (VC) (www.boku.ac.at/ iam/pbiotech/phytopath/col.html). Numerous culture media were compared for multiplication of donor plants before and during thermotherapy, and most importantly for meristem regrowth. Strong regrowth of excised meristems depends on culture conditions, light conditions, and temperature regimes. Also changes of culture vessels from large to small tubes to 24 well microtiter plates considerably improved the survival rate. Regrowing shoots were finally placed on the initial medium for shoot multiplication. The hormone composition was optimized, and the use of sorbitol instead of sucrose (Wilkins and Dodds 1983) had a favorable influence on the persistence of cultures during the heat treatment. 2. Propagation of Malus Cultivars and Rootstocks In Vitro. Apple cultivars were established in vitro from actively growing buds (Laimer et al. 1988; Laimer da Camara Machado et al. 1991), and subcultured on MS medium (Murashige and Skoog 1962) supplemented with 0.55 mm myo-inositol, 20 g L-1 sucrose and 0.8 percent purified agar and 1.6 ~M BA and 0.05 ~M IBA. The cultures were kept at 22°C ± 2°C with a light intensity of 50 ~Mol m-2 S-l provided by cool white fluorescent tubes (Sylvania GTE Standard F 58/133) with a 16 h photoperiod. The virus infection status of the in vitro apple cultivars was repeatedly determined by ELISA (Knapp et al. 1995a). Malus cultures should be placed into thermotherapy 18 days after the last subculture. Directly after thermotherapy, meristems were dissected and cultured for 14 days on MS

212

M. LAIMER

medium supplemented with 1 mg L-1 BA and then changed to the proliferation medium again. 3. Propagation ofPrunus Cultivars and Rootstocks In Vitro. Prunus cultivars and rootstocks were established in vitro as described (Weiss et al. 1993, Knapp et al. 1995a). Prunus armeniaca was subcultured in a proliferation medium based on macro- and microelements of Lloyd and McCown (1980) supplemented with 100 mg L-1 myo-inositol, 20 g L-1 sucrose, 0.8 percent purified agar and 7.4!J-M 2iP, 1.8!J-M BA, and 2.5 !J-M IBA (Weiss et al., 1993). Prunus cultures were placed into thermotherapy 18 days after the last subculture. Directly after thermotherapy, meristems were dissected and cultured for 14 days on MS medium supplemented with 1 mg L-1 BA and then changed to the proliferation medium again.

G. Virus Distribution Throughout In Vitro Host Plants

Knowing the distribution patterns ofPPV, PNRSV, ASGV, and ACLSVin their respective in vitro host plants (Malus domestica and Prunus spp.) allows the determination of the temporal and spatial expression of selected fruit tree viruses during subculture cycles. lmmuno-tissue printing was used as a rapid and accurate immunological method for diagnosis and localization of ACLSV, ASGV, and PPV to assess the phytosanitary status of meristems. Single ACLSV infected shoots of different apple cultivars were used to investigate ACLSV distribution in in vitro stems (Knapp et al. 1995b). ASGV did not occur naturally as a single infection in apple cultivars; therefore shoots originally infected with both ACLSV and ASGV, which had undergone elimination treatments and where only ACLSV was eliminated, were examined for ASGV by means of immuno-tissue prints. ACLSV could be readily found within the enlarged base of in vitro shoots of apple. Highly infected tissue was interspersed with completely healthy tissues. The enlargement was caused by the wounding response following original shoot excision and tissue organization was less regular. As viruses move from cell to cell via plasmodesmata, tissue organization plays an important role (Hull 1989). In cross sections in the basal to middle shoot region of apple tissue cultures ACLSV was evenly distributed within the area of vascular bundles, accumulating in xylem and phloem. From field sampling it is known that ACLSV aggregates in cambium tissue (Fuchs et al. 1988). Cortical parenchyma contained less virus than the epidermis. Distribution of ACLSV decreased toward the shoot tip. However, intense signals were observed by ITP within the epi-

4. DETECTION AND ELIMINATION OF VIRUSES AND PHYTOPLASMAS

213

dermis, especially in the area of leaf onset. Virus was detected in localized spots within the cortex and rarely in the vascular bundles, which are less mature toward the shoot tip (Knapp et al. 1995c). ASGV was distributed unevenly in the stems but could be detected by ITP at levels, where samples gave negative ELISA readings. ASGV was usually found along the vascular bundles, in the shoot tips, epidermis, and cortex. Strong signals were found within the youngest leaflets near the meristem-the meristem itself being free of ASGV. This again indicates the importance of small meristems as explants (Knapp et al. 1995c). PPV was detected in all types of tissues but occurred in irregular patterns (Knapp et al. 1995c). This explains the problems of detecting PPV in in vivo plants. H. Thermotherapy Conditions

Plant species may tolerate the influence of prolonged heat treatments to a variable extent (Tables 4.3, 4.4). Several woody species, such as Prunus, are very sensitive to heat, and their exposure to elevated temperature results in poor survival (Stein et al. 1991; Spiegel et al. 1993). In vitro shoots of Prunus die when treated following the protocol established for Malus species. To overcome this problem temperatures for Prunus were set for 12 h 38°C with light and 8 h at 36°C in darkness. A temperature of 36°C appears to be sufficient to inhibit viral replication and still allow plants to recover over night. The duration of thermotherapy was adjusted to or determined by the vitality of the cultures (Table 4.4). Sensitive species or cultivars tend to browning or even necrosis, thus decreasing the subsequent survival rate of prepared meristems. Since virus replication increases rapidly after plants are returned to room temperature (Dawson et al. 1978), test tubes with heat-treated shoot bundles should be taken individually for meristem preparation in order to allow a maximum of ten minutes at room temperature before meristems are dissected from the shoots. I. Elimination Success

At the lAM around 60 apple, plum, and apricot cultivars have been subjected to thermotherapy treatment. Various combinations of temperature adaptation and duration have been applied (Tables 4.3, 4.4). A stepwise temperature adaptation (TA) (four days at 28°C, two days at 31°C, two days at 35°C) at the beginning of thermotherapy was also undertaken to try to improve the survival rate of meristems. Success

N

.....

*'"

Table 4.3 . Elimination rates of several pome fruit viruses from different apple cultivars after in vitro thermotherapy treatment and meristem preparation. *

No. treated shoots

Viral load before Apple cultivar

ASPV

Arlet

ASGV

ACLSV

MS

LS

Day of thermotherapy

-

++

39

12

18

No. of prepared meristems

No. of surviving meristems

MS

LS

MS

LS

37

11

4

4

Champagner Renette

+

+

++

17

15

24

9

4

2

2

Elstar

-

-

++

30

40

28

28

39

2

3

Fuji

+

++

43

-

21

19

Jonagold

+

++

40

25

25

40

9

6

1

Jonica Kronprinz Rudolf

-

+

32

13

33

15

10

2

++

36

17

28

26

3

-

-

-

1

-

ASPV MS

2/2

ACLSV

ASGV

LS

MS

LS

MS

LS

-

-

-

1/4

2/4

2/2

0/2

1/2

2/2

2/2

-

-

-

2/2

3/3

-

-

III

-

5/6

III

0

III -

-

2/2

9

0

-

-

9/9

Landsberger Renette

-

++

14

9

21

14

9

9

5

MacIntosh

-

++

24

5

14

22

1

1

1

Maschanzker

+

-

++

34

40

47

32

1

10

0

Summerred Summerred

-

++ ++

++

30 27

10 40

23 21

24 11

2 20

1 3

1 17

-

Virus-free lines after thermoptherapy

5/6

-

-

6/10

* Special care was dedicated to record the fate of meristems from main shoot (MS) or lateral shoots (LS).

III

9/9

3/5

III

III

7/10 Oil

Oil

3/3

15/17

III

III

Table 4.4. Elimination rates of several stone fruit viruses from different Prunus cultivars after in vitro thermotherapy treatment and meristem preparation from main shoot (MS) or lateral shoots (LS).

No. treated shoots

Viral load before thermotherapy

Prunus cultivar

PPV

Bergeron

+

Brompton

-

Buhlers Zwetschge

N

>-'

U1

PDV

PNRSV

ACLSV

MS

Day of thermoLS therapy

-

20

20

No. of No. of prepared surviving meristems meristems MS

LS

15

3

5

+

-

+

28

20

22

19

-

+

+

40

46

20

14

Cafona

+

+/-

Dr. Mascle

+

-

Hauszwetschge Hungarian Best Koroszer Weichsel

-

20

Virus-free lines after thermoptherapy PPV

MS

LS

MS

LS

1

1

1/1

1/1

3

-

-

-

9

8

4/4

+/-

28

13

21

14

8

4

5

-

+

40

85

20

10

23

-

4

-

+

-

16

39

21

1

9

1

6

-

++

-

-

12

20

19

0

11

0

5

-

36

27

18

6

4

5

1

-

30

13

20

9

11

2

4

++

-

Kozlienka

-

++

Kraska

+

-

+/-

Marille Viessling

+

-

-

-

San Castrese

+

-

Spatbluhende Koch

+

-

32

20

19

16

12

27

-

32

30

-

20

20

9

47 4

1

-

1

1/1

15

-

54

-

4

27

12

3

7

1

5/5

MS

MS

4/4

5/5

MS

-

2/3

9/9

8/8

9/9

8/8

-

-

4/4

5/5

-

3/4

-

5/5 8/8 1/1

1/1

-

ACLSV

LS

1/1

6/6

2/2

4/4

-

8/8

5/5

4/4

717

LS

3/3

3/4

8

PNSRV

PDV

1/1

LS

216

M. LAIMER

rates in elimination of pathogens show high variation and depend on plant species, pathogen, and the type of infection (single or mixed) under investigation. By comparing titers of ACLSV, ASGV, and PPV in leaves of single in vitro shoots of different apple and apricot cultivars before and after variable periods of thermotherapy followed by meristem preparation there are several noteworthy observations: 1. virus particles persist and replicate over the entire shoots during thermotherapy since they can be detected by ELISA even after three weeks of heat treatment; 2. there is a distinct difference in the success rate depending on the duration of the heat treatment, that is, the longer, the better, provided the plant genotype allows it; 3. since heat treatment was adapted to the physiological state of the plant material, this difference is logically due to genotypic variation among the cultivars; 4. mixed infections are more difficult to eliminate, confirming that double infections may cause greater than additive effects; S. the time frame required for reliable testing for the absence of eliminated viruses can be reduced by at least six months by using PCR instead of ELISA detection. The elimination of the filamentous viruses ACLSV, ASGV, and ASPV from apple cultures yielded quite different results among cultivars, indicating again the specificity of the host-pathogen interactions. ACLSV is present in 28 accessions in Malus and Prunus of the Vienna Collection (www.boku.ac.at/iam/pbiotech/phytopath/col.html#aclsv). Of these in vitro cultures were established and 12 accessions of Malus were subjected to various therapy procedures, four cultivars containing ACLSV as single infection and eight cultivars containing ACLSV in a mixed infection. When present as a single pathogen, ACLSV was easily eliminated, but slightly more resistant to elimination treatments, when present in mixed infections (Table 4.4). The distribution pattern of ACLSV further explains the ease by which this virus is eliminated through meristem preparation (Knapp et al. 1995a). ACLSV accumulates in stems of tissue cultures in a typical pattern, that is, in decreasing concentrations toward the apex. In stems of main shoots, it appears more frequently than in sterns of newly formed axillary shoots. ASPV was present in the VC (www.boku.ac.at/iam/pbiotech/phytopath/col.html#aspv) in 17 accessions in vitro, but only twice as a single infection. These were obtained after thermotherapy treatment of

4. DETECTION AND ELIMINATION OF VIRUSES AND PHYTOPLASMAS

217

multiple infections, where it had persisted, or as defined single infections kindly provided by J. Kummert (Gembloux). With eight of these cultures (two single, six mixed infections) thermotherapy experiments were conducted, and ASPV was easily eliminated (Table 4.3). ASGV was the most recalcitrant pathogen to eliminate from Malus cultures in the whole program. It was present in the VC in seventeen accessions, eight as mixed infections together with ACLSV and ASPV and nine as single infections as the remaining virus from previous elimination experiments (www.boku.ac.at/iam/pbiotech/phytopath/col.html #asgv). From a pomological point of view, this plant material should not be included in further propagation schemes. However, for improving our understanding of viruses, we considered these accessions valuable tools for elimination studies. Eight of these cultures (four mixed, four single infections) were submitted to thermotherapy treatment and meristem preparation (Table 4.3,4.5). The percent of shoots freed from ASGV was rather low. Heat apparently did not effectively inhibit ASGV replication and distribution throughout the plant. Even explants from growth during the last week of the treatment were not free of the virus. It is still unclear how ASGV migrates into the shoot apex, possibly through meristematic cells in the cambium in dividing zones. It appears noteworthy, however, that meristems of lateral shoots often yielded shoots free of ASGV. These axillary shoots were formed only during the first week of the heat treatment or

Table 4.5. Elimination results of viruses from tissue cultures with single and mixed infections. Infection status

Apple cultivars

Single ACLSV

Arlet Elstar Kronprinz Landsberger Reinette

ACLSV mixed with ASPV

ACLSV mixed with ASGV and ASPV

Observations ACLSV is eliminated easily

Fuji Jonagold Jonagored Maschanzker

ACLSV is eliminated easily

Champagner Reinette Gelber Bellefleur Rubinette Summerred

ASPV sometimes persists ASGV is hardly eliminated

ASPV sometimes persists

ACLSV is eliminated easily

218

M. LAIMER

during the week of temperature adaptation. In fact, most of the ASGVfree meristems originated from lateral shoots obtained during the one week of stepwise temperature adaptation. The isometric viruses of the Ilarvirus genus are readily detected by the available protocols and easily eliminated for stone fruit plants by the described procedures. ApMV as single infection in Malus tissue cultures was lost over the years in all plantlets indicating that the virus is not very stable. In stone fruits virus elimination studies of single infections of PNRSV and PDV were conducted at the lAM with Prunus instititia and Prunus cerasus, respectively. The limiting factor, however, in virus therapy for stone fruits appears to be related to survival after thermotherapy due to the sensitive reaction of the plantlets exposed to higher temperatures (Table 4.4). When the physiological growth characteristics of apricot are considered (Costes et al. 1995), the results obtained are not surprising (Table 4.4), namely that in most cases the meristems of lateral shoots grow better and yield a higher percentage of virus-free plants than meristems from the main shoots exposed to heat therapy. PNRSV is present in the VC in fifteen accessions in vitro (ten single, five mixed infections) from different origins throughout Europe. Infected Prunus insititia Kozlienka was used as positive control and as model for PNRSV elimination. PNRSV was easily eliminated by the described approach both from single and mixed infections. PDV is present in the Vienna Collection in eight accessions, four of which were established in vitro (www.boku.ac.at/iam/pbiotech/ phytopath/col.html#pdv). Of the three single infections (one was mixed with ACLSV and PNRSV) the Prunus cerasus cultivar Koroszer Weichsel has been our model to study the behavior of the virus in elimination treatments, which yielded convincing success rates (Table 4.4). V. ELIMINATION OF PHYTOPLASMAS

So far, the successful elimination of PD and other phytoplasmas has involved the use of tetracycline or heat (Nyland 1975; Davies and Clark 1994). Routine elimination ofphytoplasmas at the lAM, involves a combination of heat therapy and meristem culture, omitting the tetracycline treatment. AP-, ESFY-, and PD-infected in vitro cultures of the VC were initially used for the improvement of the detection system. AP is present in the VC as in vitro cultures in four accessions from different origins (www.boku.ac.at/iam/pbiotech/phytopath/col.html#ap). even though AP-infected plants often have strongly reduced growth habit. Thermotherapy and meristem preparation efficiently eliminate

4. DETECTION AND ELIMINATION OF VIRUSES AND PHYTOPLASMAS

219

Table 4.6. Elimination results of phytoplasmas from tissue cultures of apple, pears, and Prunus. No. of prepared meristems

No. of surviving meristems

Phytoplasma-free lines after thermotherapy

Species

Pathogen

MS

LS

MS

LS

MS

LS

Pyrus Prunus Malus

PD ESFY AP

49 21 22 53

19 5 18

46 20 14 21

10 3 9 3

45 19 13 21

10 3 9 3

7

AP, PD, and ESFY. Of the 21 main shoot meristems infected with AP, all were cured (Table 4.6). PD is present in the VC in four accessions, two of which have been established in vitro (http://www.boku.ac.at/iam/pbiotech/phytopath/ col.html#pd). Thermotherapy and meristem preparation were carried out, meristems were regrown to plantlets, and the expected success rate of elimination was confirmed, for example, from 46 main shoot meristems from pear infected with PD, 45 were free from phytoplasma, while all 10 meristems from lateral shoots were cured (Table 4.6). ESFY is present in the VC in twelve accessions, nine of which have been established in vitro (www.boku.ac.at/iam/pbiotech/phytopath/ col.html#esfy). Of fourteen main shoot meristems of Prunus thirteen were ESFY-free, while again all nine meristems from lateral shoots were pathogen-free (Table 4.6). VI. INDEXING, MASS PROPAGATION, AND GERMPLASM CONSERVATION Plantlets obtained from meristem tip culture are propagated in vitro as mericlones (Boxus and Druart 1986). As soon as possible, plantlets from each mericlone are individually indexed. These tests are repeated several times before deciding if a mericlone is virus-free. Indexing can be done by the use of indicator plants, ELISA, or PCR tests. The major advantage of PCR tests over ELISA testing is the considerable gain of time considering that evaluations by ELISA might take one and a half to two years until the initially reduced virus titer reaches a reliable detection level again. By applying PCR, test results concerning the health status of a culture can be obtained within six months after heat treatment and meristem preparation.

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Virus-free clones are propagated by axillary branching for delivery to nurserymen (Boxus and Druart 1986; Rosati and de Paoli 1992). According to international regulations, virus-free elite plants are to be kept under conditions to avoid re-infection by air and soilborne vectors, for example, in an insect-proof screenhouse. As a result ofthe sanitation program that includes in vitro therapy and disease indexing, a number of virus-tested pome and stone fruit trees (cultivars and rootstocks) have been obtained. They are maintained in an insect-proof screenhouse at the lAM (Tables 4.7, 4.8). Currently, protocols for cryopreservation are being adapted to allow a long term storage of this valuable plant material.

Table 4.7.

List of virus tested apple trees at the lAM.

Apples Arlet Baumann Reinette Champagner Reinette Delbard Estival Elstar Fuji Gala Royal Gelber Bellefleur Golden Delicious Grosser Bohnapfel Ilzer Rosenapfel Jonagold Jonagored Jonathan Jonica Kronprinz Rudolf Landsberger Renette Lord Lambourne McIntosh Roter Boskoop Roter Trierscher Weinapfel Rubinette Steirischer Maschanzker Summerred Welschbrunner Rootstocks M7 M9 M25 M26 M27 MMlll

In vitro + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

In vivo + + + + + + + + + + + + + + + + + + + + + + +

4. DETECTION AND ELIMINATION OF VIRUSES AND PHYTOPLASMAS Table 4.8.

221

List of virus tested stone fruit trees at the lAM.

Stone fruits Plum Bluefree Brompton Biihlers Friihzwetschke Cacaks Friihe (Rana) Cacaks Beste (Najbolja) Hanita Hauszwetschke Italienische Zwetschke President Stanley Valor Apricot Bergeron Harcot Luizet Orangered Priana Rouge de Sernhac Peach Dixired Red Haven Cherry Bigarreau Burlat Bigarreau Moreau Kor6szer Weichsel Ozark Premier Sunburst Van Rootstocks F12/1

GF8-1 Myrobolan B Prunus serotina ssp. capuli Prunus insititia Kozlienka

In vitro

In vivo

+ + + + + + + + + + +

+

+ + + + + +

+ + + + +

+ +

+ +

+ + + + +

+ + + +

+

+ + +

+ + + + +

+ + + + +

VII. CONCLUSIONS

This review summarizes 15 years experience of the Plant Biotechnology Unit of the lAM in the field of pome and stone fruit tree virus and phytoplasma elimination by tissue culture techniques. vVe have found that the most effective method for the elimination of viruses from a propagation chain of vegetatively propagated plant material is a combination of thermotherapy followed by shoot tip culture (Walkey 1991).

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As has been stated (Mink et al. 1998), the in vitro system is most convenient for large scale virus elimination, multiplication of virusfree shoots, and the collection of virus-free germplasm. Similar as with in vivo thermotherapy, but even in shorter time frames, it is possible by repeated screening of progenies within the first six months to recover virus-free plants, which then can be rapidly propagated for further use, even if only a reduced number of meristems survive all handling steps. Little is known of the behavior of fruit tree viruses in in vitro systems. Factors such as random selection of healthy buds as starting explants, especially with unequally distributed viruses like PPV (Marenaud and Massonie 1977; Casper and Meyer 1981) and ACLSV (Gilmer et al. 1971; Fridlund 1973, 1983) have to be taken in account. Nevertheless, experience with over 50 fruit tree cultivars showed that random negative selection is a very rare event. It has been reported that distribution of virus infection decreases with time within a cultivar collection (Spiegel et al. 1996). However, this behavior, being dependent on the cultivar and the pathogen, is not a general rule. In the case of 'Champagner Renette' apple, for example, after four years of cultivation neither ASGV nor ACLSV were spontaneously eliminated in any of the shoots. In contrast, ApMV spreads very slowly, if at all, in orchards (Dhingra 1972) and is known to have rather unstable particles (Fulton 1972). Therefore, its rare occurrence and its loss in vitro is not surprising. Verification of the virus-free status of plants after thermotherapy procedures is only achievable through a careful screening procedure. Elimination methods like thermotherapy and meristem preparation may reduce the virus concentrations to levels where normal detection methods become too insensitive. Improved diagnostic tools can considerably enhance progress in this respect. The reliable detection of viral pathogens in fruit trees is an essential requirement for any sanitation program. Two-Step and DAS-ELISA with polyclonal antisera are the most important procedures for large-scale routine diagnosis and detection of many virus isolates, as they are available commercially. Immuno-tissue printing is more reliable than ELISA for the diagnosis of ASGV. Because of the extremely localized and limited occurrence of ASGV in stem tissues, ELISA might provide false negatives. The described localization of ASGV in young primordial leaves demonstrates that meristem dissection has to be carried out with high precision, as leaf primordia must not be included in the explant for effective elimination of this virus.

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From our experience different criteria need to be applied to determine the best virus testing approach. If a certain plant material (be it in vivo or in vitro) is to be tested for the presence of one or only a few viruses or phytoplasmas (and antisera are available), IC-PCR is the method of choice since it combines the rapid extraction advantages of serology with the sensitivity of PCR detection. However, if the target is to verify the presence of a higher number of pathogens, a purification procedure of nucleic acids offers the advantage that only one sampling step of plant material is required, since it yields sufficient material to carry out and repeat several tests. We compared different RNA and DNA purification protocols and found the method of Bertheau et al. (1998 and of Kobayashi et al. 1998), involving the use of silica gel for a rapid purification of nucleic acids, to be the most appropriate technique, for the detection of viruses and phytoplasmas (Heinrich et al. 2001). Results from the virus elimination experiments with single and mixed infections of ASGV and/or ACLSV within in vitro shoots mimic experiences with these viruses under field conditions in the summer (Fuchs 1980), where at higher temperatures symptoms are masked, both visually and serologically. ACLSV is more strongly affected by elevated temperatures than ASGV (Welsh and Nyland 1965; Campbell 1968). ASGV is well known for its stability under higher temperatures (Lister 1970b). This property and its ability to accumulate in shoot tip regions may very well explain its recalcitrance to heat therapy. It is apparent that most of the serious virus disease problems around the world are the direct or indirect result of human activity (Thresh 1982) and that no technological developments in the field may be as efficient as an intelligent avoidance strategy. Production and distribution of virus free or virus-tested planting material of fruit trees is a technical challenge, but it also has political and socioeconomic implications. Just consider how often material of a chosen cultivar, once it is produced by one or another technique, is classified by the growers to be out-of-date. Few consider how long it takes to breed a new cultivar and how much time is required to make new accessions virus-free. The use of virus-and phytoplasma-tested planting material continues to be of major importance, if production of fruits should be accomplished in a sustainable way (Engel 1990). Virus and phytoplasma elimination in pome and stone fruit trees is a complex art, requiring elaborate techniques and skills and plenty of practical handling experience. Despite the considerable progress made in the last years, there is still great need to improve our knowledge of the process.

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Ten Houten, J. G., F. Quak, and F. A. van der Meer. 1968. Heat treatment and meristem culture for the production of virus-free plant material. Neth. J. Plant Pathol. 74:17-24. Teycheney, P. Y., G. Tavert, R. Delbos, M. Ravelonandro, and J. Dunez. 1989. The complete nucleotide sequence of plum pox virus RNA (strain D). Nucleic Acids Res. 17:10115-10116. Thresh J. M. 1982. Cropping practices and virus spread. Annu. Rev. Phytopathol. 20:193-218. Trifonov, D. 1969. Lokalisierung und Wanderung des Scharkavirus (plum pox virus) in Pflaumenbaumen. Zbl. Bakt. Abt. II. 123:340-347. Trifonov, D. 1974. Susceptibility to the plum pox virus of twelve varieties ofthe cultivar Kjustendilska sinya sliva. Publ. House Bulg. Acad. Sc. Virology. Sofia. 131-138. Veronesi, F., A. Bertaccini, A. Parente, M. Mastronicola, and M. Pastore. 2000. PCR indexing of phytoplasma-infected micropropagated periwinkle treated with PAP-II, a ribosome inactivating protein from Phytolacca americana leaves. Acta Hort. 530:113-119. Walkey, D. G. A. 1991. Production of virus free plants. p. 270-292. In: Applied plant virology. Wiley, New York. Waterworth, H. K 1993. Processing of foreign plant germplasm at the National Plant Germplasm Quarantine Center. Plant Dis. 77:854-860. Waterworth, H. K, and R. W. Fulton. 1964. Variation among isolates of necrotic ringspot and prune dwarf viruses isolated from sour cherry. Phytopathology 54:1155-1160. Weiss, H., V. Hanzer, K Knapp, A. da Camara Machado, H. Katinger, and M. Laimer da Camara Machado. 1993. In vitro Vermehrung von Prunus armeniaca. Vortr. Pflanzenziicht. 27:322-326. Welsh, M. F., and G. Nyland. 1965. Elimination and separation of viruses in apple clones by exposure to dry heat. Can. J. Plant Sci. 45:443-454. Wetzel, T., T. Candresse, M. Ravelonandro, R. P. Delbos, H. Mazyad, A. Aboul-Ata, and K J. Dunez. 1991. Nucleotide sequence of the 3'-terminal region of the RNA of the El Amar strain of plum pox potyvirus. J. Gen. Virol. 72:1741-1746. Wilkins, C. P., and J. H. Dodds. 1983. Tissue culture conservation of woody species. p. 113-136. In: J. H. Dodds (ed.), Tissue culture of trees. AVI Publ. Co., Westport, CT. Yamaga, H., and T. Munakata. 1991. Production of virus-free apple planting stock by meristem culture. ASPAC Food and Fertilizer Technology Center Japan, Technol. Bul. 126:11-17. Yanase, H., K. Sawamura, G. I. Mink, and A. Yamaguchi. 1975. Viruses causing apple topworking disease (Taka Tsugi Byo) in Japan. Acta Hort. 44:221-229. Yoshikawa, N., and T. Takahashi. 1988. Properties of RNAs and proteins of apple stem grooving and apple chlorotic leaf spot viruses. J. Gen. Virol. 69:241-245. Yoshikawa, N., K Sasaki, M. Kato, and T. Takahashi. 1992. The nucleotide sequence of apple stem grooving capillovirus genome. Virology 191:98-105. Yoshikawa, N., H. Matsuda, Y. Oda, M. Isogai, T. Takahashi, T. Ito, and K. Yoshida. 2000. Genome heterogeneity of apple stem pitting virus in apple trees. NCBI Acces. No. AB045371. Zawadzka, B. 1980. The response of several plum cultivars to infection with plum pox virus. Acta Phytopath. Acad. Sci. Hung. 15: 215-221.

5

Pear Fruit Volatiles Francesca Rapparini and Stefano Predieri* Istituto di Biometerologia-Firenze, Sezione di Bologna Via Gobetti 101 40129 Bologna, Italy

1. INTRODUCTION II. ANALYSIS OF CHEMICAL COMPOSITION A. Sample Preparation B. Isolation and Concentration of Pear Volatiles C. Identification and Quantification of Compounds D. Determination of Organoleptic Value of Identified Volatiles E. Volatiles Found in Pears III. BIOGENESIS A. Lipid-derived Volatiles B. Amino Acid-derived Volatiles C. Carbohydrate-derived Volatiles D. Other Biosynthetic Pathways E. Ester Formation IV. FACTORS AFFECTING QUALITATIVE AND QUANTITATIVE EMISSION OF PEAR VOLATILES A. Genetic Differences B. Environmental Effects 1. Preharvest Factors 2. Maturity at Harvest 3. Storage Conditions C. Fruit Physiology 1. Intra-fruit Volatiles Location 2. Ripening 3. Senescence 4. Disorders

*The authors acknowledge Graziella Cristoferi, James P. Mattheis, Patrizia Salamone, Ester A. Sztein, and David Sugar for encouragement and assistance with this manuscript.

Horticultural Reviews, Volume 28, Edited by Jules Janick ISBN 0-471-21542-2 © 2003 John Wiley & Sons, Inc. 237

F. RAPPARINI AND S. PREDIERI

238

V. VOLATILE COMPOUNDS' INFLUENCE ON QUALITY A. Fresh Fruit B. Processed Products VI. SUMMARY AND CONCLUSIONS LITERATURE CITED

ABBREVIATIONS AND ACRONYMS AAT ADH AEDA AVG CA FID GC GCO HPLC HPLS IR LOX MA MD-GC MEP MS NA NAA NADH NMR OUV PDC PID RI

SDE SFE SPME TD TLC Uo

UV

alcohol acyltransferase alcohol dehydrogenase aromatic extract dilution analysis aminoethoxyviny19lycine controlled atmosphere flame ionization detector gas chromatography gas chromatography olfactometry high pressure liquid chromatography hydroperoxide lyase infrared (spectroscopy) lipoxygenase modified atmosphere multidimensional gas chromatography methylerythritol phosphate mass spectrometry normal atmosphere naphthalenacetic acid nicotinamide adenine dinucleotide nuclear magnetic resonance odor activity value pyruvate decarboxylase photoionization detector retention index simultaneous distillation-extraction supercritical fluid extraction solid-phase microextraction thermal desorption thin layer chromatography odor unit ultraviolet (spectroscopy)

5. PEAR FRUIT VOLATILES

239

I. INTRODUCTION

The pear (Pyrus spp., Rosaceae) is one of the most delectable fruits and its particular aroma, determined by volatile compounds, is a key quality factor. The genus Pyrus consists of more than 22 primary species and cultivars that are divided into those from the West (European or French pears), which combine a buttery juicy texture with rich flavor and aroma, and those from the East (Asian, Chinese or Japanese pears) which are characterized by a crisp texture and sweet but subacid flavor. European pears are considered cultivars of P. communis 1., whereas Asian cultivars are derived from P. pyrifolia (Burm.) Nakai, P. ussuriensis Max., and P. x bretschneideri Redh. In France and England P. nivalis Jacq. is used for the production of an alcoholic cider (perry). The earliest written record of pear culture in Europe comes to us from Homer, who about 3000 years ago in The Odyssey refers to pears as one of the "gifts of the gods" which grew in the garden of Alcinous (Hedrick et al. 1921; Janick 2002). Historic records of pear cultivation in Asia date from about 2000 years ago (Shen 1980). Among deciduous tree fruits, pear, with more than 12 million tonnes produced annually worldwide, is currently surpassed only by apple in economic importance. Major improvements have been achieved in the last few decades both in pear cultural practices and in technologies for fruit preservation. World pear production is now based on a limited number of well known traditional cultivars with distinct quality traits. Development of improved cultivars will depend on combining yield, disease resistance, and improved quality, especially texture and flavor. The first chemical investigation on pear volatiles was published by Harley and Fisher in 1927. Since then, methods of increasing reliability for the determination of the chemical identity of pear volatiles have been developed. Research on pear fruit volatile flavor components was initially focused on 'Bartlett' (syn. 'Williams'; 'Williams' Bon Chretien') (Jennings et al. 1960; Jennings 1961; Jennings and Creveling, 1963; Jennings and Sevenants 1964; Heinz et al. 1964; Heinz et al. 1965; Jennings et al. 1964; Heinz and Jennings 1966; Heinz et al. 1966; Romani and Ku 1966; Creveling and Jennings 1970; Jennings and Tressl 1974), a major cultivar in many pear production areas due particularly to its fine aroma. These studies identified decadienoate esters as 'Bartlett' pear characterimpact compounds, defined by Nursten (1970) as "compounds that give a food product its distinctive aroma." Contributory compounds, defined by Crouzet et al. (1997) "responsible for a typical contribution or note," were also identified.

240

F. RAPPARINI AND S. PREDIERI

Paillard (1990) published a comprehensive review of whole pear aroma that includes volatile constituents. Most recently, a number of investigations, including the extensive studies of Suwanagul and Richardson (1998a,b), have widened the interest over several European and Asian pears and different Pyrus species. To date, more than 50 cultivars have been studied and more than 300 compounds have been identified, including hydrocarbons, aldehydes, alcohols, esters, ketones, and sulphur compounds. However, studies relative to the volatile components of pear are still relatively few as compared to apple, peach, strawberry, and banana (Suwanagul and Richardson 1998a,b). Studies on the correlations between chemical and sensorial information could provide significant advances in the knowledge about the relationships between volatiles and pear eating quality. Several attributes, including appearance, flavor, flesh texture, and grit (stone cell) content determine pear quality. Flavor perception, the process linking plant biochemistry with the physiology and the psychology of the consumer (Beaudry 2000), is a major aspect for food preference. Pear flavor depends on a delicate balance of sugars, acids, phenolics, and aromatic compounds (Bell et al. 1996), but a number of additional factors, texture for example, influence perception. Flavor is perceived in combination by taste and smell, defined by Rothe (1988) as "the chemical senses," that are complementary in their contribution to overall flavor. Taste is a sensation assessed through the contact of water-soluble compounds with the mouth and tongue and is perceived as the intensity of sweetness, acidity, saltiness, bitterness, and umami. Aroma, sensed through olfactory receptors that bind volatiles emitted by food, plays a dominant role in flavor delineation (Tucker 1993; Kays and Wang 2000) and in most temperate fruits flavor is closely related to aroma. Odor perception takes place in the upper part of the nasal cavity only, the so-called regia aJfaetaria (Rothe 1988). This area, of about 10 cm 2 , contains some 10 million receptors that bind volatiles emitted by food. There are two ways for odorous substances to reach olfactory receptors: via the breathing stream, directly into the nasal cavity, or through the nasopharynx, that connects the mouth with the nasal cavity. The volatiles perceived in both of these ways are important determinants of fruit eating quality. Consumers first make nondestructive (smelling) then destructive (chewing) quality evaluations and then all the sensory inputs are integrated into a decisive judgment of quality (Abbott et al. 1997). Because aromatic volatiles are the substances that

5. PEAR FRUIT VOLATILES

241

define the distinctive pear fruit flavor (Bell et al. 1996), studies on volatiles can provide an important contribution to an improved knowledge of overall pear quality and consumer preference. The capacity that the individual fruit has for expressing its genetic potential for volatile production is determined by preharvest and postharvest conditions. These include genetic traits, cultural practices, harvest maturity, and storage regime. An expanded knowledge of factors contributing to volatile formation and emission by pear fruits would be of benefit to the entire pear industry. Volatiles are of high importance in determining product characterization and overall quality of both fresh and processed pears. Volatiles playa major role in determining the quality of new products, such as fresh-cut fruit (Schlimme 1995; Gorny et al. 1998; Senesi et al. 1999) and in the development of new packaging technologies (Kjaersgaard et al. 1997; Chen and Varga 1999). Studies on volatile compounds are of importance for the monitoring of developmental processes such as ripening, senescence, the onset of disorders, and off-flavors (Richardson and Kosittrakun 1995). There is an increasing interest in volatiles produced during postharvest, principally in relation to superficial scald or anaerobic respiratory metabolism (Ke et al. 1990; Chen et al. 1993; Lange 1997; Zoffoli et al. 1998). Research on volatiles emitted by pear leaf and flower has received some attention in relation to genetics or ecological issues (Quamme 1984; Miller et al. 1989; Baraldi et al. 1999; Scutareanu et al. 1996; Drukker et al. 1995,2000; Knight and Light 2001; Light et al. 2001). This review presents a comprehensive summary of the research conducted on pear fruit volatiles as components of aroma, as markers of fruit physiological status, and as factors affecting product quality. We will describe the evolution of the analytical techniques that allowed the present knowledge of volatile biogenesis, emission patterns, and rates. Factors that affect volatile emission, such as genetic traits and pre- and postharvest conditions will also be discussed. II. ANALYSIS OF CHEMICAL COMPOSITION

Several different approaches have been applied in the analysis of pear volatiles (Table 5.1). Comparison of results from different reports is complicated because the composition of the extract is highly dependent on the analytical procedures employed (Heinz et al. 1966; Takeoka et al. 1992; Giintert et al. 1998).

N

fl::>

N

Table 5.1.

Analysis of volatile compounds of pear fruits.

Year

Authors

Substrate used for extraction

1927

Harley and Fischer

Sliced and intact fruit

2

1938

Tindale et al.

3

1950

Mattus

4

1953

Fidler

5

1954

Gerhardt

6

1954

7

1955

Code

Extraction method

Analysis

Species

Cultivars

Dynamic headspace, steam distillation

Chemical reactions

Pyrus communis

Bartlett

Pyrus communis

Bartlett

Headspace

Chemical reactions

Pyrus communis

Bartlett

Adsorption

Chemical reactions

Pyrus communis

Conference, Doyenne du Comice

Intact fruit

Headspace

Chemical reactions

Pyrus communis

Anjou, Bartlett

Leonard et al.

Pulp

Steam distillation

Chemical reactions

Pyrus communis

Bartlett

Luh et al.

Blended canned fruit

Steam distillation, solvent extraction, derivatives formation

Chemical reactions

Pyrus communis

Bartlett

Chemical reactions

Pyrus communis

Bose, Doyenne du Cornice, Passe Crassane

Intact fruit

8

1956

Serini

Pulp

9

1958

Claypool et al.

Blended canned fruit

Steam distillation

Chemical reactions

Pyrus communis

Bartlett

10

1960

Jennings et al.

Essence

Solvent extraction

GC

Pyrus communis

Bartlett

11

1961

Buttery and Teranishi

Static headspace

GC

Pyrus communis

Anjou, Doyenne du Cornice

N

H::>o

w

Pyrus communis

Bartlett

GC, IR, UV

Pyrus communis

Bartlett

Solvent extraction

UV

Pyrus communis

Bartlett

Essence

Solvent extraction

GC, IR

Pyrus communis

Bartlett

Jennings et al.

Essence

Solvent extraction

GC,IR

Pyrus communis

Bartlett

1964

Spanyar et al.

Blended fruit

Steam distillation and solvent extraction

GC

Pyrus communis

19

1964

Heinz et al.

Intact and blended fruit

Headspace, solvent extraction

UV,GC

Pyrus communis

Bartlett

20

1965

PhanChon-Ton

Intact fruit

Dynamic headspace

GC

Pyrus communis

Bartlett, Belle Epine du Mas, Beurre Clairgeau, Beurre Diel, Beurre Hardy, Comptesse de Paris, Doyenne du Cornice, Louise-Bonne, Packham's Triumph, Passe Crassane

21

1966

Heinz and Jennings

Essence

Adsorption, derivatives formation

GC,IR, NMR,MS

Pyrus communis

Bartlett

22

1966

Heinz et al.

Blended fruit, essence

Solvent extraction, steam distillation, adsorption

GC,UV

Pyrus communis

Bartlett

12

1961

Jennings

Essence

Solvent extraction

13

1962

Drawert

Concentrate

14

1963

Jennings and Creveling

Essence

Solvent extraction

15

1964

Heinz et al.

Essence

16

1964

Jennings and Sevenants

17

1964

18

GC GC

N >I;>. >I;>.

Table 5.1.

(continued) Substrate used for extraction

Extraction method

Analysis

Species

Cultivars

Code

Year

Authors

23

1966

Romani and Ku

Intact fruit

Static headspace

GC

Pyrus communis

Bartlett

24

1967

Giannone and Baldrati

Fruit puree, nectars

Static heads pace, distillationextraction, derivatives formation

GC,TLC

Pyrus communis

Bartlett, Bose, Curato, Diel, Olivier de Serres, Passe Crassane

25

1968

PhanChon-Ton

Intact fruit

Static headspace

GC

Pyrus communis

Bartlett, Passe Crassane

26

1969

Fidler and North

Adsorption

Chemical reaction

Pyrus communis

Bose, Conference, Doyenne du Cornice

27

1969

Gasca et al.

Juice concentrate

GC

Pyrus communis

28

1970

Creveling and Jennings

Essence

Solvent extraction

GC, IR, UV, NMR

Pyrus communis

Bartlett

29

1970

Paillard et al.

Intact fruit

Headspace

GC

Pyrus communis

Passe Crassane

30

1973

Scotts and Wills

Cored sliced fruit

Vacuum sublimation

GC

Pyrus communis

Bartlett

31

1974

Jennings and Tressl

Intact fruit

Dynamic headspace

GC

Pyrus communis

Bartlett

N ..,.

CJ1

32

1974a

Strandjev

Essence

Solvent extraction

GC

Pyrus communis

Bartlett, Passe Crassane

33

1974b

Strandjev

Essence

Solvent extraction

GC

Pyrus communis

Bartlett, Passe Crassane

34

1977

Ghena et al.

Juice

Solvent extraction

GC

Pyrus communis

Bartlett

35

1977

Quamme and Marriage

Blended fruit

Solvent extraction

UV, IR, LC

Pyrus communis

Anjou, Aurora, Barseck, Bartlett, Bose, Clara Frijs, Corneille, Courielle, Dr. Jules Guyot, Ewart, Flemish Beauty, HW602, Kieffer, Yakima, Laxtons progress, Mac, Magness, Max Red, Maxine, Merton Pride, Moonglow, NY 8760, Parbarton, Pierre, Progress, Russett Bartlett, Seckel, Stewarts Bartlett, Surecrop

36

1978

Janes and Frenkel

Homogenate

Static headspace

GC

Pyrus communis

Bose

37

1981

Russel et al.

Blended fresh and canned fruit

Simultaneous distillation/ extraction

GC, HPLC-UV

Pyrus communis

Bartlett, Kieffer, Magness

38

1982

Strandjev

Essence

Solvent extraction

GC

Pyrus communis

Beurre Williams, Passe Crassane

39

1983

Romani et al.

Intact fruit

Static headspace

GC,MS

Pyrus communis

Bartlett

N >f;:.

0)

Table 5.1.

(continued)

Code

Year

Authors

40

1984

Quamme

41

1985

Berger et al.

Substrate used for extraction

Extraction method

Analysis

Species

Cultivars

Blended fruit and shoot

Solvent extraction, simultaneous distillation/ extraction

UV, HPLC-UV

Pyrus communis

Anjou, Aurora, Barseck, Bartlett, Beurre Superfine, Bose, Clara Frijs, Courielle, Dr. Jules Guyot, Ewart, Harvest Queen, Highland, HW603, HW606, HW607, Kieffer, Yakima, Laxtons Progress, ~ac, ~agness, ~ax Red, ~axine, ~oonglow, NY 8760, Parbarton, Russett Bartlett, Seckel, Stewarts Bartlett, Surecrop

Homogenate

Solvent extraction

GC/GC-~S/

Pyrus communis

Bartlett

UV

Pyrus communis

Anjou

GC

Pyrus communis

Bartlett

Pyrus communis

Bartlett, La France

Pyrus communis

Bartlett

GCO 42

1990

Chen et al.

Intact fruit

43

1990

Ke et al.

Juice

44

1990

Shiota

Peeled and cored sliced fruit

45

1991

Berger

Solvent extraction

Simultaneous distillation/ solvent extraction

GC,

GC

GC-~S

N

~

'1

46

1991

Rizzolo et a1.

Sliced flesh

Static heads pace

GC, GC-MS

Pynzs communis

Passe Crassane

47

1992

Horvat et a1.

Blended peeled fruit

Simultaneous steam distillation/ solvent extraction

GC, GC-MS

Pyrus pyrifolia

Chojui, Hosui, Kosui, Ya Li, Shinko

48

1992

Nanos et a1.

Juice, suspension cultured fruit cells

Static headspace

GC

Pynzs communis

Bartlett

49

1992

Takeoka et a1.

Blended and intact fruit

Simultaneous vacuum distillation/ extraction, dynamic headspace

GC, GC-MS

Pynzs pyrifolia

Seuri

50

1993

Chen et a1.

Intact fruit

Solvent extraction

UV

Pynzs communis

Anjou

51

1993

EccherZerbini et a1.

Sliced fruit flesh

Static headspace

GC, GC-MS

Pynzs communis

Conference, Doyenne du Cornice

52

1994

Avelar et a1.

Intact fruit, pulp

Dynamic headspace, supercritical fluid extraction

GC, GC-MS

Pynzs communis

Rocha

53

1994b

Ke et a1.

Juice

Static headspace

GC

Pynzs communis

Bartlett

54

1995

Imayoshi et a1.

Intact fruit, blended peel and pulp

Dynamic headspace, steam distillation

GC, GC-MS

Pynzs x bretshneideri

Ya Li

N

"'"

~

Table 5.1.

(continued)

Substrate used for extraction

Extraction method

Analysis

Species

Cultivars

Code

Year

Authors

55

1995

Richardson and Kosittrakun

Intact fruit

Static headspace

GC

Pyrus communis

Anjou, Bartlett

56

1996

Suwanagul

Intact fruit

Dynamic headspace, SPME

GC, GC-MS, GCO

Pyrus communis

Anjou, Bartlett, Bose, Doyenne du Cornice, Forelle, Packham's Triumph, Seckel, Vicar of Winkfield

57

1997a

Beurle and Schwab

Blended cored fruit

Solvent extraction

GC, GC/MS, MDGC

Pyrus communis

Alexander Lucas, Bartlett, Gute Luise, Gellerts, Madame Vertl:'3, Vereinsdechant, Packham's Triumph

58

1997

Chen and Varga

Intact fruit

Solvent extraction

UV

Pyrus communis

Anjou

59

1997

De Vries et al.

Intact fruit

Headspace

Laser-based photoacoustic spectroscopy

Pyrus communis

Conference

60

1997

Kjaersgaard et al.

Juice

Dynamic headspace (purge and trap)

GC, GC-MS

Pyrus communis

Clara Frijs

61

1997

Lange

Peeled fruit tissue

Static headspace

GC

Pyrus communis

Bartlett

62

1997

Recasen et al.

Juice

GC

Pyrus communis

Conference

N

~ (.0

63

1997

Yang and Lee

64

1998

EccherZerbini and Grassi

Pulp

Headspace

65

1998

Giintert et al.

Pulp

Simultaneous distillationl extraction dynamic

66

1998

Xu et al.

Flesh

67

1998

Oshita et al.

68

1998

69

Pyrus pyrifolia

Niitaka

Pyrus communis

Conference

GC, MS, NMR, IR, GCO

Pyrus communis

Bartlett

Distillation

GC-MSMDGC

Pyrus x bretshneideri

Ya Li

Intact fruit

Headspace

Electric odor sensor

Pyrus communis

La France

Rizzolo et al.

Pulp

Solvent extraction

GCIGCO

Pyrus communis

Doyenne du Cornice

1998

Zoffoli et al.

Peel discs

Solvent extraction

UV

Pyrus communis

Anjou, Bartlett, Packham's Triumph

70

1999

Chervin et al.

Peeled and cored fruit

GC, enzymatic assays

Pyrus communis

Packham's Triumph

Juice Intact, blended fruit

Static headspace

GC

Pyrus seratina

Niitaka, Shinsui

SPME

GC

Pyrus communis

Packham's Triumph

Pyrus communis

Conference

Pyrus communis

Anjou

71

1999

Park et al.

72

2000

Chervin et al.

73

2000

EccherZerbini et al.

74

2000

Ju and Curry

GC

Static headspace

Intact fruit, tissue discs

SPME

GC, GC-MS

N

c.n

0

Table 5.1.

(continued)

Code

Year

Authors

Substrate used for extraction

75

2000

Ju et a1.

Blended flesh

Static headspace

GC

Pyrus x bretshneideri

Ya Li, Laiyang Chili

76

2000

Lo Scalzo et a1.

Fruit peel discs

Solvent extraction

GC-MS

Pyrus communis

Conference

77

2000

Oshita et a1.

Flesh, intact fruit

Static headspace

GC, electric odor sensor

Pyrus communis

La France

78

2001

Ju et a1.

Intact fruit

SPME

GC-MS

Pyrus communis

Bartlett

79

2001

Pinto et a1.

Juice

Static headspace

GC

Pyrus communis

Blanquilla

80

2001

Kharlamov and Burrows

Intact fruit

81

2001

Rapparini and Predieri

Sliced flesh

Pyrus communis

Harrow Sweet

Extraction method

Analysis

Species

Cultivars

Laser-based photoluminescence spectroscopy Dynamic headspace

GC/MS

Ge, gas chromatography; GCO, gas chromatography olfactometry; IR, infrared spectroscopy; MD-GC, multidimensional gas chromatography; MS, mass spectrometry; NMR, nuclear magnetic resonance; SPME, solid phase microextraction; TLC, thin layer chromatography, UV, ultraviolet spectroscopy.

5. PEAR FRUIT VOLATILES

251

Difficulties in fruit volatile isolation and analysis may arise for several reasons including concentration level of the volatile compounds, complexity of the aroma mixture, physical and chemical diversity, and instability (Parliment 1998). Pear flavor constituents are usually present in extremely low concentrations (parts per million, billion, or trillion) relative to the total fruit weight (Takeoka et al. 1992; Suwanagul and Richardson 1998b). Pear volatiles are extremely complex mixtures and may consist of hundreds of individual components, and physical and chemical properties of the different volatiles vary. Flavor composition usually includes a wide variety of different chemical classes (saturated and unsaturated hydrocarbons, organic acids, esters, aldehydes, alcohols, ketones, oxides, terpenes, and sulphur compounds) that cover a wide range of polarities, solubilities, volatilities, and pH values (Suwanagul and Richardson 1998b), thus the extraction and separation can be difficult. A further problem complicating the study of pear aroma is the formation of artifacts as a consequence of the instability of many volatile components, which may be oxidized by the air, and/or degraded by heat or extreme pH values. Other chemical processes, including photodecomposition, adsorption, vaporization, and fermentation due to microbial action, can also occur in the sample between the time of collection and analysis (Takeoka and Full 1997). Long sampling times increase the possibility that these processes may occur during sample isolation (Heath and Reineccius 1986). Because many pear volatile compounds are present in trace amounts, additional volatiles can be present as impurities due to contamination from surfaces that the sample contacts (Takeoka and Full 1997) or to residue of pesticides applied in the orchard (Nijssen 1991). In addition to chemically-induced changes, the possibility of secondary volatile production via enzymatic reactions must be considered. When fruit is cut, crushed, homogenized, or blended, certain enzymatic processes may be activated, some of which are extremely rapid once cellular disruption begins. This leads to the production of many volatiles, which normally occur only in trace amounts or not at all in intact cells (Heath and Reineccius 1986; Takeoka and Full 1997). A. Sample Preparation

Studies on pear volatiles can be divided in two groups according to the physiological state of the starting fruit sample: (1) Examinations of the volatiles released from intact fruit. This methodology considers only the odor compounds normally produced in the living pear tissue and

252

F. RAPPARINI AND S. PREDIERI

perceived directly in the nose before consumption. Sampling from intact fruits also allows for time-course studies (Mattheis et al. 1991); (2) Methods in which fruits are fragmented, blended, or homogenized to a lesser or greater degree, prior to or during the analysis. Cell disruption removes barriers to diffusion and thus allows for the determination of volatile composition and concentration inside the fruit tissue. However, because previously compartmentalized enzymes and substrate mix, new volatiles are formed. The ability to form volatiles after cell disruption can change during fruit ripening, apparently because of changes in enzyme and substrate availability (Baldwin et al. 2000). This procedure mimics the release of aromatic volatiles in the mouth during chewing and thus reflects the aroma perceived in the nose retro-nasally (Rothe 1988; Chervin et al. 2000). In order to study whether particular volatiles are present in the intact fruit or whether they are only formed following cell disruption, different methods of enzyme inactivation can be applied, including addition of either methanol or saturated solution of calcium chloride, and rapid heating by microwave (Schreier 1984; Takeoka et al. 1992; Buttery and Ling 1993). To be as close as possible to the development of volatiles released upon chewing, the enzyme inhibitor can be added immediately after pulp maceration (Chervin et al. 2000). The presence of the peel in the analyzed sample also constitutes an important factor, since a different volatile profile has been found between epidermal tissue (peel) and parenchima tissue (pulp) (Berger 1991; Chervin et al. 2000; Lo Scalzo et al. 2002). B. Isolation and Concentration of Pear Volatiles

Most of the methodologies employed in pear volatile studies involve classical procedures of flavor isolation: distillation and/or solvent extraction, and concentration of headspace volatiles. These procedures utilize differences in vapor pressure (distillation and headspace) or solubilities in different solvents (extraction). Direct solvent extraction has been widely used to isolate fruit volatiles of various pear cultivars (Gasca 1969; Creveling and Jennings 1970; Strandjev 1974a,b; Quamme and Marriage 1977; Quamme 1984; Berger et al. 1985a; Chen et al. 1993; Avelar et al. 1994; Beuerle and Schwab 1997a; Rizzolo 1998; Zoffoli et al. 1998; Cigic and Zupancic-Kralj 1999; Lo Scalzo et al. 2002). It has proven to be an effective extraction method yielding a large number of analytes, including highly water-soluble flavor constituents, which are typically poorly recovered by distillation and headspace analysis (Takeoka and Full 1997). However, the extract may contain semi- and nonvolatile constituents (Rothe 1988). Solvent choice

5. PEAR FRUIT VOLATILES

253

is an important factor to consider for optimum recovery of pear volatiles (Leahy and Reineccius 1984). Solvents are usually selected because of their selectivity and boiling point. The most frequently used volatile organic solvents in preparation of pear volatile extracts have been ethyl chloride and diethyl ether. Less polar solvents include pentane, isopentane, hexane, iso-octane, dichloromethane, alone or in combination. Diethyl ether has been widely employed as its lower boiling point permits excess solvent to be easily removed and it has a high extraction capacity for a broad spectrum of volatile compounds (Schultz et al. 1977). Distillation is among the oldest methods used for selective extraction of volatile compounds from pear aqueous matrices (Table 5.1). The advantage over other isolation methods (e.g., solvent extraction) is the separation of non- or semivolatile materials, normally present in high amounts, from highly volatile compounds (Schreier 1984). This technique often requires an additional step in which samples must be extracted from the distillate and concentrated so as to reach a detectable level (Coulibaly and Jeon 1996). This can involve solvent extraction and concentration, adsorption techniques, or freezing. The most frequently used distillation method for isolating pear volatiles has been steam distillation (Harley and Fisher, 1927; Leonard et al. 1954; Luh et al. 1955; Claypool et al. 1958; Spanyar et al. 1964, 1965; Heinz et al. 1966; Paillard et al. 1970; Imayoshi et al. 1995). The steam and volatiles are usually condensed in a series of cooled traps. The contents of the traps are combined and solvent extracted. This methodology is timeintensive and is subject to artifacts from solvent and/or for heat-sensitive compounds (Rothe 1988). Vacuum distillation method has been applied in several studies of pear volatiles, to minimize artifact formation by thermal degradation or hydrolysis, but is semiquantitative at best (Gasca et al. 1969; Giintert et al. 1998). As an alternative, simultaneous distillation-extraction (SDE) in a specialized apparatus such as the Licken/Nickerson (L/N) system has been used to produce the organic concentrates from pear fruits (Russell et al. 1981; Quamme 1984; Shiota 1990; Horvat et al. 1992; Takeoka et al. 1992; Giintert et al. 1998). Even though this technique exposes the sample to high temperatures and is known to produce thermally generated artifacts, it has been used in fruit flavor research as it can simulate the composition of volatiles that would be formed during processing such as canning (Takeoka and Full 1997). Using SDE, volatiles can be quickly extracted with a small amount of solvent in a single step within a shorter time as compared to lengthy distillation followed by extraction (Coulibaly and Jeon 1996). Heinz et al. (1966) compared solvent extraction, steam distillation and adsorption methods to analyze essences deriving from 'Bartlett'

254

F. RAPPARINI AND S. PREDIERI

pear processing. The major qualitative difference between the isolation methods was found to be in the relative proportion of high- to lowboiling point compounds. They found that the direct solvent extract and steam-distilled samples tend to contain a lower portion of low-boiling point compounds compared to charcoal absorption samples. The highboiling point compounds were similar among all extraction methods. Gtintert et al. (1998) found that the composition of the extract obtained by simultaneous distillation-extraction under vacuum is similar to that obtained by vacuum steam distillation (syn. vacuum headspace sampling method), suggesting that the former is the closest alternative to the latter method. In almost every case, distillation and solvent extraction methods require not only large amounts of sample, but also large volumes of organic solvents in certain steps of the preparation. The problems related to excessive use of solvent have been pointed out by several workers (Jennings 1961; Schreier 1984; Rothe 1988; Shiota 1990; Takeoka et al. 1992), and include concern for environmental pollution, an increased probability of sample contamination due to solvent-borne impurities, the risk of masking of volatile compounds by the solvent peak, and the loss of analytes. Extraction with liquid carbon dioxide (C0 2 ) or supercritical fluid extraction (SFE), is a more selective and efficient alternative to avoid problems encountered in extraction with low-boiling point organic solvents, and has been applied in isolation of volatiles from several fruits including pears (Polesello et al. 1993; Avelar et al. 1994). A major advantage of SFE using supercritical CO 2 is the low extraction temperature. Furthermore CO 2 is inexpensive, nonflammable, nonexplosive, chemically inert, readily available in a pure state, and leaves no toxic residue in the extract (Maarse 1991). Some authors, however, express doubts about the ability of this solvent to extract all compounds quantitatively (Taylor and Linforth 1994). Analyses of volatile components in the vapor phase above the matrix sample (headspace) were performed in pears as early as 1927 by Harley and Fisher. The headspace sampling system (Fig. 5.1) is becoming a powerful technique not only for the analysis of fruit samples (Dirinck et al. 1984; Ibanez et al. 1999), and food samples such as olive oil (Rapparini and Rotondi 2002), but is also widely employed in ecological and environmental studies (Bicchi and Joulain 1990; Charron et al. 1996, Ruther and Hilker 1998; Jacobsen 1997; Takeoka and Full 1997; Hewitt 1998; Baraldi et al. 1999; Butrym and Hartman 1999; Mendes et al. 2000; Rapparini et al. 2000a; Rapparini et al. 2001), investigations on tissue cultures physiology (Predieri et al. 1999; Rapparini et al. 2000b), in forensic science (Goldbaum et al. 1964; Pohl and Keller 1985), and in perfume research (Bedoukian et al. 1979). Headspace sampling tech-

5. PEAR FRUIT VOLATILES

Fig. 5.1.

255

Headspace sampling of sliced pears (Photo F. Rapparini)

nique has been reviewed elsewhere (Schaefer 1981; Jacobsen 1997). Some advantages are the relatively small amount of sample required, the reduced probability of artifact formation and sample loss, and the simplicity of sampling, that allows for minimum time-gap from sampling to chemical analysis (Teranishi 1998). This sampling procedure is the most direct way of qualitatively and quantitatively measuring volatile compounds released from the sample matrix, that is the part directly detected by smell (Maarse 1991). This is much more meaningful than total volatile analysis for correlating chemical and sensory analyses (Dirinck et al. 1984; Teranishi 1998; Ibanez et al. 1999). Headspace sampling of pear volatiles has been reported for intact, sliced, or crushed pears, as well as whether aqueous or alcoholic pear distillates were used (Table 5.1). Because volatiles are present at low levels in the gas phase above the sample, a concentration procedure is necessary. Enrichment can be achieved by the use of two major techniques: static and dynamic headspace sampling.

256

F. RAPPARINI AND S. PREDIERI

Using the static headspace technique, the sample is enclosed in a gastight container and, after equilibration, a small volume of the accumulated vapors present in the gas phase over the matrix (headspace) is collected with a syringe and directly injected into a gas chromatograph. This technique is preferred when automation is required, as in quality control methods or in sample screening (Dirinck et al. 1984; Manura and Overtone 2001). Direct analysis of equilibrium headspace vapor has been employed widely to isolate pear volatiles (Buttery and Teranishi 1961; Romani and Ku 1966; Giannone and Baldrati 1967; Paillard et al. 1970; Janes and Frenkel 1978; Romani et al. 1983; Rizzolo et al. 1991; Eccher-Zerbini et al. 1993; Richardson and Kossittrakun 1995; Lange 1997; Eccher-Zerbini and Grassi 1998; Chervin et al. 2000; EccherZerbini 2000; Oshita et al. 2000; Pinto et al. 2001). Giannone and Baldrati (1967) found that directly injecting headspace samples from pear fruits allowed for the detection of low-boiling point compounds not detected with solvent extraction methodologies. However, this technique did not allow for detection of high-boiling point compounds due to insufficient partition into the gas headspace volume. Romani and Ku (1966) encountered similar limitations while isolating pear volatiles from 'Bartlett' fruits. Furthermore, Heath and Reineccius (1986) showed that only the most abundant volatiles can be analyzed, suggesting that this method is not adequate for isolation of trace volatiles. Therefore, in such a system, it becomes difficult to establish whether the additional volatiles analyzed are due to increased concentration in the headspace or are new products appearing as a consequence of altered metabolism in a closed system (PIotto 1998a). The dynamic headspace sampling technique or a dynamic flowthrough system allows for detection of high-boiling point compounds, such as those characteristic of pear fruits (decadienoate esters), and trapping of a much larger volume of headspace volatiles in order to obtain sufficient material for analysis (Dirinck et al. 1984). In this trapping system, the headspace vapors are purged with a gas stream (clean air or nitrogen) continuously flowing over or through the sample (purge and trap technique). Volatile compounds are concentrated from this headspace gas volume at the outlet of the container, generally by liquid absorption, solid adsorption, and cooling (cryogenic method) (Schaefer 1981). Solid adsorbents include charcoal or synthetic porous polymers such as the Porapak series, the Chromsorb series, and Tenax (Paillard et al. 1970; Jennings and Tressl 1974; Takeoka et al. 1992; Imayoshi et al. 1995; Suwanagul and Richardson 1998b; Rapparini and Predieri 2002). The relevant properties of the different adsorbents have been investigated in a number of studies (Matisova and Skrabakova 1995; Takeoka and Full 1997; Brancaleoni et al. 1999; Manura 2001). None of these

5. PEAR FRUIT VOLATILES

257

adsorbents is perfect in trapping all volatile compounds with the same efficiency and they all exhibit some selectivity; therefore, the selection of the adsorbents depends on the chemical properties and concentrations of the compounds of interest as well as on the purity of the sample (Heath and Reineccius 1986). Pear volatiles trapped on the adsorbents are eluted with solvents, or by thermal desorption, and subsequently analyzed by gas chromatography either on-line or off-line. Thermal desorption provides better recovery than solvent elution of all trapped compounds while avoiding co-elution of low-boiling point compounds with the solvent (Wampler 1997). Thermal desorption also limits the possibility of artifact formation originating from interactions between solute and solvent. However, only solvent desorption allows multiple injections from a single sample (PIotto 1998a). The first study of pear volatiles reported using dynamic headspace methods was carried out by Pan-Chon-Ton in 1965 who isolated volatiles from pear fruits of various cultivars. Jennings and Tressl (1974) used adsorption on Porapak Q followed by elution with pentane prior to gas chromatographic analysis. In the last 30 years, similar methods have been extensively used for studying pear volatiles (Paillard et al. 1970; Rizzolo et al. 1992; Takeoka et al. 1992; Avelar et al. 1994; Kjaesgaard et al. 1997; Giintert et al. 1998; Suwanagul and Richardson 1998; Cigfc and Zupancic-Kralj 1999; Chervin et al. 2000; Rapparini and Predieri 2002). The dynamic enrichment system offers many advantages over static headspace measurements, since the former concentrates the analytes to detectable levels over a wide range of concentration and molecular weight (Suwanagul1996; Rapparini and Predieri 2002). As air is flushed through the sampling container, the fruit is maintained under aerobic conditions, thus limiting the formation of artifacts (PIotto 1998a). This method is nondestructive, and sampling from intact fruits using this collection procedure allows for time-course studies. Both dynamic and static headspace methodologies allow scientists to design and modify the headspace apparatus to best accomplish specific experimental needs. However, several experimental parameters, including volume of air to be sampled, volume of the headspace system, sampling time and speed, and carrier flow rate (Bicchi and Joulain 1990), influence headspace composition, and hence, the analytical results and their reliability and consistency. The effects of these factors may be worthy of advanced investigations and should be taken into account when comparing results from different experimental systems and/or laboratories. Despite these problems and although solvent extraction remains a commonly applied method in pear aroma studies, a number of papers indicates that headspace sampling is an increasingly widespread technique and a promising research tool for the examination of pear fruit volatiles.

258

F. RAPPARINI AND S. PREDIERI

Recently, a relatively new absorption technique called solid-phase microextraction (SPME), developed by Pawliszyn and coworkers (Arthur and Pawliszyn 1990; Arthur et al. 1992; Potter and Pawliszyn 1992), is increasingly being applied in fruit flavor studies (Yang and Peppard 1994). It consists of a fused-silica fiber coated with a polymeric stationary phase that can be placed completely in the sample (immersion sampling) or in the headspace of the sample (Roberts et al. 2000). The volatile organic analytes are absorbed and concentrated in the coating and then thermally desorbed inside the injector port of a gas chromatograph (Zhang and Pawliszyn 1993). The detection limits of the headspace SPME technique have been claimed to be at the subpicogram level (Zhang and Pawliszyn 1995). In the last decade this method has been applied in fruit aroma studies including pear (Yang and Peppard 1994; Ibanez et al. 1998; Chervin et al. 2000; Ju and Curry 2000; Ju et al. 2001). Suwanagul and Richardson (1998a) have discussed advantages and disadvantages ofthis technique while isolating headspace volatiles of ripening pears. Static heads pace trapping of compounds by SPME over a fixed time allowed the authors to accurately follow the quantitative changes of particular volatile compounds such as a-farnesene or various decadienoate esters without excessive losses due to oxidation. SPME has proven to be a fast, simple, repeatable, and inexpensive method for the extraction, identification, and quantification of organic compounds from pears. This technique has the advantage over various solvent extractions or headspace methods of reducing losses of compounds due to extract concentration or to purged air streams, and of avoiding volatiles masking by solvent. C. Identification and Quantification of Compounds

Once pear volatiles have been collected, chemical separation and detection is used for qualitative and quantitative analysis. Until the advent of gas chromatography (GC), studies concerned with nonethylene emissions from pears were seriously handicapped and the qualitative and quantitative analysis done at that time were mainly based on classical organic chemistry techniques (Harley and Fisher 1927; Mattus 1950; Gerhardt 1954; Luh et al. 1955; Serini 1956). The decisive breakthrough in pear aroma analysis was closely related to the introduction of gas chromatography in the 1950s. The development of this technique has vastly improved the analysis of pear volatiles, resulting in more than 300 compounds being identified to date (Table 5.2). The advantages of GC include high separation effect; more flexibility of the separation conditions; a very low detection limit (from 10-5 to 10-12 ) (Scott 1998) which

5. PEAR FRUIT VOLATILES

259

is as low as human sense organs can detect; relatively rapid and reproducible analysis with a high degree of significance (Rothe 1988). The power of gas chromatography has been enhanced by the development of capillary columns, programmed temperature regimes, the use of organic polymers as stationary phases, and the introduction of more sensitive detection methods (nuclear magnetic resonance, mass spectrometry, Fourier transform infrared spectroscopy) (Rothe 1988).

Table 5.2.

Volatile compounds identified in pear fruits.

Compounds

References (Table 5.1 code)

Alcohols

Aliphatic alcohols Methanol Ethanol

n-Propanol 2-Propanol (iso-propanol) n-Butanol 2-Butanol 2-Methyl-l-propanol (iso-butanol) n-Pentanol (amyl alcohol) 2-Pentanol 2-Methyl-l-butanol 2-Methyl-2-butanol 3-Methyl-l- butanol (iso-pentanol) 3-Methyl-3-buten-2-ol n-Hexanol (E)-2-Hexen-l-ol 2-Methyl-l-pentanol n-Heptanol 3-Ethyl-l-pentanol n-Octanol n-Octen-3-ol (E)-2-0cten-l-ol

Octane-l,3-diol

7,9,24,25,32,33,34,38,70 13,21,24,25,27,29,30,32,33,34,38,43,46,48, 51,53,54,55,56,59,60,61,62,63,64,70,71,73, 75,77,79,81 21,24,25,27,56,65,81 13,27 18,21,22,24,25,29,30,32,33,34,38,44,46,51, 52,54,56,65,68,77,81 13,27 13,25,27,44,52,56,65,81 21,24,25,27,32,33,34,38,44,46,51,52,54,56, 68,81 13 27,44,52,54,56,65,68,81 27,51 13,24,27,46,51,54,65,81 68 13,21,22,39,44,46,49,51,52,54,56,65,66,68, 72,81 54,65,68 54 21,44,49,56 81 18,21,44,46,49,51,52,54,56,65,68,72,81 65 54 57,65

(continues)

260

Table 5.2.

F. RAPPARINI AND S. PREDIERI (continued)

Compounds

References (Table 5.1 code)

(Z)-5-0ctene-l,3-diol 2-Ethyl-l-hexanol 6-Methyl-5-hepten-2-ol n-Nonanol n-Decanol (E)-3-Decen-l-ol (Z)-3-Decen-l-ol (Z)-4-Decen-l-ol (E)-2-Decen-l-ol (E,E)-2,4-Decadienol n-Dodecanol n-Hexadecanol 2,3-Butylenglycol 2,6,10-Trimethyldodeca-2,7 -(E), 9-(E),11-tetraen-6-ol

57 14,44,52,54 54,72 54,56 49,54 49,54 54 49,54 54 54 54, 72 44

Aromatic alcohols Benzenethanol Phenylethanol

8

76 68 72

Aldehydes Aliphatic aldehydes Methanal (formaldehyde) Ethanal (acetaldehyde) Propanal Butanal Pentanal Hexanal 2-Hexenal (E)-2-Hexenal (E,E)- 2,4-Hexadienal Heptanal (E)-2-Heptenal 2-Methyl-2-pentenal (E,E)-Ethyl-4-pentenal Octanal (E)-2-0ctenal Nonanal n-Nonenal

24,32,33,34,38 1,2,7,23,24,27,30,32,33,34,36,38,43,46,48, 51,53,55,59,61,62,63,64,70,71,73,77,79 24,25,27,52 20,81 81 24,27,44,46,47,49,52,54,56,65,68,72,81, 27,56 44,47,49,52,54,68,72,81 81 81 68 68 52 81 72,81 44,72,81 81

261

5. PEAR FRUIT VOLATILES 2-Nonenal (E)-2- Nonenal

(E,Z)-2,6-Nonadienal Decanal 2,4-Decadienal (E,E)- 2,4-decadienal (Z,E)- 2,4-decadienal

2-Butyl-2-octenal

47 68 68 81 44,49 49,52,54,68 68 47

Aromatic aldehydes Benzaldehyde

47,60,81

Phenylacetaldehyde

49

Ketones Acetone 2-Butanone (methylethyl ketone) 3-Hydroxy 2-butanone (acetyl methyl carbinol; acetoin) 2-Pentanone (methylpropyl ketone) 1-Penten-3-one 2-Methylcydopentanone 2-Heptanone 6-Methyl-5-hepten-2-one 2-Undecanone 3,4-epoxy-3-ethyl-2-butyl ketone 2,3-Butanedione (diacetyl) Geranylacetone

24,25,46 24,44,46 7,8,44,52,65 24,68 52 68 68,81 56,72,81 68 66 7

81

Esters

Formates Methyl formate

25,27

Ethyl formate

13,25,32,33,34,38,54

iso-Propyl formate

27

Butyl formate Hexyl formate

81 81

Acetates Methyl acetate Ethyl acetate

13,21,23,25,29,31,33,38,39,46,52,77 13,18,21,22,23,24,25,27,30,31,32,33,34,38, 39,44,46,49,51,52,53,54,60,77,81

Propyl acetate

21,22,23,24,25,27,29,31,32,33,34,38,39,44, 49,52,54,56,65,81

Butyl acetate

18,21,22,23,24,25,27,29,30,31,32,33,34,38, 44,45,46,49,51,52,54,56,60,65,68,72,77,81 (continues)

262

Table 5.2.

F. RAPPARINI AND S. PREDIERI (continued)

Compounds

References (Table 5.1 code)

iso-Propyl acetate Pentyl acetate (amyl acetate)

20 18,20,21,22,23,24,25,29,31,32,33,34,38,44, 46,49,51,52,54,56,60,65,68,72,81

iso-Butyl acetate (2-methylpropyl acetate) tert-Butyl acetate 3-Methylbutyl acetate Uso-pentyl acetate; iso-amyl acetate) 2-Methylbutyl acetate 3-Methyl-2-butenyl acetate Hexyl acetate

(E)-2-Hexenyl acetate 4-Hexen-l-01 acetate 3-Hexen-l-ol acetate (Z)-3-Hexen-l-01 acetate (Z)-3-Hexenyl acetate 2-Methylpentyl acetate 5-Hexenyl acetate Heptyl acetate 3-Hepten-l-ol acetate Octyl acetate 4-0cten-l-ol acetate 3-0cten-l-ol acetate 2,4-0ctadien-l-ol acetate (E)-2-0ctenyl acetate Nonyl acetate Decyl acetate (E)-3-Decenyl acetate Prenyl acetate Phenylethyl acetate p-Phenylethyl acetate 2-Phenylethyl acetate

24,25,32,33,34,38,44,46,49,51,52,54,56,60, 68,81 27 27,29,44,49,54,56,81 44,49,54,81 49 16,21,22,23,24,25,29,30,31,32,33,34,38,39,44, 45,46,47,49,51,52,54,56,60,65,68,72,77,78,81 54,65 56 81 56 54 56 44 21,49,52,54,56,68,72,81 56 21,44,49,54,56,60,72,81 56 56 56 54 49 49 49,54 54 54, 72 81 49,54,56

Propanoates Methyl propanoate Ethyl propanoate Ethyl-2-propenoate Propyl propanoate

56 24,25,27,46,49,54,56,66 54 25,27,32,33,34,38,56

263

5. PEAR FRUIT VOLATILES

Butyl propanoate Hexyl propanoate Ethyl-2-methyl propanoate Ethyl-2-methyl propenoate 3-Methylbutyl-2-methylpropanoate 1-Methylbutyl-2-methyl propanoate Pentyl-2-methylpropanoate Hexyl-2-methylpropanoate 2-Methylhexyl propanoate Butanoates Methyl butanoate Methy1-iso-butanoate Methy1-4-oxytransbutenoate Ethyl butanoate Ethyl-iso-butanoate Ethyl-2-butenoate Ethyl-(E)-2-butenoate Methyl-2-methylbutanoate Ethyl-4-oxytransbutenoate Ethyl-3-hydroxybutanoate Ethyl-3-acetoxybutanoate Propyl butanoate Ethyl-2-methyl butanoate Ethyl-(E)-2-methyl-2-butenoate Butyl butanoate Butyl-iso-butanoate iso-Butyl-iso-butanoate 1-Methylpropyl butanoate 2-Methylpropyl butanoate Propyl-2-methyl butanoate Methyl-2-ethyl-2-methyl butanoate Pentyl butanoate Butyl-2-methyl butanoate Hexyl butanoate Hexyl-iso-butanoate Hexyl-2-butenoate 3-Methylbutyl-3-methyl butanoate Heptyl butanoate

56,81 49,56,78,81 49,54 54 56 56 56 56 56 49,54,56,81 27 21 27,45,47,49,54,56,66,81 25 49 54 49 21 54 54 49,56,81 49,54,56,66 54 49,56,72,78,81 25 27 56 49,81 56 68 56 56,81 49,54,56,72,78,81 81 56 56 56 (continues)

264 Table 5.2.

F. RAPPARINI AND S. PREDIERI (continued)

Compounds

References (Table 5.1 code)

1-Methylhexyl butanoate Hexyl-2-methyl butanoate Octyl butanoate Butyl-(E)-2-methylbutyl-2-enoate

56 49,56,81 49,68 44

Pentanoates Methyl pentanoate 3-Methyl pentanoate Ethyl pentanoate Butyl pentanoate Hexyl pentanoate 3-Methyl fluorine pentanoate

49 66 47,49,54 81 81 66

Hexanoates Methyl hexanoate Ethyl hexanoate Ethyl-(E)-2-hexenoate Ethy1-(E)-3-hexenoate Ethyl-(Z)-3-hexenoate Ethyl-3-hydroxyhexanoate Propyl hexanoate 1-Methylethyl hexanoate Butyl hexanoate Butyl-2-hexenoate Butyl-3-hexenoate Butyl-4-hexenoate 2-Methylpropyl hexanoate Pentyl hexanoate 2-Methylbutyl hexanoate Hexyl hexanoate

49,54,56,81 44,47,49,52,54,56,66,68,72,81 49,54 54 54 49,54,66 49,56,81 56 49,56,60,72,78,81 56 56 56 49,56 49,56,81 56 49,56,72,78,81

Heptanoates Methyl heptanoate Ethyl heptanoate

49,56 49,54,56

Octanoates Methyl octanoate Methyl-(E)-2-octenoate Methyl-(Z)-3-octenoate Methyl-3-hydroxyoctanoate Ethyloctanoate Ethyl-(E)-4-octenoate

21,49,56,72,81 21,49,56,65 56,65 21,44,57,65 18,21,49,54,56,65,66,72,81 49

Z65

5. PEAR FRUIT VOLATILES Ethyl-(E)-Z-octenoate Ethyl-(Z)-Z-octenoate Ethyl-(Z)-3-octenoate Ethyl-3-hydroxyoctanoate Ethyl-5-(Z)-3-hydroxyoctenoate Ethyl-3-acetoxyoctanoate Propyl octanoate Butyl octanoate Z-Methylpropyl octanoate 3-Methylbutyl octanoate

21,45,49,54,56,65 56 65 Zl,44,45,54,57,65 57 65 49,56

Hexyl octanoate

56 49 49 49,56

Nonanoates Ethyl nonanoate

49,56

Decanoates Methyl decanoate Methyl-(E)-Z-decanoate Methy1-4-decanoate Methy1-(Z)-4-decanoate Methyl-(Z)-?-decanoate Methyl decenoate Methyl-Z-decenoate Methyl-4-decenoate Methyl-(E)-Z-decenoate Methyl-(Z)-?-decenoate Methyl-(Z)-4-decenoate Ethyl decanoate Ethyl-(Z)-4-decanoate Ethyl decenoate Ethyl-4-decenoate EthyI-(E)- Z-decenoate Ethy1-(E)-3-decenoate Ethy1-(Z)-4-decenoate Methyl- Z,4-decadienoate Methyl-(E,Z)-Z ,4-decadienoate Methyl-(E,E)-Z,4-decadienoate Methyl-(Z,Z)-Z,4-decadienoate Ethyl-2,4-decadienoate Ethyl-(E,Z)-Z,4-decadienoate Ethy I-(E,E)- Z,4-decadienoate Ethyl-(Z,Z)-Z ,4-decadienoate Propyl-(E,Z)-Z,4-decadienoate Butyl-(E,Z)-Z,4-decadienoate

34 56 Z2 34 7Z 56 56 21,38,56,65 3Z 21,3Z,33,38,44,46,49,65,81 21,ZZ,3Z,33,34,38,44,49,54,56,65,66,72,81 34 38 56 Zl,33,44,56,65,81 49 21, ZZ, 32, 33,38,44,45,46,49, 54, 81 49 16,Zl,Z2,3Z,33,34,38,44,46,56,65,7Z,81 Zl,ZZ,32,33,34,38,44,46,56 56 49 17,Zl,ZZ,44,45,46,49,54,56,65,72 21,ZZ,32,33,34,38,44,46,54,56 56 65,Z8 65,Z8

Zl,Z2,49, 56,65, 7Z,81

(continues)

266

Table 5.2.

F. RAPPARINI AND S. PREDIERI (continued)

Compounds

References (Table 5.1 code)

Hexyl-(E,E)-2,4-decadienoate Hexy1-(E,Z)-2 ,4-decadienoate Ethyl decatrienoate Ethyl-(E,E,Z)-2,4,7-decatrienoate Ethyl-(E,Z,Z)-2,4, 7-decatrienoate

28 65,28 49 28,56 65

Other esters C 12-CI8 Ethyl dodecanoate 2,4,6-trimethyl dodecanoate Methyl-(E)-2-dodecenoate Ethyl-ll-dodecenoate Ethyl-(?)-dodecenoate Ethyl-(E)-2-dodecenoate Ethyl dodecadienoate Methyl-(E,Z)-2,6-dodecadienoate Ethyl-(E,Z)-2,6-dodecadienoate

49 66 28 65 56 28 28 28 28

Ethyl-(E,Z,Z)-2,6,9-

dodecatrienoate Methyl tetradecanoate Ethyl tetradecanoate Methyl-(?)-tetradecenoate Methyl-(Z)-5-tetradecenoate Ethyl-(?)-tetradecenoate Ethy1-(Z)-5-tetradecenoate Methyl-(Z,Z)-5,8-tetradecadienoate Ethyl-(Z,Z)-5,8-tetradecadienoate Methyl-(E,E,Z)-2,4,8-tetradecatrienoate Eth yl-(E,E,Z)- 2,4 ,8-tetradecatrienoate Methyl hexadecanoate Ethyl hexadecanoate Methyl hexadecenoate Methyl-(Z)-7-hexadecenoate Ethyl hexadecenoate Methyl hexadecadienoate Methyloctadecanoate Methyl-(Z)-9-octadecenoate Methyl-l0-octadecenoate

28 56 28,49,56 56 28 56 28,65 28,56,65 28,45,56,65 28 28 28, 76 28,49,66,76 28 56 28 56 28 28 56

267

5. PEAR FRUIT VOLATILES Methyl-2,4-octadienoate Ethyl-2,4-octadienoate Methyl-2,4,6-octatrienoate Dibutyl phthalate Diethyl carbonate Ethyl benzoate Butyl benzoate Hexyl benzoate Ethyl tiglate Methyllinoleate Methyllinolenate Ethyllinolenate Methyl oleate Hydrocarbons Methyl benzene (toluene) Dimethyl benzene (xylene) n-Undecane n-Pentadecane n-Hexadecane n-Heptadecane n-Octadecane n-Nonadecane n-Methylnaphtalene Valencene Undecatetraene 1-(E,Z)-3,5-Undecatriene 4-Methyl cyclohexene 1-Methyl-4-(1-methylethyl)1,3-cyclohexadiene Benzeneacetonitrile 4-Allylanisole (estragole) 4-Propenylanisole (anethole) Tributylmethyl borane Terpenes Limonene ~-Phellandrene

3-Carene a-Farnesene (Z)-a-Farnesene (E,E)-a-Farnesene (E,Z)-a- Farnesene (E,E)-3 ,6-a-Farnesene

56 56 56 44 54 47,49,54 56 56 49 45, 76 45, 76 76 45 44,47 47 44,81 47,49,66 49 47,49,66 49 47,49 49 49 72 41 81 56 81 60,81 81 66 44,52,54,60 56 56 31,42,44,45,47,49,50,52,54,58,60,69,74,76,78 44 56,72,81 56,72,81 65 (continues)

268 Table 5.2.

F. RAPPARINI AND S. PREDIERI (continued)

Compounds

References (Table 5.1 code)

Copaene (E)-Linalool oxide (Z)-Linalooloxide a-Terpineol Terpinen-4-o1 (E,E)- Farnesol (E,E)-3 ,6-Farnesol Eugenol

72 65 65,81 44,49,54 44,54 44 65 49,68

Acids Acetic acid 2-Methyl butanoic acid 3-Methyl butanoic acid Hexanoic acid Nonanoic acid Hexadecanoic acid

54,72 65 65 54 72 44

Sulphur compounds Ethylmethylthio acetate Ethyl-3-methylthio propanoate 3-Methylthiopropyl acetate Ethyl-3-methylthio-(E)2-propenoate Ethyl-3-methylthio-(Z)2-propenoate 3-Methylthio-propanal (methional)

54 49,54,65 49,54 49,54 54 65

Miscellaneous ~-Damascenone

3,4-Dehydro-~-ionol

2-Ethyl furan Pentyl furan Biphenyl 2-Methoxy-3-iso-propyl pyrazine Diethyl phosphine 3-Hepta alkyne-2,6-diketone5-methyl-5-(1-methyl ester) 1-Methyl-4-(1-methylethylidene)-cyclohexane 1,3-Dioxanes

65 65 81 47,60 49 60 66 66 56 57

5. PEAR FRUIT VOLATILES

269

The relatively good separation obtained by GC has obviated the application of high pressure liquid chromatography (HPLC) in studies of fruit volatiles, although HPLC is normally applied for the separation of nonvolatile compounds. The direct use of HPLC as a prefractionation technique for pear volatiles has been undertaken only sporadically (Russell et al. 1981; Quamme 1984; Zoffoli et al. 1998). The use of HPLC in isolation of fruit volatiles is not useful for the total analysis of the complex mixture of pear volatiles, but has proven to be a ready tool to identify and quantify some important volatile compounds of pear aroma, such as decadienoates (Russell et al. 1981; Quamme 1984), a-farnesene, and conjugated trienes (Zoffoli et al. 1998). Indeed, these compounds are characterized by a relatively high degree of unsaturation (Crombie 1955), and thus can be spectrophotometrically detected and quantified by HPLC with an UV detector between 200 and 300 nm (Jennings and Creveling 1963; Heinz et al. 1964; Heinz et al. 1966; Creveling and Jennings 1970; Quamme and Marriage 1977; Chen et al. 1990; Chen et al. 1993). HPLC, in contrast to GC, prevents thermal generation of artifacts (Kubeczka 1981). The identification of pear volatiles separated by gas chromatography has been mainly achieved by using their chromatographic retention indices. The Kovats Retention Index (RI) system (Kovats 1965), has been often used for the identification of a large number of volatiles (Jennings and Shibamoto 1980) including those released from pear fruit (Russell et al. 1981; Takeoka et al. 1992; Avelar et al. 1994; Suwanagul and Richardson 1998b). However, Kovats RI alone is not definitive because ofthe possibility of compounds exhibiting the same retention on a given column under a given set of chromatographic conditions. Farkas et al. (1994) published a study useful for the adjustment of operational conditions, enabling high reproducibility of standard relative retention indices for flavor compounds. Kovats RI is generally used as a complementary criterion to reinforce identification determined by spectroscopic techniques such as mass spectrometry (MS). Mass spectral fragmentation pattern is highly characteristic and, when coupled with Kovats RI, provides a powerful tool for compounds identification. Jennings and Shibamoto (1980) have compiled the mass spectra of about 700 aroma volatiles including some found in pear. The GC-MS combination is by far the most applied technique for structural elucidation and reliable identification of aromatic volatile compounds in food and beverages (Maarse 1991), including pears (Shiota 1990; Rizzolo et al. 1991; Horvat et al. 1992; Takeoka et al. 1992; Kjaersgaard et al. 1997; Giintert et al. 1998; Chervin et al. 2000; Oshita et al. 2000; Lo Scalzo et al. 2002; Rapparini and Predieri 2002). Elucidation and confirmation of pear aroma compounds have also utilized nuclear magnetic resonance (NMR) and UV and IR spectroscopic

270

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methods. On the basis of gas chromatographic studies, and on infrared and ultraviolet spectroscopy, Jennings (1961) and Jennings and Creveling (1963) isolated and characterized several alcohols and acids from freshly prepared essence hydrolysates of 'Bartlett' pear esters. Further studies identified (sometimes tentatively) a few of the major volatile compounds of 'Bartlett' pear by using repetitive gas chromatography, infrared, ultraviolet and mass spectroscopy, nuclear magnetic resonance, and melting points (Jennings and Sevenants 1964; Jennings et al. 1964; Heinz and Jennings 1966; Creveling and Jennings 1970). More recently Giintert et al. (1998) applied the same spectroscopic techniques to identify volatile compounds isolated from pear fruits. The use of simple procedures such as acidic or alkaline extraction, hydrolysis, formation of intensely coloured derivatives, hydrogenation, or ozonolysis, as separation techniques prior to gas chromatographic analysis provided additional information on the chemical classes involved (Luh et al. 1955; Jennings 1961; Jennings et al. 1964; Heinz and Jennings 1966; Giannone and Baldrati 1967; Gasca et al. 1969; Creveling and Jennings 1970). Many important volatiles of pear aroma are enantiomeric and in many cases their biosynthesis is stereoselective (Kim and Grosch 1978; Kim and Grosch 1981; Gargouri and Legoy 1998). Determination of the enantiomeric composition of volatile compounds could provide important insights into their biosynthesis. There has been great progress in the stereochemical analysis of enantiomers due to the introduction of modified chiral capillary columns (especially cyclodextrins) and multidimensional gas chromatography (MD-GC). These methods provide useful information not only for achieving a better and accurate characterization of volatiles, but also for gaining a deeper insight into their biosynthetic origins. Only few authors have applied these methodologies to identify some volatiles in pear fruits (Takeoka et al. 1992; Beuerle and Schwab 1997b). Quantification of the identified pear volatiles has been mainly performed by calculating the peak area in the chromatogram obtained with a flame ionization detector (FID) (Romani and Ku 1966; Phan-Chon-Ton 1968; Giannone and Baldrati 1967; Jennings and Tressl1974; Russell et al. 1981; Romani et al. 1983; Shiota 1990; Horvat et al. 1992; Avelar et al. 1994; Imayoshi et al. 1995; Beuerle and Schwab 1997a; Kjaersgaard et al. 1997; Lange 1997; Suwanagul and Richardson 1998b; Chervin et al. 2000; Ju and Curry 2000; Ju et al. 2001). In most cases, the quantification was approximate because no FID response was determined, and only in recent years more accurate measurements have been performed. Quantitative analysis has been performed primarily by using different internal standard methods (Heinz et al. 1965; Jennings and Tress11974; Shiota 1990; Rizzolo et al. 1991; Kjaersgaard et al. 1997; Lange et al. 1997; Suwanagul and Richardson 1998b; Chervin et al. 2000) and/or by using standards

5. PEAR FRUIT VOLATILES

271

(Rizzolo et al. 1991; Avelar et al. 1994; Richardson and Kosittrakun 1995; Park et al. 1999; Ju et al. 2001; Pinto et al. 2001; Rapparini and Predieri 2002). A stable isotope-labeled analogue of the analyte is the most accurate internal standard method when a MS detection system is used (Grosch and Schieberle 1988; Butrym and Hartman 1999). Regardless of the method used, collection and analysis of accurate, truly representative volatile samples is difficult. Extraction and concentration are usually prerequisites to the actual analysis of the volatiles themselves and are difficult procedures compared to chromatographic analysis of the volatile extract (Taylor and Linforth 1994). An important factor in properly interpreting any chromatogram is to understand how the flavor volatiles were concentrated and delivered into the instrument. Therefore, a combined utilization of techniques that have differing chemical affinities for pear volatiles is advisable (Paillard et al. 1970; Rizzolo 1988). Volatile emission from pear fruits has also been studied by laserbased spectroscopic techniques, which utilize changes in physical properties of volatiles. De Vries and coworkers (1997) showed that a laser photoacoustic system can be used to detect and monitor minute changes in concentrations of some low-boiling point volatile compounds emitted by pears, such as ethanol and acetaldehyde. Kharlamov and Burrows (2001a,b) applied the laser photoluminescence spectroscopy, based on the ability of organic volatiles emitted by fruit to luminescence when irradiated by the laser beam. They observed qualitative and quantitative changes of the photoluminescence spectrum during fruit ripening. These nonintrusive techniques could provide a real-time monitoring system of volatiles emitted by fruit. Among the nondestructive assessments of volatiles present in the headspace over a fruit sample, electronic odor detection systems, called "electronic noses," which are based on new chemical sensors, are increasingly being applied (Craven et al. 1996; Abbott et al. 1997). These instruments, utilizing differences in the electrochemical properties of volatiles, are based on the adsorption and subsequent desorption of the compounds released by the sample onto an array of semi-conducting polymer or metaloxide sensors, each characterized by its own degree of reactivity and selectivity (Oshita et al. 1998; Sinesio et al. 2000). This system could be more sensitive and simpler than headspace/GC analysis (Oshita et al. 2000; Young et al. 1999), and find application for discriminating one sample from another based on classes of volatiles, rather than for identification/quantification of individual substances (Maul et al. 1998). Several authors tested electronic noses on different fruits such as strawberry, blueberry, peach, melon, tomato, and apple (Hetzroni et al. 1994; Benady et al. 1995; Simon et al. 1996; Maul et al. 1998;

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F. RAPPARINI AND S. PREDIERI

Brezmes et al. 2000; Sinesio et al. 2000; Di Natale et al. 2001) as well as on pears (Oshita et al. 1998, 2000; Brezmes et al. 2000). Sinesio et al. (2000) found qualitative similarities between the response of an electronic nose system and human sensory measurements in the evaluation of volatiles released by tomato. Oshita et al. (1998, 2000) suggested a strong relationship between the results obtained in pear by headspace GC and the electronic odor detection system used. The electronic nose has been evaluated for nondestructive monitoring of pear fruit ripening process, showing the ability of discriminating fruit of different ripening stages (Oshita et al. 1998, 2000; Brezmes et al. 2000). The low capacity of discriminating odorant molecules, together with the variability due to environmental factors, such as temperature and humidity, limit the application of this technology (Craven et al. 1996). However, if improved, and properly applied, the electronic nose may become a nondestructive technique for monitoring the physiological state (ripening and deterioration) of fruit as well as to replace or supplement classical quality control practice in food chemistry and industry (Oshita et al. 2000; Young et al. 1999; Bremzes et al. 2000). D. Determination of Organoleptic Value of the Identified Volatiles A comprehensive characterization of the volatile profile of fruit includes qualitative and quantitative chemical analysis, together with studies on sensory evaluation of the identified compounds. Chemical data alone does not provide an indication of the relative contribution of the volatiles to the overall aroma in terms of sensorial intensity. Rouseff and Leahy (1995) stress how the most difficult problem is to interpret which combination of components in what proportion is responsible for the perceived aroma. Many of the volatile components are not flavor-active, and other compounds present even in trace amounts can have significant effects (Teranishi et al. 1981). The olfactory system is extremely sensitive; it can detect odors in parts per trillion whereas receptors on the tongue can detect flavors compounds in parts per hundred (Baldwin et al. 2000). Scientific use of human perception and description ability is the basis for a fruitful correlation with analytical data. Chemical, biochemical, and physical interactions of the food constituents occur in the mouth or nose and together with psychological factors, affect perception and evaluation. These interactions make the relationship between instrumental and sensory data intrinsically incomplete (PIotto 1998a; Buettner and Schieberle 2000). Various studies conducted on pear investigated the correlation between chromatographic results and perceived aroma, and led to the

5. PEAR FRUIT VOLATILES

273

identification of pear character impact and contributory compounds. Jennings et al. (1960) showed that after fractionating aqueous essences of processed pears, five fractions contributed significantly to the desirable pear aroma, while four possessed undesirable aromas. Hexyl acetate was reported to be a contributory flavor compound, and methyl-(E,Z)2,4-decadienoate a "character impact compound" for 'Bartlett' pears. Subsequently, also ethyl-(E,Z)-2,4-decadienoate was recognized as a flavor constituent of 'Bartlett' pear aroma and it was shown that the odors of the pure synthetic methyl, ethyl, propyl, butyl, pentyl, and hexyl esters of (E,Z)-2,4-decadienoic acid are described by sensory panelists as possessing powerful pear aromas. The aroma intensity of 'Bartlett' pear essence, as determined by sensory evaluation, correlates very closely with the intensity of absorbance at 263 to 267 nm, which is associated with esters of 2,4-decadienoic acid (Heinz et al. 1964). Studies performed on processed fruits of several pear cultivars show that high concentrations of decadienoate esters are characteristic of 'Bartlett' pear and other cultivars with 'Bartlett'-like flavor (Quamme and Marriage 1977; Quamme 1984). Suwanagul (1996) combined pear flavor intensity evaluation and volatile emission quantification by SPME on 'Bartlett,' 'Doyenne du Cornice,' and 'Anjou' pears. Pear flavor intensity score, was found to have extremely high positive correlations to the release rates of various esters. On the other hand, highly significant negative correlations were observed between pear flavor intensity and the concentration of a compound with a mild oxidized or cooked note (methyl-(E)- 2-octenoate) and two isomers of a-farnesene. The study of odor intensity of pear volatiles has also been approached by means of calculated threshold values of single identified compounds. A known concentration of the aromatic compound is diluted with a neutral agent (Le., water or air) until the detection limit for human sense is reached (Rothe 1988). This threshold value indicates the minimum concentration needed to produce an olfactory response. It is negatively correlated to aroma intensity of volatile compounds, so it can be used as an index of sensory impact. The most frequently reported method consists of calculating the ratio of each single compound concentration to its odor threshold value. Compounds in excess of their threshold value are considered to make contributions to flavor, whereas those below their threshold are thought to have little or no effect. This parameter has been referred to as "aroma value" (Rothe and Thomas 1963), "unit flavor base" (Keith and Powers 1968), "odor unit" (Do) (Teranishi et al. 1991), and "Odor Activity Value" (OAV) (van Gernert 1994). Guadagni et al. (1966), Buttery et al. (1987), Teranishi et al. (1991), and PIotto et al. (1998b) attempted to calculate the odor unit of volatile compounds in

274

F. RAPPARINI AND S. PREDIERI

different food samples. Based on the Uo values Takeoka et al. (1992) found the following compounds to be important in the Asian pear 'Seuri' (P. pyrifolia [Burm.] Nak.) aroma: ethyl 2-methyl butanoate, ethyl hexanoate, ethyl butanoate, ethyl 2-methyl propanoate, hexyl acetate, ethyl heptanoate, hexanal, ethyl pentanoate, and ethyl propanoate. Ethyl(E,Z)-2,4-decadienoate was a relatively small contributor to the aroma of'Seuri' fruit, conversely to what was observed in 'Bartlett' by Jennings and Sevenants (1964). Another method to evaluate the odor significance of individual compounds and to estimate their relative contribution to overall fruit aroma is gas chromatography-olfactometry (GCO). In this method, the effluent of the GC column is split, with one portion of the eluted volatiles flowing to the instrumental detector (generally FID), whereas the remaining effluent is directed to a "sniff port" where individual peaks are tested by a human assessor (Rothe 1988). Volatile compounds are smelled at the exact time they enter the GC detector, and the assessor rates their odor intensity and defines sensory attributes. The GCO technique has been applied in flavor research to identify odor-active compounds in flavor extracts, to describe odor quality of aroma components, and to quantify the odor significance of a compound in flavor systems (da Silva et al. 1994). Advantages and disadvantages of this technique and its different applications have been widely discussed (Acree 1993; Grosch 1993; Acree and Barnard 1994; van Gernert 1994). GC sniffing studies performed by Berger (1991) and Suwanagul (1996) on 'Bartlett' fruits confirmed the odor impact of hexyl acetate and decadienoates (Heinz et al. 1964; Jennings and Sevenants 1964; Jennings et al. 1964), and several other compounds described as having "pear-like" or "fruity" aroma were also identified. Using the GCO, Suwanagul (1996) identified in 'Doyenne du Cornice' and 'Anjou' nine pear-like aroma compounds identical to those found in 'Bartlett,' plus a number of contributory flavor compounds exhibiting slight differences among the three pear cultivars. The efficiency of GCO was highlighted by Rizzolo (1998), who analyzed the odor profile of 'Doyenne du Cornice' using GC/FID, GC/PID (photoionizator detector) and GCO. Of these methods, only GCO detected a compound present at very low concentration that the author indicates as the one responsible for the characteristic aroma ofthis cultivar and tentatively identifies by GC/MS as methyl-2-ethyl-2-methyl butanoate. The use of GCO without the quantification of the assessor response is limited to screening odor active volatiles in a complex sample (PIotto et al. 1998b). A number of applications combine GCO technique with methods able to determine the odor intensity of the chromatographic resolved compounds (Maarse and van der Heij 1994). The most widely used are CharmAnalysis™ (Acree et al. 1984), aromatic extract dilution analysis

5. PEAR FRUIT VOLATILES

275

(AEDA) (Grosch 1994), and asme (the Greek word for smell) (da Silva et al. 1994). CharmAnalysis™ and AEDA are based on dilution techniques and on the determination of the odor-detection threshold values of the compounds eluted from theGC column. Both methods, as odor units, have been criticized because they assume additivity of odor-active chemicals and do not consider synergism or antagonism between compounds (Forss 1981). These techniques also assume a linear correlation between odor activity (sensory perception) and component concentration, ignoring the exponential relationship between these two variables as postulated by Stevens' law (Frijters 1978; Buttner et al. 1999). The asme method, on the other hand, is based on psychophysical estimation of the individual odor intensity of volatiles according to Stevens' law (da Silva et al. 1994). With asme, trained subjects, sniffing the GC effluent mixed with humidified air, directly record the odor quality, intensity and duration of each odoractive compound. Some applications of CharmAnalysis™ (Cunningham et al. 1986; Young et al. 1996) have already been reported in studies on apple volatiles. asme has been successfully applied in the identification of odor-active compounds emitted by fruits of apple cultivar 'Gala' (PIotto 1998a; PIotto et al. 1998b; PIotto et al. 2000). E. Volatiles Found in Pears

Volatiles emitted by pear fruits are primarily esters, alcohols, hydrocarbons, and aldehydes, and many are high molecular weights (Table 5.2). Unsaturated aliphatic compounds are the primary contributors to pear aroma (Paillard 1990). Aliphatic esters are qualitatively and quantitatively the dominant compounds in pear profiles, as in most fruits (Paillard 1990; Sanz et al. 1997). Jennings and Creveling (1963) found that the disappearance of ester compounds from the alkaline hydrolysate of pear essence resulted in the disappearance of desirable pear aroma. Suwanagul (1996) reported that all odor-active compounds identified by SPME sampling of 'Bartlett,' 'Doyenne du Cornice,' and 'Anjou' were esters. When various European cultivars were studied, esters accounted for as little as 60 percent to as high as 99 percent by dynamic headspace analysis (Suwanagul and Richardson 1998b; Rapparini and Predieri 2002). Similar results were obtained in the Asian pear' Seuri,' characterized by a volatile profile dominated by esters, accounting for 66 percent to 97 percent of the headspace vapors (Takeoka et al. 1992). Pear volatiles typically comprise a wide variety of esters, and those formed from even-numbered carboxy chains such as acetic, butanoic, hexanoic, octanoic, decanoic acid, or dodecanoic acid, and ethyl, butyl, or hexyl alcohol, are more typical than esters containing odd-numbered

276

F. RAPPARINI AND S. PREDIERI

chains. Acetate and butanoate esters are the most prevalent volatile compounds in pears. In some European pears, butyl acetate and hexyl acetate are the major acetate esters (Shiota 1990; Rapparini and Predieri 2002), while in Asian pear 'Ya Li' (P. x bretschneideri Redh.) acetate esters are minor components. Furthermore, esterified fatty acids with up to C18 are widely present in European pear aroma profiles, and are saturated and unsaturated with two, or even three, double bonds. The methyl to hexyl esters of (E,Z)-2,4-decadienoic acid are character-impact compounds of 'Bartlett' pear, and other esters (Jennings and Sevenants 1964) including hexyl acetate, 2-methylpropyl acetate, butyl acetate, butyl butanoate, pentyl acetate, and ethyl hexanoate contribute to pear aroma. All these compounds possess a strong "pear-like" aroma (Suwanagul 1996). Additional compounds, ethyl octanoate and ethyl-(E)-2octenoate, contribute to floral, sweet, or fruity aromas (Table 5.3). Table 5.3.

Odor characterization of some volatiles identified from pear fruits.

Compounds Alcohols n-Propanol n-Butanol n-Pentanol (amyl alcohol) 2-Methyl-l-butanol 3-Methyl-3-buten-2-ol n-Hexanol

Odor descriptions

(E)-2-Hexen-l-ol n-Heptanol n-Octanol

Oxidized pear,4 aldehyde 4 Medicinal,5 metallic 7 Roasted 7 Shunky,5 fresh cheese 7 Leafy/ green 7 Oxidized,4 soapy,4 fresh rose,6 fresh grass,6 engine 7 Urinous 7 Potato pee14 Earthy,6 citrus-like 7

Aldehydes Hexanal (E)-2-Hexenal (E)-2-Heptenal 2-Methyl-2-pentenal

Fruity 7 Seasoned cheese 7 B uggy 7 Green,! sharp 7

Ketones 2-Pentanone (methylpropyl ketone) 2-Heptanone 2-Methylcyclopentanone

FloraF Spicy 7 Butter-like 7

Esters Propyl acetate Butyl acetate

Floral,4 estery4 Fruity,3,4,5,6 very fruity,3 estery,4,6 pear,5,6 flora1,5 sweet,5,6 bubble gum,5 very perfume,5 geranium-like 7

277

5. PEAR FRUIT VOLATILES iso-Propyl acetate Pentyl acetate (amyl acetate) iso-Butyl acetate (2-methylpropyl acetate) iso-Pentyl acetate (3-methylbutyl acetate; iso-amyl acetate) Hexyl acetate 4-Hexen-1-01 acetate Heptyl acetate Octyl acetate 4-0cten-1-01 acetate 2-Phenylethyl acetate Ethyl propanoate Ethyl butanoate Ethyl-2-methyl butanoate Butyl butanoate 1-Methylpropyl butanoate 3-Methylbutyl-3-methyl butanoate Ethyl hexanoate Butyl hexanoate 2-Methylpropyl hexanoate Pentyl hexanoate Hexyl hexanoate Methyl-(EJ-2-octenoate Ethyl octanoate Ethyl-(E)-2-octenoate Ethy1-(Z)-3-octenoate Methy1-4-decenoate Ethyl decanoate Ethyl-4-decenoate Ethyl-(Z)-4-decenoate Methyl-(E,Z)-2,4-decadienoate EthYl-(E,Z)-2,4-decadienoate Ethyl-(E,E)-2,4-decadienoate Ethyl-(Z,Z)-2,4-decadienoate Propyl-(E,Z)-2,4-decadienoate Butyl-(E,Z)-2,4-decadienoate Ethyl-2,4-octadienoate Methyl-2,4,6-octatrienoate 1-(E,Z)-3,5-undecatriene

Pear,4,5,6 fruity,4 estery,4.6 sweet,5 candy,6 floral,6 rancid 7 Pear,4 apple,4 fruity,4 bubble gum,5 sweet,5 floral,6 mushroom 7 Candy,4,5 perfume 4,5,6 Pear,:J,4,5,6 floral,4 sweet,4,5 fruity,6 very fruity,4 estery, 6 clove-like 7 Mushroom4,5,6 Fermented 7 Chemical4, solvent-like 4, rancid perfume 6 Perfume,4,6 candy,5 sweet 5 Sweet,5 cand y 5 Fruity,4 caramel,4 milky4 Fruity,4,5,6 very fruity,3 estery,4 floral,5,6 cand y 5 Fruity,5 sweet,5 cand y 5 Pear,4 estery4 Bubble gum,4 candy4 Bubble gum,4 candy,4 sweet,4 floral 4 Pear,4,5 floral,4 fruity,4,5 estery,4 sweet,5 cooked pear,6 oxidized,6 medicine,6 legume 7 Cooked artichoke 6 Mushroom 4 Lemon,6 citrus,6 green (citrus-lea£)6 Floral,4,5,6 candy 6 Oxidized,4 cooked fruit 4 Floral,4,6 sweet,4 cooked apple,5 fruit y 6 Flora1,6 pear:J Fruity,4,5 sweet 5 Anise 6 Fermented food 4 Citrus 5 pear:J Floral,4 estery,4 pear,3,5 fruity,5 ripe pear,6 very fruit y 6 Pear,1,3,4 pear peel,4 green,4,5 cooked pear fruit,5 ripe pear,6 pear blend,6 fruit y 6 Alcohol,4,6 fermented food,4,5,6 sourS Pear,4,6 fruit y4,6 Pearl Pearl Mushroom4 Perfume 5 Balsamic,2 strong fruit y 2

1, Jennings et al. 1964; 2, Berger et al. 1985b; 3, Berger et al. 1991; 4, Suwanagul 1996 ('Bartlett' pear fruits); 5, Suwanagul 1996 ('Cornice' pear fruits); 6, Suwanagul 1996 ('Anjou' pear fruits); 7, Rizzolo 1998

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Alcohols constitute the second largest category of pear volatile compounds. Depending on the cultivar, alcohols account for 1.5 to 14 percent of the total volatiles collected by headspace sampling of pear fruits (Suwanagul and Richardson 1998b; Rapparini and Predieri 2002). Straight-chain aliphatic and saturated alcohols are detected using headspace or distillation methods. Unsaturated C6 alcohols, which have powerful aroma notes, are produced by Asian pears (Imayoshi et al. 1995). Straight-chain saturated aldehydes identified among pear volatiles are generally accompanied by the corresponding alcohols. Numerous authors have identified acetaldehyde and the C6 aldehydes hexanal and (E)-2-hexenal, both generally formed by enzymatic reactions when cellular structures are disrupted and considered responsible for "green" flavor (Drawert et al. 1974; Imayoshi et al. 1995). These aldehydes are major components in solvent extracts from pulp, but not in headspace samples of intact pear fruit (Imayoshi et al. 1995). Acetaldehyde is often considered to be a fermentation product, mostly found when physiological alteration of the fruit occurs, for example under unfavorable storage conditions (Paillard 1990). It is also produced by damage or cutting, and the quantities emitted are proportional to the area of fruit flesh exposed (Paillard 1990). Apart from 3-hydroxy-Z-butanone (acetoin), only a few ketones, mostly straight-chain, have been reported in solvent extracts and headspace samples. Some hydrocarbons, such as benzene, toluene, and xylene can be considered to be pollutants from anthropogenic activities (Kupiszewska and Pilling 1994), and should be viewed with caution as contamination compounds from the environment. Terpenes, which can provide fruity aromas, are mainly represented in pear fruit by limonene and farnesene. Alpha-farnesene has been found to be present in high concentration in Asian pear peel (Shiota et al. 1981) and in 'La France' pear fruits (Shiota 1990), and its (E) isomer has also been indicated as a characteristic compound of Pome fruits (Paillard 1990). Both isomeric forms of this compound were not found to possess any odor characteristics in the GCO tests (Suwanagul 1996). Conjugated trienes, which are considered as oxidation products of the terpene afarnesene, have been isolated in pear fruits by some authors (Chen et al. 1993; Chen and Varga 1997; Zoffoli et al. 1998; Chervin et al. 2000). Among these trienes, a Cl l hydrocarbon, 1-(E,Z)-3,5-undecatriene, which is characterized by a balsamic, pleasant odor with strong fruity undernotes, possesses an ultra-low odor detection threshold and thus may contribute to the overall pear aroma even at low concentrations (Berger et al. 1985a). Several oxygenated terpenoid volatiles, which are important in the flavor of other fruits, have been identified in pears and are mainly repre-

5. PEAR FRUIT VOLATILES

279

sented by monoterpene alcohols, such as a-terpineol, farnesol, and eugenol even their quantities were much lower than those of aliphatic or olefinic alcohols. These monoterpene alcohols are widely used in artificial flavors and fragrances because of their characteristic floral notes (Arctander 1969). Sulphur-containing compounds having unique odor properties such as methyl-3-methylthio-3-propanoate (Berger 1991), have also been identified in pear (Takeoka et al. 1992; Imayoshi et al. 1995; Oshita et al. 1998). Other volatile compounds present in trace amounts and/or in a restricted range of cultivars, and belonging to more infrequent chemical families, should nevertheless be noted. They may play important roles in pear aroma when their odor threshold is low, or when the odor characteristic is distinct from the fruity note imparted by esters (Table 5.4). III. BIOGENESIS

The biogenetic pathways of most of the aromatic components have been described in detail for several fruits such as apple (Yahia 1994; Fellman et al. 2000), tomato (Buttery and Ling 1993; Baldwin et al. 2000), strawberry (Forney et al. 2000b), and banana (Tressl and Drawert 1973), while information for pear is still scarce. European pears, apple, peach, and banana, are typical climacteric fruits. In the preclimacteric stage, ethylene (CzH z) induces biochemical, physical, and chemical changes resulting in increased protein synthesis and changes in enzyme activity (Schreier 1984). These processes lead to an accumulation of metabolites and substrates, such as fatty acids and amino acids, for production of volatiles (Drawert 1974). Furthermore, early in the ripening process, an increase in permeability of cell membranes occurs (Ben-Arie et al. 1979; Brady 1987). This process, that continues through fruit senescence, is characterized by the breakdown of cell wall components and membranes, leading to a loss of compartmentation and an increase in substrates for volatile synthesis (Paliyath and Droillard 1992). During ripening, the metabolism of the fruit changes mainly to catabolic pathways (Tressl and Albrecht 1986), and high molecular weight structures, such as polysaccharides, lipids, proteins, and amino acids, are enzymatically converted to volatile compounds (Schreier 1984; Heath and Reineccius 1986). Extensive studies have demonstrated that the formation of volatiles in various ripening fruits such as pear, apple, and banana is initiated by the climacteric rise in respiration and reaches a maximum during the postclimacteric ripening phase (Heinz et al. 1965; Romani and Ku 1966; Tressl and Jennings 1972; Jennings and Tressl 1974). In 'Bartlett' pears, volatile formation

280

F. RAPPARINI AND S. PREDIERI

Table 5.4.

Odor threshold of some volatiles that have been identified in pear fruits.

Compound

Hexanal

~o

2-Methylpropyl acetate

4.5"·c, 5", 20\ 161

>-\0

3-Hydroxy-2-butanone

Butyl acetate

Odor threshold in water (ppb)

Chemical structure

HO

IO~O

66 b.
~oyo

65 i

0

Hexyl acetate

~O~

115\ 2 b."

0

Butyl butanoate

~O~

100"

0

Ethyl hexanoate

Ethyloctanoate

~O~ ~o~ 0

I b.l,3 g

9i

0

Methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl(E,Z)-2,4-decadienoate

1-(E,Z)-3,5-Undecatriene

~ O-R R= - CH,. - C,H,.. - C,H,•• - C,H 9 •• - C,H ll •• - C6 Hp •

~

Ethyl= 1001

0.0 1-0.02a in air

a-Famesene

aBerger et al. 1985b; bButtery et al. 1982; CButtery et al. 1987; dFlath 8t al. 1967; eGuadagni et a1. 1963; fHall and Anderson 1983; gPyysalo et al. 1977; hTakeoka et a1. 1990; iTakeoka et a1. 1992; iTakeoka et al. 1996.

5. PEAR FRUIT VOLATILES

281

proceeds in a dynamic way during ripening: butyl acetate, hexyl acetate, and a-farnesene, are produced at rates subject to cyclic variations that are out of phase. This suggests that various control mechanisms alternatively activate or inhibit the different pathways leading to production of esters and terpenes. Volatile aromatic compounds produced by fruit are derived primarily from three major plant metabolic pools: lipids, particularly fatty acids; protein, particularly free amino acids; and carbohydrates, particularly monosaccharides and disaccharides (Salunkhe and Do 1976; Sanz et al. 1997). Biosynthesis is further complicated by the fact that some of these volatiles are formed in the intact fruit during ripening ("primary" aroma compounds), while others are produced only when fruit tissue is disrupted, either by slicing, crushing, or even during chewing ("secondary" or "technological" aroma compounds) (Drawert 1974; Schreier 1984). Cellular disruption permits the mixing of the enzyme and substrate, which normally have different subcellular location, thereby resulting in generation of "new" volatiles. A. Lipid-derived Volatiles Many aroma compounds in fruits and plant materials are generated from lipid metabolism (Tressl and Albrecht 1986). The lipid content of pear fruit ranges from 0.3 to 0.7 percent of fresh weight in the peel and pulp, respectively (Kolesnik et al. 1989). The lipids present in 'Bartlett' and 'Bere Ardanpon' pears are characterized by high levels (80 to 90%) of unsaturated fatty acids, which are found in higher concentration in the peel than in the flesh (Kolesnik et al. 1989). Russell et al. (1981) evaluated the role of fatty acids in pear flavor synthesis, incubating unsaturated fatty acids, such as oleic, linoleic, and linolenic acids, with pear puree. Sensory evaluation, ultraviolet spectroscopy, and liquid and gas chromatography showed that flavor compounds were generated when fatty acids were added to 'Bartlett' pear puree, and thus oleic, linoleic and linolenic acids appear to be precursors of the characteristic flavor compounds of 'Bartlett' pear. Synthesis of some flavor compounds also occurred when 'Kieffer' pears were incubated with 'Bartlett' pear enzymes. Although the resulting profile of flavor constituents was not characteristic of either 'Bartlett' or 'Kieffer' pears, a slight increase in the concentration of those compounds most prominent in the 'Bartlett' pear essence was detected.

282

F. RAPPARINI AND S. PREDIERI

Production of volatile flavor compounds from lipids is assumed to occur via two major oxidative pathways: ~-oxidation and the lipoxygenase (LOX) pathway (Sanz et al. 1997). It has been suggested that ~ oxidation is the main metabolic pathway producing primary aromatic compounds in fruits, whereas the LOX pathway may account for the widest variety of secondary aroma compounds from fatty acids in disrupted plant tissues (Schreier 1984). In pear fruits, ~-oxidation is considered to be the most important pathway leading to production of volatiles. Jennings (1961), Jennings et al. (1964), Heinz and Jennings (1966) and Creveling and Jennings (1970) found the fatty acid moieties of volatile esters isolated from ripe 'Bartlett' pears to be straight-chains with even numbers of carbon atoms. The chemical structure, position of the double bonds, and substitution patterns have been established and are consistent with derivation of these products from ~-oxidation of the unsaturated fatty acids found in pear mitochondrial particles (Romani et al. 1965). Jennings and Tressl (1974) proposed a possible ~-oxidation pathway for the biogenesis of mono-, di-, and tri-unsaturated esters specific to 'Bartlett' pear from unsaturated fatty acids (Fig. 5.2). Oleic and linoleic acid are metabolized to various shorter chain-length acyl-CoAs by losing two carbons during each oxidation cycle. Subsequently, these oxidation products react with various alcohols to yield esters. During this process, isomerization may occur to yield various (E), or (Z) isomers. The decadienoate esters are believed to derive via ~-oxidationof linoleic acid (Heinz and Jennings 1966). This pathway has also been reported to operate in other fruits such as apple (Paillard 1979; Bartley et al. 1985; Beuerle and Schwab 1999), banana, and strawberry (Tressl and Albrecht 1986). The ~-oxidation of polyunsaturated linoleic and linolenic fatty acids, as well as their oxygenated derivatives in apple fruit has also been proposed as a possible pathway for the biosynthesis of volatile Cs-diols such as octane-1,3 -diol and its unsaturated analog (Z)-5-octene-1,3-diol (Beuerle and Schwab 1999). Both compounds have been identified in relatively high amounts in pear fruits of several cultivars (Beuerle and Schwab 1997a). Due to their chemical structures, both diols are considered to be intermediates of fatty acid metabolism. Radioactive labeling experiments in apple fruits support this hypothesis, indicating that linoleic acid and linolenic acid are the natural precursors of octane-1,3diols and (Z)-5-octene-1,3-diol, respectively (Beuerle and Schwab 1997b). Derivatives of Cs-diols, 1,3-dioxanes, identified in pear fruit extracts (Beuerle and Schwab 1997a) are formed from octane-1,3-diol, (Z)-5-octene-1,3-diol, (R,Z)-3,7- and (R,S)-3,7-octane-1,3,7-triol, and the

r -

Unolcyl-CoA -

c,pO

!

""'-S-CoA

-2CH 2

~

~C""'-S_COA

!

-2C1/2 -2CH 2 c,pO

~""'-S-COA

!

R-OH~COASI/ ~C~ Methyl and Ethyl cis-5. cis-8 Tetradccadienoate

,pO

,pO

~C""'-o_R

~C""'-S_COA /i-ciS, t::.2-trans

Enoy/-eoA

R-OH

Isomerase

~C,pO

-2CH 2

1

~C--:7°

""'-S-coA

""'-O-R

FAD~

R-OH~COASH

FADH 2

~ """

c~

""'-O-R

CoASH

""'-S-CoA

Acyl-eoA Dehydrogenase

~C--:70

""'- S-CoA

R-OH~COASH ~C,pO ""'-O-R

N

~

Fig. 5.2.

Biogenesis of decadienoate esters

284

F. RAPPARINI AND S. PREDIERI

appropriate aldehydes and ketones in apple and cider (Kavvadias et al. 1999). Although ~-oxidation is considered to be the main pathway for pear volatile biogenesis, it is generally assumed that the widest variety of fruit flavor compounds formed from lipids is generated via the lipoxygenase (LOX) pathway (Heath and Reineccius 1986). Heinz and Jennings (1966) suggested the possible involvement of the LOX pathway in pear aroma biosynthesis as a plausible alternative to ~-oxidation for the production ofCs and ClO ester moieties. Many of the aliphatic esters, alcohols, acids and carbonyls found in fruits are generated from the oxidation of free fatty acids. This LOX biosynthetic system has been demonstrated to act not only in green leaves, as described by Hatanaka (1993), but also in numerous fruits such as tomato (Galliard and Matthew 1977), apple (Fellman et al. 2000), strawberry (Latrasse 1991), and olive (alias et al. 1995). Gardner (1991) provided a comprehensive review of the plant lipoxygenase pathway and its physiological significance. The LOX pathway is considered to be active mainly upon disruption of plant tissue, when polyunsaturated free fatty acids are released from phospholipids by endogenous lipolytic enzymes, acyl hydrolases, and phospholipases. Thus, polyunsaturated fatty acids of plants, such as the membrane-predominating linoleic and linolenic acids, are transformed in the presence of oxygen into enantiomeric hydroperoxide fatty acids with (Z,E)-diene conjugation by the action of lipoxygenase (Gardner 1991). The fatty acid hydroperoxides are then enzymatically converted to aldehydes and oxoacids. Depending on the type of lipoxygenase and plant tissue, either 9- or 13-hydroperoxides or a mixture of both, are produced. Grosch et al. (1977), while incubating pear homogenates with linoleic and linolenic acids, found that the ratio of 13- to 9hydroperoxide was in favor of the 9-isomer. However, in a subsequent study, lipoxygenase purified from pear fruits was found to oxidize linoleic acid to 13- to 9-hydroperoxide in a ratio of 7:3 (Kim and Grosch 1978). The conversion of hydroperoxides to aldehydes and oxoacids represents another enzyme-mediated step at which substrate selectivity is expressed. Among the hydroperoxide metabolizing enzymes, hydroperoxide lyase (HPLS) appears to be the only one to have been investigated in detail in several fruits (Gardner 1991). Hydroperoxide lyases are grouped into three classes based on substrate specificity: 9-, 13-, and nonspecific hydroperoxide lyases. A HPLS isolated from pear fruits has been found to be specific for 9-hydroperoxides, which are cleaved to (Z)3-nonenal and 9-oxononanoic acid, while a small amount of (E)-3nonenal was also reported (Kim and Grosch 1981; Gargouri and Legoy 1998). Because the analytical procedure used by Gargouri and Legoy

5. PEAR FRUIT VOLATILES

285

avoids enzymatic volatile transformation, they suggested that the isomerization of the (Z)-3-double bond to the conjugated (E)-2-derivative is mostly due to nonenzymatic rearrangement. The LOX pathway should be active not only upon disruption of plant tissue, but also in ripening and senescing fruits. Since cell walls and membranes become more permeable to different substrates during these physiological stages the chance of reaction between enzymes and substrates increases, leading to the possibility of emission of volatiles by this pathway (Paliyath and Droillard 1992; Sanz et al. 1997). The synthesis of pear volatile esters from unsaturated fatty acids could explain the superior flavor developed by fruits following low temperature storage which stimulates increased production of the unsaturated acids (Zill and Cheniae 1962; Creveling and Jennings 1970). Romani et al. (1965) observed that, although long-chain fatty acids exist in pears, the unsaturated C18 :2 and C18 :3 fatty acids in pear mitochondrial particles decrease relative to saturated homologous CI6 and CI8 fatty acids during ripening at 20°C. Creveling and Jennings (1970) suggested that during cold storage there may be an accumulation of the unsaturated Cl8 -carbon fatty acids in membrane lipids that may increase the pool of substrates available for volatile production by esterification during subsequent ripening. B. Amino Acid-derived Volatiles

Amino acids also represent an important source of volatile compounds contributing to the primary aroma of fruits (Schreier 1984; Tressl and Albrecht 1986). Changes in free amino acid composition occur during ripening, when characteristic aroma is produced in most fruits (Sanz et al. 1997). In general, the metabolism of amino acids generates alcohols, carbonyls, acids, esters (either aliphatic, branched, or aromatic), and sulphur compounds (Sanz et al. 1997). Although there are no studies on amino acid-derived volatiles in pears, research carried out on other fruits can provide useful information on this biosynthetic pathway. Radioactive labeling experiments on banana (Myers et al. 1970; Tressl and Drawert 1973), tomato (Yu et al. 1968), and apple (Hansen and Poll 1993) have shown that alanine, valine, leucine, phenylalanine, and aspartic acid are converted to branched chain alcohols, esters, and acids. The initial steps in amino acid catabolism are deamination followed by decarboxylation giving rise to aldehydes. Various reductions, oxidations, and esterifications then can lead to alcohols, carboxylic acid, or esters, respectively (Drawert 1975; Heath and Reineccius 1986). The aromatic amino acids tyrosine and phenylalanine may be precursors of some minor volatile compounds identified in pear fruit aroma.

286

F. RAPPARINI AND S. PREDIERI

Phenylalanine is converted to ~-phenylethanol, ~-phenylethyl acetate, and ~-phenylethyl butanoate in banana in the same way as described above for aliphatic amino acids (Tressl and Drawert 1973). Both phenylethanol and its esters have been found as minor components in pear fruits. Phenylalanine is the main product of shikimic acid metabolism and is critical in the biogenesis of phenolic compounds. The mechanism involves the transformation of phenylalanine into activated cinnamic acids, which can be further reduced, oxidized, esterified, or isomerized to generate different volatile compounds (Schreier 1984). Among these cinnamic-derived compounds, the allyphenol eugenol and volatile compounds derived from benzoic acids such as benzaldehyde and benzenethanol, have been identified in pear fruit aroma. C. Carbohydrate-derived Volatiles

Although one may consider that all plant volatile flavors ultimately derive from carbohydrates, as they are the primary compounds produced by photosynthesis, only a few classes of volatile aromatic components in fruits originate directly from carbohydrate metabolism (Sanz et al. 1997). These include terpenes and furanones, both classes produced by pear fruits. Terpenes, mainly monoterpenes and sesquiterpenes such as a-farnesene, are derived from mevalonic acid through two independent pathways: the cytosolic acetate-mevalonate pathway (sesquiterpenes) and the alternative, nonmevalonate 1-deoxy-D-xylulose-5-phosphate pathway, also referred to as the methylerythritol phosphate (MEP) pathway (isoprene, mono- and diterpenes) (Eisenreich et al. 1998; Lichtenthaler 1999). Additional interconversion of terpenes can occur via several pathways. D. Other Biosynthetic Pathways

The intermediate carbonyls produced by the aforementioned pathways can be enzymatically converted to the corresponding alcohols by alcohol dehydrogenase (ADH). The ability of some fruits to convert aldehydes to alcohols has been demonstrated (Hamilton-Kemp et al. 1996; Yamashita et al. 1977), and ADH has been isolated and characterized from various fruits (Bartley and Hindley 1980; Longhurst et al. 1990; Mitchell and Jelenkovic 1995; Chervin et al. 1999). This enzyme not only leads to aroma changes by supplying the precursors for the highly flavored esters, but also maintains the fruit tissues in a reduced state, which is important because many oxidation processes are necessary for aroma production (Yahia 1994). ADH plays an important role as enzymic sequence of lipoxygenase and hydroperoxide cleavage activity in the formation of volatile C6 com-

5. PEAR FRUIT VOLATILES

287

ponents in disrupted plant material (e.g. during fruit processing) (Schreier 1984). These compounds are considered to be responsible for the "green" odor notes in plant products (Hatanaka 1993). Together with the enzyme pyruvate decarboxylase (PDC), ADH is responsible for acetaldehyde and ethanol production (Ke et al. 1994a). Acetaldehyde is synthesized through the decarboxylation of pyruvate by PDC, and then reduced to ethanol by ADH using nicotinamide adenine dinucleotide (NADH) as a cofactor. Ethanol is the major end product of anaerobic metabolism (fermentation) (Davies 1980) and, together with acetaldehyde, accumulates in pears under imposed hypoxia and poor gas exchange in ripening tissues (Ke et al. 1990; Nanos et al. 1992; Ke et al. 1994a; de Vries et al. 1997; Lange 1997; Pinto et al. 2001). Ke et al. (1994a) suggested that low O 2 and/or high CO 2 concentrations may induce fermentation in pear fruits by one or more of the following means: (1) increased amounts of PDC and ADH due to de novo biosynthesis; (2) PDC and ADH activation caused by decreased cytoplasmatic pH; and (3) PDC and ADH activation and more rapid fermentation due to increased substrate and/or cofactor concentrations (pyruvate, acetaldehyde, or NADH). 'Bartlett' pears under hypoxia had higher ethanol concentrations and increased ADH activity (Nanos et al. 1992; Ke et al. 1994a). The increase in PDC and ADH activities under limited O 2 as an indirect response to decreased cellular pH has also been suggested in strawberry fruit (Ke et al. 1994b) and other plant tissues (Davies 1980; Roberts 1989). Subsequent studies carried out by Chervin et al. (1999) indicate that ADH does not limit ethanol production during pear ripening and that induction of the expression of this enzyme may be regulated at the posttranscriptionallevel. Increased transcription and translation found for ADH isozymes may be the cause of higher levels of ADH activity when anoxic conditions are present. E. Ester Formation

Although esters are qualitatively and quantitatively one of the main groups of volatile compounds in fruit aroma, there are few reports on the biochemical aspects of ester formation in fruit. However, this biosynthetic pathway has been well characterized for microorganisms (Sanz et al. 1997). Various esters are formed after incubation of whole fruit or tissue slices with aldehydes, alcohols, or acids as reported in banana (Deda et al. 1971), strawberry (Yamashita et al. 1977), and apple (Knee and Hatfield 1981; Bartley et al. 1985). Two enzymes seem to be involved in regulating the level of net ester production in fruit: alcohol acyltransferase

288

F. RAPPARINI AND S. PREDIERI

(AAT) and esterase. AAT catalyses the esterification of alcohols and acids by transferring an acyl moiety of an acyl-Coenzyme A (CoA) to the corresponding alcohol, while esterase hydrolyses esters. Two factors determine in large part the volatile ester composition in fruit: availability of the substrates, acyl-CoAs and alcohols, and the properties of the AAT and esterase enzymes (e.g., substrate specificity). The amount of enzyme activity present is also a determining factor. Esterase has received little attention compared to AAT, and has been described and isolated in few studies on fruit (Knee and Hatfield 1981; Goodenough and Entwistle 1982; Goodenough 1983; Goodenough and Riley 1985; Knee et al. 1989). In apple fruits, the hydrolysis of esters by esterase has been considered to play an important role in the production of volatiles, since its activity could be responsible of increased precursor availability, in particular alcohols, for subsequent metabolic processes (Goodenough 1983; Fellman et al. 2000). Esterase activity is particularly high in the peel of apple fruit such that the volatiles diffusing out of an apple are enriched in alcohols (Knee 1993). Goodenough and Entwhistle (1982) isolated an esterase of high molecular weight whose activity increased during fruit development and ripening. However, in subsequent studies, Knee et al. (1989) measured a constant esterase activity. AAT has been the subject of several reports and has been isolated, purified, and characterized in different fruits (Harada et al. 1985; Perez et al. 1993; Olias et al. 1995). Suwanagul (1996) showed that AAT activity purified from 'Bartlett' pear increases with the onset of fruit ripening and then decreases. Maximum activity corresponded to optimum eating ripeness of the pears as measured by flesh firmness. Pear AAT exhibited alcohol substrate specificity. Enzyme activity increased as the alcohol carbon number increases from 1 to 6 and then declined. No enzyme activity was detected when secondary alcohols were tested, while the enzyme was more active with straight-chain alcohols as substrate than with branched-chain alcohols with the same carbon number. The alcohol substrate specificity of AAT observed by Suwanagul (1996) is consistent with pear volatile profile: the higher activity of pear AAT enzyme for hexyl alcohol is expected, as hexyl acetate is a dominating ester in pear aroma, and low specificity for secondary alcohol is consistent with the lack of esters with secondary alcohols in pear volatile profiles. On the other hand, the acyl-CoA substrate specificity of pear AAT is inconsistent with pear volatile profiles. A higher enzyme activity for lower carbon number of acyl moieties was observed. These findings are a curious feature of pear AAT because the 'Bartlett' impact compounds (2,4 decadienoate esters) are likely derived from the unsaturated ten carbonyl acyl moiety.

5. PEAR FRUIT VOLATILES

289

IV. FACTORS AFFECTING QUALITATIVE AND QUANTITATIVE EMISSION OF PEAR VOLATILES A. Genetic Differences Although all Pyrus species basically produce the same aroma compounds, as analytical techniques have become more sensitive and the number of identified volatiles increases, differences among cultivars are becoming more apparent. In early study, Giannone and Baldrati (1967), demonstrated differences in the aroma profiles of 18 compounds of the European pears 'Bartlett,' 'Bose' (syn. 'Beurre Bose,' 'Kaiser'), 'Diel,' 'Olivier de Serres,' 'Curato,' and 'Passe Crassane.' More recently, Suwanagul (1996) and Suwanagul and Richardson (1998a,b) studied emission of volatiles by eight pear cultivars. Thanks to the improved efficiency of the analytical methodology available to these authors, they could delineate a high-definition volatile profile for each cultivar and detect consistent differences among cultivars. In fact, on a total of 112 volatile compounds identified, over 40 percent were reported for the first time in pear fruit, thus significantly increasing the possibility of discriminating among cultivars by aroma analysis. While all the cultivars tested were found to produce decadienoate esters, the differences involved the total amount of volatiles produced and the relative contribution of individual compounds to the volatile profiles. Among the cultivars tested, 'Doyenne du Cornice' had the highest emission rate with 42 l-lg/kg/100L of air, while 'Seckel' emission was 60 times lower (0.7 l-lg/kg/100L of air). Hexyl acetate was the ester present in the highest amount in 'Bartlett,' 'Packham's Triumph,' 'Anjou,' 'Doyenne du Cornice,' and 'Forelle.' In the case of 'Bose' and 'Vicar of Winkfield,' the main volatile was butyl acetate, while for 'Seckel' it was ethyl-(E,Z)-2,4decadienoate. Differences among cultivars also have been found in other classes of volatiles. Octane 1,3 dial and (Z)-5-octene-1,3-diol were found in seven pear cultivars ('Packham's Triumph,' 'Alexander Lucas,' 'Gute Luise,' 'Gellerts,' 'Madame Verte,' 'Vereinsdechant,' and 'Bartlett') (Beuerle and Schwab 1997a). However, 'Vereinsdechant' and 'Bartlett' synthesized also ethyl-3-hydroxyoctonanoate, and only 'Bartlett' produced methyl3-hydroxyoctonanoate and ethyl-(Z)-5-hydroxyoctonanoate. The two European pears most commonly grown in]apan, 'Bartlett' and 'La France,' showed many differences in volatile composition and quantity (Shiota 1990). 'La France' produced primarily acetates oflow molecular weight: ethyl acetate, propyl acetate, butyl acetate, and hexyl acetate, which accounted for about 70 percent of the total. Alcohols constituted the majority of the remaining volatiles. Ethyl-(E,E)-2,4-decadienoate was

290

F. RAPPARINI AND S. PREDIERI

produced by 'La France,' but not its isomer, (E,Z)-2,4-decadienoate, an important component of 'Bartlett.' 'La France' also emitted 5-hexenyl acetate, an unusual component of fruit aroma, hence the "bleu cheese" note that is a sensory trait characteristic for this cultivar. Another typical 'La France' aroma trait, the "peely fresh green" note was attributed to a-farnesene. Takeoka et al. (1992) studied the volatiles emitted by the Asian pear 'Seuri' and detected a wide variety of esters including propanoates, butanoates, 2-methyl butanoates, pentanoates, hexanoates, heptanoates, and octanoates. Ethyl-2-methyl butanoate was found to be an important contributor to the aroma. Several other esters contributed significantly to perceived aroma, but ethyl-(E,Z)-2,4-decadienoate was not among them, a clear difference between Asian and European pears. Horvat et al. (1992) characterized the volatile profile of the Asian pear cultivars 'Chojui,' 'Hosui,' 'Kosui,' 'Shinko,' and 'Va Li.' Of the 17 compounds identified, only five (toluene, ethyl butanoate, hexanal, (E)-2hexenal, and a-farnesene) were found in all cultivars, and consistent differences in the relative amounts of individual compounds were observed. Conversely 2-butyl-2-octenal was the main volatile in 'Chojui' and 'Shinko' (respectively 31.9% and 22.5% of the total volatiles) but was not detected in the other cultivars. These differences suggest that high variability in volatile production is present among Asian pear cultivars. Imayoshi et al. (1995) found ethyl acetate (31.85%), ethyl butanoate (18.05%), and ethyl hexanoate (4.27%) as the major esters emitted by 'Va Li,' yielding an ester profile quite different from European pears (Shiota 1990; Suwanagul and Richardson 1998b). Nonetheless, ethyl (E,Z)-2,4-decadienoate was found. A sniffing study of these volatiles indicated that some quantitatively minor components, the unsaturated alcohols (E)-3-decenol and (Z)-4-decenol, are important components of characteristic fruity aroma of 'Va Li.' Bell et al. (1996) state that the study of flavor compound synthesis and genetic control is an important objective for pear breeding work, since a fine aroma is a key for the success of any new cultivar. Quamme and Marriage (1977) and Quamme (1984) studied volatile profiles of several pear cultivars and selections on the basis of emission by canned fruits. Their primary goal was to determine if a high decadienoate content could be used as a marker in cross-breeding programs to select pears for processing. The relationship between the amount of decadienoate esters and the desired 'Bartlett'-like aroma was studied by comparing sensory tests and analytical measurements. Of 29 genotypes tested, only mutants of 'Bartlett,' the selection 'HW-606' and the cultivars 'Harvest Queen,' 'Laxton's Progress,' and 'Surecrop' were characterized by high deca-

5. PEAR FRUIT VOLATILES

291

dienoate ester levels. Sensory evaluation of these genotypes indicated all as having a 'Bartlett'-like aroma, with the exception of 'Surecrop.' This suggests that high production of decadienoate esters alone does not result in 'Bartlett'-like aroma. All the tested cultivars and selections that proved to have a 'Bartlett'-like aroma had 'Bartlett' as a parent, and Quamme (1984) concluded that the probability of selecting seedlings with 'Bartlett' flavor without using 'Bartlett' as a parent was extremely low. Russell et al. (1981) studied 'Bartlett' volatiles to study the inheritance of aromatic traits and breeding pears combining resistance to fire blight (Erwinia amylovora) and a satisfactory flavor. As a preliminary step, a sensory analysis of several cultivars was performed, and three cultivars that were highly, intermediately, or poorly flavored were identified, namely 'Bartlett,' 'Magness,' and 'Kieffer,' respectively. Following this assessment, the volatile composition was studied in detail. 'Bartlett' was clearly different from the other cultivars, suggesting its complex volatile profile is the basis of its unique flavor. In fact 'Bartlett' produces both the greatest variety and the highest concentration of high-boiling point flavor compounds. In contrast, 'Magness' and 'Kieffer' have a greater concentration of low-boiling point constituents. The comparison of the results obtained from the analysis of the latter two cultivars is an example of the imperfect correlation between volatile profiles and sensory perception. In fact, despite the sensory rating, 'Kieffer' had more volatile constituents than 'Magness.' The feasibility of early selection for fruit quality, based on decadienoate emissions from vegetative organs of prefruiting seedlings has been evaluated (Quamme 1984). The juvenile period of pear seedlings deriving from cross-breeding entails a minimum waiting period of four years, but usually much longer, before flowering (Zimmerman 1977), thus prefruiting screening for traits correlated to fruit quality would be useful. Unfortunately, decadienoate esters were not detected in essences extracted from actively growing or dormant shoots of 'Bartlett', thus an early selection based on decadienoate ester content of the leaves and stem tissue was not feasible (Quamme 1984). To our knowledge, specific studies on the genetic basis of pear aroma have not yet been conducted. However, low heritability has been estimated for flavor traits, and the major genetic determinants of flavor have been judged to be of an additive nature (Bell and Janick 1990). Analytical studies on volatiles of 'Bartlett' mutants were reported only by Quamme (1984). The three mutants 'Parbarton,' 'Stewart's Bartlett,' and 'Maxred' scored a similarity to typical 'Bartlett' aroma higher than the original cultivar, although having lower levels of decadienoates. Mutants described as having qualitative changes in perceived aroma have been

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F. RAPPARINI AND S. PREDIERI

reported, mostly for 'Bartlett.' They were either spontaneously originated such as 'Bhiel,' 'Cinnamon' (Sansavini et al. 2000), or were induced by y-ray mutagenesis (Predieri and Zimmerman 2001) such as 'William Ramada' described in Sansavini et al. (2000). 'Bartlett' mutants with changes in aroma intensity, as perceived by taste panelists, were reported also by Predieri and Bogoni (1998). Nearly all these mutants of 'Bartlett,' and of other cultivars described by Le Lezec (1998), associated changes in aroma to an increase in fruit russet. A correlation between volatile emission and skin characteristics can be hypothesized, since pear volatile production is highest in the skin and nearby tissues (Berger 1991; Chervin et al. 2000); the skin is reported to be the main barrier to diffusion of gases to or from cortical cells (Knee 1991), and the cuticle affects volatile emission (Baur et al. 1996). The metabolic pathways responsible for the synthesis of aroma compounds are complex and often integrated with aspects of primary and secondary metabolism. However, with the help of appropriate technologies, new breakthroughs in the understanding of these processes could be achieved. Studies to identify genes involved in production of volatile compounds in fruits are reported for tomato (Griffiths et al. 1999) and strawberry, where a gene controlling aroma synthesis has recently been identified (Aharoni et al. 2000).

B. Environmental Effects 1. Preharvest Factors. The effects of preharvest factors on pear quality have been investigated (Wrolstad et al. 1991) and differences in flavor among production areas were reported as early as 1958 by Claypool et al. However, comprehensive studies on the effect of environmental factors on volatiles production have not been reported for pear as they have for other fruit crops including apple (Yahia 1994; Ferrandino et al. 1999; Fellman et al. 2000; Forney et al. 2000a). Several environmental and cultural factors affect fruit development in the orchard and create the potential for volatile emission during ripening. These include water, sunlight, nutrient availability, and crop load (Mattheis and Fellman 1999). Each fruit species can have a unique response to preharvest conditions, and this determines the efficacy of a specific cultural technique or the excellence of a particular production area. In 'McIntosh' apple high nitrogen treatments were found to increase volatile production (Somogyi et al. 1964); while in 'Anjou' (syn. 'Beurre d'Anjou,' 'd'Anjou') pear, flavor was reported to decrease with increased nitrogen fertilization (Raese 1977). 'Bartlett' fruit quality was unaffected by nitrogen fertilization (Shackel et al. 1999).

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2. Maturity at Harvest. The capacity of fruit to produce volatiles is heavily regulated by harvest date. Delaying harvest can increase aroma (Nielsen 1993). The use of early harvested (mature-green) 'Bartlett' fruit as compared to fruit harvested later (partially-ripe) for fresh-cut processing resulted in slices that were less susceptible to browning, but with eating quality compromised by lack of juiciness and fruity aroma (Gorny et al. 2000). Early-harvested 'Cascade' pears were incapable of developing acceptable flavor (Ma et al. 2000). Ester production was observed to increase with harvest date in 'Doyenne du Cornice' but did not show a clear trend in 'Conference' (Eccher-Zerbini et al. 1993). Suwanagul (1996) found no differences in 'Bartlett' pears harvested at different maturity stages (early, optimum, and late); while, for 'Packham's Triumph' and 'Anjou,' higher emissions were recorded for fruit harvested early. Specific studies need to be conducted to properly identify the optimum harvest date for each cultivar, in order to couple high flavor volatile emission with avoidance of storage disorders. However, harvest date is often not sufficient for indicating fruit physiological state at harvest. An analysis of relationship between maturity at harvest and potential volatile emission requires the availability of reliable maturity indices. Pears are usually harvested commercially before they become ripe for eating. Some fruit quality traits (Le., lower content of stone cells), together with fear of loss of fruits through abscission, senescence, pathogen attack, wind, or hail storms encourages growers to harvest less mature fruits (Knee 1993). Pears harvested when they are immature fail to ripen properly and to express their full flavor potential. Development of usable maturity indices has progressed further for apple than for pear (Kingston 1992). Wrolstad et al. (1991) report that the most used index for commercial harvest is days from full bloom, as a rough indicator to be tuned with the integration of fruit testing. Easy to measure fruit parameters correlated to fruit maturity such as titratable acidity are useful for apple (Lau 1988) but not for pear (VangdaI1982). Pear soluble solids content at harvest can provide useful information (Vangda11982, 1985), but it is influenced by a number of factors causing variation between and within trees, including position of fruit on the tree (Wang 1982; Roelofs and de Jager 1997; Benitez et al. 1998; EccherZerbini et al. 2000). Fruit color changes are correlated to fruit ripening in Asian pears (Wrolstad et al. 1991) but are not reliable indicators for European pears (Wang 1982; VangdaI1985). Fruit flesh firmness is more directly correlated to pear maturity (Wang 1982), and is commonly used for the determination of optimal harvest date for each cultivar (Wang 1982; Wrolstad et al. 1991; Bertolini and Folchi 1993; Richardson and

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Gerasopoulos 1994), although the effectiveness of this parameter could be increased by integrating it with other indices (Mann and Singh 1985; Nielsen 1993). Volatiles may also playa direct role in the issue of indices of maturity, or better, of the determination of physiological maturity, which was defined by Watada et al. (1984) as "the stage of development when a plant part will continue ontogeny even if detached." Volatile production can be evaluated for monitoring fruit development by nondestructive methods and predicting appropriate harvest date. Abbott et al. (1997) describe relatively low-cost electronic sniffers, that can be taken to the field for sensing fruit volatile production prior to harvesting. At present, a major disadvantage for the use of such sensors is the long time required to perform a single measurement, and then purge the sensor to perform the next. However, this nondestructive screening approach may be useful for future commercial operations once analysis times become rapid enough to permit real-time analysis on industry and sorting lines (Maul et al. 1998). Encouraging results have been reported in apple (Song et al. 1997) and could potentially apply to pear. 3. Storage Conditions. In all major pear-growing areas, the bulk of the crop is stored for several months to maintain availability and organize marketing as economically desirable. For many pear cultivars, storage at -1°C is so effective in delaying ripening that no other treatment is necessary, so fruits are stored in normal atmosphere (NA) (Knee 1993). However, controlled atmosphere (CA) is widely used, and recommended CA conditions for the most important pear cultivars have been reported by authors such as Bertolini and Folchi (1993) and Richardson and Kupferman (1997). Atmosphere modification, the manipulation of O 2 and CO 2 levels, affect the production of volatile esters of a number of fruits including pear (Beaudry 1999; Pint6 et al. 2001). Aroma compounds that confer characteristic odors are generally suppressed by storage in a low O 2 atmosphere. Beaudry (1999) ascribes this effect to the influence of O2 concentration on responses to ethylene in climacteric fruits and on oxidative processes, including respiration. Brackmann et al. (1993), while discussing the suppression of aroma production after CA storage in apple, stated that the problem is not fully understood despite a number of studies that have found several connections with fruit physiology, including respiration, availability of alcohols as precursors, lipid metabolism and synthesis, and changes in sensitivity to ethylene. In pear, Chervin et al. (1999, 2000) noticed a slight decrease in aroma intensity of 'Packham's Triumph' fruits after short-term storage under low O 2 concentration, as compared to fruits stored in NA. The perceived

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decrease in aroma was measured on whole fruits as a consistent reduction in emission of ethyl and methyl decadienoate esters. By grouping the obtained data according to their alcohol or acyl precursors, no differences in either methyl or ethyl groups were found. These authors postulated that the observed low presence of decadienoates was due to reduced synthesis of the decadienoate moiety. They also indicated two steps of decadienoate moiety synthesis, via lipoxygenase, requiring Oz availability and thus possibly affected by imposed hypoxia. Besides their work on whole fruit, the experiments of Chervin et a1. (2000) also included emissions from crushed, homogenized pear flesh, and reported the reduction in two additional esters, heptyl and phenylethyl acetate, possibly due to residual effects oflow Ozlevels on the disrupted tissues. Technologies used in storage could also affect volatile emission. Fruit surface coatings (Amarante and Banks 2001) affect skin gas permeability, consequently generating modifications in water loss, volatile emission, and fruit internal atmosphere. Alterations in fruit physiology resulting from the application of coatings are aimed at achieving beneficial effects analogous to those obtained with the use of controlled atmosphere. However, it should be considered that any modification of fruit internal atmosphere may induce anaerobic respiration resulting in development of off-flavors (Meheriuk and Sholberg 1990). Storage conditions immediately preceding retail marketing (shelf-life) can influence volatile production. In experiments on 'Decana d'Inverno' ('Doyenne d'Hiver') pears, Grazianetti (1998) observed that after six months storage in CA, prolonging storage with 15 days in NA caused a decrease in basic quality traits, including aroma. The use of modified atmosphere (MA) packaging technology, as a means to slow the processes of ripening and senescence at various steps of storage, transport, and marketing, can affect volatile production (Mattheis and Fellman 2000). Changes in apple ester production have been reported (Brackmann et a1. 1993). Experiments on 'Conference' (Geeson et a1. 1991a) and 'Doyenne du Cornice' pears (Geeson et a1. 1991b) have shown unsatisfactory results in terms of flavor and aroma production by MA-stored pears. Kjaersgaard et a1. (1997) evaluated volatile emission of 'Clara Frjis' pears previously held in closed, perforated, or open polyethylene bags. Pears in closed bags had higher production of off-flavor compounds (ethanol and ethyl acetate) and pears in open bags had higher production of volatiles (excluding ethanol and ethyl acetate). Flavor volatiles showed a characteristic "climacteric-like" peak. Sensory evaluation indicated that fruit held in perforated or openbags had the highest rating of fruity flavor and recorded the highest production of acetate esters. Toivonen (1997) highlights the importance of

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the study of nonethylene volatiles occurring in packaged products, including the approach of investigating selective absorbing substances able to remove only off-flavor volatiles. C. Fruit Physiology 1. Intra-fruit Volatiles Location. Studies on volatiles emitted by various fruit species have shown differences in volatile production among different fruit tissues (Paillard 1981), with production determined to be highest in the skin and nearby tissues. In studies conducted on apple, Guadagni et al. (1971) observed peels producing higher concentrations of esters than whole peeled or unpeeled fruits. In 'Bartlett' pears a higher amount of volatiles were detected in epidermal tissue than in parenchyma (Berger 1991). This pattern was coincident with more pronounced differences in the concentration of some esterified unsaturated CI8 fatty acids (methyl oleate, linoleate, linolenate), which can be important biogenetic precursors of volatiles. In 'Packham's Triumph,' Chervin et al. (2000) found that flavor volatile compounds were located mainly in the pear skin, since they did not detect esters other than acetates in crushed pear flesh lacking skin. In Asian pear 'Ya Li,' Imayoshi et al. (1995) found differences in the total number of volatiles present in pulp (68), in peel (77), and those released by intact fruits (44). Alphafarnesene was the most abundant volatile emitted by intact fruits (6.2%), while it was a minor component of volatiles in pulp. On the other hand, two of the major components of pulp, hexanol and hexanal, were not found among those emitted by intact fruits. However, it must be considered that these compounds can be enzymatically derived by peeling and slicing during sample preparation of pulp. 2. Ripening. European pear is a climacteric fruit. It is so defined because it exhibits a peak in respiration and ethylene production during its ripening process. When the fruit reaches physiological maturity and ripening processes start, respiratory activity increases considerably, increasing CO 2 evolution. Ethylene emission follows a pattern similar to the one of CO 2 , and is a major factor in regulating fruit ripening. A complete report on ethylene is out of the scope of this review, since ethylene biosynthesis and its role in fruit ripening is a regular subject of comprehensive reviews (Brady 1987; Abeles et al. 1992; Tucker 1993; Oetiker and Yang 1995; Fluhr and Mattoo 1996; Lelievre et al. 1997a; Yueming and Jiarui 2000; Giovannoni 2001), including most of the studies conducted on pear as related to endogenous fruit production and exogenous treatments (Hansen 1967; Wang et al. 1971; Looney 1972; Wang et al. 1972; Sfakio-

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takis and Dilley 1974; Chen and Mellenthin 1981; Knee et aL 1983; Blankenship and Richardson 1985; Wang et al. 1985; Yoshida et al. 1986; Knee 1987; Toumadje and Richardson 1994; Puig et al. 1996; Chen and Varga 1997; Elgar et al. 1997; Gerasopulos and Richardson 1997; Lelievre et al. 1997b; Agar et al. 1999, 2000). Ethylene will be herein considered only in its relationship to other pear volatiles. The sharp increase in climacteric ethylene production is considered as controlling the initiation of aroma biosynthesis and of many other biochemical and physiological processes (Lelievre et al. 1997a). Initiation of ripening is induced by a threshold concentration ofintemal ethylene (Chen and Mellenthin 1981), reported for pear to be 40 J.lL/L (Tucker 1993). However, ripening should be considered as a coordinated, rather than as a linked, set ofbiochemical pathways (Tucker 1993; Giovannoni 2001). Watada et al. (1984) define ripening as the stage "when a commodity possesses characteristic aesthetic and/or food quality." Volatile emission in pear fruits and its connection with ripening has been most studied using 'Bartlett.' In one of the earliest studies conducted on pear, Luh et al. (1955) showed that volatiles of 'Bartlett' included methyl alcohol, total carbonyl compounds, acetoin (3-hydroxy-2-butanone), diacetyl (2,3-butanedione), and that ester content gradually increased with ripening. Claypool et aL (1958) found volatile reducing substances to increase greatly during ripening of 'Bartlett' fruits, with higher emission by fruits ripened at 20°C as compared to the emission by those ripened at 25 and 30°C. Heinz et aL (1965) studied the emission trend of decadienoate esters as related to the climacteric peak of respiration. The increase of decadienoates continued as ethylene and CO 2 production declined. One to two days after the climacteric peak the maximum emission of decadienoates was detected and fruits were rated as having the highest eating quality. Jennings (1972) found that in fruits ripening at 20°C the maxima of CO 2 and ethylene production preceded the highest production of decadienoate. Jennings and Tressl (1974) observed that production of volatile organic compounds by 'Bartlett' pears ripening at 25°C exhibited cyclic variations, especially in the level of the major estersacetates and decadienoates-and the terpene a-farnesene. In particular, the production of the decadienoates was observed to start in the preclimacteric ripening phase, reaching the first maximum at the climacteric rise of respiration. These findings have been confirmed by Shiota (1990) who reported that the emission of volatiles from 'Bartlett' fruit does not increase linearly from an unripe stage to fully ripe stage, although having a maximum just before the ripe stage. The delay of fruit ripening on the tree induced by exogenous growth regulators may reduce premature abscission and provide flexibility for

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scheduling of harvest, packing, and cold storage (Clayton et al. 2000). Some studies have shown how these treatments can have negative effects on fruit quality and flavor volatiles. Romani et al. (1983) found that a preharvest treatment with 400 ppm aminoethoxyvinylglycine (AVG), applied to inhibit ethylene synthesis, thereby delaying the ripening of 'Bartlett' pears, resulted in a prolonged preclimacteric phase of fruits stored at 20°C. A marked suppression (about 50%) of the production of volatiles including methyl, ethyl, and hexyl acetate was also observed. However, in a more recent study conducted by Clayton et al. (2000), AVG was used in combination with naphthalenacetic acid (NAA) , at concentrations lower than those used by Romani et al. (1983), resulting in good quality parameters, although specific evaluation of aroma or measurement of volatile emission has not been reported. Treatments with ethylene have shown a potential for synchronizing fruit ripening (Puig et al. 1996); and, although no associated studies on volatile emission were performed, fruits subjected to sensory analysis received high ratings for flavor. Gamma-irradiation, applied to inhibited pear ripening, also reduced production of 2,4-decadienoate esters, resulting in flavorless and abnormal fruit (Heinz et al. 1965). Shiota (1990) investigated the changes in the composition of volatile components of 'La France' pear fruits during maturation, and found that the concentrations of esters, especially ethyl, propyl, butyl, and hexyl acetates, increased markedly with fruit maturity. When these results were compared with the aroma volatiles of 'Bartlett' pears at similar maturity stages, a greater release of esters from 'La France' pear fruits was observed. Suwanagul (1996) followed volatile emission from 'Bartlett,' 'Packham's Triumph,' and 'Anjou' pears during ripening at 20°C. While esters and alcohols increased with ripening, a concomitant decrease of hydrocarbons and a-farnesene was observed. If pears reach horticultural maturity during storage and possess the prerequisites for utilization by consumers (Watada et al. 1984), the final steps before actual fruit consumption can be crucial for optimizing emission of volatile flavor compounds. Rapparini and Predieri (2002) showed that variations in volatile emission of 'Harrow Sweet' pears are dependent on shelf-life duration (ripening for four or five days at 25°C following cold storage) and fruit temperature during consumption (fruit kept at 19, 25 or 32°C for 90 minutes prior to volatile analyses and taste panel). In general, 'Harrow Sweet' exhibited progressively higher rates of volatile release with increasing temperature at both shelf-life durations. This was expected, since the release of these compounds is primarily driven by their vapor pressure, and the temperature rise

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accelerates enzymatic reactions correlated with volatile synthesis and release. The aroma profile of 'Harrow Sweet' was characterized by the presence of decadienoate esters, the emission of which was found to be close to that observed in 'Bartlett' (tested as standard), especially when fruits were held at 32°C for 90 minutes prior to analyses (Fig. 5.3). This result coincided with a higher perception of the characteristic pear aroma, as evaluated by sensorial analysis. These findings point out the important role of adequate inhome handling practices to maximize pear volatile emission in order to optimize eating quality of each cultivar. 3. Senescence. Watada et al. (1984) define senescence as "those processes that follow physiological maturity or horticultural maturity and lead to death of tissue." Although Brady (1987) points out that the distinction between ripening and senescence has never been finely defined, as related to pear fruits, it appears that monitoring of volatiles production can provide some helpful insight toward the characterization of horticultural maturity, ripening, and senescence of pears. Despite the numerous changes affecting flavor and texture that determine the limits of fruit acceptability, some volatiles, such as ethanol and acetaldehyde, can help in understanding and monitoring the passage from maturity to senescence. Pear fruit produce acetaldehyde and ethanol as a normal event during ripening and senescence (Nanos et al. 1992). Ripe fruit contain small amounts of ethanol and acetaldehyde as part of their aroma (Ritenour et al. 1997); and a small amount of these volatiles is important for flavor development and sensory quality of a number of products (Paz et al. 1981). Changes in volatile concentration of ripening 'La France' pears were studied after different shelf-life periods by Oshita et al. (2000). After a period of cold storage, fruits were analyzed immediately (unripe) or were held at 20°C for one (ripe) or five days (overripe). All of the six volatile compounds identified in unripe pears increased with progression to the ripe stage, when an additional compound, methyl acetate, appeared in the profile. Advancing to overripeness, ethanol, acetaldehyde, and ethyl acetate continued to increase; while an opposite trend was observed for butyl acetate, hexyl acetate, and butanol, suggesting that their peak emission was associated with the ideal fruit ripeness. Chervin et al. (1999) analyzed emission of ethanol, acetaldehyde, and methanol by 'Packham's Triumph' fruits ripening at 20°C after cold storage. The three volatiles started to rise markedly between days four to seven, a reasonable period of shelf-life for reaching optimal eating quality. The emission of these compounds then increased at a faster rate with the onset of fruit senescence.

w

0 0

AbundariI:.

13

36e+07

32e+07

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24e+07

12 2e+07

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10 14

16 4000000

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Fig. 5.3. Total ion chromatogram (TIC) obtained from the TD-GC-MS (Thermal Desorption Gas Chromatogram Mass Spectrometry) sis of the headspace of 'Harrow Sweet' pear unpeeled fruit slices. TO Cold Trap injector (Chrompack, Middleburg, The Netherlands) conbackflush: 20ml/min for 5 min, desorption: 250°C for 5 min, cold trap low P: -150°C Cold trap high P: 230°C, injection: 230°C for 1 min. GC (HP 5890, Hewlett Packard, Palo Alto, USA) conditions: HP-l column 60m x 0.25 mm 1.D.,0.25!lm film ofpolymethylsiloxane; carrier gas flow: helium, 10 ml/min; oven program: initial: 40°C; 7 min, ramp: 5°C/min, final: 240°C; 10 min; detector: 280°C. MS conditions (HP 5970): Electron Impact (EI) ionization (70 eV);Scan range: 20-350 amu. 1: ethanol; 2: methyl acetate; 3: n-propanol; 4: n-butanol; 5: propyl acetate; 6: iso-butyl acetate; 7: hexanal; 8: butyl acetate; 9: (E)-2-hexenal; 10: n-hexanol; 11: 2-methyl-l-butanol acetate; 12: pentyl acetate; 13: hexyl acetate; 14: heptyl acetate; 15: hexyl butanoate; 16: methyl-(E,Z)-2,4-decadienoate; 17: ethyl decanoate; 18: a-farnesene. In the box mass spectrum of methyl-(E,Z)-2,4-decadienoate.

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These results show some of the promising perspectives of the use of volatiles emissions coupled with sensory analysis. One of the interesting aims could be an "instrumental" help in defining the boundaries of pear fruit optimal state for consumption (or eating quality), a definition encompassing several specific physiological, physical-chemical and sensory attributes. In fact, as related to volatiles, it corresponds to the period of ripening that starts when fruit develop an optimal sensory equilibrium of character impact and contributive compounds and lasts until the senescence stage when concentrations of the compounds responsible for off-flavors exceed the sensory threshold. 4. Disorders. Disorders can be seen as sudden and undesired hastenings

of senescence that compromise the achievement and/or reduce the duration of acceptable horticultural maturity. Disorders affecting pear fruit during storage were reviewed by Raese (1989) and are listed by Richardson and Kupferman (1997) as scald (superficial and senescent), internal browning or breakdown, brown heart, pithy brown core, core flush, cavity formation, and chilling injury. The incidence of disorders is determined by preharvest factors (e.g., maturity at harvest, fruit mineral content, environmental conditions during fruit development) and by storage conditions, particularly when CA is used. Inception of disorders is often signaled by accumulation and/or emission of volatile compounds. Volatiles such as ethanol and acetaldehyde have been widely studied for their correlation with disorders in fruits. It is recognized that their accumulation may contribute to the development of internal disorders (Toivonen 1997). Harley and Fisher (1927) associated pear scald and breakdown in 'Bartlett' with accumulation of acetaldehyde. Tindale et al. (1938) observed a continuous increase in ethanol content during ripening of 'Bartlett'; acetaldehyde increased during cold storage and subsequent ripening, and achieved its maximum concentration at the onset of core breakdown. An increase in acetaldehyde and ethanol concentration, and in the related enzyme ADH activity has been associated with brown core development in 'Blanquilla' pears (Pinto et al. 2001). Low O 2 and/or high CO 2 are beneficial for improving poststorage fruit quality but may induce anaerobic respiration and production of undesirable volatiles including ethanol and acetaldehyde (Ke et al. 1990). The production of these compounds from pear fruits can be enhanced by hypoxia with the risk of negative effects on fruit quality (Nanos et al. 1992; Richardson and Kosittrakun 1995). Anaerobic conditions can negatively affect fruit flavor by increasing sugar and/or acid loss and enhancing off-flavor production. Off-flavors originate presumably from the

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alteration of typical fruit flavor and aroma compounds, and from the production and accumulation of ethanol and acetaldehyde (Mattheis and Fellman 2000). However, the importance of these two volatiles as a direct cause of off-flavors is still to be verified (Yahia 1994). Richardson and Kosittrakun (1995) doubted the connection between ethanol and acetaldehyde and off-flavors, because anaerobiosis-induced off-flavors disappeared when fruit was stored in air without much change in ethanol and acetaldehyde concentration. The same authors observed large differences in anaerobic responses between the cultivars 'Bartlett' and 'Anjou.' In fruits stored under low O 2 concentration at O°C, 'Bartlett' developed off-flavors after five to eight days, while'Anjou' did not during a 20-day period. At 20°C, 'Anjou' off-flavors were detected, but always at lower levels than those of 'Bartlett' stored under the same conditions. Risks for fruit quality loss related to anaerobiosis should be avoided through the storage of fruit of sound physiological state and the application of appropriate storage techniques for the use of CA or MA (Mattheis and Fellman 2000). In CA-stored 'Conference' fruits, acetaldehyde and ethanol contents were observed to increase rapidly only with the onset of senescence (Recasens et al. 1997). Ke et al. (1990) observed in 'Bartlett' an increase in ethanol with 0.25 kPa 02; while fruit stored in 0.5 or 1 kPa O 2 had about the same ethanol content of fruits kept in air. Short-term exposure to concentrations of 50 and 80 kPa CO 2 increased ethanol and acetaldehyde concentrations, while 20kPa CO 2 did not. For Asian pears that, unlike European pears, ripen on the tree the use of CA is aimed at maintaining high at-harvest quality. Under these conditions ethanol and acetaldehyde have been studied in relation to onset of skin blackening. Yang and Lee (1997) observed 'Niitaka' fruit during 90-day storage at -0 to 1°C, under different CA conditions. The highest emissions of ethanol and acetaldehyde were detected from fruit stored in 3 kPa CO 2 and 20 kPa 02' an environment that caused the maximum incidence of skin blackening. Pears stored under CA conditions of 1.2 kPa or 3 kPa CO 2 , and 1.2 kPa or 3 kPa O2 had no symptoms of disorders and exhibited low concentrations of ethanol and acetaldehyde. Park et al. (1999) compared 'Niitaka' and 'Shinsui' stored at O°C or 5°C in air or under three CA treatments (2 kPa O 2 + 0 kPa CO 2 ; 2 kPa O 2 + 2.5 kPa CO 2 ; 2 kPa O 2 + 5 kPa CO 2 ). In 'Shinsui', acetaldehyde and ethanol concentrations were unaffected by atmosphere, while increasing temperature caused a three- to sevenfold increase in acetaldehyde and a six- to ninefold increase in ethanol levels, depending on storage conditions. For all treatments, 'Niitaka' generally had much lower concentrations of both volatiles than 'Shinsui.'

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Although the mechanism of scald development is complex and still not completely elucidated, it is generally accepted that the sesquiterpene a-farnesene and its oxidation products (conjugated trienes and 6-methyl5-hepten-2-one) play an important role in the development of superficial scald of apple (Huelin and Coggiola 1970; Anet and Coggiola 1974; Mir and Beaudry 1999; Rowan et al. 2001) and pear fruit (Chen et al. 1990; Chen et al. 1993; Zoffoli et al. 1998). The production of a-farnesene increases during storage, and decreases, due to oxidation, when the disorder appears. Zoffoli et al. (1998) observed postharvest evolution of afarnesene in 'Bartlett,' 'Anjou,' and 'Packham's Triumph.' The amount was minor at harvest, then increased during cold storage to reach a peak after two to three months. Afterward, the concentration in pear peel declined with a pattern dependent on cultivar and maturity stage. Alpha-farnesene and conjugated trienes in the peel tissue of'Anjou' fruit were suppressed by low oxygen in CA storage. This effect of low oxygen in CA was observed on 'Anjou' also by Chen and Varga (1997). Chervin et al. (2000) observed that low O 2 storage significantly reduced level of a-farnesene and other terpenes (undecatetraene and copaene) as well as of esters in 'Packham's Triumph.' V. VOLATILE COMPOUNDS' INFLUENCE ON QUALITY

A. Fresh Fruit

Quality is a human construct comprising many properties or characteristics, including sensory properties, nutritive values, chemical constituents, functional properties, and defects (Abbott 1999). Quality requirements of pear fruit have been recently outlined by Wrolstad et al. (1991), Neri and Brigati (2000), and Eccher-Zerbini (2002) in relation to ripening, softening, flavor volatiles, and taste. Pear fruit quality should be evaluated on the basis of the specific and transitory use of the product by different users, in relation to the distinct steps from research to production and consumption (breeder, grower, packing house and storage managers, retailer, consumer). However, as noted by Shewfelt (1999), the term quality is frequently used in postharvest studies but rarely defined. The author underlines the risk of mixing and confusing different concepts and perspectives under the definition of quality, thus limiting the possibility of improving the quality of products delivered to the consumer, and suggests that scientists conduct their research with a consumer-oriented approach. A major issue for consumer-oriented research on pears is to outline all the passages from biochemical composition of fruits, to correlations between analytical and sensorial traits,

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and finally to the importance of sensory traits to determine fruit quality. Most analytical studies performed on pear quality are based on flesh firmness, soluble solids content, and titratable acidity; while volatiles determinations are less commonly carried out. Aroma assessment is seldom performed and often "hidden" in sensory evaluation judgments as a nondetermined component of flavor (Kappel et al. 1995; Elgar et al. 1997; Chen and Varga 1999; Ma et al. 2000). However, high correlations have been found between fruit acceptance and perceived aroma (Quamme and Marriage 1977; Suwanagul 1996; Predieri et al. 2002). This suggests that studies on volatiles emission can reasonably contribute to a better understanding of pear quality parameters. Shewfelt (1999) addresses two major points that may highlight the difficulty in correlating volatile determination with quality: (1) volatiles, as compared to other quality-related traits such as sugars and acids, are less readily quantifiable; (2) it is problematic to determine whether large differences in predominant volatile compounds affect flavor perception any more than small differences in compounds present in trace amounts. Furthermore, smelling through the front of the nose may produce a different experience than when the aroma is perceived in the mouth during chewing, a process that changes temperature, viscosity, and polarity of the food, and alters aroma release and perception (Baldwin et al. 2000). These factors complicate the design of experimental procedures able to correlate volatile analyses with a prediction of consumer acceptance and perceived quality. Some advances in the studies on the correlations between instrumental and sensorial analyses in pear may derive from GCO techniques such as CharmAnalysis™ and Osme. Sensory science could aid our understanding of the relationships among volatiles, sensory traits, and pear quality. Some help could come from the use of more consistent methods for sensory analysis, including the improvement ofterms ubiquitous in literature on pear, but somehow seem obsolete and vague such as 'Bartlett'-like or "eating quality." Reports should always indicate whether the assessors are "consumer" or "selected and trained expert judge," in order to better evaluate the responses. When expert assessors are available, they should be required to judge not only in terms of "flavor" but specifically for pear traits affecting eating quality. Predieri et al. (2002) indicate that the seven parameters allowing for a complete description are juiciness, acidity, sweetness, pear aroma, astringency, flesh firmness, and texture. Additional parameters could be the evaluation of off-flavor and overall acceptance. Furthermore, it is well known that terms used in sensory evaluation are of high importance for the output of data. We think that it is important

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to emphasize that some problems in comparing data from international sources could come from the use of different languages. For example when the Italian sapore, Spanish sabor or French saveur, are translated to the English flavor the technical sensory meaning of these words becomes slightly different. On the other hand the terms for sweetness (Italian dolcezza; Spanish dulzor; French douceur) or aroma (the same in Italian and Spanish, arome in French) have a higher interlinguistic consistency. Thus the definition and use of more specific terms will help in many ways, including the writing of an international review such as the present one. B. Processed Products

One of the major aims of pear processing is to retain volatiles in processed products; several factors, including cultivar traits, fruit physiological state and processing technologies, affect the quality of the final product. 'Bartlett' has been the preferred cultivar for canning worldwide because of its unique traits, including the permanence of its flavor components after processing (Wrolstad et al. 1991). Quamme and Marriage (1977) studied the relationship of aroma volatile compounds to canned fruit flavor among several pear cultivars, finding high correlations between aroma and flavor, indicating the importance of volatiles in canned pear flavor. Measurements of decadienoate equivalents revealed that all cultivars having a 'Bartlett-type' flavor were characterized by the same moieties and relative amounts of decadienoate esters. Kappel and Quamme (1987) reported that the selection 'HW-606', that has been found comparable to 'Bartlett' in quality of canned halves, gave disappointing results when processed as puree, indicating that cultivars can have traits influencing fitness for processing with specific products. Strandjev (1982) evaluated the influence of different temperature treatments and of different sterilization times on the qualitative and quantitative composition of aromatic substances of 'Bartlett' and 'Passe Crassane' fruits. Relatively low temperatures (98°C) and short periods of sterilization (20 minutes) provided the best conditions to avoid excessive loss of aromatic volatile substances of pureed pear fruits. Processed product contained the typical pear aroma, likely due to the presence of the high-boiling point esters, which are typical of pear aroma. Leonard et al. (1976) described the effect of the capture and adding back of pear volatiles lost during process of puree preparation in open systems. Use of a closed system resulted in a pear puree with higher flavor intensity. However, the most acceptable product was the puree with only 50 percent of the essence added back.

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The puree with 100 percent restored essence was judged to be too intense. This shows that volatiles, although possibly highly correlated to pear flavor pleasantness, should always be considered in relation to the other flavor components. Wrolstad et al. (1991) point out that decadienoates are susceptible to oxidation, due to their high degree of unsaturation. This may be important for maintaining flavor in products derived from processed fruits during storage. The quality of alcoholic beverages prepared from pears requires a well defined aroma stable over a long period. De Vincenzi et al. (1987), reviewing volatile compounds in food, list 41 volatile compounds for pear brandy, including 18 esters. Bricout (1977) found three groups of compounds that contribute to aroma: the (E,Z)-2,4decadienoates esters; compounds that increase during fermentation, such as hexanal; and other compounds deriving from fermentation of sugars that are not found in fruit. Cigfc and Zupancic-Kralj (1999) studied aromas in pear brandy as related to sunlight irradiation. Sensorial differences were found between brandy stored in colorless or green bottles. Headspace sampling of brandy stored in green bottles indicate that ethyl-(E,Z)-2,4-decadienoate (68 ppm), ethyl-(E,Z)-2,4 decadienoate (21 ppm) and methyl-(E,Z)-2,4-decadienoate were present. In colorless bottles the amount of ethyl- and methyl-(E,Z)-2,4-decadienoate was lower, while other isomers of 2,4-decadienoates were found at higher concentrations. Further experiments, conducted by exposing brandy to UV light, showed the progressive transformation of ethyl- and methyl-(E,Z)2,4-decadienoate (responsible for pleasant aroma) into their (Z,E) and (E,E) isomers (producing a less pleasant odor and taste). The experiment showed the importance of protecting pear brandy from sunlight and is an interesting example of the importance of processing and storage in the production of high quality goods. VI. SUMMARY AND CONCLUSIONS

The pear is a complex fruit; its journey from orchard to the consumer's mouth and sensory perception is long and full of twists and turns. Volatiles play two major roles related to pear fruit physiology and quality: (1) markers of physiological activity detectable by nondestructive tests; (2) key components of fruit flavor. Regarding their role as markers, volatiles can be evaluated as indicators of fruit development and physiological maturity. As related to maturation during storage and the ultimate reaching of horticultural maturity, studies on volatiles as markers of appropriate fruit activity or

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senescence have already found a niche both in research and applied studies, and further advances can be anticipated. Aroma is a major factor in the determination of pear fruit flavor, and a more detailed knowledge of volatiles will most certainly benefit the producers and consumers of this fruit crop. Research efforts should be focused on biosynthesis and metabolism of pear volatiles, in particular on the manipulation of the character-impact and contributory compounds emission. To pursue the fulfillments of product-oriented quality the relationship between instrumental and sensory studies should be a major point of investigation. To obtain high correlations with sensory results, instrumental flavor characterization should pay major attention to both the isolation procedures used and to the identification of relevant components to be quantified. Instrumental analysis is an important tool in quality protocols of food producers, that monitors parameters determining the flavor of the final product. The availability of effective instrumental volatile analysis coupled with appropriate sensory analysis could contribute to quality-oriented decisions including selection of cultivars, cultivation and harvesting techniques, storage conditions, processing, and consumer information on eating quality. While basic quality parameters, for example, sugar content, may be more readily achieved, aroma could be the critical factor differentiating top quality fruits from standard quality products. In specific, volatiles could be evaluated as quality markers of new cultivars, typical production area and/or particular cultural technique (i.e., organic). As a better understanding of the genetic and biochemical factors that influence or control metabolism and biosynthesis of aroma compounds is achieved, better cultural and postharvest methodologies could be developed both to optimize flavor of standard pear cultivars and to make pear breeding more successful. Increased performance of analytical instruments and sensors, advances in biotechnology, and development of powerful computer programs will make identification and quantification of important chemical components possible. These individual components can then be studied in their correlation with sensory descriptors to correlate to enzymes, and gene products. Ultimately, the identification and isolation of genes involved in the production of aroma volatiles may lead to environmental and genetic manipulation for pear quality improvement. Integrating the fields of plant breeding, molecular biology, postharvest physiology, sensory evaluation, and food and consumer science should provide new opportunities for making pear quality more predictable and to optimize flavor at the time of consumption.

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6

The Physiology of Flowering in Strawberry Rebecca 1. Darnell Daniel J. Cantliffe Daniel S. Kirschbaum Horticultural Sciences Department University of Florida PO Box 110690

Gainesville, FL 32611

Craig K. Chandler Gulf Coast Research and Education Center University of Florida 13138 Lewis Gallagher Road Dover, FL 33527

I. INTRODUCTION II. FLORAL MORPHOLOGY III. ENVIRONMENTAL EFFECTS ON REPRODUCTIVE GROWTH A. Light B. Temperature C. Light and Temperature Interaction D. Chilling E. CO 2 Concentration IV. FLORAL INDUCTION MODELS A. Single Factor Control Model 1. Floral Promoter 2. Floral Inhibitor 3. Identification of Floral Promoter/Inhibitor B. Multifactor Control Model V. GENETICS OF FLORAL INDUCTION VI. CONCLUSIONS LITERATURE CITED

Horticultural Reviews, Volume 28, Edited by Jules Janick ISBN 0-471-21542-2

© 2003 John Wiley & Sons, Inc.

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I. INTRODUCTION

The cultivated strawberry (Fragaria x ananassa Duch.) is a widely adapted small fruit grown from the low-latitude tropics and subtropics (Mexico, Colombia, Costa Rica, Guatemala) to high-latitude, cold, continental areas (Poland, Russia, Germany, Belgium, Scandinavia). Cultivation area is over 200,000 ha worldwide and estimated world production in 2000 was over 3 million tonnes (FAG 2001). About 27 percent of the total production is in the United States, with significant production in Spain (12%), Japan (7%), Poland (6%), Mexico (5%), and Korea (5%) (FAG 2001). Numerous authors have described the environmental control of flowering in strawberry, particularly the effects of daylength and temperature (Darrow 1966; Larson 1994; Hancock 1999). This chapter's objective is to integrate information available on the strawberry flowering response, much of which is now dated, with new information on the physiology and genetics of the flowering process. II. FLORAL MORPHOLOGY

The cultivated strawberry is a hybrid between F. virginiana Duch. and F. chiloensis (L.) Duch. The history of this chance hybrid is reviewed extensively by Darrow (1966). Plants are primarily hermaphroditic, although staminate and pistillate genotypes exist (Hancock 1999). Both F. virginiana and F. chiloensis are trioecious, with different populations exhibiting staminate, pistillate, or hermaphroditic plants. The strawberry is a perennial plant that is usually described as herbaceous, although it is a true woody plant, as evidenced by the production of secondary xylem in roots and crowns (Darrow 1966; Esau 1977). The plant body is comprised of a compressed stem, or crown, from which arise leaves, runners, roots, axillary crowns, and inflorescences. The strawberry inflorescence is a modified stem terminated by the primary flower (Fig. 6.1). Arising laterally from this stem are usually two secondary flowers, from which four tertiary flowers may arise, followed by eight quaternary flowers. Individual flowers are typically comprised of ten sepals, five petals, 20 to 30 stamens, and 60 to 600 pistils, depending on the flower order (Hancock 1999). Flower induction is the event that initiates the transition of a vegetative apex to a floral apex in response to an environmental or developmental cue. Induction is usually determined after the fact, by documenting the beginning of floral initiation, which is the first anatomical change observed in the apex undergoing transition. Because of the difficulty in separating floral induction from initiation, these words are

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Receptacle

I

Fig. 6.1. A typical strawberry inflorescence with primary, secondary, tertiary, and quaternary flowers. (From Dana 1980.)

often used synonymously. For the purposes of this chapter, "induction" will be used to indicate "induction/initiation" and no attempt will be made to differentiate between the two processes. The anatomical transition from a vegetative to a floral apex begins as a broadening and raising of the apical meristem, followed by the formation of the primary flower and first bract primordium (Taylor et al. 1997). This is followed by the appearance of the secondary flower primordium and the formation of individual primary floral organs. Sepal primordia develop first, in a ring on the periphery of the flower primordium (Taylor et al. 1997). This is followed by the centripetal initiation of petal, stamen, and lastly, carpel primordia. III. ENVIRONMENTAL EFFECTS ON REPRODUCTIVE GROWTH

Strawberry reproductive growth can be markedly altered by environmental conditions such as light, temperature, preplant chilling, and CO 2 concentration.

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A. Light Photoperiod and light quality have profound effects on strawberry flowering, while light intensity exerts a lesser effect. Strawberries are classified according to their photoperiodic flowering response: short day (SD) or Junebearing plants initiate flower buds under short days; long day (LD) or everbearing plants initiate flowers under long days; and dayneutral (DN) plants are relatively insensitive to daylength with respect to flower induction. Some authors consider day-neutral genotypes as a class of everbearers (Nicoll and Galletta 1987); however, others consider true day-neutral genotypes genetically and physiologically distinct from the other classes (Ahmadi et al. 1990). Most authors agree that categorization is difficult due to the continuum of photoperiodic responses observed in different genotypes. The photoperiodic response is quantitative (facultative), and the response curve varies for cultivars within a class. In general, SD genotypes initiate flowers when the photoperiod is less than 14 h, while LD genotypes generally require photoperiods greater than 12 h (Darrow 1936). Downs and Piringer (1955) found that increasing the photoperiod from 11 to 17 h increased flower bud induction by 2-, 5-, and 20-fold in three different LD cultivars. In contrast, floral induction in DN genotypes occurs independently of daylength. Durner et al. (1984) reported that inflorescence number was similar under photoperiods of 9 or 16 h for two DN cultivars. The minimum number of photoinductive cycles required for flower bud induction in strawberry ranges from 7 to 24 (Hartmann 1947a; Heide 1977; Hancock 1999). 'Missionary' required four to seven inductive 10h photoperiodic cycles to start flower induction, although maximum flowering occurred with 21 cycles (Hartmann 1947b). Went (1957) found 9 to 15 short day cycles were needed to induce flowering in 'Marshall.' The SD cultivar 'Sparkle' was induced to flower with 12 to 15 cycles under an 8-h photoperiod (Moore and Hough 1962). In general, as temperature increases, the number of required inductive cycles increases (Ito and Saito 1962). Photoperiod perception in most plants, including strawberry, occurs in the leaf. Hartmann (1947a) found a positive correlation between the percentage of leaf area exposed to SD photoperiods and inflorescence number per plant in the SD cultivar 'Missionary.' Jonkers (1965) reported that inflorescence number in runner plants of a SD cultivar increased as the number of fully expanded leaves increased from zero to three. Although the photoperiod response of flower bud induction in strawberry is well described in terms of daylength, little is known regarding the effect of light quality. Vince-Prue and Guttridge (1973) compared SD

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strawberries grown under inductive 8-h days with plants subjected to an 8-h extension with either red or far-red light. They found that floral induction occurred as readily under the red light extension as under the SD control, whereas the far-red extension delayed induction (Table 6.1). This inhibition was not observed when far-red light was applied only during the second half of the 16-h night. Red light, however, which had no effect when applied during the first half of the 16-h night, completely inhibited floral induction when applied during the second half. Thus, the inhibitory effect of red light increased, while the inhibitory effect of far-red light decreased, as the night proceeded. This pattern of response differs from that of most other SD plants, and, in fact, resembles more closely the pattern of response found in LD plants. There are other reports of far-red light inhibition of flower induction in strawberry (Kadman-Zahavi and Ephrat 1974; Guttridge 1985). Reproductive development in strawberry is also affected by light intensity. These effects appear to be mediated primarily by effects on CO 2 assimilation. Light saturation curves indicate that strawberry leaf CO 2 assimilation increases with increasing photosynthetic photon flux, reaching light saturation at 500 to 700 Jlmol m-2 S-1 (Chabot 1978; Ceulemans et al. 1986). Dennis et al. (1970) found that inflorescence number in a DN cultivar increased as light intensity increased from ~220 to ~430 Jlmol m-2 S-1. Similarly, reproductive biomass of F. vesco L., the alpine strawberry, increased significantly as light intensity increased from 22 to 150 Jlmol m-2 S-1 (Chabot 1978). Under field conditions, 60 percent shade during the entire growing season reduced total yield the following season by Table 6.1. Percentage of strawberry plants flowering after exposure to red and far-red irradiation treatments for 18 or 21 cycles. Treatment Daylengtha

Flowering plants (%) Irradiation b

18 cycles

21 cycles

SD + Ext.

R FR

100 0

100 40

Ext. + SD

R FR

0 60

0 100

SD

100

100

LD

0

0

aSD + Ext = 8-h photoperiod + 8-h daylength extension at end of photoperiod; Ext. + SD = 8-h daylength extension prior to beginning of 8-h photoperiod; SD = 8-h photoperiod; LD = 24-h photoperiod. bDaylength extension with either red (R) or far-red (FR) light. Modified from Vince-Prue and Guttridge (1973).

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R. DARNELL, D. CANTLIFFE, D. KIRSCHBAUM, AND C. CHANDLER

20 to 45 percent (Ferree and Stang 1988). Light intensity also directly affects flower development. Smeets (1976) reported that very low light intensities (less than ~10 /-lmol -2 S-l) for several days prior to anthesis induced stamen abortion and reduced yield in 'Glasa.' B. Temperature

Although research on temperature and photoperiod interactions abounds, few studies have measured the direct effect of temperature alone on strawberry reproductive development. At high latitudes where long photoperiods prevail and in tropical to equatorial latitudes, where short photoperiods prevail, temperature is the major factor influencing the flowering response in strawberry. In general, temperatures above 28°C inhibit flowering in SD and DN cultivars of F. x ananassa and F. vesca (Ito and Saito 1962; Chabot 1978; Durner and Poling 1988; Okimura and Igarashi 1997). Heide (1977) found that flower number in SD cultivars was significantly less at 24°C compared with 18°C. Day/night temperatures of 26°/22°C, respectively, suppressed flowering in DN and SD F. x ananassa genotypes compared with 18°/14° or 22°118°C (Durner et al. 1984), while flowering in F. vesca was inhibited at temperatures of 30°/20° and 40 0 /30°C compared with 20 0 /10°C (Chabot 1978). Night temperatures of 10°C promoted floral induction compared with either 5° or 13°C for several SD cultivars (Kawakami et al. 1990). Manakasem and Goodwin (1998), using scanning electron microscopy, reported that floral induction in the SD cultivar 'Torrey' was more dependent on minimum temperature than on daylength or maximum temperature; in the DN cultivar 'Aptos,' floral induction occurred regardless of daylength and was much less sensitive to temperature than was the case with 'Torrey.'

c. Light and Temperature Interaction Photoperiod and temperature interactions on strawberry flowering have been reviewed by Guttridge (1969, 1985), Strik (1985), Durner and Poling (1988), and Darnell and Hancock (1996). For many SD cultivars, the short day requirement for floral induction can be overridden by low temperatures. Darrow (1936) exposed plants of nine SD cultivars to a combination of three photoperiods (less than 13.5, 14, and 16 h) and three temperatures (12°, 15.5°, and 21°C). Maximum flower number occurred under the shortest photoperiods and the two lowest temperatures. The longer the photoperiod, the lower the temperature needed to maximize flower number. Hartmann (1947a,b) found that several SD cultivars flowered under either long (15 h) or short (10 h) photoperiods if temperatures were maintained at 15.5°C, while at 21°C, only those plants under the short

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photoperiod flowered. Jonkers (1965) obtained more flowers at 15° than at 21°C in 'Talisman' under short days, but failed to induce flowers at 15°C under long days. Optimum flower induction occurred with 12-h photoperiods at 18°C for several SD cultivars (Heide 1977). However, as temperature decreased to 12°C, induction occurred in some cultivars even at photoperiods of 16 and 24 h. Went (1957) exposed the SD cultivar 'Marshall' to several combinations of temperatures (ranging from 6° to 20°C) and photoperiods (ranging from 8 to 24 h). Plants initiated flowers at all temperatures under an 8-h photoperiod, while under 16 or 24-h photoperiods, flowering occurred only at 6° or 10°C. Durner et al. (1984) described the flowering response to different photoperiod and temperature combinations in SD, LD, and DN genotypes (Table 6.2). They found that flowering in SD cultivars was induced under short photoperiods (i.e., 9 h) and cool temperatures (18°/14°C day/night); however, at higher temperatures (22°/18°, 26°/22°, or 30 0 /26°C), no flower induction occurred regardless of photoperiod. This same high temperature limitation to flower induction was evident in the LD and DN cultivars under short photoperiods, although the limitation was not as marked as that observed in SD plants. These studies suggest that flower induction in strawberry is photoperiodically insensitive at low (~10° to 15°C) and high (~25°C) temperatures. At low temperatures, most genotypes initiate flowers regardless of photoperiod, while at high temperatures, the flowering response is almost completely inhibited (Table 6.3). Table 6.2. Average number of inflorescences per plant for Junebearing, everbearing, and day-neutral strawberries grown under two photoperiods at four temperatures for three months (n 12).

No. inflorescences per plant Junebearing

Everbearing

Day/night temperature (oC)

SDa

18/14

2.1a b

2.0a

2.0a

22/18

0.3b

o.lb

0.5b

26/22

O.Ob

o.ob

O.Ob

30/26

o.Ob

o.ob

o.ob

NIa

SD

NI

Day-neutral SD

NI

OAb

3.3b

4.3ab

1.7a

1.3c

5.0a

0.2b

O.Oc

4.9a

O.ob

O.Oc

OAc

(JSD, short day (9-h light period at 320 ~mol m-2 S-1); NI, night interruption (9-h light period with 3-h night interruption at 40 ~mol m- 2 S-1 in the middle of the dark period). bMean separation within strawberry types by Duncan's multiple range test, 5 percent level. Modified from Durner et al., 1984.

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R. DARNELL, D. CANTLIFFE, D. KIRSCHBAUM, AND C. CHANDLER

Table 6.3. Flower initiation response of short day, long day, and day-neutral strawberry genotypes to short photoperiods (SD) and long photoperiods (LD) under three different temperature ranges. Response Photoperiod

Low (10° to 15°C)

Medium (15° to 25°C)

Short Day Genotypes SD

+a

LD

+

+

Long Day Genotypes SD

+

LD

+

+

Day-neutral Genotypes SD

+

+

LD

+

+

fI+, flower initiation; -, no flower initiation.

D. Chilling

The question of whether strawberry buds enter endodormancy (physiologically imposed dormancy within the dormant structure) is still open to debate. Strawberries do, however, require exposure to low temperatures (0° to 7°C) in order to promote maximum vegetative growth, and the chilling requirement is cultivar dependent in F. x ananassa (Piringer and Scott 1964; Voth and Bringhurst 1970; Durner et al. 1986; Darnell and Hancock 1996). In general, leaf number, leaf size, and runner number increase with time of exposure to chilling (Bailey and Rossi 1965; Tehranifar and Battey 1997). Although chilling stimulates the development of previously initiated inflorescences (Tehranifar and Battey 1997), flower induction is inhibited (Avigdori-Avidov et al. 1977; Larson 1994; Tehranifar and Battey 1997), delayed, or both (Durner and Poling 1987), resulting in a reduction in early yield in SD cultivars (Albregts and Howard 1977, 1980).

E. CO 2 Concentration

There are only a few studies on the effects of CO 2 enrichment on strawberry flower induction. Hartz et al. (1991) found no effect of increased CO 2 concentration (700 to 1000 !lmol mol-I) supplied during flower

6. THE PHYSIOLOGY OF FLOWERING IN STRAWBERRY

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induction on flower or inflorescence number of two SD cultivars. They attributed this to the complex physiology of flower induction in strawberry and the wide number of environmental factors that influence it. The enrichment studies were done under plastic tunnels, and although air temperatures were not reported, maximum temperatures in a related study with vegetable crops reached 41°C. Thus, any potential effect of CO 2 enrichment on flower induction in strawberry may have been overridden by excessively high temperatures. Deng and Woodward (1998) reported greater flower number in SD strawberry plants exposed to 56 Pa CO 2 from initial planting until fruit harvest, compared with the ambient CO 2 control. However, it is unclear from their work whether high CO 2 increased flower induction or development of previously initiated flowers. IV. FLORAL INDUCTION MODELS

Although floral induction has been studied in many plants, the process itself is still poorly understood. Studies have led to two broad models for the control of flowering: single factor and multifactor. The single factor control model is based on the view that a single factor-a floral promoter ("florigen") or a floral inhibitor-is responsible for induction. This model has been supplanted by the multifactor control model, which proposes that a number of factors, both promoters and inhibitors, regulate flowering (Bernier 1988). A. Single Factor Control Model 1. Floral Promoter. Several studies reported in the 1930s (Chajlachjan 1936; Borthwick and Parker 1938; Hamner and Bonner 1938) proposed that the biosynthesis of a "florigenic" substance occurred when plants were exposed to inductive photoperiods. In the 1940s, plant physiologists began to seek "florigen" in many photoperiodically sensitive plants, including strawberry. Hartmann (1947a) studied flower induction in SD plants with various proportions of the leaf area exposed to both short and long photoperiods. The number of infloresences formed was directly proportional to the amount of leaf area exposed to short days. Hartmann speculated that after perceiving enough short day cycles, the leaves of strawberry released a florigenic substance that was translocated to the meristem, which then caused a transition from vegetative to reproductive growth. He proposed that when more leaves were exposed to SD, the substance was released in larger amounts (Le., the response to photoperiod was quantitative).

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R. DARNELL, D. CANTLIFFE, D. KIRSCHBAUM, AND C. CHANDLER

Additional experiments further supported the presence of a florigenic substance (Hartmann 1947a). Mother plants of 'Missionary' were placed under an inductive 10-h photoperiod, while their attached daughter plants were placed under a noninductive 15-h photoperiod. Daughter plants grown under long photoperiod, but attached to mother plants grown under short photoperiod, flowered. Hence, Hartmann proposed that a floral signal was translocated from mother to daughter plants. Guttridge (1956) reported that flowering occurred in defoliated daughter plants grown under continuous light if they were attached to mother plants grown under an inductive (i.e., 9-h) photoperiod, again supporting the idea of a translocatable floral promoter. However, detached, defoliated daughter plants grown under continuous light were not included as controls in the experiment, and subsequent work (Thompson and Guttridge 1960) indicated that fully defoliated (detached) strawberry plants could flower under 24-h photoperiods. This finding casts doubt on the previous conclusion regarding the transmission of a floral promoter. Kirschbaum (1998) examined temperature effects on flower induction in SD 'Sweet Charlie.' Attached mother-daughter plants were exposed to separate day/night temperatures of 20 0 /16°C or 30 o /26°C for 15 cycles under 12-h photoperiods. After completion of the temperature treatments, plants were transferred to 15-h photoperiods and 30°C to allow rapid development of flowers. When daughter plants were grown at 20 o /16°C, all attached mother plants flowered regardless of the temperature at which the mother plants were grown (Table 6.4). When daugh-

Table 6.4. Temperature effects on flowering in attached mother-daughter plants of the SD strawberry 'Sweet Charlie' (n = 6). Attached mother (M)-daughter (D) plants were exposed to combinations of 20 o /16°C or 30 o /26°C day/night temperatures. Flowering plants (%) Plant Daughter Mother

DL-ML(J

DL-MHb

DfrMLc

DfrMHd

0

17

50

0

100

100

67

17

(JD L-M L, both plants at 20 o /16°C. bDL-MH, daughter plant at 20 o /16°C, mother plant at 30 o /26°C. cDwM L, daughter plant at 30 o /26°C, mother plant at 20 o /16°C. dDwM H, both plants at 30 o /26°C. Modified from Kirschbaum, 1998.

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ter plants were grown at 30 o /26°C, however, flowering in mother plants was inhibited, again regardless of the temperature at which the mother plants were grown. Although the flowering response of the daughter plants was inconsistent, possibly due to their age (Jonkers 1965), the response of the mother plants suggests that the high temperature inhibition of flowering in SD strawberry can be overcome by the transmission of some substance (floral promoter?) formed under low (inductive) temperatures. Alternatively, it might be argued that a floral inhibitor is synthesized in daughter plants under high temperatures, resulting in a reduction in flowering of attached mother plants grown under inductive temperatures (compare DH-MLwith DL-ML). 2. Floral Inhibitor. Although results from some studies give support to the floral promoter hypothesis, several studies suggest that floral induction in strawberry is possible only if flower inhibitors are removed from the leaves (Guttridge 1959a,b; Leshem and Koller 1964; Jahn and Dana 1966). This hypothesis assumes that under long photoperiods, a floral inhibitor is present in high enough concentrations to inhibit flower induction in SD plants. Under short photoperiods, inhibitor concentration decreases, allowing flower induction to occur. In DN cultivars, this inhibitor system is either inactive or nonexistent (Guttridge 1968). Several lines of evidence support the floral inhibitor hypothesis. Jonkers (1965) exposed mother plants of SD 'Deutsch Evern' to short or long photoperiods, and placed attached daughter plants in the opposite photoperiod. Daughter plants grown under long photoperiods did not flower, regardless of the photoperiod to which the mother plants were exposed. As expected, daughter plants grown under short photoperiods flowered, however, flower number was reduced and delayed in daughter plants when the mother plants were grown under long compared with short photoperiods. Similarly, flower induction occurred earlier in detached versus attached daughter plants grown under short photoperiods when they were attached to mother plants grown under long photoperiods (Leshem and Koller 1964; Jahn and Dana 1966). These results support the hypothesis that in SD plants, a floral inhibitor is synthesized under long photoperiods. In strawberry, this inhibitor can be translocated from the mother plant to the daughter plant, delaying or partially inhibiting the flowering response even when the daughter plants are grown under inductive conditions. Results from studies on the effects of leaf age on flower induction in SD plants also support the floral inhibitor hypothesis. Thompson and Guttridge (1960) found that mature leaves delayed flower induction in the SD 'Talisman.' Three groups of plants, those with only young leaves, those

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R. DARNELL, D. CANTLIFFE, D. KIRSCHBAUM, AND C. CHANDLER

with only mature leaves, and intact controls, were grown under inductive SD photoperiods. Plants with only mature leaves were delayed in flower induction by two plastochrons (the time interval between the origination of two successive leaf primordia) relative to intact plants and plants with only young leaves. The authors concluded that mature leaves synthesized a flower inhibitor, and when only mature leaves were present, the inhibitor was translocated to the apex and inhibited flowering. However, in intact plants in which both mature and young leaves were present, the inhibitor was translocated to young leaves, thus reducing the amount of inhibitor reaching the apex and allowing flowering to occur more readily. In the same work, the authors reported that removal of mature leaves increased the range of photoperiods at which flower induction occurred compared with the intact control. Plants lacking mature leaves flowered at photoperiods between 10 and 16 h, while control plants flowered only under a 10- to 12-h photoperiod. Additionally, fully defoliated plants flowered under 24-h photoperiods. Removal of mature leaves apparently removed the inhibitor and allowed floral induction in SD plants under long photoperiods. The hypothesis that flowering in strawberry is associated with a translocatable flowering-inhibiting and vegetative growth-promoting substance seems to have more scientific support than the flowering promoter hypothesis. In SD plants, the inhibitor would be synthesized in fully expanded leaves and translocated to the apex. Long photoperiod and chilling would promote synthesis of the inhibitor, which would accumulate to high levels in the apex, inhibiting flower induction. According to this hypothesis, under short photoperiods, the biosynthesis of the flower inhibitor would be minimized, reducing inhibitor levels in the apex and allowing induction to occur. Although many studies have described the flowering response in SD strawberry in terms of floral inhibitors, there is essentially no evidence for transmission of a floral inhibitor (or promoter) in LD or DN genotypes. 3. Identification of the Floral Promoter/Inhibitor. Since the advent of the single factor control model for flowering, numerous efforts have been made to elucidate the nature of the flowering inhibitor or promoter. Much of the work focused on the role of hormones in flowering, since, for photoperiodically sensitive plants, the flowering signal is translocated from the perceiving organs (leaves) to the apex. Gibberellins are the class of hormone most heavily implicated in the flowering process in strawberry, with lesser emphasis on cytokinins. Studies of the other major hormones-auxin, abscisic acid (ABA), and ethylene-have focused on strawberry fruit development and postharvest physiology,

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rather than flowering. Thus, little information is available for those hormones. More extensive reviews on hormonal patterns during strawberry reproductive growth can be found in Guttridge (1969) and Reid (1983).

Gibberellins. Gibberellins (GA) are probably the hormones most studied with regard to their effects on strawberry, and have been associated with both the promotion of vegetative growth and inhibition of floral induction (Guttridge and Thompson 1959; Guttridge 1985). However, flower development and therefore yield may also be enhanced by GA. The effect of GA on flowering in strawberry has been illustrated by examining the response of strawberry to exogenous GA applications and by analysis of endogenous GA-like substances in plants at critical stages of development. In general, application of GA mimics the effects of LD on vegetative and reproductive growth in SD strawberry (Thompson and Guttridge 1960; Porlingis and Boynton 1961; Guttridge and Thompson 1964). Floral induction under inductive SD conditions is inhibited by exogenous applications of GA 3 or GA 4 + 7 applied during the flower induction stage (Porlingis and Boynton 1961; Guttridge and Thompson 1964; Weidman and Stang 1983). Avigdori-Avidov et al. (1977) found that GA 3 application to SD cultivars grown under short days was as inhibitory to flowering as growing plants under long days. Similarly, flowering in DN cultivars is also inhibited by GA 3 (Guttridge and Thompson 1964; Dennis and Bennett 1969; Kender et al. 1971). Although floral induction in SD and DN strawberry is inhibited by exogenous GAs, flower development appears to be enhanced. Spray applications of GA 3 applied to field grown SD plants in November increased fruit number and total yield the following spring (Singh et al. 1960). This response was likely due to promotion of flower development by GA 3 , not promotion of flower induction, since plants had previously been exposed to four weeks of inductive short days in the field prior to treatment. Smith (1960) and Lesham and Koller (1964) observed an increase in early yield in GA 3 -treated SD plants compared with a nonsprayed control, with no effect on total yield. This was attributed to accelerated peduncle elongation and flower in Lesham and Koller 1964. Choma and Himelrick (1984) found that GA 3 applied to field-grown SD and DN strawberry cultivars four weeks after planting in the spring increased yield the following year. The yield increase was correlated with increased runner production the previous year and therefore an increase in plant population. There was no effect of GA on yield in a LD cultivar, presumably because runner production was not enhanced. Lopez-Galarza et al. (1989) noted earlier fruit production after treating

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R. DARNELL, D. CANTLIFFE, D. KIRSCHBAUM, AND C. CHANDLER

tunnel-grown SD cultivars with GA 3 in the winter, with no effect on fruit weight or total yield. Endogenous levels of certain GA-like substances increase in strawberry plants in response to increasing photoperiod. Uematsu and Katsura (1983) found higher GA l9 -like levels in crowns and leaves of 'Hokowase' plants subjected to 14-h photoperiods for nine days than in plants under 11-h photoperiods for the same period. Concentrations of GAl' GA 8 , and GA l9 were higher in strawberry petioles under LD compared with SD (Wisemann and Turnbull 1999). Unfortunately, neither study looked at correlations between endogenous GAs and flowering. Leshem and Koller (1966) detected no GA-like substances in leaves of SD strawberry during floral induction; however, levels of presumed GA 3 and GA 7 increased significantly during peduncle elongation. Increased levels of endogenous GAs in apices of chilled versus nonchilled SD plants were correlated with reduced flower induction in the chilled plants (Avigdori-Avidov et al. 1977). If GAs inhibit flower induction in SD cultivars, thus mimicking the effects of LD, then one might expect that GAs would promote induction in LD strawberry cultivars, as it does in many other LD species (Thomas and Vince-Prue 1997). However, Kender et al. (1971) found that exogenous GA application inhibited flowering in LD strawberries. The role of GAs in strawberry growth and development has been further illustrated by the application of GA-biosynthesis inhibitors. AMO1618 increased flower induction in SD cultivars grown under noninductive long days (Avigdori-Avidov et al. 1977). Applications of paclobutrazol increased total yield in SD cultivars when applied four to five weeks after transplanting in the fall (Deyton et al. 1991; Bish et al. 1996). This was attributed to a source-sink effect, either by promoting photosynthesis, thereby increasing assimilate availability (Deyton et al. 1991) or by decreasing vegetative growth, resulting in increased assimilate partitioning to reproductive growth (Bish et aI., 1996). On the other hand, paclobutrazol applied just prior to bloom reduced yield in strawberry by inhibiting pollen germination and therefore fruit number (McArthur and Eaton 1987). Thus, inhibitors of GA synthesis do not appear to affect flowering by reducing GA content.

Cytokinins. Although cytokinins are suspected to be involved in the process of flower induction in strawberry and other species, evidence for such involvement is minimal. Exogenous cytokinin increased inflorescence number in both SD and LD strawberry cultivars, presumably by increasing branch crown formation (Waithaka and Dana 1978; Weidman and Stang 1983).

6. THE PHYSIOLOGY OF FLOWERING IN STRAWBERRY

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Yamasaki and Yamashita (1993) found changes in the cytokinin composition in strawberry crown apices during flower induction. Plants of the SD cultivar 'Toyonoka' were analyzed for cytokinins during inductive conditions. Apices were also dissected and examined microscopically for evidence of induction. Zeatin and zeatin riboside were detected in apices before the beginning of SD treatments. Just prior to the first morphological changes observed in the apex as it underwent transition from vegetative to reproductive growth, the authors observed an increase in zeatin and a decrease in zeatin riboside. Following induction, the concentration of zeatin riboside increased again. The authors speculated that such changes in cytokinin concentration played an important role in flower induction. However, no definitive evidence was offered for this conclusion and changes observed may simply have been correlative rather than cause and effect. Changes in cytokinin composition during flower induction have also been reported in other species. For example, isopentenyladenine, isopentenyladenosine, and zeatin riboside concentrations were lower before than after flower induction in Sinapsis alba L. (Bernier et al. 1993), Litchi chinensis Sonn. (Chen 1991), Euphoria longana Lam. (Chen et al. 1997), and the hybrid orchid Aranda 'Noorah Alsagoff' (Arachnis hookeriana Rchb. x Vanda hookeriana Rchb.) (Zhang et al. 1995), but no causal relationship was shown.

Other Hormones. In addition to GAs and cytokinins, other hormones have been implicated in the flowering response of strawberry, including auxins, ABA, ethylene, and polyamines. Moore and Hough (1962) reported that auxin levels in the apex of strawberry plants grown under inductive conditions decreased after 15 inductive cycles, but increased soon thereafter. The authors considered this fluctuation to be a consequence rather than a cause of floral induction. Other experiments have shown that application of auxins to leaves of SD strawberries early in the inductive short day cycle reduce both flower and achene number (Tafazoli and Vince-Prue 1978), suggesting that auxin is inhibitory to flowering. The effects of exogenous ABA on flowering of strawberry are inconsistent. EI-Antably et al. (1967) reported that exogenous ABA promoted flowering in SD cultivars grown under long days, but reduced petiole length and number of stolons in LD cultivars. Conversely, Kender et al. (1971), using both exogenous ABA and analyzing endogenous inhibitors (presumed to be ABA), concluded that ABA was not involved in the flower induction process in strawberry. Ethylene application during floral induction increased fruit number and yield in SD strawberry (Blatt and Sponagle 1974). Similarly, Cain

340

R. DARNELL, D. CANTLIFFE, D. KIRSCHBAUM, AND C. CHANDLER

et al. (1983) reported that ethylene increased fruit number and yield in field grown DN cultivars. They attributed this to an increase in crown number and therefore an increase in the sites for inflorescence induction; however, they did not confirm this morphologically. Polyamines may also be involved in floral induction in strawberry. Tarenghi and Martin-Tanguy (1995) detected putrescine, spermidine, and spermine in the shoot apices of SD strawberries during floral induction and prior to floral emergence. Polyamine biosynthesis and flowering were inhibited in plants treated with DL-a- difluoromethyl-ornithine (DFMO), a specific inhibitor of putrescine biosynthesis. Exogenous application of putrescine restored the flowering response. Although specific changes in hormone composition, or levels, or both are associated with the flowering process in several different plant species, there is no compelling evidence implicating hormones as the sole floral promoter or inhibitor. Instead, current thinking has led away from the single factor control model, and toward the multifactor control model. While not eliminating the hypothesis that hormones are involved in the flowering process, the multifactor control model also implicates other factors as playing significant roles in flowering.

B. Multifactor Control Model In many species, flowering can be induced by a variety of environmental/chemical signals. This observation led Bernier et al. (1981) to propose that multiple factors, both promoters and inhibitors, interact to regulate floral induction. Later, Bernier et al. (1993) proposed a multifactor control model for flowering based on studies of the LD species Sinapsis alba. In this model, inductive photoperiods cause a rapid but transient translocation of sucrose from leaves to root and shoot apices (Fig. 6.2). The increase of sucrose in the roots triggers cytokinin translocation to the apex, via the xylem. The accumulation of both sucrose and cytokinin in the apical meristem then induces the transition from vegetative to floral growth. More recent work with a diversity of photoperiodically sensitive plants supports this model (Bernier et al. 1998). Using single cycle LD (Sinapsis alba, Arabidopsis thaliana 1., and Lolium temulentum 1.) and SD (Xanthium strumarium 1.) plants, the authors showed that floral induction in all species caused several changes in the content of the phloem sap exported from mature leaves. In all cases, the first change to occur was an increase in sucrose concentration, and this clearly preceded any activation of the apical meristem, as evidenced by an increase in cell division. Thus, the increased sucrose concentration in the phloem was not a source-sink effect (i.e., higher demand by the apices), sug-

341

6. THE PHYSIOLOGY OF FLOWERING IN STRAWBERRY

•

1.1>

>

•

LD WCtPtlon I

1

Mature

leaf

Fig. 6.2. Diagram of the model for the control of transition to flowering in Sin apsis alba involving sucrose and cytokinins. Step 1 (wavy arrow), perception of LD induction by mature leaves; Step 2 (solid arrow), starch mobilization in leaves and stem followed by transport of sucrose in the phloem to both the apical meristem and roots; Step 3 (dashed arrow), transport in the xylem from roots to leaves of zeatin riboside ([9R]Z and isopentenyladenine riboside ([9RliP); Step 4 (dotted arrow), transport in the phloem from leaves to the apical meristem of isopentenyladenine (iP). (From Bernier et al. 1993.)

gesting that sucrose acts as a floral signal. In both S. alba and X. strumarium, the increase in sucrose export preceded the increase in cytokinin concentration in the xylem. In fact, in S. alba, phloem disruption between the mature leaf and root prevented sucrose translocation to the roots and also eliminated the stimulation of cytokinin export

342

R. DARNELL, D. CANTLIFFE, D. KIRSCHBAUM, AND C. CHANDLER

from roots to the apical meristem via the xylem. Increased export of polyamines from the mature leaves, which coincides with the increased export of cytokinin from the roots, may also be related to flower induction in S. alba, since polyamines appear to interact with cytokinins in the control of the cell division cycle (Bernier et aI. 1993). Although not specifically mentioned, Bernier's model implies that under noninductive conditions, various flowering inhibitors (e.g., GAs in strawberry?) are present that prevent changes in the chemical constituents that lead to flowering. Although chemical changes (particularly changes in sucrose and cytokinin) in phloem sap concentration prior to floral induction have been reported in both SD and LD plants, the evidence is insufficient to conclude that these chemicals are floral signals. Verification lies with studies to test the effects of applied sucrose and cytokinins, as well as genetic studies using flowering mutants and transgenic plants. V. GENETICS OF FLORAL INDUCTION

Although several genes involved in fruit development, flavor, and cold tolerance in strawberry have been identified (Hokanson and Maas 2001), identification of genes involved in the flowering process in strawberry has not yet been achieved. However, multiple genes that control flowering in Arabidopsis and pea have been identified. In Arabidopsis, flowering involves the expression of floral meristem identity genes, such as LEAFY (LFY) , APETALA1, and 2 (AP1, AP2), CAULIFLOWER (CAL), and UNIDENTIFIED FLORAL ORGANS (UFO) (Koornneef et aI. 1998; van Nocker 2001). There appear to be at least four pathways that control expression of these genes and therefore flowering in Arabidopsis. Two appear to be regulated developmentally; while two are regulated environmentally (i.e., photoperiod and vernalization). The photoperiodic promotion pathway begins with daylength perception by photoreceptors, such as phytochrome (principally PHYA and PHYB) and cryptochrome (CRYl and CRY2) (Levy and Dean 1998). Daylength perception initiates a transduction or input pathway that apparently entrains the circadian clock, resulting in output pathways that generate overt rhythms, that is, photoperiodic control of flowering (McClung 2001). Components of the downstream signaling pathways from PHY and CRY are unknown, although several intermediates are implicated in an Arabidopsis circadian system model proposed by McClung (2001). Light quality also influences flowering through effects on the circadian clock (van Nocker 2001). However, light is not required for floral induction, since Arabidopsis can be induced to flower in complete darkness when sufficient

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carbohydrates are provided to the meristem (Koornneef et al. 1998; van Nocker 2001). Vernalization also induces flowering in Arabidopsis, through the expression of vernalization genes (VRNl and VRN2) and a transduction pathway that may involve GA synthesis, activity, or both (Koornneef et al. 1998). However, Arabidopsis mutants defective in GA biosynthesis or perception still exhibit normal flowering responses to vernalization (van Nocker 2001), arguing against the involvement of GA as a component of the transduction pathway. Work with late-flowering ecotypes of Arabidopsis revealed the presence of a dominant allele at the FRIGIDA (FRI) locus that is responsible for delayed flowering (Battey 2000). These effects can be overcome by vernalization. A second locus, FLOWERING LOCUS C (FLC), has been identified that also delays or represses flowering. FLC is hypothesized to be the primary flowering inhibitor in Arabidopsis, whose synthesis is promoted by FRI (Michaels and Amasino 1999), and may be inhibited by VRN2 (Sheldon et al. 1999), although evidence for this is equivocal. The octoploid nature of the commercial strawberry makes genetic analysis of flowering complex, and, as stated earlier, no genes involved in flowering of strawberry have as yet been identified. However, Battey et al. (1998) are using positional cloning to isolate genes involved in flowering of F. vesca. This wild species has both a continually flowering form (F. vesca f. semperflorens) and a seasonal flowering form (F. vesca f. vesca). A single gene controls this trait and the dominant allele confers seasonal flowering in F. vesca f. vesca (Brown and Wareing 1965). This is in contrast to work with F. x ananassa, which suggests that the dominant allele confers photoperiodic insensitivity (Ahmadi et al. 1990) or that flowering in F. x ananassa is a quantitative trait (Hancock et al. 2001) On the other hand, F. vesca has several characteristics that make it an attractive system for studying flowering in strawberry; it is diploid (2n = 14), has a short generation time (approximately four months), a clear distinction between vegetative and reproductive growth, and a described transformation system (EI Mansouri et al. 1996; Battey et al. 1998). Furthermore, the physiology of the flowering processes in F. vesca and F. x ananassa is similar enough to imply a common molecular basis (Battey et al. 1998). Since the data support the hypothesis that floral induction in strawberry occurs after removal or repression of a floral inhibitor, Battey (2000) speculates that a homologue of FLC may be involved in the inhibition of flowering in strawberry and other plants. Under conditions of warm temperatures and long photoperiods, this repressor gene becomes active in SD plants, and floral inhibition occurs (Fig. 6.3). Battey further proposes that expression or activity of the FLC homologue in

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Warm, long days

I Cool, short days I t-------

Flower repressor gene active

I Flower :epr~ssor I t------gene InactIve

Cold ----------1 Reactivation of flower repressor-! gene

Inhibitor

II

No inhibitor

Vegetative growth, flower emergence and fruiting

II

Flower initiation

I

Sep

Nov

!~C2r~~S_e~ inhibitor

Dormancy

Jan

Feb

I

Mar

Apr

May

Jun

Jul

Aug

Oct

I t------,Dormancy

Dec

Fig. 6.3. The physiological and genetic control of the perennial cycle in the Junebearing strawberry. (Months applicable in N. Hemisphere.) (From Battey et al. 1998.)

strawberry may be inhibited by homologues of genes identified in Arabidopsis that are involved in the promotive photoperiodic pathway. Thus, under cool temperatures and short photoperiods, the repressor gene is inactivated and floral induction occurs. However, this hypothesis has not been verified. As well, the roles of winter chilling, low temperatures, and other environmental factors in regulating expression of flowering genes in strawberry, how these genes interact, and the components of the signal transduction pathway(s) are still unknown. Work is continuing, however, and recent research using intersimple sequence repeat PCR primer-pair combinations has identified two markers located within 2.2 eM of the seasonality locus inF. vesca (Cekic et al. 2001). This represents the first step toward isolation of the seasonal flowering gene by map-based cloning. VI. CONCLUSIONS

The transition from vegetative to reproductive growth in strawberry involves a series of consecutive stages beginning with floral induction. In general, flower induction in strawberry is controlled primarily by the interaction of photoperiod, temperature, and genotype. The effects of these factors on flowering have been described in many studies, and flowering can be manipulated based on information from such studies. Despite our ability to regulate flowering, the connection between the environmental signals and flower induction has not been elucidated. The evidence supports the idea of the transmission of a floral inhibitor between leaves and apices or between mother and daughter plants, rather than the transmission of a florigenic substance, although the lat-

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ter theory has not yet been discarded. However, regulation of flowering by a single factor fails to account for the complexities of flowering, which is inducible by a variety of signals. Clearly, the unraveling of the flowering process in strawberry will not be achieved by continuation of descriptive studies on environmental influences on flower induction and development. Rather, it awaits the identification of the genes involved in the flowering process. Both the octoploid nature of the cultivated strawberry and the difficulty in manipulating it in vitro present obstacles to standard molecular biology approaches (Hokanson and Maas 2001). F. vesca, a diploid with similar flowering physiology to F. x ananassa, offers a simpler plant system with which to work. However, genetic analysis of flowering in F. vesca may have limited applicability to the more complex octoploid, F. x ananassa, and the usefulness of this approach in unraveling the flowering process in F. x ananassa remains to be seen. LITERATURE CITED Ahmadi, H., R. S. Bringhurst, and V. Voth. 1990. Modes of inheritance of photoperiodism in Fragaria. J. Am. Soc. Hort. Sci. 115:146-152. Albregts, E. E., and C. M. Howard. 1977. Effect of planting date and plant chilling on growth and fruiting responses of three strawberry clones. Proc. Fla. State Hort. Soc. 90: 278-280.

Albregts, E. E., and C. M. Howard. 1980. Effect of pre-transplant chilling and planting date on the growth and fruiting response of the 'Dover' strawberry. Proc. Fla. State Hart. Soc. 93:239-241.

Avigdori-Avidov, H., E. E. Goldschmidt, and N. Kedar. 1977. Involvement of endogenous gibberellin in the chilling requirements of strawberry (Fragaria x ananassa Duch.). Ann. Bot. 41:927-936. Bailey, J. S., and A. W. Rossi. 1965. Effect offall chilling, forcing temperature, and day length on the growth and flowering of Catskill strawberry plants. Proc. Am. Soc. Hart. Sci. 87: 245-252.

Battey, N. H. 2000. Aspects of seasonality. J. Expt. Bot. 51:1769-1780. Battey, N. H., P. LeMiere, A. Tehranifar, C. Cekic, S. Taylor, K. J. Shrives, P. Hadley, A. J. Greenland, J. Darby, and M. J. Wilkinson. 1998. Genetic and environmental control of flowering in strawberry, p. 111-131. In: K. E. Cockshull, D. Gray, G. B. Seymour, and B. Thomas (eds.). Genetic and environmental manipulation of crops. CAB, New Yark. Bernier, G. 1988. The control of flaral evocation and morphogenesis. Annu. Rev. Plant Physiol. Plant Mol. BioI. 39:175-219. Bernier, G., L. Corbesier, C. Perilleux, A. Havelange, and P. Lejeune. 1998. Physiological analysis ofthe floral transition, p. 103-109. In: K. E. Cockshull, D. Gray, G. B. Seymour, and B. Thomas (eds.). Genetic and environmental manipulation of crops. CAB, New York. Bernier, G., A. Havelange C. Houssa, A. Petitjean, and P. Lejeune. 1993. Physiological signals that induce flowering. Plant Cell 5:1147-1155. Bernier, G., J. M. Kinet, and R. M. Sachs. 1981. The physiology of flowering, Vol. II. CRC Press, Boca Raton, FL.

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Bish, E. B., D. J. Cantliffe, and C. K. Chandler. 1996. Strawberry plug transplants: regulation of growth and production. Proc. Fla. State Hort. Soc. 109:160-164. Blatt, C. R, and A. G. Sponagle. 1974. Effects of several growth regulators on runner plant production, yield and fruit maturity of the strawberry. Can. J. Plant Sci. 54:873-875. Borthwick, H. A., and M. W. Parker. 1938. Influence of photoperiods upon differentiation of meristems and the blossoming of Biloxi soybeans. Bot. Gaz. 99:825-839. Brown, T., and P. F. Wareing. 1965. The genetic control of the everbearing habit and three other characters in varieties of Fragaria vesca. Euphytica 14:97-112. Cain, N. P., R P. Ormrod, and D. Evans. 1983. Yield responses of strawberry to fall application of ethephon. Can. J. Plant Sci. 63:1093-1095. Cekic, c., N. H. Battey, and M. J. Wilkinson. 2001. The potential ofISSR-PCR primer-pair combinations for genetic linkage analysis using the seasonal flowering locus in Fragaria as a model. Theor. Appl. Genet. 103:540-546. Ceulemans, R, W. Baets, M. Vanderbruggen, and 1. Impens. 1986. Effects of supplemental irradiation with HID lamps, and NFT gutter size on gas exchange, plant morphology and yield of strawberry plants. Scientia Hort. 28:71-83. Chabot, B. F. 1978. Environmental influences on photosynthesis and growth in Fragaria vesca. New Phytol. 80:87-98. Chajlachjan, M. H. 1936. On the hormonal theory of plant development. Compt. Rend. (Doklady) Acad. Sci. U.RS.S. 3:442-447. Chen, W. S. 1991. Changes in cytokinins before and during early flower bud differentiation in lychee (Litchi chinensis Sonn.). Plant Physiol. 96:1203-1206. Chen, W. S., K. L. Huang, and H. C. Yu. 1997. Cytokinins from terminal buds of Euphoria longana during different growth stages. Physiol. Plant. 99:185-189. Choma, M. E., and D. G. Himelrick. 1984. Responses of day-neutral, June-bearing and everbearing strawberry cultivars to gibberellic acid and phthalamide treatments. Scientia Hort. 22:257-264. Darnell, R L., and J. F. Hancock. 1996. Balancing vegetative and reproductive growth in strawberry, p. 144-150. In: M. V. Pritts, C. K. Chandler, and T. E. Crocker (eds.), Proc. IV North American strawberry conference. Univ. of Florida, Gainesville, FL. Darrow, G. M. 1936. Interrelation of temperature and photoperiodism in the production of fruit-buds and runners in the strawberry. Proc. Am. Soc. Hort. Sci. 34:360-363. Darrow, G. M. 1966. The strawberry. Holt, Rinehart and Winston, New York. Deng, X., and F. 1. Woodward. 1998. The growth and yield responses of Fragaria ananassa to elevated CO 2 and N supply. Ann. Bot. 81:67-71. Dennis, F. G. Jr., and H. O. Bennett. 1969. Effect of gibberellic acid and deflowering upon runner and inflorescence development in an everbearing strawberry. J. Am. Soc. Hort. Sci. 94:534-537. Dennis, F. G. Jr., J. Lipecki, and C. Kiang. 1970. Effect of photoperiod and other factors upon flowering and runner development of three strawberry cultivars. J. Am. Soc. Hort. Sci. 95:750-754.

Deyton, D. K, C. K Sams, and J. C. Cummins. 1991. Strawberry growth and photosynthetic responses to padobutrazol. HortScience 26:1178-1180. Downs, R J., and A. A. Piringer. 1955. Differences in photoperiodic responses of everbearing and June-bearing strawberries. Proc. Am. Soc. Hort. Sci. 66:234-236. Durner, E. F., J. A. Barden, D. G. Himelrick, and E. B. Poling. 1984. Photoperiod and temperature effects on flower and runner development in day-neutral, Junebearing, and everbearing strawberries. J. Am. Soc. Hort. Sci. 109:396-400. Durner, E. F., and E. B. Poling. 1987. Flower bud induction, initiation, differentiation, and development in the 'Earliglow' strawberry. Scientia Hort. 31:61-69. Durner, E. F., and E. B. Poling. 1988. Strawberry developmental responses to photoperiod and temperature: a review. Adv. Straw. Prod. 7:6-14.

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Durner, E. F., E. B. Poling, and E. A. Albregts. 1986. Early season yield responses of selected strawberry cultivars to photoperiod and chilling in a Florida winter production system.]. Am. Soc. Hort. Sci. 111:53-56. El-Antably, H. M., P. F. Wareing, and]. Hillman. 1967. Some physiological responses to D, L abscisin (dormin). Planta 73:74-90. EI Mansouri, I.,]. A. Mercado, V. Valpuesta,]. M. Lopez-Aranda, R. Pliego-Alfaro, and M. A. Quesada. 1996. Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L. Plant Cell Rep. 15:642-646. Esau, K. 1977. Anatomy of seed plants. 2nd ed. Wiley, New York. FAO. 2001. FAOSTAT Agricultural Statistics Database. http://www.fao.org. Ferree, D. c., and E. ]. Stang. 1988. Seasonal plant shading, growth, and fruiting in 'Earliglow' strawberry.]. Am. Soc. Hort. Sci. 113:322-327. Guttridge, C. G. 1956. Photoperiodic promotion of vegetative growth in the cultivated strawberry plant. Nature 178:50-51. Guttridge, C. G. 1959a. Evidence for a flower inhibitor and vegetative growth promoter in the strawberry. Ann. Bot. 23:351-360. Guttridge, C. G. 1959b. Further evidence for a growth-promoting and flower-inhibiting hormone in strawberry. Ann. Bot. 23:613-621. Guttridge, C. G. 1968. Hormone physiology of growth regulators in strawberry, p. 157-169. In: Plant growth regulators, monograph no. 21. Soc. Chern. Ind., London. Guttridge, C. G. 1969. Fragaria, p. 247-267. In: L. T. Evans (ed.). The induction of flowering. Cornell Univ. Press, Ithaca, New York. Guttridge, C. G. 1985. Fragaria x ananassa, p. 16-33. In: A. H. Halevy (ed.). Handbook of flowering. Vol. III. CRC Press, Boca Raton, FL. Guttridge, C. G., and P. A. Thompson. 1959. Effect of gibberellic acid on length and number of epidermal cells in petioles of strawberry. Nature 183:197-198. Guttridge, C. G., and P. A. Thompson. 1964. The effect of gibberellins on growth and flowering of Fragaria and Duchesnea. ]. Expt. Bot. 15:631-646. Hamner, K. c., and]. Bonner. 1938. Photoperiodism in relation to hormones as factors in floral initiation and development. Bot. Gaz. 100:388-431. Hancock,]. F. 1999. Strawberries. CABI, New York. Hancock, ]. F., ]. ]. Luby, A. Dale, A. Callow, S. Serce, and A. EI-Shiek. 2002. Utilizing wild Fragaria virginiana in strawberry cultivar development: Inheritance of photoperiod sensitivity, fruit size, gender, female fertility and disease resistance. Euphytica (in press). Hartmann, H. T. 1947a. Some effects oftemperature and photoperiod on flower formation and runner production in the strawberry. Plant Physiol. 22:407-420. Hartmann, H. T. 1947b. The influence of temperature on the photoperiodic response of several strawberry cultivars grown under controlled environment conditions. Proc. Am. Soc. Hort. Sci. 50:243-245. Hartz, T. K., A. Baameur, and D. B. Holt. 1991. Carbon dioxide enrichment of high-value crops under tunnel culture. ]. Am. Soc. Hort. Sci. 116:970-973. Heide, O. 1977. Photoperiod and temperature interactions in growth and flowering of strawberry. Physiol. Plant. 40:21-26. Hokanson, S. c., and]. L. Maas. 2001. Strawberry biotechnology. Plant Breeding Rev. 21: 139-180. Ito, H., and T. Saito. 1962. Studies on the flower formation in the strawberry plants. I. Effects of temperature and photoperiod on the flower formation. Tahoku]. Agri. Res. 13:191-203. ]ahn, O. L., and M. N. Dana. 1966. Dormancy and growth of the strawberry plant. Proc. Am. Soc. Hort. Sci. 89:322-330. ]onkers, H. 1965. On the flower formation, the dormancy and the early forcing of strawberries. Meded. Landbouwhogesch. Wageningen 65-6:1-59.

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Kadman-Zahavi, A., and E. Ephrat. 1974. Opposite response groups of short-day plants to the spectral composition of the main light period and end-of-day red or far red irradiation. Plant Cell Physiol. 15:693-699. Kawakami, T., H. Aoki, and T. Toki. 1990. Method of early fruit maturing using low night temperatures and short day conditions during the propagation of strawberries. Bul. China Pref. Agri. Expt. Sta. 31:55-72 (English summary). Kender, W. J., Carpenter, S., and J. W. Braun. 1971. Runner formation in everbearing strawberry as influenced by growth-promoting and inhibiting substances. Ann. Bot. 35:1045-1052.

Kirschbaum, D. S. 1998. Temperature and growth regulator effects on growth and development of strawberry. MS Thesis, Univ. of Florida, Gainesville, FL. Koornneef, M., C. Alonso-Blanco, A. J. M. Peeters, and W. Soppe. 1998. Genetic control of flowering time in Arabidopsis. Annu. Rev. Plant Physiol. Plant Mol. BioI. 49:345-370. Larson, K. D. 1994. Strawberry, p. 271-297. In: B. Schaffer and P. Andersen (eds.). Handbook of environmental physiology of fruit crops. Vol. I: Temperate crops. CRC Press, Boca Raton, FL. Leshem, Y., and D. Koller. 1964. The control of flowering in the strawberry Fragaria ananassa Duch. I. Interaction of positional and environmental effects. Ann. Bot. 112:569-578. Leshem, Y., and D. Koller. 1966. The control of flowering in the strawberry Fragaria ananassa Duch. II. The role of gibberellins. Ann. Bot. 30:587-595. Levy, Y. Y., and C. Dean. 1998. The transition to flowering. Plant Cell 10:1973-1989. Lopez-Galarza, S., B. Pascual, J. Alargada, and J. Maroto. 1989. The influence of winter gibberellic acid applications on earliness, productivity and other parameters of quality in strawberry (Fragaria x ananassa Duch.) cultivation on the Spanish Mediterranean coast. Acta Hort. 265:217-222. Manakasem, Y., and P. B. Goodwin. 1998. Using the floral status of strawberry plants, as determined by stereomicroscopy and scanning electron microscopy to survey the phenology of commercial crops. J. Am. Soc. Hort. Sci. 123:513-517. McArthur, D. A. J., and G. W. Eaton. 1987. Effect offertilizer, paclobutrazol, and chloromequat on strawberry. J. Am. Soc. Hort. Sci. 112:241-246. McClung, C. R. 2001. Circadian rhythms in plants. Annu. Rev. Plant Physiol. Plant Mol. BioI. 52:139-162. Michaels, S. D., and R. M. Amasino. 1999. FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11 :949-956. Moore, J. N., and L. F. Hough. 1962. Relationships between auxin levels, time of floral induction and vegetative growth of the strawberry. Proc. Am. Soc. Hort. Sci. 81:255-264. Nicoll, M. F., and G. J. Galletta. 1987. Variation in growth and flowering habits ofJunebearing and everbearing strawberries. J. Am. Soc. Hort. Sci. 112:872-880. Okimura, M., and I. Igarashi. 1997. Effects of photoperiod and temperature on flowering in everbearing strawberry seedlings. Acta Hort. 439:605-607. Piringer, A. A., and D. H. Scott. 1964. Interrelation of photoperiod, chilling, and flower cluster and runner production by strawberries. Proc. Am. Soc. Hort. Sci. 84:295-301. Porlingis, I., and D. Boynton. 1961. Growth responses of the strawberry plant Fragaria chiloensis var. ananassa, to gibberellic acid and to environmental conditions. Proc. Am. Soc. Hort. Sci. 78:261-269. Reid, J. H. 1983. Practical growth regulator effects on strawberry plants-a review. Crop Res. 23:113-120.

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Sheldon, C. S., J. K Burn, P. P. Perez, J. Metzger, J. A. Edwards, W. J. Peacock, and K S. Dennis. 1999. The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 11:445-458. Singh, J. P., G. S. Randhawa, and N. L. Jain. 1960. Response of strawberry to gibberellic acid. Indian J. Hort. 17:21-30. Smeets, L. 1976. Effect of light intensity on stamen development in the strawberry cultivar 'Glasa.' Scientia Hort. 4:255-260. Smith, C. R. 1960. Effect of autumn applications of potassium gibberellate on fruit production of the strawberry. Nature 187:620. Strik, B. C. 1985. Flower bud initiation in strawberry cultivars. Fruit Var. J. 39:5-9. Tafazoli, K, and D. Vince-Prue. 1978. A comparison of the effects of long days and exogenous growth regulators on growth and flowering in strawberry, Fragaria x anon ossa Duch. J. Hort. Sci. 53:255-259. Tarenghi, K, and J. Martin-Tanguy. 1995. Polyamines, floral induction and floral development of strawberry (Fragaria x anon ossa Duch). Plant Growth Reg. 17:157-165. Taylor, D. R., P. T. Atkey, M. F. Wickenden, and C. M. Crisp. 1997. A morphological study of flower initiation and development in strawberry (Fragaria x anonossa) using cryoscanning electron microscopy. Ann. Appl. Bioi. 130:141-152. Tehranifar, A,., and N. H. Battey. 1997. Comparison of the effects ofGA 3 and chilling on vegetative vigour and fruit set in strawberry. Acta Hort. 439:627-631. Thomas, B., and D. Vince-Prue. 1997. Photoperiodism in plants. Academic Press, San Diego. Thompson, P. A., and C. G. Guttridge. 1960. The role ofleaves as inhibitors of flower induction in strawberry. Ann. Bot. 24:482-490. Uematsu, Y., and N. Katsura. 1983. Changes in endogenous gibberellin level in strawberry plants induced by light breaks. J. Japan. Soc. Hort. Sci. 51:495-511. van Nocker, S. 2001. The molecular biology of flowering. Hort. Rev. 27:1-39. Vince-Prue, D., arid C. G. Guttridge. 1973. Floral initiation in strawberry: spectral evidence for the regulation of flowering by long-day inhibition. Planta 110:165-172. Voth, V., and R. S. Bringhurst. 1970. Influence of nursery harvest date, cold storage, and planting date on performance of winter California strawberries. J. Am. Soc. Hort. Sci. 95:496-500. Waithaka, K., and M. N. Dana. 1978. Effects of growth substances on strawberry growth. J. Am. Soc. Hort. Sci. 103:627-628. Weidman, R. W., and K J. Stang. 1983. Effects of gibberellins (GA 4 + 7 ), 6-benzyladenine (6BA) and promalin (GA 4 + 7 + 6-BA) plant growth regulators on plant growth, branch crown and flower development in 'Scott' and 'Raritan' strawberries. Adv. Straw. Prod. 2:15-17. Went, F. W. 1957. Environmental control of plant growth. Chron. Bot., Waltham, MA. Wisemann, N. J., and C. G. N. Turnbull. 1999. Endogenous gibberellin content does not correlate with photoperiod-induced growth changes in strawberry petioles. Austral. J. Plant Physiol. 26:359-366. Yamasaki, A., and M. Yamashita. 1993. Changes in endogenous cytokinins during flower induction of strawberry. Acta Hort. 345:93-99. Zhang, N., Yong, J., Hew, c., and X. Zhou. 1995. The production of cytokinin, abscisicacid and auxin by CAM orchid aerial roots. J. Plant Physiol. 147:371-377.

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Flower and Fruit Thinning of Peach and Other Prunus Ross E. Byers Department of Horticulture Virginia Polytechnic Institute and State University Alson H. Smith, Jr., Agricultural Research and Extension Center 595 Laurel Grove Road Winchester, VA 22602 Guglielmo Costa Dipartimento di Colture Arboree University of Bologna Via Fanin 50 Bologna, 40126, Italy Giannina Vizzotto Dipartimento di Produzione Vegetale e Tecnologie Agrarie Udine University Via delle Scienze 208 Udine, 33100, Italy

1. INTRODUCTION

A. Fruit and Flower Thinning as Horticulture Practices B. Economic Benefits of Flower versus Fruit Thinning 1. Pollination, Fertilization, Ovule Abortion, and Compatibility 2. Competitive Assimilate Fruit Drop II. REPRODUCTIVE PHYSIOLOGY A. Flower Development B. Chilling C. Fruit Set

Horticultural Reviews, Volume 28, Edited by Jules Janick ISBN 0-471-21542-2 © 2003 John Wiley & Sons, Inc. 351

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III. ABSCISSION IV. THINNING PRACTICES A. Dormant pruning B. Flower Bud Inhibition or Abortion 1. Gibberellic Acid 2. Oils 3. Ethephon/GA Sprays at Leaf Fall and During Dormancy C. Flower Thinning 1. Mechanical Flower Removal 2. Chemical Flower Removal D. Fruit Thinning 1. Manual Fruit Thinning 2. Chemical Fruit Thinning E. Chemical Application Technology 1. Chemical Flower Thinning 2. Fruit Thinning F. Combinations of Flower and Fruit Thinning V. FUTURE PROSPECTS LITERATURE CITED

I. INTRODUCTION

A. Flower and Fruit Thinning as Horticultural Practices

Flower and fruit thinning of Prunus are commercially practiced in an effort to maximize crop value by optimizing fruit size, color, shape, and quality, to promote return bloom, and to maintain tree growth and structure (Farley 1923; Havis 1962; Byers and Lyons 1984 a,b; Webster and Andrews 1986; Byers 1989 a,b). In addition, thinning promotes more uniform annual bearing, which optimizes the use of labor and field, packaging and storage equipment. This chapter will emphasize flower and fruit thinning of peach and nectarine (P. persica), since the need to reduce whole tree photosynthetic demand of the fruit is much greater than for smaller stone fruit species where the ratio of total fruit weight per tree to total leaf area is low, such as with sweet cherry (P. avium) and tart cherry (P. cerasus). Certain European plum (P. domestica) and Japanese plum (P. salicina) cultivars set very heavy crops that can dramatically affect tree structure, even though the fruits are typically much smaller than peach fruits. The need for thinning other Prunus species, such as apricot (P. armeniaca) or sweet cherry, has been influenced by market prices for larger fruit, but tree structure is seldom an issue. In many regions, most sweet cherry cultivars require cross-pollination to set fruit and thus do not set as well as do peach cultivars, which are self-fruitful. As breeding programs introduce more self-fruitful cherry cultivars, the need for commercial fruit thinning will likely become more important.

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Typically, the longer unwanted fruits remain on the tree, the greater the negative effect they will have on fruit and leaf size, tree growth, flower bud differentiation, flower bud hardiness, the next season's crop potential, and tree survival (Shoemaker 1933; Byers and Lyons 1985a,b; Byers et al. 1985, 1990; Myers 1986; Byers and Marini 1994). Estimating the maximum numbers of fruit to be retained to harvest requires an integrated knowledge of market price structure for different sized fruit, the genetic potential of a cultivar for fruit size and yield, and the effect of cultural practices (pruning, fertilization, irrigation, soils, scoring) on crop value. The optimum fruit size and yield for fresh versus processed markets may differ substantially by geographic region. For fresh peach fruit, the optimum diameter depends on customer preference and may range from 6.4 to 8.8 em. Typically, processors want fruit 6 to 8 em in diameter and uniform yellow flesh color for canning. The optimum date for thinning usually occurs before the demand for carbohydrates exceeds supply (Connors 1919; Costa et al. 1986; DeJong and Goudriaan 1989). Fruit growth kinetics based on daily demand for photosynthates, expressed as increase in dry weight per degree day (sink strength), may be useful in establishing an appropriate thinning date to achieve a specific fruit size. Environmental and cultural conditions (i.e., cultivar selection, light, water, soil, pruning, thinning, and fertilization practices) affect yield and fruit quality. The low tree densities (250 trees/hal typical in the eastern United States, allow a maximum fruit load of 500 to 700 peaches when nonirrigated, and perhaps 20 percent more when irrigated. These numbers could vary 10 to 30 percent or more, depending on environment and cultural practices. In irrigated areas of California or Australia with high light intensity, higher numbers are typical, particularly where largefruited cultivars can be grown or where smaller fruit are desirable for the processing market. In high density orchards when up to 1500 plus trees/ha are planted, trees must be irrigated to guarantee marketable fruit size. The major cullage factors for stone fruit are small fruit size and, secondly, insufficient color. Inadequate fruit thinning soon after bloom is considered responsible for small fruit size (Havis 1962). Typically, alternate bearing is not a problem with most stone fruits (exceptions are sweet cherry and some European plums), unless trees are cropped excessively or growth is extremely vigorous (Dorsey 1935). Fruit size, color, shape, flower bud differentiation, natural fruit set, and thinning response differ among cultivars. Recognition of these individual characteristics is extremely important for maximizing crop value. Certain peach cultivars produce large fruits (e.g., 'J. H. Hale' and 'Glohaven') while others produce smaller ones (less than 6.35 em in diameter) despite moderate croploads (e.g., 'Georgia Bell'). Furthermore, return bloom may be heavy on some cultivars (e.g., 'Biscoe,' 'Madison')

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354

but poor on others (e.g., 'Redglobe,' 'Blake') despite a moderate crop. Therefore, thinning practices must be selected for each individual cultivar within each geographic region and market. B. Economic Benefits of Flower versus Fruit Thinning

Bloom thinning peach trees can result in a 7 to 30% increase in fruit size and yield when compared to hand-thinning fruit 40 to 50 days after full bloom (AFB) (Fig. 7.1). The effect of thinning on the following year's 250..,..---------------,

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Fig. 7.1. The influence of time of hand removal of flowers and fruits from 'Redhaven' peach trees on number of fruit per bushel (A), yield (B), and time of fruit maturation (C) (from Havis 1962).

355

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS Table 7.1. Calculated costs and returns in relation to fruit size of peach with 296 trees/ha and 700 fruit/tree (Byers 1989). Fruit diameter (cm)

No. fruit/ 35.24L (bushel)

Price !I 17.24 kg/ carton ($)

Crop return/ha ($)

Packing and transportation costs ($/ha)

Net profit/ha ($)

1.45-5.07 5.08-5.70 5.71-6.34 6.35-6.98 6.99-7.61 7.62-8.26

338 253 185 143 112 85

3 6 9 12 15 18

681 1812 3672 6348 10095 15390

1795 2057 2428 2851 3355 3992

-1114 - 245 + 1244 + 3497 + 6740 + 11398

flSoutheastern Peach Report, Vol. VI, No. 40, August 4,1987, Columbia, Sc.

crop has not been closely studied, but some increase in yield and size has been reported (Byers 1989a,b). However, the major economic effect of thinning is related to influences on the current season's leaf surface and crop load as the fruits compete for photosynthates. Trees with genotypes that mature earlier in the season, produce and set a higher percentage of flowers per tree, or produce smaller fruit usually derive a greater economic benefit from bloom thinning (or early fruit thinning) than late maturing genotypes. The cost of bloom thinning coupled with hand fruit thinning may result in annual costs similar to hand-thinning alone 40 to 50 days AFB (Byers 1989a,b). Alternatively, partial bloom thinning may increase crop value one to three times and profits several times because of increased fruit size, yield, and price (Table 7.1). Since prices usually increase with fruit size, adequate bloom thinning, followed by handthinning, irrigation, and other cultural practices may greatly increase profitability. II. REPRODUCTIVE PHYSIOLOGY

A. Flower Development

An understanding of reproductive phenology of Prunus is required to understand the ramifications of adjusting the crop load for maximum economic value. Typically, Prunus sp. differentiate very high numbers of flower buds but cultivars within species may differ widely (Table 7.2). Flower bud induction and differentiation begins soon after bloom and continues over a considerable length of time during the summer, as long as the shoots are actively growing. In peach, Dorsey (1935) determined that axillary flower buds were differentiated at eight nodes in the

356 Table 7.2.

R. BYERS, G. COSTA, AND G. VIZZOTTO

Fruit set of various stone fruit species. Fruit set References

Species

(%)

Almond

14-57

Apricot

0-60

Egea et al. 1991; Lichou et al. 1995; McLaren et al. 1995; Mahanoglu et al. 1995a,b

Cherry Sweet

0-40

Bargioni 1978; Webster et al. 1979; Roversi and Ughini 1985; Godini et al. 1997

0-27 2-50

Montalti and Selli 1984 Montalti and Selli 1984

Tart Self-pollination Cross-pollination Peach Clingstone Freestone

36-85 40-95

Kester and Griggs 1959; Socias i Company and Felipe 1987; Vasilakakis and Porlingis 1984; Kester 1994; Godini 1997

Filiti et al. 1982; Visai et al. 1985; Weinbaum and Erez 1983 Filiti et al. 1982; Visai et al. 1985; Weinbaum and Erez 1983

Nectarine

28-85

Filiti et al. 1982; Visai et al. 1985; Weinbaum and Erez 1983

Plum Open pollination Cross-pollination

16-55

Keulemans 1991 Keulemans 1991

tip and as growth continued, more buds were differentiated. However, under favorable conditions, peach and nectarine trees may set so much fruit that flower bud initiation may be inhibited at all nodes as the tip is growing. As the shoot grows, each node goes through a period when flower bud initiation is possible, but if cropload is too heavy or if growth is extremely vigorous, differentiation may not occur. Thinning (or harvesting) a substantial part of the crop reduces the competition between flower buds and developing fruits, shoots (Fig. 7.2A and 7.2B), and roots. Flower bud differentiation may be initiated again, if growth of the tip produces new nodes, but fruit removal will not cause initiation of new flower buds at nodes previously differentiated. In early maturing cultivars, sufficient growth may occur after harvest to allow sufficient numbers of buds to be formed during the remainder of the season. Flower initiation at the basal nodes is evident microscopically by about 50 days after bloom, and at subsequent nodes into late summer. The number of flower buds at each node and the total number of nodes with flower buds can be significantly modified by internal and external factors. Inter-

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

BLOOM THINNED

I

REDHAVEN

HAND THINNED

HAND THINNED

357

BLOOM THINNED

I CRESTHAVEN

I

Fig. 7.2. Bloom thinning increased leaf size, fruit size, and shoot growth of Redhaven peach trees (A), and increased flower bud numbers of Cresthaven trees (B) especially at the first 5 nodes. Since the basal flowers are the last to open, they may be less subject to injury from late bloom freezes (Byers et al. 1990).

nal factors include low nutritional status (C/N ratio) or high gibberellin (GA) content that inhibits flower bud formation (Zeevaart 1983; Goldsmith and Monselise 1970) while external factors include the time of thinning (Byers et al. 1990), climatic conditions (temperature, light, water), inorganic nutrition, and the application of plant growth regulators. Leaf nitrogen and dry matter content of shoots in one year are positively correlated with flower production per unit shoot length during the subsequent year (Marini and Sowers 1990). In both peach (Johnson et al. 1992) and cherry (Kenworthy 1974) any stimulation of growth caused by water, nitrogen, pruning, or other cultural management techniques may increase the ratio of vegetative to flowering buds. In contrast, low vigor (e.g., drought, poor soil aeration, root or trunk injuries, viruses, etc.) may favor flower bud initiation. Flore (1994) pointed out the inverse relationship between vigor and flower bud initiation in fruit crops; however, a heavy crop will inhibit both tree growth and flower bud formation.

B. Chilling In perennial woody plants, chilling is required for normal shoot growth and flowering in the spring (Weinberger 1950; Brown 1958; Monet and Bastard 1968; Legave 1975, 1978; Lam-Yam and Parisot 1990). If the

358

R. BYERS, G. COSTA, AND G. VIZZOTTO

chilling requirement has not been adequately met, the flower bud may die and abscise or flowerslfruits may abscise; thus the next season's growth, flowering, and yield may be compromised. When stone fruit trees are grown commercially under subtropical and tropical conditions' the winter chilling requirement may be partially overcome by chemical and cultural practices. The practices used to break physiological dormancy (Erez and Lavi 1984; Edwards 1987, 1990; Erez 1995) may greatly influence the final result of early flower and fruit thinning. The preceding season's chilling temperatures and duration playa key role in shoot vigor and speed of bud-break (Fuchigami and Nee 1987). The chilling period necessary to satisfy the rest requirement may vary greatly between species and cultivars (approximate temperatures between O°C and 7°C with a duration from 200 to 1000 hr); but its influence on final fruit set is unpredictable. Several methods (e.g., Utah Chill model, Asymcur) in different growing areas have been proposed to determine the time and temperatures that satisfy the chilling requirement (Bennett 1950; Doorenbos 1953; Samish 1954; Gurdian and Biggs 1964; Nienstaedt 1966; Richardson et al. 1974; Nooden and Weber 1978; Erez et al. 1979; Samish and Lavee 1982; Erez 1995). Even though much progress has been made in the selective breeding of new low-chilling peach cultivars, artificial techniques to break rest are still needed. Preconditioning floral buds in the autumn by cultural techniques reduces the need for chilling. Typically in warm winter growing regions, chemical sprays, mineral oils, cyanamide (Erez 1987; De Benito 1990), oil-cyanamide, and other chemicals (thiourea, potassium nitrate, gibberellic acid, cytokinins, paclibutrazole, and Armobreak (a surfactant made of a unique group of fatty amines) have been used to break dormancy, thus compensating for insufficient chilling (Saure 1985). C. Fruit Set

The number of fruit set per tree depends on the number of flowers per unit length of wood, the amount of fruiting wood, climatic conditions during pollination/ fertilization (Byers and Marini 1994), adequate chilling, pruning, and many other environmental and cultural factors. In peach, fruit set between seasons has been as few as 10 percent of the flowers or as great as 85 percent (r. e. Byers unpublished). Obviously, the amount of natural fruit set may greatly influence the numbers of fruit that need to be removed to optimize crop value. Fruit set is extremely variable among Prunus species and cultivars. In addition, reported values differ considerably for different environmental conditions (Table 7.2). The number of reproductive sinks early in the season can be extremely high in stone fruit, up to 50,000 flowers per tree in sweet cherry, and 20,000 in peach. These flow-

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

359

ers and fruit represent a tremendous demand on the tree reserves before adequate leaf surface develops to support shoot and fruit growth (Keller and Loescher 1989). Furthermore, the number of peach fruits on an unthinned and unpruned tree is often too high for the tree to support, resulting in small fruit and limb breakage (Southwick et al. 1996a,b). In some peach cultivars, fruit set may be so great that shoot growth, and leaf number, and fruit enlargement are so inhibited that trees may appear to have very few leaves even as late as 50 days after full bloom (AFB). 1. Pollination, Fertilization, Ovule Abortion, and Compatibility. Pollination, followed by fertilization of the ovule, is required for fruit set in most stone fruit cultivars. Unfertilized fruit typically abscise, but some cultivars retain a small percentage until harvest. In some peach cultivars, these fruit mature later than the main crop and are referred to as "buttons." If temperatures are below 12° to 15°C during bloom and/or the 7 to 10 days following bloom, poor pollination and pollen tube growth will occur, resulting in a low percentage of fertilized ovules, and a large number of fruit abscising within 50 days AFB. Fruit from unfertilized flowers will start slowing in growth rate about 15 to 25 days AFB and will typically abscise before the pit lignifies (pit hardening stage) (r. e. Byers, unpublished). The fruit and seed developing from nonfertilized flowers may not be distinguishable from those from fertilized flowers for the first 25 days AFB. Workers hand-thinning peach, nectarine, plum, and apricot trees within 40 days AFB, and particularly within 15 to 25 days AFB, may find it difficult to visually differentiate between fruit that will drop naturally and those that will remain on the tree, as ovules of unfertilized fruit mayor may not have turned brown. Application of chemical sprays that inhibit pollination and fertilization increase the number of "buttons"; therefore, assessment of the degree of thinning by these chemicals may be difficult, and might complicate follow-up handthinning in the first 25 days AFB. Since weather conditions during the pollination and fertilization period may cause 50 to 80 percent of the fruit to remain unfertilized, workers that are hand-thinning at bloom may over-thin. To avoid this, partial bloom thinning (i.e., leaving two to four times as many flowers as needed), followed by fruit thinning 35 days AFB is typically practiced. This maximizes the fruit size and yield benefits of early crop reduction without danger of over-thinning. Peach and nectarine cultivars are typically self-compatible and frequently are not as subject to alternate bearing as other stone fruit species if good cultural practices are used. When weather is poor for cross-pollination, self-incompatible cultivars (e.g., 'J.H-Hale' peach, 'Ruby Red' nectarine) set poorly, resulting in considerable financial loss.

360

R. BYERS, G. COSTA, AND G. VIZZOTTO

Apricot, European plum, Japanese plum, and sweet cherry require fruit thinning only when fruit set is very high and/or the price for larger fruit greatly increases crop value. Thinning of the fruitlets for these species may be practiced, to increase fruit size, to avoid branch breakage or stimulate flower initiation (Webster and Andrews 1986). Unfortunately, predicting final fruit set at bloom is frequently difficult. Thompson and Liu (1973) proposed that erratic fruit setting of 'Italian' prune induced by cool weather was the result of ovule abortion. Breeding of cultivars with enhanced ovule longevity and consistent annual production may increase the need for thinning (Hanson and Breen 1985; Sun et al. 1991). Apricot cultivars are generally considered self-compatible (Bailey and Hough 1975; Limongelli and Cappellini 1978; Cappellini and Limongelli 1981), but some cultivars have incompatibility problems in some areas (Lamb and Stiles 1983; Nyujt6 et al. 1986); thus single-cultivar apricot orchards should beavoided in these regions (Burgos et al. 1993). 'Canino' apricot produces self-compatible pollen, but the pollen germinates very poorly (Mahanoglu et al. 1995a,b). In the self-fertile 'Rouge de Roussillon,' pollination and fertilization do not limit fruit set; but poor fruit set may occur because of embryo sac abnormalities during seed formation (Lichou et al. 1995), or fruit competition during cloudy weather. In plum, some cultivars yield inconsistently because of poor pollination, low pollen viability, slow pollen tube growth, poor nutritional status of the tree, and/or short embryo sac longevity as a consequence of unfavorable environmental conditions, especially cloudy weather. Cross-pollination with suitable cultivars is therefore recommended (Keulemans 1991). In sweet cherry (Looney 1988), sour cherry, and almond (P. amygdaJus), the number of fruit/shoot is very high, but fruit thinning is not commercially practiced because fruit weight/cm 2 trunk cross-sectional area is still low relative to that in peach or apple. However, areas where set is high, crop value can be increased substantially by early flower or fruit thinning to increase fruit size. Hand-thinning is considered too costly for these fruits because fruit numbers per tree is very high and trees are often very large. In these crops, chemical thinning technology is needed, particularly as high fruit-setting cultivars are introduced. A study of the most important cultivars in Italy indicated that only 'Montmorency,' 'Schattenmorelle,' 'Nabella,' and 'Northstar' were selfcompatible (Montalti and Selli 1984). Standard sweet cherry and almond cultivars are self-incompatible. Because of incompatibility, the most important commercial sweet cherry cultivars require specific pollinizers (Bargioni 1978; Lugli and Sansavini 1997). Almond cultivars are typically self-incompatible. To achieve a commercial fruit set (30% or higher) pollinizers are required (Kester and Griggs 1959). In both almond

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

361

and sweet cherry, full or partial self-compatibility, late bloom, and high fruit set are the main objectives of breeding programs (Kester 1994; Godini et al. 1997). The rapid increase of the almond industry in California may have resulted from the introduction of self-compatible cultivars. As such programs become more successful, the need for thinning all stone fruits will likely increase. Very early flowering (almond < apricot < plum < sweet cherry < tart cherry < peach = nectarine) and consequent susceptibility to freeze damage is also a major influencing fruit set factor. Similarly, winter hardiness and winter freeze injury of species and cultivars may differ. In one study, cultivars of freestone peach were more winter hardy (70% of the cultivars examined during a spring freeze damage were resistant) in comparison with cultivars of nectarine and genetic clingstone peach (15 to 16% resistant) (Filiti et al. 1982). 2. Competitive Assimilate Fruit Drop. Abscission and inhibition of growth of young fruit may result from strong competitive influences from other fruit, shoots, roots, xylem, phloem, and other cellular growth within the tree (Costa et al. 1983; Byers et al. 1984b; Byers et al. 1985; DelValle et al. 1985). A shortage of metabolites during certain early stages of fruitlet development may reduce their sink strength and further reduce fruit growth at later stages when metabolite supply may not be limiting. Competing sinks may use most of the available metabolites and ultimately reduce tree growth, fruit growth, flower bud differentiation, and/or cause abscission of weaker fruit. Under optimal environmental conditions and cultural management, metabolite supply is mainly determined by intersink competition, which may be reduced by the removal of a portion of the developing fruitlets (thinning). If there is substantial competition for the current season's photosynthates, the smaller and weaker fertilized fruit may abscise from the tree at about 45 days AFB. This drop is usually referred to as "June drop" even though it may not occur at that time of year. A significant "June drop" indicates that the fruit were competing significantly with each other and that the remaining fruit will be smaller at harvest than if no such drop had occurred. Fruit lost to "June drop" or hand-thinning represents a loss of photosynthetic reserves, which cannot be regained. Fruit drop caused by poor pollination and fertilization may occur 30 to 45 days AFB and can be confused with drop caused by competition between fruit. Since light is required for photosynthesis, three to four days of cloudy weather between 35 to 50 days AFB may reduce the supply of photosynthate causing fruit abscission in stone fruits (Byers and Lyons, Jr. 1984; DelValle et al. 1985). Since prolonged cloudy periods do not usu-

362

R. BYERS, G. COSTA, AND G. VIZZOTTO

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DAYS AFTER FULL BLOOM

Fig. 7.3. The influence of shading entire 'Cresthaven' peach trees at various times after full bloom on fruit abscission (Byers et al. 1985). Mean separation by Duncan's multiple range test, 5 percent level.

ally occur at this time in the season, fruit drop would not occur until a week or so later. Thinning induced by shading (Fig. 7.3) will increase the average size of the remaining fruit at harvest because of the crop reduction and selective abscission of smaller fruit. If a significant reduction in flowers or fruit number has occurred in the first 20 days AFB, "June drop" would not be expected, since fruit competition would not be sufficient to cause abscission. III. ABSCISSION

Abscission involves a series of programmed events that culminate in altered cell morphologies and weakening of tissue, which may be in response to tissue damage or a part of normal physiological development (Bleecker and Patterson 1997). Anatomically, abscission is the consequence of the dissolution of the middle lamella between adjacent cells in a specialized zone consisting of one or more layers of small, isodiametric cells, which are densely cytoplasmic. The fracture usually occurs in the plane of the middle lamella (Addicott 1982; Sexton and Roberts

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

363

1982). In peach, the fruit abscission layer develops across the vascular bundles leading in part to the separation of vascular strand (Tirlapur et al. 1995), and the final separation may take place by mechanical fracturing, as in tart cherry (Stosser et al. 1969). Typically, the major enzymes involved in cell wall dissolution are exo- and endo-polygalacturonase (PG), and endo-J3-1,4-glucanase (EG) (Ramina et al. 1993; Sexton 1995). Although the final result is the same for leaf and fruit abscission, the induction mechanisms differ for the two organs. In peach fruit pedicels, cell wall digestion starts from the middle lamella, and subsequently extends to the entire parietal mass. In leaf pedicels, cell enlargement occurs after activation of the abscission zone (AZ), involving the separation layer of the fruit, and cells of the adjacent tissues at the adaxial side in a proximal position (Rascio et al. 1985; Ramina et al. 1993). Bonghi et al. (1992) found that EGase activity was high in peach leaf abscission zones but much lower in fruit abscission zones, whereas PG activity was not detected in abscission zones of leaves, but was present at very high levels in those of fruit. These differences in hydrolytic activities (enzymes) are of potential practical use because they may permit chemical stimulation of fruit abscission without leaf abscission (Hadfield and Bennett 1998). Abscission is restricted to specific locations in peach and nectarine fruits (Fig. 7.4). Three abscission zones (AZ1, AZ2, and AZ3) exist between the fruit and the stem (Nelson et al. 1984; Rascio et al. 1985), and are recognizable before the onset of cell wall dissolution and are consistent with the target-cell hypothesis proposed by Osborne (1979). Flowers and small fruits (early June drop) separate from the tree at the basal abscission zone (AZ1) between pedicel and peduncle, while AZ2 (located between the flower receptacle) and the peduncle at AZ3 (between the fruit and the receptacle). These zones are activated in mid(AZ2) and late (AZ3) June drop (Rascio et al. 1985). The AZ3 resembles that in sweet cherry (Wittenbach and Bukovac 1972) and plum (Simons and Chu 1975). The hormonal regulation of the early fruit abscission process differs from senescing pedicels of ripening fruit, even though both may be influenced by ethylene (Sexton and Roberts 1982; Abeles et al. 1992). Ethylene biosynthesis increases before abscission of many organs, but abscission can occur without a rise in ethylene biosynthesis. Inhibitors of ethylene biosynthesis or action may interfere with both ethyleneinduced abscission and also abscission caused by other stresses (Reid 1985). The AZ3 zone does not selectively produce ethylene upon abscission induction, but ethylene biosynthesis may occur in nonzone tissue. The

364

R. BYERS, G. COSTA, AND G. VIZZOTTO

Fig. 7.4. In peach, abscission may occur at the base of the peduncle [abscission zone (AZ1)] if the bud, pistil, or fruit has been injured before bloom or up to approximately 30 days AFB. Abscission at AZ2 may occur because of injury, exposure to ethylene, or fruit competition mid-season, or at AZ3 late in the "competitive fruit abscission" ("June drop") stage or at harvest.

specific abscission response of AZ3 may be due to changes in ability of ethylene to activate specific genes and to stimulate the secretion of cellwall degrading enzymes, such as EG in the separation zone (Sexton et al. 1985; Ruperti et al. 1998). Abscission zone response to ethylene is required in the fruit pedicel in a manner that may be analogous to ripening fruit. The presence of ethylene alone is not sufficient to induce abscission (Lanahan et al. 1994), but ethylene may induce cell wall hydrolysis responsible for the degradation of the middle lamella and the loosening of the primary cell wall of the separation layer cells. Ethylene regulates at the transcriptional and/or translational level the activity of the main enzymes involved in the process (Ruperti et al. 1998). Abscissic acid (ABA) and ethylene can influence auxin synthesis and transport and may markedly counteract the suppression of abscission by auxin (Sexton et al. 1985). The progress of abscission is determined by the accelerating effects of ethylene on the influence of auxin concentration. Ethylene, through its effect on chlorophyll degradation (Choe and Wang 1986) and ABA, by increasing photorespiration (Popova et al. 1987), may reduce the production and availability ofleaf assimilates. In addition, the availability of minerals and nutrients translocated in the

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

365

respiration stream may be reduced due to the restricted transpiration rate. These imbalances and localized deficiencies in dry matter may be critical factors in hastening abscission of leaves and fruits (Noga and Bukovac 1990). Ethylene application also inhibits growth in peach (Ramina et al. 1986) and may be the result of processes previously induced by ethylene (Bleecker and Patterson 1997). IV. THINNING PRACTICES

A. Dormant Pruning

Dormant pruning is frequently used to reduce the current season's flower bud complement; and if substantial pruning is not done before growth begins, photosynthetic reserves stored in the roots, trunk, and limbs may be wasted on the development of flowers, shoots, and fruit (Fig. 7.5).

Fig. 7.5. In the year following a freeze that defruited trees, tree growth (A) flower numbers (B), tree density (C), and fruit number (D) was several times greater than in trees carrying a typical crop. If trees are allowed to flower without substantial dormant pruning, the greater competition between fruits may cause a greater than normal fruit drop about 45 days AFB. Unpruned trees will also reduce spray penetration of chemical bloom thinners so that chemical deposits from adjoining row sprays will lower the thinning response.

366

R. BYERS, G. COSTA, AND G. VIZZOTTO

Shoemaker (1933) and Marini (2000) suggested that substantial pruning might be used as an early season thinning technique. A grower may choose to prune twice; the first time to remove major bull canes over 1 m in length and the second to provide tree containment. A second pruning may be used after the potential of winter killing of flower buds has passed as a flower bud thinning technique. However, Marini's (2000) work dearly shows that a freeze after winter pruning in 1997 improved crop value. The freeze in 1997 partially thinned the trees and thus allowed greater yields of more marketable fruit on trees pruned less severely (Table 7.3). In 1998, no freeze occurred, thus pruning to limit the number of bearing shoots to only 73 shoots per tree resulted in higher yields of larger fruit and increased crop value (Table 7.3). Even though dormant pruning can be used as an early thinning technique, the necessity for further thinning after pruning may be substantial in some regions. When bloom thinning, a crop loss, or a partial crop loss occurs, detailed pruning for partial thinning may be even more imperative because the number of flower buds/tree may be four times, and bearing shoots/tree twice, that of trees thinned 45 days AFB (Byers 1990). In addition, pruning or chemical flower bud inhibition allows additional time to further reduce the potential crop prior to bloom and before bloom or post thinning practices are initiated.

Table 7.3. The effects of retaining various numbers of shoots per tree on fruit set, yield, fruit weight, and crop value of 'Norman' peach trees (Marini 2000). No. fruit per tree

Shoots/tree

Before thinning

Removed in thinning

Harvested

Yield (kg/tree)

FW/fruit (g)

Crop value ($/tree)

73 110 146 220

296 352 432 510

27 58 81 168

1997 (Freeze) 269 294 351 342

91.6 97.1 111.6 108.0

156 151 146 147

34.90 32.87 37.67 37.54

73 110 146 220

720 910 920 1600

227 415 518 1180

1998 (No freeze) 560 480 420 430

59.0 55.8 42.6 44.5

123 118 113 111

33.00 28.00 21.00 22.00

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

367

B. Flower Bud Inhibition or Abortion 1. Gibberellic Acid. Gibberellic acid (GA 3 ) inhibits flower bud formation in a wide variety of woody fruit trees including cherry, peach, nectarine, apricot, and almond (Zeevaart 1983). Goldsmith and Monselise (1970) determined that GA 3 application increased cellular division and expansion in the sub-apical zone, which subsequently increased internode length in citrus and reduced the number of flower buds at each node. The increased proportion of vegetative buds to flower buds resulted in a greater leaf to fruit ratio in the following year. Byers (1990) also found increased shoot length, shoot number, and bud numbers per centimeter of shoot length. In stone fruit, both GA 3 application time (Fig. 7.6) and rate per ha influence the number of flowers initiated (Hull and Lewis 1959; Bradley and Crane 1960; Stembridge and LaRue 1969; Byers et al. 1990). GA 3 sprays applied at full bloom through mid-season typically inhibit flowering in peach, but applications later in the season induce abortion of previously differentiated buds (Stembridge and LaRue 1969). GA 3 application prior to leaf fall caused some delay of bud development (Fig. 7.7B), flowering, and increased bud hardiness the following spring (Proebsting and Mills 1964; Stembridge and La Rue 1969). A wide window of opportunity exists for either rate or timing of GA 3 sprays for reducing flower bud numbers by varying degrees (Edgerton 1966; Brown et al. 1968; Stembridge and La Rue 1969; Corgan and Widmoyer 1971; Intrieri and Sansavini 1972; Byers et al. 1990; Southwick and Yeager 1991; Oliveira and Browning 1993; Southwick et al. 1995b; Lemus 1996; Costa and Vizzotto 2000). In California, application of GA 3 in mid-June was less effective than when applied from mid-June to early July (Southwick et al. 1996b). The time of GA 3 spray application can have a major effect on bud development along specific shoot sections. GA 3 applied when the largest shoots are about 10 to 15 cm in length (30 to 70 days AFB) will inhibit flower bud development at the base and mid-sections of the shoots, with maximum effect at about 50 days AFB (Byers 1990). Since greater numbers of flower buds are produced at the basal nodes of shoots, and on short, weak shoots that have many buds and shorter internodes, GA 3 can specifically reduce these buds by timing the spray at the time they are being differentiated (Byers et al. 1990). GA 3 , under the name of Release LC (Valent Biosciences), is currently registered only in California. This material could be particularly useful in inhibiting flower-bud formation after a crop loss, or on certain cultivars that have an extraordinarily large number of buds such as 'Bisco'

R. BYERS, G. COSTA, AND G. VIZZOTTO

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Influence of time of GA;) application on numbers of flowers and fruit per shoot, and on individual fruit volume in the subsequent year (Byers et al. 1990).

Fig. 7.6.

or 'Redhaven.' Additionally, GA 3 could be used in conjunction with a regular bloom thinning program where winter injury to flower buds is not a significant problem. GA 3 might also be used to reduce flower bud numbers at the base of shoots so that rope thinning would be more uniform and effective (Byers et al. 1990) (Fig. 7.8ABC). Since the effect of GA 3 is time- and concentration-dependent, a wide window exists for reducing flower bud numbers by 50 to 75 percent. In addition, the influence of environment and adjuvants on absorption and activity of GA 3 has

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

369

Fig. 7.7. Influence of soybean oil (8 %) applied in the dormant season on killing of a peach pistil (A) (note flower bud began enlarging with dead pistil). Previous season GA;{ spray delayed development of peach pistil in the subsequent season (B) resulting in lower percentage of flowers setting fruit. Injury to the peach pedicel (C) following application of ammonium thiosulfate. Dead flowers 30 days after bloom still attached to the shoot (D) caused by ammonium thiosulfate phytotoxicity to the pediceL At 25 days AFB unfertilized fruits (E) are difficult to distinguish from larger fertilized fruit (note development of button nonfertilized fruits that will abscise about 50 days AFB).

370

R. BYERS, G. COSTA, AND G. VIZZOTTO

not been adequately studied. An additional benefit of preharvest GA 3 applications is maintenance of flesh firmness and a slight delay in ripening (Southwick and Yeager 1995). On apricot, the effect of GA 3 is concentration-, time- and formulationdependent (Southwick and Yeager 1995; Southwick et al. 1995a). Over a three year period, the highest GA concentration used strongly reduced flower numbers the subsequent season. GA 3 greatly reduced handthinning time and increased fruit size and firmness; however, the percentage of flowers setting fruit was not affected (Southwick et al. 1997). On prune, GA 3 improved fruit set (Webster and Goldwin 1981) and quality (Boyhan et al. 1992) in the year of application and inhibited flowers (Webster and Goldwin 1981). In several climatic regions, flower bud inhibition may not become widely accepted because of the potential risk of subsequent damage to buds by winter or spring freezes. This may further reduce cropping to sub-optimal levels (Byers et al. 1990). 2. Oils. Vegetable and petroleum oils applied as a dormant spray at 75 to 110 L/ha (single or multiple applications) are known to reduce tree and flower bud respiration, delay bud break, and kill 40 to 60 percent of the flower buds (Fig. 7.7A) (Call and Seeley 1989; Deyton et al. 1992; Myers et al. 1996). However, considerable research is still needed to determine the proper time, rate, and number of applications needed. Where winter temperatures drop below -15°C, the risks of dormant oil may be detrimental to tree and flower bud survival, fruit size, and tree growth.

3. Ethephon/GA3 Sprays at Leaf Fall and During Dormancy. Autumn sprays of GA 3 and ethephon on peach, apricot, and sweet and tart cherry can delay blossoming and increase floral bud hardiness (Proebsting and Mills 1973; Browne et al. 1978; Soni and Yousif 1978; Walser et al. 1981; Buban and Turi 1985). Crisosto et al. (1989) reported that application of ethephon during several different leaf-fall stages delayed 'Redhaven' peach bloom the following year. However, ethephon application at the 10 percent leaf drop stage reduced flower and fruit number by almost 50 percent, whereas later applications had little effect. Bud hardiness, measured as the percentage of bud survival, was greatest following ethephon treatment at 50 percent leaf drop. Fruit set was not affected by any of the ethephon treatments. In other experiments, GA 3 and ethephon sprays applied in October or November killed peach flower buds (Williams 1989). This effect was

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

371

Fig. 7.8. Stationary (A) or rotating (B) rope drags are an effective and rapid method for peach flower bud removal. As a result of bloom thinning, basal buds (C) are difficult for rope drags to thin due to adjacent twigs that hold ropes away from buds. High pressure water streams (D) can be directed to narrow crotch areas that can not be thinned by rope drags (Byers, unpublished).

related both to date of application and concentration of the chemicals, as previously shown by Stembridge and La Rue (1969). Gianfagna et al. (1986) found that 100 and 200 mg/L of ethephon applied in autumn delayed flowering of peach several days, but killed some flowers. Postharvest sprays of GA3 applied to 'Patterson' apricot reduced flower number (Southwick and Yeager 1991) and, in some experiments, reduced fruit set. Similar effects were obtained with ethephon on 'Shirokaga' and 'Inazumi' Japanese apricot (Frunus mume) (Paksasorn et al. 1995). High-volume sprays of ethephon to 'Victoria' plum, just prior to leaffall, delayed blossoming by several days the following spring. However, blossom quality and fruit set were frequently poorer on the ethephonsprayed trees than on the controls; the addition of GA 3 overcame the effects on flower quality and increased yields (Webster 1984a, 1984b; Webster and Andrews 1986).

372

c.

R. BYERS, G. COSTA, AND G. VIZZOTTO

Flower Thinning

Flower or early fruit removal will conserve photosynthetic reserves (stored from the previous season or produced in the current season) by reducing competition between all organs throughout the tree. For this reason, thinning may increase vegetative shoot growth, flower bud differentiation, flower bud hardiness, fruit size, and yield (Byers 1989a, 1989b; Byers et al. 1990; Byers and Marini 1994). When a peach tree is bloom-thinned (or the crop is lost due to a freeze), two to five times more flower buds/unit length of wood plus additional shoot length (Fig. 7.2) are produced compared with trees hand-thinned 40 to 50 days after bloom (Byers and Lyons 1984a; Byers and Marini 1994). Therefore, the need for early thinning may be even greater in the years following a partial or complete early season crop loss. After a freeze or when trees are bloom-thinned, the greatest increase in flower bud numbers occurs near the base of the current season's shoots and on short shoots throughout the tree canopy (Byers et al. 1990). Fruit that develop at these locations are typically the smallest fruit on the tree; and, if not removed at bloom, will result in a large number of small fruit. Elimination of the crop by a spring freeze would cause substantially more shoot growth and flower bud numbers than would bloom thinning. Since basal flower buds on the shoot are the last to open in the spring, they may provide some protection from late spring frosts (Byers and Lyons 1985a; Byers 1989a, 1989b; Byers et al. 1990). In any case, no more than 3000 to 6000 flower buds should remain on a normal-sized peach tree after bloom. If peach trees are not thinned or are thinned very late (60 days AFB) extreme competition between fruit (Fig. 7.2A and 7.2B) may have a major effect on vegetative growth and flower bud differentiation so that the subsequent season's crop may be compromised (Byers and Lyons 1985a; Myers 1986; Byers et al. 1990; Byers and Marini 1994). Overcropped treesmay cause reduced tree vigor, smaller crops of more undersized fruit, and trees more susceptible to disease, cold injury, and a shortened tree life (Shoemaker 1933). Heavy detailed pruning in the winter plus flower thinning in the spring can reduce flower bud numbers and prevent over-cropping, small fruit size, and poor shoot growth. Heavy nitrogen fertilization of trees cannot compensate for the loss in photosynthetic reserves from over-cropping. If not thinned adequately, many peach and nectarine cultivars may exhibit biennial bearing habit. However, because trees will respond to good cultural management practices, commercial orchards typically will produce an adequate number of flower buds each year and thus mask the alternate bearing cycling.

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

373

Annual pruning, thinning, fertilization, and cultural practices are critical to maximizing crop value (fruit size x yield x price), by assuring adequate vegetative growth, flower bud numbers, and carbon reserves for the next season. 1. Mechanical Flower Removal.

Hand or Brush Thinning. Hand or brush thinning (e.g., plastic or wire brushes, limb switches, gloves, etc.) of peach and nectarine has been practiced for over 75 years. Mechanical flower removal, unlike chemical thinning allows visual determination of the number and location of flowers throughout the canopy immediately after thinning. Rope Drags. Tractor-mounted, fixed or rotating rope curtain drags, have been used to remove a large number of flowers very cheaply from peach and nectarine trees (Baugher et al. 1988; Baugher et al. 1990, 1991). Rope strands 2.5 to 3.2 cm in diameter and 450 to 500 cm in length are hung over a horizontal bar at 5-cm intervals and dragged over a tree in full bloom (Fig. 7.8A & 7.6B). This removes the flowers most effectively in the tops and periphery of the tree. Dragging ropes in opposite directions two to four times increases uniformity and the degree of thinning with each pass. Rope spacing may be varied to change the amount of thinning during each pass. Rotating rope drags and spiked-drum, impact shakers (Glenn et al. 1994) are other variations of the rope curtain drag. Unfortunately, flower buds in narrow angle crotches in the top and sides of the tree are not adequately thinned by the ropes. Since flower thinning increases basal bud development at nodes one through five, flower thinning the previous season will cause additional clustering of flower buds in crotches at the junction of current season and two-yearold wood (Fig. 7.8C). In addition, rope drags are more effective on trees with flat tops which may not be the most productive fruiting system. Water Thinning. High pressure streams of water (Fig. 7.8D) from single or multiple spray gun nozzles mounted on a sprayer or tractor can effectively remove flowers from stone fruit species that have short, inflexible stems (Byers 1990). Irrigation nozzles (40 mm diameter) with plastic water straighteners provide a water stream with adequate force if pump pressure of 31.6 to 38.7 kg/cm 2 is maintained (Byers 1990). A single nozzle will require 4.5 to 5.7 L/minute; thus a substantial pump and 14,000 to 19,000 L of water are required for full-size trees that have been adequately pruned in the dormant season. This method may not be practical, since labor costs and the amount of water required may become prohibitive, particularly for dense, nonpruned trees.

R. BYERS, G. COSTA, AND G. VIZZOTTO

374

2. Chemical Flower Removal. The primary disadvantage of flower thinning is the potential for subsequent killing of flowers or fruit by spring frosts, and the uncertainty of favorable environmental conditions for pollination, fertilization, and fruit set (Batjer 1965). Since most chemical thinning agents rely on an interference with the natural process of pollination and fertilization, cutting the stigma and one half the style off demonstrated the importance of natural fruit set and the interfering treatment (Fig. 7.9). Even though many researchers indicated that bloom thinners might have limited commercial value, the economic impact of early thinning must be weighed against several factors. These include the probability of a local freeze, earliness of bloom, value of crop in relation to costs, later fruit hand-thinning costs, availability of labor, and the potential for biennial bearing for each cultivar.

Elgetol. Sodium 4,6-dintro-ortho-cresylate (DNOC), known under its commercial name Elgetol, became the first important apple flower thinning agent (Batjer and Thompson 1948; Williams 1979) and remained so for over 40 years until registration was canceled in 1990 by the Environmental Protection Agency. Elgetol thinning at bloom in the western United States had been considered a part of a total thinning program that was essential for maximizing fruit size and for providing an adequate return bloom for the next season (Williams 1993, 1995). Hildebrand (1944) showed that Elgetol inhibited fruit set when applied as late as 32 h after

'Sa-

100 90

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80

c

70

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-o-Control ..... Pink -.-Bloom ~ Petal Fall (63%) ~ Petal Fall (74%) ---t:r Petal Fall (92%) -+- Petal Fall (100%) ""'*- Shuck Split

50

~ 40

~c

~

et

95% Confidence Interval

Stigma Cut Off

30 20 10

o

-3

18

35

50

62

Days After Full Bloom Fig. 7.9.

Effect of time of stigma removal on fruit set of peach (R. E. Byers, unpublished).

375

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

pollination. Microscopic observation showed pollen tube growth was approximately halfway down the style 32 h after pollination (Hildebrand et al. 1944). When Elgetol was applied just before wet and humid periods, it caused fruit and foliage injury and erratic results; thus, it was used very little in the eastern United States where humid conditions prevail. In addition, re-wetting and poor weather conditions during pollination and fertilization resulted in variable fruit set and unpredictable responses related to the Elgetol application. Cancellation of Elgetol registration caused universities and chemical companies to seek a replacement for it (Williams 1994). Consequently, the potential for development ofimproved chemical thinning agents caused a resurgence of interest in flower thinning in the United States (Byers 1997). Prior to 1991, investigation of new flower thinning agents was a low priority in the eastern United States due to erratic thinning and injury to leaves caused by Elgetol. In the 1980s, fertilizers, surfactants, and desiccants were investigated for flower thinning of peach trees in the United States (Table 7.4). Several of these chemicals are effective bloom thinners for apple including endothall, ammonium thiosulfate (ATS), and long chained fatty acids (Byers and Lyons 1985; Williams 1993; Williams 1995; Southwick et al. 1995a,b; Bound and Jones 1997, Byers 1997; Fallahi 1997). Table 7.4. Effects of airblast spray applications of several flower thinning agents and surfactants on 'Redhaven' peach fruit set (Byers and Lyons, Jr. 1985).

Treatment Control Hand thinning ATS + no surfactant ATS + Spray-Aide® ATS + Spray-Aide® ATS + Spray-Aide® ATS + X-77® ATS + Spray-Aide® ATS + Dithane Z-78® CC-42 (polyoxypropylene ammonium chloride) DuPont WK® Endothall®+ X-77® Endothall®+ X-77® Methyl oleate+ Na C0 3 NH 4 N0 3 + X-77® SN-50 (oxyalkylated alcohol)

No. fruit/cm 2 limb cross sectional area

Fruit diam. (cm)

0 0 30 mIlL 30 mIlL + 1.25 mIlL 30 mIlL + 2.5 mIlL 30 mIlL + 5 mIlL 30 mIlL + 5 mIlL 30 mIlL + 5 mIlL + 9.6 giL 25 mIlL

16.2 aO 5.0 d 3.8 de 3.7 de 2.7 de 2.7 de 2.8 de 3.7 de

4.80 e 5.13 de 6.32 a 6.12 ab 6.45 a 6.50 a 6.60 a 6.20 a

11.7 bc

5.54 cd

25 mIlL 0.75 mIlL + 5 mIlL 0.75 mIlL + 2.5 mIlL 20 mIlL + 38.4 giL 120 giL + 5 mIlL 25 mIlL

1.0 e 9.7 cd 9.5 cd 14.8 ab 2.3 e 6.7 d

6.32 5.44 5.61 5.13 6.38 6.02

Formulation ratelL

a d bcd de a abc

°Means separation within columns by Duncan's new multiple range test, 5 percent level.

376

R. BYERS, G. COSTA, AND G. VIZZOTTO

Fertilizers. Of the fertilizers evaluated as flower thinners for peach, ammonium thiosulfate (ATS) (National Chelating Corp.) was among the most effective and easiest to use since it was formulated as a liquid (Byers and Lyons 1984a, 1985a,b). However, urea, ammonium nitrate, calcium nitrate, and many other phytotoxic liquid fertilizer formulations were found effective. The use of ATS fertilizer for flower thinning has been commercially practiced without registration in peach and apple orchards in the United States. Applications made in the later stages of apple bloom (90% open flowers) caused more russetting or "marking" of the apple, but most of these chemicals were more effective and less injurious to peach, nectarine, and other Prunus since stone fruit are protected by the corolla from direct spray contact at bloom (Byers 1997). Many caustic chemical sprays may interfere with ovule fertilization if applied soon after flower opening, but the mode of action also includes injury (Fig. 7.7C) to the pedicel (Erez 1975; Byers and Lyons 1985a,b). After several years of experience, the effective airblast spray rate of ATS was found to be approximately 3.1 L/ha (Byers and Lyons 1985a,b; Byers 1999). The addition of a surfactant did not improve ATS effectiveness. When applied with an airblast sprayer, ATS was most effective when about 70 to 90 percent of the flowers had opened (Byers 1999). ATS burns blossoms and young shoots, especially when applied with certain fungicide tank mixes (Olien et al. 1995). In the case of a long bloom period, one application of ATS was insufficient to cause adequate thinning. Two applications, spaced one to three days apart, and timed at 30 percent open flowers and again at about 95 percent open, substantially increased thinning efficacy (Byers 1999; r. e. Byers unpublished). However, three applications caused substantial shoot injury and defruiting of trees when applied within a five-day flower-opening period (r. e. Byers unpublished). Because of potential defruiting and injury, extreme caution should be used when making multiple applications. A possible explanation for injury is that the chemical degradation between applications is not complete, thus a second or third application might add to the previous chemical deposits on flowers and foliage. Additionally, the rate of chemical degradation as influenced by environmental conditions may vary considerably. Thiourea and/or urea (10 to 12% w/w) applied during bloom resulted in thinning, but applications at the beginning of bud swell and postbloom also caused thinning in early ripening cultivars (Di Marco et al. 1992; Erez 1975). Surfactants. Several surfactants including Dupont WK, Witco SN-50, and Ortho X-77 are effective as bloom thinners (Byers and Lyons 1984a, 1985a,b). Effectiveness is positively correlated with increased phyto-

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

377

toxicity; thus, the initial research involved screening surfactants for phototoxicity. One of the most extensively studied surfactants is a fatty amine polymer called ArmoThin (Akzo Nobel Corp.), which has been tested on several stone fruit species with positive result (Costa et al. 1995; Southwick and Fritts 1995; Costa et al. 1996; Lemus 1996, 1998; Lichou et al. 1996; Southwick et al. 1995a,b; Byers 1999). This compound induces early anther dehiscence, a marked reduction in pollen germination, and reduced pollen tube growth in the stylar tissue soon after germination (Costa et al. 1995). The data on pollen tube growth suggests that the compound was more effective the sooner it was applied after flower opening (Baroni et al. 1995). Application of 2 to 3 percent Armothin when 70 to 80 percent of the flowers have opened has provided good thinning of several different cultivars in several climatic areas. However, much of this work does not report the tree size or chemical rates/ha, both of which influence the deposit and action on the flower (Byers and Lyons 1985a,b).

Desiccating Agents. Endothall (Elf Atochem, Inc.) is among the most promising of this class of chemical bloom thinners and is very effective at very low rates (Byers and Lyons 1984a, 1985a,b). In addition, Wilthin (Entech Corp.) has an EPA registration for use on peach and apple trees in the United States. Our research indicates that a surfactant must be used with this material to be effective (Byers 1999). However, additional testing ofWilthin is needed to determine the necessary rates since recommended label rates frequently may be too low for adequate effectiveness. Dormex (DK International, Inc.) is another promising chemical that is not registered for thinning stone fruit but is currently used for breaking dormancy. Treatment with hydrogen cyanamide (Dormex) or ArmoBreak, after the chilling requirement is met, may stimulate flower bud abscission or inhibit flower opening. As a rule, these chemicals are applied when the chilling requirement is not completely met. Normally, under such conditions, hydrogen cyanamide, nitrogen or surfactant mixtures are applied 40 to 60 days before expected bud-break. In nectarine, applications near to bloom (less than 40 days) may inhibit flower-bud burst (Fallahi et al. 1990). In apricot, application of thiourea, KN0 3 , and Erger D (a mixture of fatty amine polymer and nitrogen compounds) six weeks before the expected bud-break, produced contradictory results. In one case, application of thiourea and KN0 3 increased expected yield but reduced fruit weight (Kuden et al. 1995). In another, Erger D increased bud mortality and fruit weight and response was increased with age of the bearing shoot. These applications also induced early maturity in apricot (Costa et al. 1998).

378

R. BYERS, G. COSTA, AND G. VIZZOTTO

Long Chain Fatty Acids. Pelargonic acid (Thinex-Mycogen Corp.), YI1066 (UAP Corp.), and several other fatty acids are potential bloom thinners because they are sufficiently phytotoxic and short-lived in plant tissues (Klein and Cohen 1995; Fallahi 1997; Byers 1999). Pelargonic acid is currently registered in the United States for stone fruit and apple flower thinning and has been used with some degree of success. In plum, several chemicals including ATS, urea, wettable sulfur, and Dormex, have been tested as bloom thinners. Urea and wettable sulfur have given positive results on several cultivars (Scholtens 1993; Webster and Hollands 1993; Balkhoven-Baart 1997). Dormex acted as a thinner although response differed with cultivar, concentration, and time of application (Fallahi et al. 1992). D. Fruit Thinning 1. Manual Fruit Thinning. Typically, hand-thinning of peach fruits is performed approximately two weeks prior to the pit hardening stage, soon after unfertilized fruit are visually smaller than fertilized fruit. In Italy hand-thinning is normally performed after the pit-hardening stage, when fruits start to grow again and after natural abscission takes place. However, because of the high demand for labor, the short time period when hand-thinning can be performed, and the high cost per tree, alternatives to hand-thinning are universally sought for commercial production (Weinberger 1941; Costa and Vizzotto 2000). Hand-thinning is generally superior to rubber-hose or mechanical shaking methods because the remaining fruits can be better spaced and selected. Knocking fruit from trees with a 40 cm rubber hose can be selective if care is taken to strike the smaller fruit. Mechanical shaking devices remove the largest fruit, thereby reducting mean fruit size, yield, and crop value (Berlage and Langmo 1982; Costa 1978). Limb and whole tree crop loads vary widely following mechanical shaking, due to shaking intensity, limb stiffness, and tree structure. Damage to trees or limbs, equipment cost and availability are also disadvantages.

2. Chemical Fruit Thinning. Chemical fruit thinning is an attractive method for reducing crop load since very little labor is required to perform the practice. However, despite extensive research with many, none has provided satisfactory results (Edgerton and Greenhalgh 1969; Buchanan et al. 1970; Aitken et al. 1972; Costa and Grandi 1974; Costa 1978; Shaybany et al. 1979; Costa and Vizzotto 2000). Furthermore, chemical injury to the tree, leaves, and fruit have been difficult to avoid. Chemical thinning of peach and other stone fruit trees has been considered more difficult than thinning of species such as apple for several

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

379

reasons. Peach flowers open nearly simultaneously; however, cool temperatures may cause the bloom period to differ substantially in length between seasons. In some apple cultivars, a fruiting spur contains a dominant "king" flower that naturally produces the largest fruit and four weaker side flowers. These two categories of flowers, "king" and "lateral," behave distinctly differently. The "king" flowers set a higher percentage of fruit, and the resulting fruit typically contains more seeds than does a lateral fruit. However, most stone fruit species normally develop only one seed per fruit, and each flower has an equal probability for fruit set. In addition, most of the hormone-type chemicals used as apple fruit thinners have been ineffective or have caused either leaf or fruit injury at the concentrations needed to cause stone fruit abscission. In peach, ethephon and other ethylene-generating compounds cause more leaf than fruit abscission, whereas in apple, rates of ethephon (up to 1500 mg/L) which cause leaf or shoot injury may be three to five times higher than those that are required for fruit abscission (300 to 500 mg/L). Auxins [l-naphthaleneacetic acid (NAA) and 1-naphthaleneacetamide (NAAm)] effective on apple typically have been ineffective or have caused fruit abnormalities, leaf abscission, and/or resulted in inconsistent results in Prunus. Since 1970, numerous investigators in several countries have evaluated (2-chloroethyl) phosphoric acid (ethephon) as a chemical fruit thinner for peach and other stone fruits. While some trials have provided positive results, the use of ethephon has not become widespread because of substantial leaf drop and variable thinning related to a number of internal and external factors (Edgerton and Greenhalgh 1969; Stembridge and Gambrell 1971; Costa 1978; Ramina 1981). Factors that may affect response include temperature on the rate of ethylene evolution (Qlien and Bukovac 1978), chemical binding of ethephon to cellular fractions (Lavee and Martin 1974a,b, 1975); fruit versus leaf abscission, fruit load, tree vigor, ratio of vegetative to reproductive sinks, planting density, environmental shading, and nutritional level (Costa and Grandi 1974; Ramina 1981; Costa et al. 1983). In some experiments, inhibitors of GA biosynthesis caused fruit abscission. Paclobutrazol also induced fruit abscission on plum when applied at 1000 or 2000 mg/L at the onset of pit hardening. Full-bloom and later sprays of paclobutrazol were ineffective (Webster and Andrews 1986). Paclobutrazol, a well-known growth retardant for stonefruit, also induces some fruit abscission if applied at the shuck-off stages (about 20 days AFB). However, such sterol inhibitors are not effective on all cultivars and under some growing conditions (Blanco 1987; Marini 1987). In apricot, several different chemicals (NAA, ethephon) and nitrogen compounds have been tested as thinners. NAA was the most effective,

380

R. BYERS, G. COSTA, AND G. VIZZOTTO

but was phytotoxic. Ethephon gave the best results in terms of fruit quantity and had no negative effect on fruits or foliage (Farmahan and Dhiman 1998). NAA and ethephon have been used on plum to induce fruit abscission. Effects were cultivar-, concentration-, and time-specific for each chemical (Webster and Andrews 1986; HarangozQ et aI. 1996). Chemicals that inhibit photosynthesis (e.g., terbacil) cause fruit abscission when applied at least 30 to 40 days AFB (Byers et aI., 1984b; DelValle et aI. 1985), but also cause chlorosis and abscission of leaves. Whole tree shading experiments show that fruit thinning can be achieved without harm to fruit or foliage (Fig. 7.3). E. Chemical Application Technology 1. Chemical Flower Thinning. Typically, recommendations for chemical thinner rates for a block of trees are extrapolated from either handgun dilute sprays to single trees sprayed to the point of drip or airblast sprays applied to trees in a single row. Airblast spray rates that cause thinning to a single row may over-thin when applied to an entire block due to the increased deposit from adjacent row drift. In order to obtain a similar deposit as a single row application (Fig. 7.10AB), a block of trees would need to be sprayed with a reduced rate/ha to obtain similar thinning. At bloom, well-pruned peach trees provide very little barrier for chemical drift to adjacent rows from airblast application, but nonpruned trees may reduce drift substantially (Fig. 7.5ABC). In one test (Fig. 7.10AB), the increased response of peach trees to airblast spray drift of ATS was proportional to the chemical deposit from adjoining rows (Byers and Lyons 1985b). Chemical deposits on peach flowers in adjoining rows were 43 percent of that of those on the sprayed row and the 25 percent on the second row; thus more chemical is deposited by multiple-row spraying than by a spray applied to one row only. If an entire block is sprayed, 68 percent of the deposit was accumulative drift from adjoining rows. Further studies indicated that water rates from 420 to 2338 L/ha did not influence efficacy of ATS if the same chemical rate/ha was used (Byers and Lyons 1985b). However, a handgun sprayer applying 1170 L/ha (Table 7.5) almost defruited the trees, whereas the airblast sprayer resulted in the desired crop load (5.5 FCSA). 2. Fruit Thinning. When chemicals were first tested for apple and/or peach thinning, most were applied with a handgun sprayer at high water volumes; thus, the amounts of chemical and water per hectare were frequently unknown and tree size was not reported. In addition, airblast

7. FLOWER AND FRUIT THINNING OF PEACH AND OTHER PRUNUS

1800

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A

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18 16.514.9

11.39.8 8.2

4.7 3.1 1.5

1.5 3.1 4.7

8.2 9.8 11.3

14.9 16.5 18

DISTANCE FROM SPRAYER (m)

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ROW OF BLOCK

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Fig. 7.10. When spraying a single middle between six rows, spray deposits (A) on flowers were determined by their distance from the sprayer (rows were located at 3.1, 9.8, and 16.5 m from the sprayed middle). Following an application of ammonium thiosulfate, fruit counts taken in the center row of a five-row wide block of peach trees showed additional thinning (B) due to drift from adjoining rows compared to spraying a single row (Byers and Lyons, Jr. 1985).

spray applications for fruit thinning were frequently not as effective as handgun applications. In apple, Sutton and Unrath (1998) demonstrated that handgun applications increased chemical deposits by 60 percent or more above treerow-volume (TRV) rates and caused more effective thinning and better disease control. A rate of 2338 L/ha (250 gal/acre) of water for mature

R. BYERS, G. COSTA, AND G. VIZZOTTO

382

Table 7.5. Effect of application method and water volume on ammonium thiosulfate (ATS) in thinning of peach fruit (Byers and Lyons 1985).

Treatment Control Hand thinned ATSo ATSo ATSo ATSo ATSo ATSo

Application method

Water applied (liter/ha)

ATS (55% a.i. formulation) (mIlliter)

No. fruit/cm 2 limb crosssectional area

Fruit diameter (cm)

20.9 ali 6.3

Airblast Airblast Airblast Airblast Airblast Handgun

2338

1590 1170 841 420 1170

15 20 30

42 84 30

5.2 b 4.0 b 5.5 b 2.8 bc 4.1 b 0.8 c

5.11 a 6.05 b 6.20 bc 5.89 b 6.20 bc 6.17 bc 6.48c

°The adjuvant Spray-Aide (5 ml/liter) was added to promote uniform coverage. ATS rate was constant at 35 liters/ha. liMean separation within columns by Duncan's new multiple range test, 5 percent level.

peach trees is a standard recommendation for dil ute airblast application in the eastern United States, although most orchards are sprayed with approximately 935 L/ha (100 gal/acre). The dosage of chemical is based on the dilute rate (chemical per 2338 L/ha). These results indicate that test reports should include information on amounts of water and chemical used per hectare, tree size, and canopy density. Tree-row-volume calibration of air-blast sprayers was an attempt to standardize the amounts of chemical and water applied per hectare (Byers et al. 1984; Herrera-Aguirre and Unrath 1980; Sutton and Unrath 1988; Byers et al. 1984b; Byers and Lyons 1985a,b; Byers 1989). HerreraAguirre and Unrath (1980) found more uniform apple thinning results by TRV calibration with ethephon, NAA + carbaryl, and NAA + ethephon, presumably because of the reduced variability. Future research reports should report method of application, chemical rate/L, tree size, and planting distances. Furthermore, with airblast applications, chemical rate/ha, water rate/ha, and phenological stage of fruit or flower development should be reported. F. Combinations of Flower and Fruit Thinning

To avoid the major disadvantage of bloom thinning, application prior to conditions that may reduce fruit set, partial flower thinning may be combined with later hand-thinning (Myers et al. 2002). For example, mechanical fixed or rotating rope drags could remove the bloom in the

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upper 60 percent of a peach tree, while another method could be used on the lower 40 percent (water thinning, hand or brush bloom thinning, chemical sprays applied with handguns). After bloom, hand or rubber hose fruit thinning (45 days AFB) could be used for additional touchup thinning. This approach has the advantage that smaller or defective fruit, or both, can be removed to produce a higher percentage pack-out. V.

FUTURE PROSPECTS

Since most Prunus species flower before the threat of spring freezes has passed, the optimum time for thinning may be approximately two weeks after bloom. At this time the fruit are not yet a serious drain on the tree's photosynthetic reserves and the chance of a spring freeze is lower. As breeding programs introduce more self-fruitful cultivars of sweet cherry, plum, and other Prunus, the need for thinning to obtain large fruit size, high yields, and therefore good prices will likely increase. Since chemical methods for flower and fruit removal are varied in effectiveness and injury to both leaves and fruit, additional physiologicalor mechanical methods should be investigated to facilitate thinning two to three weeks after bloom. Unfortunately, fruit do not naturally abscise during this period, and in most Prunus sp., unfertilized fruit cannot be visually distinguished from fertilized fruit. Therefore, along with mechanical methods for thinning, a simple method is needed for determining the percentage of unfertilized fruits so that the amount of fruit that should be removed can be determined. Research efforts in the past have been centered on chemical inhibition of flowering, flower thinning, or fruitlet thinning after the fruit have become a significant drain on current season's photosynthates. Limiting photosynthesis during specific periods of fruit development can stimulate fruit abscission without leaf injury; however, these periods may be too late to maximize fruit size and yield. Studies on ovule or abscission zone development and the effects of injuring the fruit at specific times when fruit thinning is needed (two to three weeks AFB) could lead to improved thinning methods Information is needed on the relative economic benefits of thinning by flower bud inhibition, pruning, flower thinning, early fruit thinning, and partial thinning at bloom followed by hand-thinning. The flowering process in nonpruned trees may be a significant drain on the tree that reduces fruit size and yield. Unfortunately, experiments that demonstrate the impact of leaf/flower or flower ratio for each stage of development (bud-break, pink, bloom, petal fall, shuck split, 10 mm ovule length, etc.) has not been done. To maximize crop value and minimum

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crop loss due to spring freezes, several adjustments in vegetative and reproductive development may be required at specific stages throughout the season.

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Olien, W. c., and M. J. Bukovac. 1978. The effect of temperature on rate of ethylene evolution from ethephon and from ethephon-treated leaves of sour cherry. J. Am. Soc. Hort. Sci. 103 (2):199-202. Olien, W. C., R W. Miller, Jr., c. J. Graham, E. R Taylor, Jr., and M. E. Hardin. 1995. Effects of combined applications of ammonium thiosulphate and fungicides on fruit load and blossom blight and their phytotoxicity to peach trees. J. Hort. Sci. 70:847-854. Oliveira, C. M., and G. Browning. 1993. Gibberellin structure-activity effects on flower initiation in mature trees and on shoot growth in mature and juvenile Prunus avium. Plant Growth Reg. 13:55-63. Osborne, D. J. 1979. Target cells-new concepts for plant regulation in horticulture. Scientia Hort. 30:31-43. Paksasorn, A., M. Masuda, H. Matsui, H. Ohara, and N. Hirata. 1995. Effect of fall ethephon application on bloom delay and fruit set in Japanese apricot (Prunus mume Sieb. et Zucc). Acta Hort. 395:193-200. Popova,L. P., T. D. Tsonev, and S. G. Vaklinova. 1987. A possible role for abscissic acid in regulation of photosynthetic and photorespiratory carbon metabolism in barley leaves. Plant Physiol. 83:820-824. Proebsting, E. 1., and H. H. Mills. 1964. Gibberellin-induced hardiness responses in 'Elberta' peach flower buds. Proc. Am. Soc. Hort. Sci. 85:134-140. Proebsting, Jr., E. 1., and H. H. Mills. 1973. Bloom delay and frost survival in ethephontreated sweet cherry. HortScience 8:46-47. Ramina, A. 1981. La dinamica della cascola alcuni aspetti fisiologici della abscissione nel diradamento chimico dei frutti di pesco (Prunus persica, 1. Batsch). I Fitoregolatori nel controllo della produzione degli alberi da frutto, Ferrara, 26 Mar:9-32. Ramina, A., G. Casadoro, and N. Rascio. 1993. Structural, biochemical and molecular aspects of abscission in peach. Acta Hort. 329:211-217. Ramina, A., A. Masia, and G. Vizzotto. 1986. Ethylene and auxin transport and metabolism in peach fruit abscission. J. Am. Soc. Hort. Sci. 111:760-764. Rascio, N., G. Casadoro, A. Ramina, and A. Masia. 1985. Structural and biochemical aspects of peach fruit abscission (Prunus persica L. Batsch). Planta 164:1-11. Reid, M. S. 1985. Ethylene and abscission. HortScience 20:45-50. Richardson, E. A., S. D. Seeley, and D. R Walker. 1974. A model for estimating the completion of rest for Redhaven and Elberta peaches. HortScience 9:331-332. Roversi, A., and V. Ughini. 1985. Allegagione del ciliegio dolce. II. Influenza dell'andamento climatico in fioritura. Revisti di Frutticoltura 1:35-43. Ruperti, B., C. Bonghi, P. Tonutti, and A. Ramina. 1998. Ethylene biosynthesis in peach fruitlet abscission. Plant, Cell Environ. 21:731-737. Samish, R M. 1954. Dormancy in woody plants. Annu. Rev. Plant Physiol. 5:183-204. Samish, R M., and S. Lavee. 1982. The chilling requirement of fruit trees. In: Proc. of XVI Int. Hort. Congr. Brussels, Belgium, Vol. 5:372-388. Saure, M. C. 1985. Dormancy release in deciduous fruit trees. Hort. Rev. 7:239-300. Scholtens, A. 1993. Alternatieven bij chemisch dunnen van pruinen. Verantwoord chemisch dunnen is mogelijk. Fruitteelt den Haag 83. 28-29. Sexton, R 1995. Abscission. p. 497-525. In: M. Pessarakli (ed.), Handbook of plant and crop physiology. Marcel Dekker, New York. Sexton, R, 1. N. Lewis, A. J. Trewavas, and P. Kelly. 1985. Ethylene and abscission. p. 173-196. In: J. A. Roberts and G. A. Tucker (eds.), Ethylene and plant development. Butterworths, London. Sexton, R, and J. A. Roberts. 1982. Cell biology of abscission. Annu. Rev. Plant Physiol. 33:33-162.

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Shaybany, B., G. Costa, S. S. Brown, G. Obenauff, G. C. Martin, and M. Gerdts. 1979. Effect of 2-(3-chlarophenoxy) propionamide applications on fruit size and maturity of peach. J. Am. Soc. Hart. Sci. 104:34-36. Shoemaker, J. S. 1933. Certain advantages of early thinning of Elberta. Proc. Am. Soc. Hort. Sci. 30:223-224. Simons, R K., and M. C. Chu. 1975. Spur/pedical abscission in plum (Prumls domestica L. cv. Stanley) morphology and anatomy of persisting and drop fruits. J. Am. Soc. Hort. Sci. 100:656-666. Socias i Company, R, and A. J. Felipe. 1987. Pollen tube growth and fruit set in a se1£compatible almond selection. HartScience 22:113-116. Soni, S. 1., and H. Y. Yousif. 1978. Inducing delay in the flowering of apricot with growthregulators. Indian J. Agr. Sci. 48:197-200. Southwick, S. M., and R Fritts. 1995. Commercial chemical thinning of stone fruit in California by gibberellins to reduce flowering. Acta Hart. 394:135-147. Southwick, S. M., K. G. Weis, and J. T. Yeager. 1996a. Bloom thinning 'Loadel' cling peach with a surfactant. J. Am. Soc. Hart. Sci. 121:334-338. Southwick, S. M., K. G. Weis, and J. T. Yeager. 1996b. Chemical thinning of stone fruits in California. GoodFruit Grower 47:34-35. Southwick, S. M., and J. T. Yeager. 1991. Effects of postharvest gibberellic acid application on return bloom of 'Patterson' apricot. Acta Hart. 293:459-466. Southwick, S. M., and J. T. Yeager. 1995. Use of gibberellin formulations for improved fruit firmness and chemical thinning in 'Patterson' apricot. Acta Hort. 384:425-429. Southwick, S. M., J. T. Yeager, and K. G. Weis. 1997. Use ofgibberellins on 'Patterson' apricot (Prunus armeniaca) to reduce hand thinning and improve fruit size and firmness: effects over three seasons. J. Hart. Sci. 72:645-652. Southwick, S. M., J. T. Yeager, and H. Zhou. 1995a. Flowering and fruiting in 'Patterson' apricot (Prunus armeniaca) in response to postharvest application of gibberellic acid. Scientia Hort. 60:267-277. Southwick, S. M., K. G. Weis, J. T. Yeager, and H. Zhou. 1995b. Controlling cropping in Loadel cling peach using gibberellin: Effects on flower density, fruit distribution, fruit firmness, fruit thinning, and yield. J. Am. Soc. Hart. Sci. 120:1087-1095. Stembridge, G. R, and C. R Gambrell. 1971. Thinning peaches with bloom and post-bloom applications of 2-chlaroethylphosphonic acid. J. Am. Soc. Hart. Sci. 96:7-9. Stembridge, G. R, and J. H. LaRue. 1969. The effect of potassium gibberellate on flower bud development in the Redskin peach. J. Am. Soc. Hart. Sci. 94:492-495. Stasser R, H. P. Rasmussen, and M. J. Bukovac. 1969. A histological study of abscission layer farmation in cherry fruits during maturation. J. Am. Soc. Hart. Sci. 94:239-243. Sun, X., A. N. Miller, M. Faust, and W. Potts. 1991. Effect offall ethephon application on flower development in 'Italian' prune. Scienta Hort. 45:199-207. Sutton, T. B., and C. R Unrath. 1988. A comparison of handgun and tree-row-volume pesticide applications. Plant Dis. 72:509-512. Thompson, M. M., and 1. J. Liu. 1973. Temperature, fruit set, and embryo sac development in 'Italian' prune. J. Am. Soc. Hart. Sci. 98:193-197. Tirlapur U. K., G. Costa, C. Malossini, and G. Vizzotto. 1995. Scanning electron microscopy, video-image analysis, and confocal imaging of changes occurring during peach fruit abscission. J. Am. Soc. Hart. Sci. 120:203-210. Vasilakakis, M. D., and 1. C. Porlingis. 1984. Self-compatibility in 'Truoito' almond and the effect oftemperature on se1£ed and crossed pollen tube growth. HartScience 19:659-661. Visai c., M. Marro, and Poma C. Treccani. 1985. Orientation and light effects on fertility and production of peach fruiting wood. Acta Hart. 173:177-182.

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Walser, R. H., D. R. Walker, and S. D. Seeley. 1981. Effect oftemperature, fall defoliation and gibberellic acid on the rest period of peach leaf-buds. J. Am. Soc. Hort. Sci. 106:91-94. Webster, A. D. 1984a. Delaying flowering and improving the yields of Victoria plum with plant growth regulator sprays. Acta Hort. 149:197-202. Webster, A. D. 1984b. The effect of plant growth regulator sprays and summer shoot tipping on the fruit set and yield of young plum trees. Acta Hort. 149:203-210. Webster, A. D., and L. Andrews. 1986. Flower and fruit thinning of Victoria plum (Prunus domestica L.) with paclobutrazol. Acta Hort. 179:703-704. Webster, A D., and G. K. Goldwin. 1981. The hormonal requirements for improved fruit setting of plum, Prunus domestica L. cv Victoria. J. Hort. Sci. 56:27-40. Webster, A D., and M. S. Hollands. 1993. Thinning 'Victoria' plums with ammonium thiosulphate. J. Hort. Sci. 68:237-245. Webster, A. D., and G. K. Goldwin, W. W. Schwabe, P. B. Dodd, and D. Pennel. 1979. Improved setting of sweet cherry cultivars, Prunus avium L. with hormone mixtures containing NOXA, NAA or 2,4,5 TP. J. Hort. Sci. 54:27-32. Weinbaum, S. A, and A. Erez. 1983. Autogamy among selected peach and nectarine cultivars. Fruit Var. J. 37:113-114. Weinberger, J. H. 1941. Studies on time of peach thinning from blossoming to maturity. Proc. Am. Soc. Hort. Sci. 38:137-140. Weinberger,J. H. 1950. Chilling requirements of peach varieties. Proc. Am. Soc. Hort. Sci. 56:122-128. Williams, M. W. 1979. Chemical Thinning of Apples. p. 270-300. In: J. Janick (ed.), Horticulture reviews vol. 1. AVI Publishing Co., Inc., Westport, CT. Williams, K. M. 1989. Peach bloom delay using fall applications of ethrel and Pro-Gibb. Acta Hort. 254:151-154. Williams, M. W. 1993. Sulfcarbamide, a blossom thinner for apples. HortTech. 3:322-4. Williams, M. W. 1994. New chemical approaches for control of biennial bearing of apples. p. 16-25. In: Paul Hedin (ed.), Bioregulators for Crop Protection and Pest Control. ACS Symposium Series 557, Am. Chem. Soc., Washington, DC. Williams, M. W. 1995. Endothall, a blossom thinner for apples. HortTech. 5:257-259. Wittenbach, V. A, and M. J. Bukovac. 1972. An anatomical and histochemical study of abscission in maturing sweet cherry fruit. J. Am. Soc. Hort. Sci. 97:214-219. Zeevaart, J. A D. 1983. Gibberellins and flowering. p. 333-374. In A Crozier (ed.), The biochemistry and physiology of gibberellins. Vol. 2. Praeger, New York.

8 The Reproductive Biology of the Lychee Raphael A. Stern MIGAL Galilee Technology Center PO Box 90000

Rosh Pina 12100, Israel

Shmuel Gazit The Kennedy-Leigh Center for Horticultural Research The Hebrew University of Jerusalem Faculty of Agriculture PO Box 12

Rehovot, 76100, Israel

1. INTRODUCTION A. History, Origin, and Dissemination B. The Lychee Industry Today C. The Confusion in Lychee Cultivar Names D. The Problem of Poor Productivity II. FLOWERING A. Flower Bud Formation 1. Effect of Shoot Maturity 2. Effect of Temperature 3. Effect of Water Stress 4. Effect of Endogenous Growth Regulators 5. Effect of Exogenous Growth Regulators 6. Effect of Pruning 7. Effect of Autumnal Girdling (Cincturing) 8. Effect of Fertilization B. Floral Development 1. The Inflorescence 2. The Flower 3. Sex Ratio

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Flowering Phenology Female Flower Development Pistil Receptivity The Male Reproductive Organs Pollen Viability Effect of Temperature Regime During Flower Development on Pollen Viability and Ovule Normality POLLINATION A. Abiotic Pollination B. Biotic Pollination C. Pollinators 1. Flies 2. Bees 3. The European Honeybee D. Pollination Rate THE FERTILIZATION PROCESS AND INITIAL FRUIT SET A. The Fertilization Process 1. Pollen Germination and Pollen-Tube Growth 2. Double Fertilization and Initial Fruit Set B. Factors Affecting Initial Fruit Set 1. Female Flower Fertility 2. The Effect of Putrescine 3. M1 and M z Pollen Fertility and Effective Pollination 4. The Effect of Se1£- and Cross-Pollination FRUIT DEVELOPMENT AND ABSCISSION A. Fruit Growth and Development 1. Morphology 2. Anatomy 3. Phases in Fruit Development 4. Fruit Growth Rate B. Pollen Parent Effect on Seed and Fruit Characteristics C. Fruit Abscission 1. Abscission Rate and Pattern 2. Abscission Reduction with Plant Growth Regulators CONCLUDING REMARKS LITERATURE CITED 4. 5. 6. 7. 8. 9.

III.

IV.

V.

VI.

I. INTRODUCTION

A. History, Origin, and Dissemination The lychee (Litchi chinensis Sonn., Sapindaceae) originated in southern China and possibly northern Vietnam, between 19 and 27°N (Plate 8.1). Wild trees still grow in elevated and lowland rainforests, especially in the Guangdong, Guangxi, and Hainan provinces, where lychee is one of the main species of the forest (De Candolle 1964; Knight 1980; Menzel

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and Simpson 1990b; Zhang 1997a). It has been under cultivation since about 2000 BCE (Ochse et al. 1961). The first written reference to lychee dates back to the time of the Han dynasty, in about 100 BCE. The firstknown monograph on a fruit crop was written on the lychee, by Tsai Hsiang in 1059 (Groff 1921). The lychee remained confined to the South China region until "recent" times, probably because of the great perishability of the seed (Popenoe 1920). It reached neighboring Burma (Mayanmar) at the end of the seventeenth century, and reached India about 100 years later (Singh and Singh 1954). The lychee reached the Southern Hemisphere (Madagascar, Mauritius and South Africa) around 1870 (Marloth 1947; Oosthuizen 1991; Milne 1999a). It was introduced by a Chinese trader to Hawaii in 1873 (Yee 1972), to Florida from India between 1870 and 1880 (Cobin 1954), and to California in 1897 (Pandey and Sharma 1989). It reached Australia around 1854 (Menzel et al. 19S8a). It was introduced to Israel in the 1930s (Oppenheimer 1947) (Plates 8.1 and 8.2). GalanSauco (19S9), Menzel and Simpson (1990b) and Menzel (2001) have summarized the worldwide dissemination of lychee. B. The Lychee Industry Today

Total world production of lychee is about 1.5 million tonnes. The major producing countries are: China, 950,000 t (Chen and Huang 2001); India, 360,000 t (Gosh 2001); Taiwan, 130,000 t (Tindall 1994); Thailand, 40,000 t (Subhadrabandhu and Yapwattanaphun 2001); and South Africa, Madagascar, Vietnam, and Pakistan, 30,000 t each (Milne 1999a; Trung 1999; Gosh 2001; Panhwar 2000). Minor producing countries include Australia (3000 t), Israel (1500 t), Mauritius and Reunion (1000 t together) and Florida (360 t) (Menzel et al. 1999; Milne 1999b; Knight 2001; J. Crane, pers. commun.). Almost all lychee production is consumed in the main producing countries themselves (Batten 19S2; Gosh 2001). Only about 0.5 percent of the world output is exported, although some countries, such as South Africa, Madagascar and Israel, export a high proportion of their production (Milne 1999b). The European community market imports 15,000 t of fresh lychee annually. C. The Confusion in Lychee Cultivar Names There is serious and widespread confusion in the naming of lychee cultivars (Galan-Sauco 19S9; Menzel and Simpson 1990b; Degani et al. 1995a; Gazit and Goren 1997; Zhang 1997c; Zee et al. 1995). Too often the same cultivar is named differently in different localities, and

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different cultivars share the same name. In South China, lychee cultivars are named differently in different regions, according to the dialect used. Alternatively, the same name may be used in different localities for different cultivars. This confusion at the source was amplified upon its dissemination to other countries. In many cases, cultivar identities were confused or lost. In some cases, the original name was replaced by a new local name. Today, there is still no reliable method of identifying lychee cultivars. Untillychee cultivars can be conclusively identified by a reliable DNA method, one should not assume that cultivars that look the same are indeed identical. To reduce the confusion, in this chapter we maintain the cultivar names used in the articles cited, sometimes adding the correct or more universally known synonym parenthetically. For Chinese cultivars in China, the official Romanization of the Mandarin dialect is used. D. The Problem of Poor Productivity

Lychee suffers from the widespread problem of low and irregular bearing (Samson 1980; Menzel 1983, 1984; Batten 1986; Joubert 1986; GalanSauco 1989; Stern et al. 1993a, 1995, 1998,2000). The average yield in many countries is about 5 t ha- 1 (Galan-Sauco 1989; Stern et al. 1998). Different factors may be responsible for this poor productivity: pests and diseases (Li 1997; McMillan 2000; He 2001), extreme temperatures (Kadman and Slor 1982; Menzel and Simpson 1994), water stress (Menzel and Simpson 1994; Menzel et al. 1995) and inadequate mineral nutrition (Menzel et al. 1992). However, even under optimal growing conditions, productivity may be poor and erratic (Batten 1986; Menzel 2001). A cool winter is essential for the production of flower buds in most lychee cuItivars (Groff 1943; Menzel 1983). Hence, most do not flower or yield consistently in southern Florida (Young and Harkness 1961; Young 1970; Davenport et al. 1999; Zheng et aI, 2001) and Hawaii (Nakata and Watanabe 1966; Nakata and Suehisa 1969). In Israel, commercial planting of lychee started in the mid-1970s. Under intensive fertigation the trees developed well (Gazit 1996). However, at maturity, the production of healthy trees was erratic; average yields were around 3 to 4 t ha- 1 (Shalem-Galon 1980; Stern et al. 1993a, 1995,1998). At the end ofthe1980s we began a comprehensive study of the reproductive biology of the lychee in Israel, with special emphasis on the factors responsible for its poor productivity (Stern 1992). Our better understanding enabled us to devise new treatments to improve productivity. Some of these treatments have also been used successfully in China (Stern et al. 2001) and South Africa (Penter et al. 2000).

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Linguistic and geographical barriers greatly hindered the transfer of lychee cultivars and information from China to other parts ofthe world. Groff (1921) breached these barriers by writing a comprehensive monograph on lychee and longan in China. Recently, in June 2000, the First International Symposium on Lychee and Longan was convened in Guangzhou, China. It was conducted in English and served as a forum for the exchange of information and ideas between scientists and professionals from China and many other lychee-growing countries (Acta Hort. 558, 2001). This chapter on reproductive biology of lychee is based mainly on information published in English, from most lychee-growing countries. For several topics it draws heavily on work conducted in Israel. II. FLOWERING Poor and erratic lychee flowering is a worldwide problem, especially in regions where the winter is not cool enough (Groff 1943; Young and Harkness 1961; Young 1970; Menzel 1983; Joubert 1986; Menzel et al. 1986; Zee et al. 1998). Only scant work has been done on the induction, initiation, and differentiation of floral buds in lychee (Banerji and Chaudhuri 1944; Pandey and Bajpai 1969; Shukla and Bajpai 1974). In most studies, the emergence of inflorescences has been used as the criterion to assess the effect of different factors on flowering. A. Flower Bud Formation Initiation and development of lychee floral buds occur in the winter, leading to anthesis in early to late spring. In its region of origin the winter is cool and almost rainless and day length is short, reaching a minimum of about 10 h (Groff 1921; Batten 1986). Lychee flowering is indeed promoted by cool and dry winters, but not by short day length. Lychee is a day-neutral plant: extremely short (8 h) or long (16 h) days do not affect its flowering at all (Nakata and Watanabe 1966; Stern 1992). 1. Effect of Shoot Maturity. Flower bud formation usually occurs in mature terminal shoots. Exposure of young immature shoots to inductive temperatures results in little or no bloom. Thus, a late autumnal flush that is not hardened off before the start of the winter is undesirable. Flower buds develop at the apex of the terminal mature shoot. However, when the flowering part of the shoot is pruned away, lateral buds may form flower buds. In Israel, such autumnal and early winter pruning resulted in a normal flowering rate; flowering rate decreased with

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pruning in mid-January and disappeared when pruning was performed in March (Goren 1990; Stern 1992). 2. Effect of Temperature. A cool winter is generally considered a prerequisite for adequate lychee flowering (Groff 1921,1943; Young 1970; Menzel 1983; Joubert 1986; Zee et al. 1998; Galan-Sauco 1989). Guangzhou, which is located at the center of the main lychee growing area in China (23°N), has a rather cool winter; the monthly average temperatures in November, December, January, and February are: minimum 15.2,10.8,8.7, and 11.0°C, maximum 24.3, 20.3, 19.1, and 19.0°C, respectively (Groff 1921). Under these climatic conditions, lychee usually blooms well. Joubert (1985) suggested that for successful flowering of lychee, the average monthly minimum temperature should drop below 14°C for one to three months. There is great variation among lychee cultivars in their need for a cool winter to induce flowering. In Thailand, cultivars have been selected for low-elevation tropical conditions, and are classified as tropicallychee. These cultivars require little or no cool period for flowering, and are successfully cultivated near Bangkok (l4°N) (Subhadrabandhu 1990; Subhadrabandhu and Yapwattanaphun 2001). Under this tropical climate, most of the commercial cultivars grown near Guangzhou, which may be classified as subtropical, will not bloom. All studies on lychee flowering have been conducted with subtropical lychee cultivars, which need a cool winter to initiate flower buds. There are significant differences among subtropical cultivars in their requirements for a cool winter. For example, in southern Florida, 'Brewster' blooms erratically, whereas 'Mauritius' tends to bloom consistently (C. Campbell 1994). The difference apparently reflects the climatic conditions under which these two cultivars were selected and cultivated. 'Brewster' ('Chen Zi') had been cultivated at 25°N near Putian, in the Fujian Province, whereas 'Mauritius' ('Da Zao') had been cultivated at 22.5 to 23.5°N near Guangzhou, in the Guangdong Province (Groff 1921, 1948; Menzel and Simpson 1990b). Under the marginal temperature regime of 20/15°C (day/night), a pronounced difference in flowering was found among seven cultivars; flowering rate fluctuated between 11 and 99 percent (Table 8.1) (Menzel and Simpson 1994). Hence, general statements about a specific temperature regime required for lychee flowering may be misleading: each cultivar may have a specific requirement. In Hawaii, 'Mauritius' (erroneously called 'Kwai Mi') tends to flower irregularly. Nakata and Watanabe (1966) found that flowers were initiated only after the ambient minimum temperature dropped for about a

399

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE Table 8.1. Effect of day/night temperature regimes on the percentage of terminal branches flowering in seven lychee cultivars. Terminal branches flowering (%) Cultivar Kwai Mai Red SoueyTung Bengal Tai So Kwai Mai Pink Salathiel Wai Chee

15/10°C

20/15°C

25/20°C

100 100 100 100 100 100 100

11

32 42 50 87 91 99

0 0 0 0 0 0 0

Adapted from Menzel and Simpson (1994).

month below 18.3°C. However, flowering intensity was rather light. Profuse flowering started about two months after plants had been kept at night at 13.9°C (in a cold room), while the daily maximum was about 27°C. Continuing with this treatment up to anthesis for an additional month and a half doubled the number of panicles per flowering terminal. Apparently, during that period, initiation oflateral buds continued. The effects of relevant temperatures on lychee flowering have been studied under two types of controlled temperature regimes: (1) The conventional way, in which day and night temperatures are kept constant (Menzel and Simpson 1988; Menzel et al. 1989; Stern 1992); (2) Mimicking the normal situation in the open: the temperature changes gradually, reaching a maximum at 12:00 h and a minimum at 06:00 h (Menzel and Simpson 1995). The results of these studies are presented in Table 8.2. A cool day/night temperature regime (15/10°C) induced full flowering in seven cultivars. Even moderate temperatures during the day coupled with a cool night (22/12°C and 20/10°C) were fully effective for the few cultivars studied. Warm days and moderate nights (25/20°C and higher) resulted in no bloom. Partial flowering occurred under regimes of moderate days and nights (20/15°C and 20/20°C) or warm days and cool nights (25/15°C and 25/10°C). 'Kwai Mai Pink' was fully induced to flower under 20/15°C (max/min) and only partially induced in the same temperature range (20/15°C) with the day/night version. This difference probably indicates that under field conditions, full induction may occur under somewhat higher temperatures relative to those needed under constant day/night regimes. Root temperature was found to have a significant effect on floral induction of 'Tai So' plants (Menzel et al. 1989). Keeping the root

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Table 8.2. Effect of different temperature regimes on the flowering of lychee cultivars (all plants were well watered). Flowering Full

Temperature regime (oC) 15/10 (day/night)

22/12 (day/night) 18/15 (day/night) 20/15 (max/min)

Partial

Bengal, Kwai Mai Pink, Kwai Mai Red, Salathiel, Souey Tung, Tai So (Mauritius), Wai Chee Floridian, Tai So (Mauritius) Wai Chee Casino, Kwai Mai Pink

20/10,20/5 (max/min) 15/5 (max/min)

Kwai Mai Pink

20/15 (day/night)

Bengal, Kwai Mai Pink, Kwai Mai Red, Salathiel, Souey Tung, Tai So (Mauritius), Wai Chee Tai So (Mauritius) Tai So (Mauritius)

20/20 (day/night) 25/15, 25/10 (max/min) 20/10,20/5 (max/min) 15/5 (max/min) None

Cultivars

25/20, 30/25 (day/night)

22/17 (day/night) 23/18 (day/night) 30/10, 30/25 (max/min) 25/20, 30/20 (max/min)

Kwai Mai Pink

Casino Casino

Source Menzel and Simpson, 1988; Menzel et aI., 1989; Chaikiattiyos et al. 1994 Stern, 1992 Chaikiattiyos et al. 1994 Menzel and Simpson 1995 Menzel and Simpson 1995 Menzel and Simpson 1995 Menzel and Simpson 1988

Menzel Menzel 1995 Menzel 1995 Menzel 1995

et al. 1989 and Simpson and Simpson and Simpson

Bengal, Kwai Mai Pink, Kwai Mai Red, Salathiel, Souey Tung, Tai So (Mauritius), Wai Chee Floridian, Tai So (Mauritius) Wai Chee Tai So (Mauritius)

Menzel and Simpson, 1988; Chaikiattiyos et al. 1994

Casino, Tai So (Mauritius)

Menzel and Simpson 1995

Stern 1992 Chaikiattiyos et al. 1994 Menzel et al. 1989

system in water at a constant hot temperature of 27.5°C, appreciably decreased the flowering of a canopy kept under the inductive regime of 15/10°C (day/night). Decreasing the root temperature to 12.5°C had the opposite effect: it caused full flowering of plants kept at the partially

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401

inductive regime of 20/20°C (day/night). However, the same low root temperature could not initiate flowering in plants kept under the noninductive regime of 30/25°C. 'Wai Chee' plants did not flower when kept for two weeks at the constant temperature of 15°C and then transferred to the noninductive regime of 30120°C. Only after four and ten weeks at 15°C, did partial (22%) and full (90%) flowering occur (Menzel and Simpson 1995). Apparently, under an optimal temperature regime for flower induction, floral initiation and development proceed at a faster rate. At 15/10°C (day/night), panicles emerged after only four weeks, whereas at 20/15°C (day/night), panicles emerged after six weeks (Menzel and Simpson 1988). Menzel and Simpson (1995) also found that 'Wai Chee' plants kept at a constant temperature of 15°C stopped flowering when exposed daily to 8 hours above 20°C (the temperature rose to a maximum of 25°C). They concluded that temperatures above 20°C reduce flowering in lychee. This general statement is not accurate. It does not take into account that tropicallychee initiates flowering under much higher daily maximum temperatures. Even subtropicallychee cultivars will initiate floral buds under higher maximum temperatures. Stern (1992) found that 'Mauritius' ('Tai So') and 'Floridian' ('Brewster') flower profusely when exposed daily for 16 h to 22°C (22/12°C, day/night) for several months (mid-October until the end of January). In India, Shukla and Bajpai (1974) detected floral buds in 'Rose Scented' and 'CalcuUia' two weeks after the daily maximum and minimum temperatures were 28 and 8°C, respectively. A cool period should be considered a prerequisite for the induction and formation of floral buds in lychee. However, inductive temperature regimes do not always lead to adequate flowering, in small plants (Table 8.2) or in mature trees. Trees in their "off" year and those that have put out a vegetative flush late in the autumn tend to have a poor bloom even though the weather is cool enough. Apparently, for the shoots to form floral buds, they have to be mature enough, with adequate starch reserves (Nakata and Watanabe 1966). 3. Effect of Water Stress. A dry autumn and winter are considered favorable for flowering in lychee, via the induction of vegetative dormancy (Chapman 1984; Galan-Sauco 1989; Menzel and Simpson 1994). However, water stress per se is neither essential nor sufficient for flower induction. Under inductive temperature regimes, full flowering occurred in well-watered plants (Table 8.2). Under noninductive temperature regimes, a period of water stress, which stopped vegetative growth, did not induce flowering in 'Kwai Mai Pink' or 'Wai Chee' plants (Menzel et al. 1989; Chaikiattiyos et al. 1994).

402

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Water stress was instrumental in promoting flowering and increasing yield under different growing conditions in Hawaii and in Israel. In Hawaii (20 0 N), under a hot and dry summer (April to November) and hot and rainy winter (December to March), lychee flowering is erratic. Nakata and Suehisa (1969) studied the effects of three irrigation strategies on growth, flowering (panicles emerged in December), and yields of eight-year-old 'Mauritius' (erroneously identified as 'Kwai Mi') lychee. The "wet" treatment maintained the soil water potential at a 0.45-m depth above -0.03 MPa from June to February. The "dry" treatment maintained an average soil water potential of about -0.5 MPa from June to August and -1.5 MPa from September to December. Heavy rains occurred in December and soil water potential rose to about -0.03 MPa. In the "covered" treatment, soil water potential declined from -0.03 MPa in October to -0.8 or -0.9 MPa during December and January (and then increased after irrigation in March to -0.03 MPa). Only 50 percent of the tagged branches flowered in the "wet" plot compared with 80 and 85 percent in the "covered" and "dry" plots respectively. The average yields were: 50, 71 and 84 kg/tree, respectively. The authors concluded that flowering is favored by a dry period before and during floral initiation. In Israel (32-33°N), under a hot and dry summer (April to October) and cool and rainy winter (November to March), lychee flourished, due to regular fertigation throughout the dry season. However, in spite of the long cool winter, flowering and yields were erratic. Autumnal water stress from early October to the start of the rainy season resulted in inhibition in vegetative growth, profuse flowering, and a dramatic increase in yield of young as well as mature 'Mauritius' and 'Floridian' ('Brewster') orchards (Table 8.3) (Stern and Gazit 1993; Stern et al. 1993a). In several trials, total cessation of irrigation was instituted for about a month, until some of the mature leaves started to show clear symptoms of damage, changing from green to yellow-bronze. At that stage, the noon leaf water potential reached a value of -2.8 MPa, compared to -1.5 MPa in control irrigated trees (Stern et al. 1998). Limited irrigation of 1 mm/day was immediately started and maintained until the beginning of the rainy season. Initiation of flower buds occurred during the cool rainy season, flowering in April. Yields were proportional to flowering intensity. Autumnal water stress has been adopted by the lychee industry in Israel and is now considered a routine method, which has the added advantage of saving about 15 percent on irrigation water. However, excessive water stress may result in a significant reduction in assimilation rate, chlorophyll destruction, root death, and leaf drop (Stern et al. 1993a; Menzel and Simpson 1994; Menzel et al. 1995; Roe et al. 1995).

403

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE Table 8.3. Effects of autumnal water stress on flowering and yield of 12-years-old 'Mauritius' and 'Floridian' lychee trees in Israel. Floridian

Mauritius Flowering intensity Irrigation regime

(0-3)0

Yield (kg/tree)h

Control Water stress

2.1 a C 3.0 b

23.7 a 60.7 b

Flowering intensity (0-3)

Yield (kg/tree)

2.5 a 3.0 b

21.3 a 30.5 b

OFlowering intensity grades: 0, no flowering; 1, poor; 2, medium; 3, full. hSpacing of 6 x 6 m (280 trees ha-1 ). cMean separation by Duncan's multiple range test, P 0.05. Source: Stern et al. 1993a.

To optimize the water stress regime and to find a reliable indicator for irrigation control during autumnal water stress, Stern et al. (1998) conducted a trial with four autumnal irrigation regimes (0.5, 0.25, 0.125, and o class A pan evaporation coefficient, which is equivalent to 100, 50, 25 and 0% of the recommended irrigation rate). All three water stress treatments stopped autumnal shoot growth, and significantly increased the flowering intensity and yield. The best results were obtained with the moderate water stress regime of 50 percent. A comparison of soil, leaf, and stem water potentials showed that plant water potential values are much more consistent than soil water potential values. Midday stem water potential differed more markedly among treatments than leaf water potential. Hence, it appeared to be the best indicator for irrigation control. It was concluded that to achieve the optimal effect of autumnal water stress, the midday stem water potential values should be between -2.0 and -2.5 MPa (compared to -1.5 MPa in the control = 100%) (Stern et al. 1998). A period of water stress prior to flower induction may increase flowering by arresting vegetative growth. This method may have practical significance in areas which have consistently dry autumns or winters (e.g., Florida, Israel, South Africa, and northern Australia), but is difficult to maintain in locations with high autumn rainfall, especially on heavy soils. 4. Effect of Endogenous Growth Regulators. Only two reports can be

found on the relationship between endogenous growth regulators and floral initiation in lychee. Chen (1990) found that one month before flower bud formation, gibberellin levels in the xylem sap drop

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R. STERN AND S. GAZIT

dramatically, whereas cytokinin-like activity increases greatly. At that same stage, no appreciable change was found in the diffusible indol acetic acid (IAA) and abscisic acid (ABA) levels from shoot tips. Liang et al. (1987) found the lowest gibberellin level in lychee shoot tips in January, during the period of floral initiation. 5. Effect of Exogenous Growth Regulators. The need to improve flowering intensity prompted researchers to study the effect of available growth regulators.

Auxins. The synthetic auxin, sodium naphthaleneacetate (SNA), was used in a series of trials in Hawaii, conducted from 1948 to 1953. It was sprayed (at 200 or 400 mg L-1) in the autumn to inhibit new vegetative growth and thereby increase flower bud formation (Nakata 1955). The spraying did not have a consistent effect on flowering. When the fall vegetative flush was arrested by dry weather, the SNA had an inhibitory effect on floral bud initiation. In contrast, it inhibited vegetative growth and increased flowering when heavy rainfall occurred in October and November. In Florida, a four-year trial with SNA (at 100 mg L-1) and 1naphthaleneacetic acid (NAA at 30 mg L-1) did not reveal any consistent positive effect on yield (Mustard et al. 1956). These results indicate that there is a limit to the ability of synthetic auxins to inhibit vegetative growth if the flush is particularly strong. Growth Retardants. The following growth retardants were sprayed in the autumn, but none consistently affected vegetative growth and/or flowering: maleic hydrazide at 500 to 3000 mg L-1 (Young et al. 1961); Alar (containing 85% 2,2-dimethylhydrazide) at 1000 mg L-1 (Chapman et al. 1980), or 1000 to 3000 mg L-1 (Stern 1992); and CCC (containing 40% 2chIoroethyl-trimethyl-ammonium chloride) at 0.5 to 1.5 percent (Stern 1992). A dramatic effect of paclobutrazol (1-(4-chlorophenyl)-4,4-dimethyl-2(lH-l,2,4-triazol-l-yl) pentan-3-0l) was found in China (Liang and Yu 1991). Mature lychee trees, which had never flowered, were induced to flower by two sprayings (first in November, followed by a second one 25 days later) with 400 mg L-1 PP333 (paclobutrazol) plus 40 mg L-1 KH ZP04 • A single spray was ineffective. Spraying flowering trees in January, when panicles were emerging, with 100 mg L-1 plus 10 mg L-1 KH ZP04 more than doubled the number of flowering inflorescences, and greatly decreased the percentage of leafy inflorescences. Several studies conducted in other countries gave inconsistent results: in subtropical southern Queensland the effects of autumnal paclobutrazol applications, as a foliar (4 g L-1) and soil drench (1 g m-z tree ground cover), on three lychee cultivars, were studied over three years at eight sites (Menzel and Simp-

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405

son 1990a). Vegetative growth was greatly reduced with all soil applications and with most foliar ones; nevertheless, flowering was consistently improved only in orchards with moderate bloom. No data was included about the effect on yield. Soil applications had a residual effect on tree growth whereas foliar applications did not. In Thailand, no significant effect on vegetative growth or flowering was found after paclobutrazol was applied in September, at up to a 16 g/tree soil drench or up to 500 mg L-1 spray, or in October, as a soil drench at up to 1.5 g m-zleaf canopy or 1500 mg L-1 spray (Chaitrakulsub et al. 1992a, 1992b). In Israel, Cultar (25% paclobutrazol) was sprayed in mid-October, at the very high concentrations of 0.5 to 2.0 percent, on mature trees carrying mature dormant shoots or a young vegetative flush, after "on" or "off" years. Spraying growing trees appreciably increased flowering intensity and yield, whereas spraying dormant trees did not (Stern 1992). This preliminary beneficial effect was not further pursued as autumnal vegetative growth is routinely checked in Israel by water stress (Stern and Gazit 1993; Stern et al. 1993a, 1998). No beneficial effect was found when Magic (5% uniconazole) was sprayed in October on dormant water-stressed trees (Stern et al. 1996a, 1997b).

Paclobutrazol Plus Ethephon. The combination of paclobutrazol spraying (at 500 to 1500 mg L-1), about two months before panicle emergence (in early November), and two subsequent sprayings in mid-November and mid-December with ethephon [(2-chloroethyl) phosphonic acid] at 300, 400, and 500 mg L-1, tripled flowering percentages in northern Thailand (Chaitrakulsub et al. 1992b). Spraying with 500 mg L-1 paclobutrazol + 4000 mg L-1 ethephon promoted flowering in unproductive 'Tai So' trees in Mauritius (Ramburn 2001). Ethephon. This ethylene-releasing product is widely used in Taiwan to suppress winter growth and to induce leaf abscission in lychee (Zee et al. 1998). A single spray application (at 5000 mg L-1) is effective for about 15 days. Hence, two to three applications may be required throughout the winter season. Ethephon (at 1000 to 3000 mg L-1 + 0.5% urea) caused defoliation of the late autumnal vegetative flush when applied in May in subtropical Australia. New buds emerged behind the damaged shoots within a few weeks, and flowered if the weather remained cool enough for flower initiation (Olesen et al. 1999). In Thailand, ethephon at 100 or 500 mg L-1 sprayed in late autumn significantly decreased flowering of '0 Hia' but did not have a significant effect on 'Hong Huay' flowering (Subhadrabandhu and Koo-Duang 1987). Kinetin. In Taiwan, kinetin (at 200 mg L-1) was sprayed on September 22 onto mature terminal shoots. Three weeks later, flower bud formation was anatomically detected in 60 percent, cO,mpared to 0 in control buds,

406

R. STERN AND S. GAZIT

and panicle emergence was advanced by one month (Chen and Ku 1988). 6. Effect ofPruning. In high-density lychee orchards, summer pruning is routinely performed after harvest, to control tree size (Goren and Gazit 1993). Under growing conditions where there is no natural or artificial check on the autumnal flush, pruning should be timed to produce a vegetative flush that will mature prior to the induction period (Olesen et al. 1999). Only a great deal of experience and some luck may consistently achieve this aim. When an undesirable late autumnal flush occurs, its removal by mechanical or chemical pruning, prior to or during the induction period, may greatly increase flowering rate (Goren 1990; R. Campbell 1994; Olesen et al. 1999). Removal of the young flush releases the buds on the proximal mature shoot from its apical inhibition. These active buds respond to the inductive regime and are transformed into floral buds. Even buds that started to grow under noninductive conditions formed normal inflorescences if transferred to inductive conditions when they were still small (less than 3 mm) (Batten and McConchie 1995).

7. Effect of Autumnal Girdling (Cincturing). Autumnal girdling (spiral or ringing) was found to promote flowering in a non- or partially inductive environment, apparently by inhibiting vegetative growth and increasing the carbohydrate level. This method is used in China (Zhang 1997b), and has been recommended for some cultivars in Hawaii (Nakata 1956; Yee 1972) and Florida (Young 1977). It is especially effective and necessary under environmental conditions that encourage late autumnal vegetative flushes (warm and rainy autumn) and thereby prevent flowering (Menzel and Simpson 1987; Li and Xiao 2001). In Mauritius (20°5), all autumnally girdled 'Tai So' branches flowered, whereas nongirdled branches did not flower at all, or flowered at a very low rate (Ramburn 2001). Girdling is also used to promote the flowering and fruiting of young trees (Chapman 1984). Autumnal girdling should be performed after the maturation of the first or second postharvest flush, about two to three months before flower initiation. The girdling should be accurate: the cut should reach the cambium all around, and its width should keep it "open" until flower bud initiation, but enable it to heal by fruit set (Menzel and Paxton 1986a, 1986b). Girdling the trunk, or all the main branches, is more effective than girdling only half of the tree. In the latter case, flowering and yield of the girdled part of the tree may greatly increase, but decrease in the nongirdled part, resulting in only a small average increase for the whole tree

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

407

(Nakata 1956; Li and Xiao 2001). Girdling starves the root system, especially when performed on the whole tree, and may cause serious damage. Weak or undernourished trees should not be girdled. Girdling does not always improve flowering and yield. Menzel and Simpson (1987) reported that, in a series of girdling trials over four years in Queensland, girdling increased flowering in 30 percent, had no significant effect in 58 percent and reduced flowering in 12 percent of cases. They concluded that girdling should be performed only when inadequate bloom is expected, provided that the trees are well-fertilized and have flushed after harvest. Girdling is performed to increase yield. Unfortunately, yield information is lacking in most trials. Though girdled branches usually outperform nongirdled branches on the same tree (Nakata 1956), only yield records for several consecutive years of girdled versus nongirdled control trees can determine the long-term effect of girdling on orchard yield. In Queensland, yield increases after girdling were reported: from 9 to 34 kg/tree and 3 to 22 kg/tree, in 'Wai Chee' and 'Tai So,' respectively (Menzel and Simpson 1987), and from 15 to 40 kg/tree in some orchards (Menzel and Paxton 1986b). In conclusion, the erratic effect of girdling does not justify its routine use. Vegetative growth can be checked by more consistent methods. 8. Effect ofFertilization. The vegetative flush is promoted by fertilization, especially by nitrogen fertilizers. Hence, stopping or reducing the use of nitrogen fertilizer toward the autumn tends to reduce or even eliminate the autumnal flush. In Queensland, autumnal flush was reduced when nitrogen levels were kept below 1.75 to 1.85 percent during the critical four weeks prior to panicle emergence (Menzel et al. 1988b). It has been suggested that nitrogen fertilizers should be applied only in spring, after panicle emergence and fruit set, and not later (Zee et al. 1998). Indeed, restricting nitrogen fertilization in Florida to January and March significantly reduced vegetative flush and increased flowering rate (Li et al. 2001). However, in Israel, high yields have been obtained even though fertigation is practiced up to the start of the autumnal water stress in October (Goren and Gazit 1996). Hence, nitrogen fertilization should be restricted only if needed to prevent the autumnal flush.

B. Floral Development 1. The Inflorescence. Shukla and Bajpai (1974) followed floral bud differentiation in lychee. They considered the appearance of primordia on the elongated main axis as the first indication of floral differentiation.

408

R. STERN AND S. GAZIT

The emerging inflorescence is similar in appearance to a vegetative flush (Plate 8.3). Only when primordia of the secondary inflorescence branches appear in the axils of the small leaves is it possible to identify the flush as a developing inflorescence. In inductive environments, buds at the terminal end of the mature current-season shoots are transformed into floral buds. Infrequently, floral buds can also develop on a previous-season branch, at the base of the withered past-season's inflorescence (Das and Choudhury 1958; Robbertse et al. 1995). The prominent terminal inflorescence is usually composed of a cluster of panicles (Fig. 8.1). Each panicle produces tens to hundreds of small flowers of three types: two functional males (M 1 and M z) and one functional female (F) (Das and Choudhury 1958; Joubert 1985; Costes 1988).

Fig. 8.1. 1988)

A schematic representation of a typical developing lychee inflorescence. (Castes

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

409

Robbertse et al. (1995) thoroughly detailed the morphology of the lychee inflorescence and the positions of the three flower types. They identified the inflorescence as a determinate, compound thyrse, because its side branches are cymose. The flowers are borne on partial inflorescences or dichasia. A typicallychee dichasium is presented in Fig. 8.2. Usually, the first flower to open (no. 1) is the M 1 ; the second to open (no. 2) are two female flowers; the third group to open (no. 3), are four M z flowers. The following eight (no. 4) buds usually remain dormant, but in richly branched inflorescences they may also develop into flowers. Many variations on this scheme have been encountered. In most of them, the M 1 flowers are absent and the female flowers occupy the first and sometimes also the second positions in the dichasium. 2. The Flower. The floral differentiation process has been described by Banerji and Chaudhuri (1944) and Shukla and Bajpai (1974). They found that flower development takes place in an acropetal manner: first the calyx, then the stamens and carpels. The lychee flower is small, 3 to 8 mm in diameter and 7 to 12 mm in length, and is borne on a short 2 to 6 mm-Iong pedicel (Liu 1954). It has a cup-shaped calyx, with four to five short, serrated sepals, and no petals. The stamens and pistil are inserted into the nectary located within the calyx. The ovules are anatropous and contain integuments, a nucellus and an obturator. The stamen has a two-celled anther, which dehisces longitudinally (Mustard et al. 1953; Liu 1954).

Fig. 8.2. Forms of lychee dichasia in lateral view with numbers showing the sequence of flower anthesis. (Robbertse et al. 1995)

410

R. STERN AND S. GAZIT

All three types of flowers (Fig. 8.3) open in succession on the same inflorescence (Liu 1954; Singh and Singh 1954; Das and Choudhury 1958; Mustard 1960; Joubert 1985; Stern and Gazit 1996). As all three types oflychee flowers have male and female reproductive organs, they may be classified as hermaphrodites. However, each type exhibits distinct degeneration of the male or female organs. They were named by Mustard (1960) as type I, II and III, according to the chronological order of their opening, and by Singh and Singh (1954) as male, hermaphrodite and pseudo-hermaphrodite. We prefer the terminology based on their sexual functionality (Male l , Female and Male z) (Costes 1988), which is clearer and more relevant, and we use it exclusively in this chapter. The following description of the three flower types (Fig. 8.3) is based on reports by Liu (1954), Mustard et al. (1953), Mustard (1960), Scholefield (1982) and Moncur (1988): (1) The malei (Mi ) flower has a rudimentary pistil, which appears as a conspicuous pink, pubescent protuberance. The ovary contains two half-formed ovules with no embryo sac. The pistil is surrounded by six to eight stamens, with hairy filaments, about 6 mm in length. The nectar disc is small; (2) The male2 (M2 ) flower has a prominent pistil with a style that ends in a two-lobed stigma. At anthesis, about 20 percent of the ovules contain a mature embryo sac. However, the pistil is nonfunctional as the lobes of the stigma do not open for pollination. It is surrounded by six to eight stamens, which are similar in appearance and function to those in M l flowers. The nectar disc is medium in size, much larger than in the M l flower; (3) The female (F) flower has a fully developed pistil, which is larger than that of M z. The pistil is composed of a two-lobed superior ovary, which contains two anatropous ovules, a short style, and a bifurcated stigma (Fig. 8.4). The surface of the ovary is pubescent with

F Fig. 8.3. The 3 lychee flower types: Female flower (F) Male 1 flower (M 1 ) and Male 2 flower (M z). Adapted after Castes (1988).

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

411

Fig. 8.4. Longitudinal section of a two days old female 'Mauritius' flower. A, pistil with a two-lobed, bi-locular ovary (Ov) (x25); B, normal ovule, composed of funiculus, obturator, two integuments and an embryo sac containing an egg apparatus and a polar nucleus (xl00); C, embryo sac, containing a normal egg cell and two degenerate synergids (x400); D, normal embryo sac, containing a normal polar nucleus, egg cell and two synergids (x400). ES, embryo sac; EC, egg cell; EA, egg apparatus; S, synergid; uS, degenerate synergid; P, polar nucleus; 01, outer integument; II, inner integument; OB, obturator; OW, ovary wall; F, funiculus. (Stern et al. 1996b)

R. STERN AND S. GAZIT

412

protuberances, that persist and give the fruit its rough surface. Usually, only one of the lobes of the ovary develops into a fruit, the other aborting. Occasionally, however, the two lobes may develop, producing two fruits, each containing a seed. The pistil is surrounded by six to eight stamens, with very short filaments (less than 1.5 mm in length). The anthers contain little viable pollen and do not dehisce; thus, functionally this flower type is female. The nectar disc is very large, much larger than in the M z flower.

a

3. Sex Ratio. The sex ratio in mature tree blooms has been found to differ widely among cultivars. The percentage ofF, M1 and Mz flowers fluctuate as follows: among eight cultivars in Florida, from 67, 11, and 22 in 'Mountain Lychee' to 14, 32, and 54 in 'Brewster' (Mustard et al. 1953); among five cultivars in India, from 49,8, and 43 in 'Calcuttia Late' to 20, 32, and 48 in 'Dheradun' (Chadha and Rajpoot 1969). Note that these results may not indicate a real difference among cultivars. 'Mauritius' sex ratio in Israel was reported in one study as 32, 34, and 34 (Stern et al. 1993a) and in another as 33, 10, and 57 (Nadler 1995); in Reunion, it was reported as 27, 33, and 40 (Costes 1988). In some studies, the two male-type flowers were not counted separately. In India, 32 percent of the flowers were female as found in 'Early Large Red' (Chaturvedi 1965). In Queensland, the percentage of female flowers was determined for five cultivars at four locations; it fluctuated from 43 to 16. However, for one of the cultivars ('Bengal'), the fluctuation in the four locations was almost as great: 43,34,26, and 20 percent (Menzel and Simpson, 1992). Wide-scale observations carried out over several years may reveal consistent differences among cultivars. In Queensland, 'Salathiel' was reported to have the highest percentage of female flowers, whereas 'Kwai Mai Pink' and especially 'Kwai Mai Red' had the lowest (Menzel and Simpson 1988). In China, 'Fei Zi Xiao' tends to produce long inflorescences with a high percentage of male flowers, whereas short inflorescences have a higher percentage of female flowers (Wang and Qiu 1997). Reducing the size of its inflorescences by pruning one and a half months prior to anthesis increased the female:male flower ratio and fruit set (Wu et al. 2001). Development of the three flower types appears to be influenced by internal as well as environmental factors. Age is one such factor: the first M 1 bloom is usually absent in young lychee plants (Goren et al. 1998). Costes (1988) found great variability in the 'Mauritius' sex ratio among inflorescences on the same tree and among trees growing at the same location. Nutritional as well as hormonal factors are probably involved.

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

413

The temperature regime during flower development strongly affects the sex ratio. When two-year-old plants of five cultivars, with emerging inflorescences (after induction at 15/10°C), were kept at 15/10, 20/15, 25/20 and 30/25°C, the lower the temperature regime the higher the percentage of female flowers. The average percentages of female flowers were: 72,49,27 and 11 percent, respectively. 'Bengal' and 'Souey Tung' did not produce any female flowers at 25/20 and 30/25°C. In contrast, 'Wai Chee' decreased its female flower production only at 30/25°C (Menzel and Simpson 1991). 4. Flowering Phenology. The lychee flowering season occurs in the

spring. In warm tropical climates, flowering occurs in January to February (Das and Choudhury 1958; Subhadrabandhu 1990), whereas in the cool subtropical climates it starts in April to early May (Northern Hemisphere) (Goren et al. 1998). 'San Yue Hong' is the first cultivar to flower; whereas 'Nuo Mi Ci' and 'Huai Zhi' are late bloomers (Wang and Qiu 1997). The typical flowering period of a cultivar extends for about three to five weeks (Chadha and Rajpoot 1969; Menzel 1984; Goren et al. 1998). Khan (1929) presented drawings of the gradual development oflychee flowers. As the flowers do not have petals, or well-developed sepals, there is no distinct time of flower opening, or anthesis. Hence, anthesis should be defined as the stage at which the flower reaches functional maturity: for the female flower it is the start of stigma-lobe spreading (Fig. 8.4A); for the male flowers it is the dehiscence of the first anther. Three distinct flowering waves can usually be observed in every lychee inflorescence; each wave consists of flowers of the same type: the first consists of male 1 (M 1 ), the second of female (F), and the third of male z (M z) flowers (Khan 1929; Mustard et al. 1953; Liu 1954; Costes 1988; Goren et al. 1998). There is hardly any overlap among these waves in the same inflorescence. However, the synchronization is not perfect among inflorescences on the same tree, even less so among inflorescences on different trees of the same cultivar. Thus, in a single cultivar plot, each flowering wave usually lasts for 7 to 12 days, and there are two periods of overlap, lasting 1 to 3 days each, between the female bloom and each of the two male blooms (Fig. 8.5). Such overlapping facilitates close-pollination, even on a single tree. Planting two cultivars with a female bloom that coincides with one of the male blooms of the other cultivar can improve pollination by ensuring pollen availability throughout the female bloom. Several irregularities have been observed in the three-wave flowering pattern. The first M 1 wave is sometimes absent, and flowering starts with

R. STERN AND S. GAZIT

414

2

2

ZZl 22

(MAURITIUS'

•••$&8&& 2 ?ZZ22?-~

15

'FLORI DIAN'

I

I

I

I

I

I

20

25

30

5

10

15

APRIL

MAY

Fig. 8.5. Typical flowering phenology of 'Mauritius' and 'Floridian' in Israel. The peak of flowering is represented by the center of the rhombus; its edges represent the start and termination of flowering. (Degani et al. 1995b)

a wave of female flowers. This is frequently found in young trees, but sometimes also in inflorescences borne on mature trees (Robbertse et al. 1995; Goren et al. 1998). Nakata (1956) reported that this occurs after girdling, or when trees have not undergone a flush for five to six months before flowering. Once, in 1998, all 'Mauritius' trees in a ten-year-old orchard skipped the M 1 bloom (Stern, personal observation). In some cultivars there is a tendency to produce a second sequence of flowering. Both female and male flowers may appear in this second bloom, but usually the M1 bloom is skipped (Mustard et al. 1953). This phenomenon has not been well documented in most cultivars. However, it is well known in 'Fei Zi Xiao' in China (Wang and Qiu 1997) and in Israel (Goren et al. 1998); in both countries this second bloom may result in a better fruit set than the earlier normal bloom. 5. Female Flower Development. An adequate number of fertile female flowers is a prerequisite for high fruit set. The problem of poor lychee productivity prompted several researchers to study the developmental anatomy (megasporogenesis and megagametogenesis) of the lychee ovule (Banerji and Chaudhuri 1944; Liu 1954; Mustard 1960; Joubert 1967, 1985, 1986; Stern et al. 1996b, 1997a). The megaspore mother cell (megasporocyte) can be identified in the nucellus about ten days before the embryo sac reaches maturity. Through meiotic divisions it forms a linear tetrad of haploid megaspores, of which the chalazal one elongates to form the embryo sac. After three mitotic divisions, a mature monosporic eight-nucleate embryo sac of the Polygonum type is formed. At maturity, the antipodals degenerate and cannot be observed after fusion of the two polar nuclei. During embryo sac development the nucellus is absorbed, except for the nucellar epidermis and nucellar cap at the micropylar end. Two integuments envelop the mature embryo sac. On

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

415

the ventral side of the anatropous ovule the outer integument is poorly developed, and in cross section appears only as a protuberance. On the outer integument an obturator is present; it consists of three to four cell layers of enlarged stigmatoid tissue, forming a ring around the micropylar end of the mature ovule. Anatomical sections of the pistil, ovule and embryo sac are presented in Fig. 8.4. During the first five days of anthesis, Mustard (1960) observed a high rate of embryo sac retardation and degeneration in 'Brewster'; about 40 percent of the ovules had a mature embryo sac, but in only about 12 percent of the ovules was it not degenerated. Stern et al. (1996b) determined the anatomical normality of ovules in two-day-old female flowers of two cultivars ('Mauritius' and 'Floridian'), in 11 orchards in Israel. They found that close to half of the ovules lacked an embryo sac, and a great majority of the embryo sacs lacked normal essential elements, particularly egg cells and synergids. The percentage of flowers with a normal ovule fluctuated among the different locations from 3 to 27 (Stern et al. 1996b). These results suggest that two days after anthesis, a large proportion of the lychee ovules are not fully mature. Although, this phenomenon is not common, it has been found in other plants (Sedgley and Griffin 1989). In order to determine whether delayed ovule maturity limits 'Mauritius' lychee productivity and to determine the optimum flower age for pollination, Stern et al. (1997a) studied the postanthesis changes in the stigmatic surface, the anatomy of the ovule, receptivity to pollination and initial set during flower development. They found that at anthesis, most 'Mauritius' ovules were still immature: two-thirds were sterile, that is, they did not have an embryo sac; in 41 percent of the flowers both ovules were sterile. A substantial increase in the presence of embryo sacs occurred and peaked two days after anthesis, with about 76 percent of the flowers having one or two ovules with embryo sacs. Only embryo sacs containing the three normal essential componentspolar nucleus, egg cell and at least one synergid-were considered fertile (Stern et al. 1996b). In most of the embryo sacs, one or more of these essential elements was lacking or, infrequently, degenerated. The presence of a normal egg cell, and even more so of a normal synergid, was the main limiting factor for fertility. On the first day of anthesis, only 6 percent of the flowers were fertile, as compared with 21 percent on the fifth day (Table 8.4) (Stern et al. 1997a.). Late ovule maturation, occurring several days after anthesis, also has been found in apricots (Burgos and Egea 1993; Burgos et al. 1995). Determinations of ovule normality or fertility based on anatomical criteria should be corroborated with functional tests. Stern et al. (1996b) used fruit set after hand-pollination as the functional criterion. A posi-

416

R. STERN AND S. GAZIT

Table 8.4. Percentage of normal flowers according to flower age (1, 3, and 5 days after anthesis) and advance in blooming period (1, 3, and 5 days from the beginning of female bloom). Normal flowers (%) Flower age (days) 1 3 5

Days after bloom started 1

3

5

Mean

3.4 7.1 17.9

3.8 16.7 23.3

10.7 19.2 22.2

6.0 aO 14.3 ab 21.1 b

°Mean separation by X2 test with Bonferroni correction, P = 0.05. Data are the means of -30 flowers per treatment. Source: Stern et al. 1997a.

tive and significant correlation was found between the percentage of flowers with a normal embryo sac (according to the criteria described in the last paragraph) and initial fruit set in the same inflorescences. Such a positive relationship between the rate of normal ovules (or embryo sacs) and fruit set has been found in several other fruit tree crops (Eaton 1959; Stosser and Anvari 1982; Furokawa and Bukovac 1989; Burgos and Egea 1993).

6. Pistil Receptivity. The female reproductive organ (pistil) is receptive when the stigma enables pollen grains to germinate, the style and ovary enable the normal growth of the pollen tubes and the ovule is mature and viable. By definition, anthesis of the female flower starts when the two stigmatic lobes start to separate. The stigma is receptive at this stage and retains full receptivity for several days. The surface of the receptive stigma is covered with long plump papillae and appears shiny white. With age, the stigma curves more and more, and its color gradually, within two to three days, turns white, matte-white, brown-white, and brown. Significant receptivity is retained (pollen germinated on about half of pollinated flowers), even when most of the papillae have collapsed and the color is brown-white. Receptivity is completely lost when all the papillae collapse and the stigma turns brown (Chaturvedi and Saxena 1965; Moncur 1988; McConchie and Batten 1989; Stern et a1. 1997a). When pollen germination or fruit set were used as the criteria for receptivity, it was found that under the cool regimes of 20/17°C or 22/17°C functional receptivity lasted for five days. However, under the hot regime of 33/27°C it was retained for only 36 h. Wetting the

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

417

stigma may induce its premature browning (McConchie and Batten 1989; Stern et al. 1997a). In lychee, ovule maturity tends to lag behind stigma receptivity. The highest percentage of mature normal ovules occurs several days after anthesis, when the stigma's receptivity is declining. Hence, to get the highest fruit set, hand-pollination should not be performed at anthesis, but several days later (Stern et al. 1997a). 7. The Male Reproductive Organs. All three flower types (Fig. 8.3) have stamens that produce pollen. The stamens of the two male flower types has a long and hairy filament with a yellow anther. In the female-type flower, the stamen has a very short filament with a normal-sized anther, which does not dehisce and release pollen. Each anther contains 1900 to 5200 pollen grains. The nondehisced anthers of the 'Mauritius' female flower contain the largest number of pollen grains, 5180 per anther (Costes 1988). Stern (1992) and Costes (1988) found that the Mz anther contains a significantly and consistently larger number of pollen grains than the M 1 anther: 4200 versus 2690 for 'Mauritius' in Reunion; 3300 versus 2000 for 'Mauritius'; and 3000 versus 1900 for 'Floridian' in Israel. In China, 2500 to 3000 pollen grains per anther, in an unspecified male flower, were reported for 'Huai Zhi' (Wang and Qiu 1997). Anthers in the same flower reach maturity and dehisce gradually, over two to three days (Das and Choudhury 1958; Chaturvedi and Saxena 1965); pollen sacs on the same anther may even dehisce at different times during the day (Shalem-Galon 1980). Under the light microscope, the lychee pollen grain is oblong when dry, becoming triangular or sometimes rectangular after swelling in glycerine or aqueous media. The average pollen grain length for ten Indian cultivars was 22 Jim, ranging from 18 to 27 Jim. Under a scanning electron microscope (SEM), 'Mauritius' and 'Floridian' pollen grains from the two male-type flowers have an elongated shape, with three elongated germination pores (Fig. 8.6). The pollen grain is about 10 Jim wide and 20 Jim long. No noticeable differences could be discerned among pollen grains of the two cultivars or of the two male types (Stern 1992). In contrast, Wang and Qiu (1997) reported that the sculpturing patterns on the pollen grain exine differ for different cultivars, and can help in cultivar identification. The released pollen grains are binucleate (Mustard et al. 1953; Liu 1954; Singh 1962; Chaturvedi and Saxena 1965). Liu (1954) identified defective nonfunctional pollen grains; they were small and often shriveled. In 'Hak Ip' ('Hei Ye') their mean size was only 18 Jim, compared to 25 Jim for plump functional pollen grains. She also

418

R. STERN AND S. GAZIT

encountered triangular pollen grains without germination pores. A considerable percentage of semi-quadrangular pollen grains (Fig. 8.6) was found in pollen from orchard trees, especially from M 1 flowers (Stern 1992; Stern and Gazit 1998). B. Pollen Viability. Pollen viability is usually determined by staining or by in vitro pollen germination. Both methods are easy to perform, but only give quantitative values of pollen non-viability, not of pollen functional viability. Stern (1992) compared staining by the Alexander (1969) method with in vitro germination, for 'Mauritius' and 'Floridian' M 1 and M z pollen. For all four pollen he found significantly higher sterility rates with the in vitro method. In vivo tests, performed by placing pollen on receptive stigma, give a good indication of pollen functional viability, but pollen may still germinate well and reach the ovary, but have lost the ability to reach the

Scanning electron micrograph of normal (bottom) and degenerate (top) 'Mauritius' lychee pollen grains (x2400). (Stern and Gazit 1998)

Fig. 8.6.

Plate 2.1. 'Ruby Balls' cactus cultivars developed for grafting by the National Horticultural Research Institute, RDA, Korea.

Seven-year-old 'Brewster' ('Floridian') lychee tree at harvest (Lavi. Israel).

Plate 8.1.

Plate 8.2.

A cluster of 'Mauri-

tius' fruits.

Plate 8.4. Plate 8.3.

Inflorescence.

A mature fruit, with a tiny

undeveloped ovary lobe near the pedicel.

Lychee inflorescence with many under-developed, two-lobed fruitlets and one normal··sized one-lobed fruitlet in the center.

Plate 8.5.

Plate 8.6.

'Mauritius' fruitlets at three stages of development: (A) 4 weeks old = 1 em. (Stern et al. 1995)

5 weeks old (2 g), and (C) 6 weeks old (4 g). Bar

(~1

g), (B)

Plate 8.7.

Young fruitlets, about six weeks old.

Plate 8.8. Longitudinally cut 'Brewster' mature fruits, shovving seed (left) and shriveled seed

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

419

embryo sac. The only reliable test for pollen viability is its ability to reach the embryo sac and perform double fertilization of the egg cell and the polar nuclei. The production of embryo and endosperm attests to the full viability of the male and female reproductive organs. Unfortunately, in some viability tests the source of the pollen, M1 or M z flowers, is ignored. We now know that there may be significant differences between the two (Stern and Gazit 1998).

Staining Tests. Pollen sterility was determined for two consecutive years by aceto-carmine staining. Great and consistent differences in sterility rate were found among 10 lychee cultivars, from 11 percent for 'Late Seedless' to 48 percent for 'Early Seedless,' with high consistency within cultivars (Singh 1962). Using the same method on 'Early Large Red' pollen, 16 percent of the pollen grains were found to be nonviable (Chaturvedi and Saxena 1965). 'Mauritius' and 'Floridian' M 1 and M z pollen were stained by the Alexander (1969) method: the percentage of sterile pollen grains was 79 for the two M 1 pollen and 25 and 17 for the M z pollen, respectively (Stern 1992). In Vitro Pollen Germination. Lychee pollen germinates readily in vitro. Singh (1962) reported the best germination rate and pollen-tube growth at 30°C, on a media of 15 percent sucrose; adding 20 mg L-1 boric acid increased the germination rate and greatly increased pollen-tube growth. He found that lychee pollen even germinates in distilled water, at a rate of about 20 percent. Other researchers found somewhat different optimal conditions. Two agreed that the optimal incubation temperature is 30°C (Shalem-Galon 1980; Stern and Gazit 1998) whereas Costes (1988) found different optima for M1 and M z pollen, 30 and 26°C, respectively. Robbertse et a1. (1992) reported 25°C as the optimal temperature for unspecified 'Mauritius' pollen. The best media have been reported to be: 24 percent sucrose plus 1 percent agar (Mustard et a1. 1953), 20 percent sucrose (Chaturvedi and Saxena 1965), the simplified version of Brewbaker's medium (300 mg L-1 Ca(N0 3 )z and 100 mg L-1 boric acid) (Mulcahy and Mulcahy 1985), amended with 20 percent sucrose (Robbertse et a1. 1992; Fivaz et a1. 1994) and 10 percent (0.3 M) sucrose plus 100 mg L-1 boric acid (ShalemGalon 1980; Xiang et a1. 2001). Using the hanging-drop method, M z pollen was usually found to germinate at a significantly higher rate than M1 pollen (Mustard et a1. 1953; Costes, 1988). Fivaz et a1. (1994) found a small, but significant advantage of M z pollen in four cultivars, but not in 'Madras' ('Bengal'). Stern and Gazit (1998) found a consistent and usually significant advantage of M z over M1 pollen. This advantage was pronounced when pollen from five

420

R. STERN AND S. GAZIT

cultivars was germinated under five different temperature regimes. Best germination rates were: 5 to 12 percent for M 1 pollen and about 55 percent for M z pollen. Pollen-tube growth rate was also faster for Mz than M 1 pollen (after 24 h of incubation at 25°C, 750 and 350 /-lm, respectively). In contrast, Singh (962) found a higher germination rate for 'Calcuttia' M 1 as compared to M z pollen (62 and 48 percent, respectively), when germinating pollen in cavity slides (Singh 1961). He did not find any differences in pollen-tube growth between the two pollen types. This inconsistency indicates the involvement of both environmental and genetic factors.

In Vivo Pollen Germination. This method does not produce quantitative data on pollen grain viability. Pollination is performed with a large quantity of pollen, in the case of lychee, hundreds of pollen grains. Only two pollen tubes have to reach and penetrate the two embryo sacs to fertilize them. Hence, the results are reported as percentage of handpollinated flowers that have pollen tubes reaching the ovary, the ovule or the embryo sac. In such tests, the condition of the female reproductive organs places an upper limit on the pollen's ability to reach its destination and produce a fruit. In the absence of a normal embryo sac, pollen-tube growth will probably be arrested. Stern and Gazit (1998) found the best temperature regimes for in vivo pollen germination and pollen-tube growth to be 22/17 or 27/22°C. After 48 h, pollen-tube reached the ovule in about 35 percent of the flowers, with no significant difference between 'Mauritius' and 'Floridian' M 1 and M z pollen. However, M z pollen was better at the final stage of reaching the embryo sac. Viability of M 1 and M2 Pollen. In almost all studies, M z pollen has been found to be more viable than M 1 pollen. The great difference in viability between the two pollen types is surprising. Genetically they are identical, so the difference must be phenotypic. No differences in the size or shape of pollen grains were discerned (Stern and Gazit 1998). However, 'Mauritius' and 'Floridian' M z pollen are found to be significantly richer than M 1 pollen in protein (8.1 and 6.7 percent, respectively) and sugars (10.9 and 9.9 percent, respectively) (Stern 1992). M z flowers also secrete significantly greater amounts of nectar and sugar than M 1 flowers (Stern and Gazit 1996). Apparently, the M z flower is a stronger sink, and its developing pollen benefits from a better supply of nutrients. 9. Effect of Temperature Regime During Flower Development on Pollen Viability and Ovule Normality. Various factors-genetic, nutritional, and environmental-have been found to be responsible for the abnor-

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

421

mal development or degeneration of reproductive organs (Levit 1980; Fahn 1990). In many plant species, nonoptimal temperature regimes (too low or too high) have been found to adversely affect the reproductive organs (Moss 1969; Kumar 1979; Argaman 1983; Sedgley et al. 1985). However, until the last decade, lychee was not included in these studies. Stern et al. (1996b) and Stern and Gazit (1998) studied the effects oftemperature regimes during flower development on lychee ovule normality and pollen viability. Two-year-old 'Mauritius' and 'Floridian' plants with emerging inflorescences were kept for about two months, until anthesis, under one of three temperature regimes: for the ovule studies at 22/12°C (cool), 27/17°C (warm), or 32/22°C (hot), and for the pollen studies at 22/17°C (normal), 27/22°C (warm), or 32/27°C (hot). The warm and, even more so, the hot temperature regimes had a pronounced detrimental effect on ovule normality (Stern et al. 1996b) and pollen viability (Stern and Gazit 1998). 'Floridian' was much more susceptible to high temperature than 'Mauritius.' After being subjected to the hot regime, 'Floridian' had nonfunctional ovules and sterile pollen, whereas 'Mauritius' had normal ovules and viable pollen. The different effects of the high-temperature regimes on the two cultivars reflect the climate in the region of their successful cultivation: 'Mauritius' ('Da Zao') is cultivated near Guangzhou (23°N), while 'Floridian' ('Chen Zi') is cultivated in Putian (25°N), a colder region. In Guangzhou the maximum and minimum temperatures during flower development (February through March) are about 20/10°C (Groff 1921). Thus, the detrimental effect of much higher temperatures on reproductive organ viability is not surprising. However, it should be kept in mind that apparently tropicallychee cultivars do not have the same susceptibility to warm temperatures. The poor and erratic bearing of 'Brewster' lychee in southern Florida appears to be not only the result of winters that are often not cool enough for flower bud initiation (Young 1970), but also of the detrimental effect of warm weather during flower development on the normal development of the reproductive organs. The fact that Mustard (1960) did not find normal synergids at anthesis probably reflects the detrimental effect of a warm winter on the normal development of 'Brewster' embryo sacs. In Queensland, early season 'Tai So' female and male flowers were infertile, the female flowers were incapable of producing fruit and the male flowers produced no pollen. Female flower infertility was also observed in early flowering 'Bengal' trees (Batten and McConchie 1992). Prolonged low temperatures during flower development may be the factor responsible for this infertility.

422

R. STERN AND S. GAZIT

III. POLLINATION

Since lychee flowers are functionally unisexual, pollination of female flowers can occur only by close- or cross-pollination. During female bloom, close-pollination is affected by pollen from the two partially overlapping male (M 1 and M z) bloom waves on the same tree and crosspollination by pollen from a different cultivar (Fig. 8.5). The lychee flowers are adapted for insect pollination (entomophilous) (McGregor 1976). The flowers produce large quantities of nectar (Fig. 8.7), which attract a large number of insect pollinators. On the other hand, they have some characteristics of wind-pollinated flowers. The flowers are small and inconspicuous and the stigma is relatively large (Fig. 8AA). The pollen tends to become dry and floats away a short time after dehiscence; on windy days it is even blown away as soon as the anthers dehisce. Batten and McConchie (1992) suggested that under windy conditions pollination is abiotic, whereas under calm conditions, bees playa role. A. Abiotic Pollination Lychee pollination by air borne pollen is apparently not fully effective. Only a negligible set occurred on a caged 'Brewster' tree in Florida (Butcher 1956), and in bagged inflorescences (Das and Choudhury 1958; Pandey and Yadava 1970) in India, Israel (Pivovaro 1974), and Australia (King et al. 1989). However, when bagging was accomplished with a 12mesh net (Pivovaro 1974) fruit set occurred, and when mosquito netting (Chaturvedi 1965) was used, initial fruit set was 15.5 percent, about onethird that of open-pollinated inflorescences. In Bihar, India, Phadke and Nairn (1974) found that bagging with voile cloth reduces the number of fruit per inflorescence from nine to two. These studies indicate that abiotic pollination occurs in lychee, but effective pollinator activity appears to be essential for adequate yield. B. Biotic Pollination Lychee flowers have exposed sexual organs (stigma and anther) and their nectar is available to all visitors. Indeed, a large number of insect species has been found visiting lychee flowers in India (Singh and Singh 1954; Pandey and Yadava 1970; Dhaliwal et al. 1977), Florida (Butcher 1956, 1957), Israel (Pivovaro 1974), South Africa (Du Toit 1994), and Australia (King et al. 1989). In general, visitor activity is much higher in the morning than in the afternoon. Pollination is performed by pollinators, which visit the two males and the female flowers, and during the

423

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

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Hour Fig. 8.7. Nectar volume (/-.tl) (A) and percentage of sugar (determined as TSS with a refractometer) (B) per 'Mauritius' lychee flower collected from the three flower types (M 1 , M z and F) during the morning hours (at around 06:00, 07:00, 08:00, 09:00, and 10:00 h). Values are means of two seasons. Nectar was collected on four days each year, 10 flowers per time point for each flower type (standing crop). Results relating to the same hour headed by different letters differ significantly by Duncan's multiple range test, P 0.05. (Stern and Gazit 1996)

visit come in contact with the reproductive organs. Unfortunately, there is only scant information in English about native visitors (pollinators) of lychee in China. Nectar is the main attractant for visitors of lychee flowers because the lychee bloom is such a rich source. Stern and Gazit (1996) found great differences among the three flower types in nectar volume, the percentage of sugars in the nectar, and the amount of sugar per flower. The

R. STERN AND S. GAZIT

424

Table 8.5. Mean amount of nectar sugar per 'Mauritius' flower in each of the three flower types during the morning hours. Nectar sugar (mg) Flower type Time

Male 1

Male z

Female

06:00 07:00 08:00 09:00 10:00 Mean

0.15 c a

0.82 c 0.72 bc 0.50 b 0.27 a 0.18 a 0.50 B

10.5 ab 12.8 b 12.2 b 10.3 ab 7.1 a 10.6 C

0.10 b 0.07 a _b

-

b

0.10 A

°Mean separation in columns (lowercase) and rows (uppercase) by Duncan's multiple range test, P = 0.05. bNo nectar could be collected. Average for two seasons (1989 and 1990). Source: Stern and Gazit 1996.

lowest values were found in M 1 flowers, medium values in M2 flowers and exceedingly high values in female flowers (Fig. 8.7, Table 8.5). A typical 'Mauritius' inflorescence contains over 100 female flowers, which are open at the same time. The tree is densely covered with such inflorescences. Thus, the 'Mauritius' lychee female bloom can be considered an exceedingly rich source of nectar, both in quantity and quality. Nectar production occurs in the morning and stops at noon. Some observers have found that no nectar is secreted in the afternoon (Butcher 1957), whereas others have reported diminished nectar production during this time of the day (Pandey and Yadava 1970). Lychee pollen dehiscence takes place throughout the day and night, mostly from the morning to 15:00 hr. (Das and Choudhury 1958; Chadha and Rajpoot 1969; Wang and Qiu 1997). Chaturvedi and Saxena (1965) observed the peak period of dehiscence (93%) from 08:00 to 10:00 hr. in 'Early Large Red.' Pollen does not serve as a major attractant for most lychee pollinators. Pandey and Yadava (1970) observed that honeybees collect pollen mainly at the beginning and end of the flowering period, probably because that is the only time male flowers from the first and second male blooms are present. In one commercial orchard in Israel, no bees with pollen loads were observed on the lychee bloom. Apparently, the bees preferred to collect pollen from nearby fields of wild mustard (Stern 2000 and 2001, personal observations).

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

425

C. Pollinators 1. Flies. Flies, especially blowflies (Calliphoridae) and hoverflies (Syr-

phidae), have been found on lychee flowers in Florida (Butcher 1957), India (Dhaliwal et al. 1977), Israel (Pivovaro 1974), and Australia (King et al. 1989). In Florida, the secondary screwworm fly (Callitroga macellaria) was found to be the most abundant and consistent visitor. 2. Bees. Bees are the most important pollinators of lychee (Groff 1943; Butcher 1956,1957; McGregor 1976; Free 1993). In China, lychee is recognized as a plant with abundant nectar, a source of high-quality honey. These adequate pollination by honeybees increased lychee yields by 2.5 to 2.9 times that of orchards not pollinated by honeybees (Chen 1993). However, only some of the several Apis honeybee species present in China are involved with lychee pollination. There is good reason to conclude that the Chinese honeybee (Apis cerana cerana), which is endemic to the lychee region of origin, is the main native pollinator of lychee. This species was domesticated about 2000 years ago and is grown on a large scale for honey production. The European honeybee (Apis melliferal was introduced to South China only about a century ago (Crane 1990; Chen 1993). We assume that these two honeybee species now serve as the main lychee pollinators in China. In India, the three local honeybee species (Apis cerana indica, A. florae, and A. dorsata) and unidentified stingless bee species (Melipona spp.) comprised 98 to 99 percent of the insects visiting lychee blooms, in two localities in Uttar Pradesh (Pandey and Yadava 1970). The stingless bees were the most active, their small size allowing them to reach the base of the flowers to become heavily dusted with pollen. Hives of the domesticated Indian honeybee (A. cerana indica) are moved to lychee orchards, because the lychee is an excellent source of goodquality honey. In the Indian Himalayas (30.5°N, 545 m altitude), the most abundant visitor was the small A. florae (50%), followed by A. cerana indica (26%). A. florae appears to be the most important pollinator there; fruit set was 25-fold higher when all insects had free access to the flowers, and 21-fold higher when large insects were excluded and only A. florae and other small insects were able to visit the flowers (Dhaliwal et al. 1977). In Pusa, Bihar, two Apis species comprised 73 percent of all visitors on lychee bloom: A. florae was again the most abundant (58%), followed by A. cerana indica (16%) (Phadke and Nairn 1974). In Queensland, a stingless bee (Trigona sp.) was the main forager in one lychee orchard (King et al. 1989). However, because of its small size, on most occasions it did not touch the stigma while visiting lychee female flowers.

R. STERN AND S. GAZIT

426

3, The European Honeybee, In many lychee-growing regions (Florida, South Africa, Israel, and Australia), the European honeybee is the most important pollinator in commercial orchards. It is strongly attracted to the lychee bloom and its hives can readily be moved to orchards in bloom. It seems to be an effective lychee pollinator, as the bee tends to touch the stigma on almost every visit to the female flower (King et al. 1989). Stern and Gazit (1996) studied the pollination rate in lychee, and the attractiveness of the three flower types to the honeybee. A significant positive correlation was found between bee density on the F, M I , and M 2 blooms and sugar percentage in the nectar (Fig. 8.8). On the other hand, a significant negative correlation was found between bee density and nectar volume per M 2 and F flowers (Stern and Gazit 1996). The level of sugar in the nectar appears to be the dominant factor attracting bees to lychee flowers. When a bee can easily fill its stomach with nectar from a few flowers on one inflorescence (Free 1993), the higher the sugar level, the higher the net energy yield per flight (Heinrich 1983). The greater attractiveness of the female bloom (Fig. 8.8) is not accidental; it reflects the fact that fruit can only set at that time. The abundance of bees in the lychee orchard during the female bloom probably has a positive effect on bee visitation to M I and M 2 blooms. The M 2 bloom was found to be much more attractive to bees than the M I bloom, apparently because it is a much richer source of nectar and sugars

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8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

427

(Fig. 8.7, Table 8.5). The rather low attractiveness of 'Mauritius' M 1 bloom to the honeybee is surprising and is difficult to explain. If this phenomenon is found to be a universal feature in lychee, it may indicate that its native pollinator (A. cerana cerana) is more appreciative of the M 1 bloom than the European honeybee. The concentrations of glucose, fructose, and sucrose in the nectar were similar in the F, M 1 , and M z flower types, 43,39, and 18 percent, respectively (Stern and Gazit 1996). This uniformity is typical for different flower types in the same plant species (Barker and Lehner 1974). The great attractiveness of the lychee bloom for the honeybee is in line with Baker and Baker's (1983) conclusion that honeybees prefer lowsucrose nectar. The amount of pollen found on bees sampled from F and M z inflorescences was higher by two orders of magnitude than that found on bees sampled during the M 1 bloom (Fig. 8.9). This large difference reflects the

15

17

19

21

April

23

25

27

29

May

Fig. 8.9. Estimated amount of pollen found (by SEM observation) on bees caught on 'Mauritius' inflorescences at the three flowering stages (M 1 , F, and M z). During the flowering of two seasons, bees were collected every other day from inflorescences at the three flowering stages, at around 07:00, 10:00, and 17:00 h; 10 bees (two bees x five trees) were collected at each time point from each inflorescence type. Values are the daily means for two years. Results are given in terms of a logarithmic scale of 0 to 3: 0 no pollen; 1 = up to 10 pollen grains; 2 = tens of pollen grains; 3 = hundreds of pollen grains. (Stern and Gazit 1996)

428

R. STERN AND S. GAZIT

pronounced differences in the quantity and quality of the nectar present inM1 and M z flowers. Bees visiting an inflorescence during its M1 bloom will be "disappointed" with the meager yield and abandon it, whereas bees visiting an inflorescence during its M z bloom will visit a large number of flowers and get dusted with abundant pollen (Stern and Gazit 1996). D. Pollination Rate

In Israel, during the first days of 'Mauritius' female bloom, pollination rates were very low (0 to 10%), in spite of the high density of honeybees on female flowers. The low density of honeybees on M 1 flowers resulted in little lychee pollen on the bees (less than 10 pollen grains per bee) (Fig. 8.9). On the day the M z bloom started, pollination rates jumped to 20 or 30 percent, reaching 80 to 90 percent a few days later. The greater attractiveness of M z flowers resulted in large amounts of pollen on the bees (hundreds of pollen grains per bee). In parallel, the number of germinating pollen grains per pollinated stigma increased from 1 or 2 to about 20 (Stern and Gazit 1996). McConchie and Batten (1991) provided some experimental information on the relationship between fruit set and pollination rate. They found that hand pollination of all female flowers on a 'Bengal' inflorescence (225 flowers) produced only nine fruit per inflorescence, whereas pollination of only 47 to 48 flowers per inflorescence (and removal of all other female flowers) yielded 13 and 23 fruit per inflorescence, for early and late-opening flowers, respectively. The detrimental effect of full pollination is surprising; it should be confirmed for 'Bengal,' and determined for other cultivars, to find out if this is a universal phenomenon in lychee or only an accidental occurrence. The significantly greater fruit set of the late-opening flowers may reflect a higher fertility rate, but may also result from the use of the potent M z pollen, versus the M 1 pollen, which is available at the end of the female bloom (Fig. 8.5). IV. THE FERTILIZATION PROCESS AND INITIAL FRUIT SET

A. The Fertilization Process 1. Pollen Germination and Pollen-Tube Growth. Lychee pollen grains

deposited on a receptive stigma germinate readily at a wide range oftemperatures, from 17 to 33°C. At 12°C 'Mauritius' and 'Floridian' pollen did not germinate at all. At 15°C, some germination and pollen-tube growth occurred (Stern 1992). Stern and Gazit (1998) found that at 17/12, 22/17 and 27/22°C, pollen tubes reached the ovaries of all pollinated flowers

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

429

in 48 h; however the ovule was penetrated in about 35 percent of the flowers only at the two warmer temperature regimes. Under the hot regime of 32/27°C, pollen tubes reached the ovary in only 20 percent of the flowers, and the ovule was not reached at all. In contrast, McConchie et al. (1991) found for 'Tai So' ('Mauritius') that at 33/27°C, pollen tubes reach the ovary in 24 h, while at 20/17°C, it takes three days. Taking into consideration the percentage of flowers with pollen tubes at the ovary they concluded that the optimal regimes for pollen-tube growth are 30/23 and 25/18°C. A week after pollination they found the highest number of ovules with a pollen-tube at 22°C. In addition, they stated that at a constant temperature above 30°C all female flowers drop off within three days of pollination. Short exposure during the fertilization process (6 hr after pollination) to the high temperature of 35°C irreversibly arrested pollen-tube growth, whereas short exposure to the low temperature of 5°C caused only a temporary delay and growth later resumed at optimal temperature (Stern 1992). Indeed, in Israel, hot spells of 35 to 40°C during female bloom have a devastating effect on lychee fruit set. Batten and McConchie (1992), on the other hand, did not find significant fruit-set diminution in flowers pollinated in the orchard at a maximum temperature of 35°C. The difference between the findings in Israel and Australia probably reflects a difference in the severity of the hot spells. 2. Double Fertilization and Initial Fruit Set. Robbertse et al. (1992) reported that the pollen tube grows along the surface of the obturator papillae to reach the entrance to the embryo sac (the micropyle). However, there is no report on the manner by which the lychee pollen tube enters the embryo sac and performs the double fertilization. The fertilization occurs two to three days after pollination (McConchie and Batten 1989; Wang and Qiu 1997). Mustard (1960) observed a zygote and nuclear endosperm as early as three days after anthesis. The first division of the diploid zygote occurs seven to ten days after pollination (Joubert 1967,1986; Wang and Qiu 1997).

B. Factors Affecting Initial Fruit Set

Successful pollination and fertilization are prerequisites for the set of a normal lychee fruit. Nonpollinated flowers of two cultivars abscised within four weeks, whereas about 10 percent of the pollinated flowers had survived at the end of this period (Stern 1992). The main factors affecting initial fruit set are: the presence of fertile female flowers; adequate pollination with effective pollen; environmental conditions favorable to pollination; the fertilization process, and fruit development.

430

R. STERN AND S. GAZIT

1. Female Flower Fertility. The presence of flowers with at least one normal ovule is a prerequisite for normal fruit set. Though lychee usually produces a large number of female flowers, most of them tend to be defective. The percentage of two-day-old normal fertile flowers in 11 orchards fluctuated between 3 and 27. After hand-pollination, the initial set of these fertile flowers was about 60 percent (Stern et al. 1996b). Usually, the final fruit set is about half of the initial set (Stern et al. 1995). The significant correlation found between the percentage of 'Mauritius' fertile female flowers and the initial and final fruit set (Stern et al. 1996b) demonstrates that under optimal pollination conditions, the percentage of fertile flowers may be the major limiting factor for a good crop in this productive cultivar. In other cultivars, shortage of fertile female flowers may be even more crucial (Mustard 1960). The fertility of 'Mauritius' female flowers was found to improve with age; initial fruit set increased from 4.0 to 13.6 percent, when pollinated on the first or fifth day after anthesis, respectively (Table 8.6). This reflected the significant increase in the percentage of anatomically normal female flowers (Stern et al. 1997a). Hence, hand-pollination for controlled progeny production, or experimental purposes, should be performed on three-to-five-day old flowers.

2. The Effect of Putrescine. Mitra and Sanyal (1990) found that spraying inflorescences of 'Bombay' twice, "before anthesis and into open flowers" with the polyamine putrescine (1,4-diaminobutane dihydrochloride) at 8 mg L-1 significantly increases initial and final fruit set, but decreased fruit weight. Stern and Gazit (2000a) found that spraying 'Mauritius' trees at the start of female bloom with 8 to 80 mg L-1 putrescine consistently increases yield, with no discernible effect on Table 8.6. Percentage of initial fruit set five weeks after hand-pollination of flowers at three ages (1, 3, and 5 days after anthesis) in three blooming periods (1, 3, and 5 days from the beginning of female bloom). Fruit set (%) Flower age (days) 1 3 5

Days after bloom started 1

3

5

Mean

3.2 6.3 12.2

3.4 7.8 14.2

5.4 11.8 14.5

4.0 aO 8.6 b 13.6 c

°Mean separation in columns by Tukey's test with Bonferroni correction, P = 0.05. Data are the means of 100 pollinated flowers per treatment. Source: Stern et al. 1997a.

431

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

fruit weight. In two commercial-scale trials, spraying with 40 mg L-l putrescine increased yield from 4 to 8 t ha-1 , and from 7 to 11 t ha-1 , with no effect on fruit weight. The consistent positive effect of putrescine calls for further trials to determine its optimal concentration for 'Mauritius' and other lychee cultivars, and the effects of other polyamines. Polyamines are known to inhibit ethylene biosynthesis (Apelbaum et al. 1981), and in this way may delay pistil senescence (Faust and Wang 1992). Stern and Gazit (2000a) suggested that the positive effect of putrescine is effected via the prolongation of pistil receptivity. The lychee pistil retains its receptivity for two to five days (Batten and McConchie 1992; Stern et al. 1997a), before reaching peak ovule maturity (Mustard 1960; Stern et al. 1997a). Prolonging pistil receptivity increases the chances of pollen grains reaching a viable embryo sac. In addition, the mature 'Mauritius' female bloom exhibits better overlap with its potent M2 bloom (Fig. 8.5). 3. M1 and M2 Pollen Fertility and Effective Pollination. Pollination with fertile pollen is essential for fruit set. Only a few studies have reported on problems related to pollination in lychee. At the start of the 'Mauritius' female bloom in Israel, pollination rate is very low (Stern and Gazit 1996). Similarily, male flowers produce no pollen in the early 'Tai So' bloom in Queensland (Batten and McConchie 1992). In both cases, the problem occurs in the M1 flowers. Stern and Gazit (1996) found that M 1 pollen is less fertile than M 2 pollen. For two consecutive years, 'Mauritius' and 'Floridian' M 1 and M 2 pollen were used to self- and cross-pollinate female flowers of both cultivars. Pollination with M 2 pollen resulted in a significantly higher final fruit set for both (Table 8.7). The greater fertility of M 2 pollen, coupled Table 8.7. Fruit set percentage after hand-pollination with M1 and Mz pollen from 'Mauritius' and 'Floridian' lychee. Fruit set (%)

Pollen source Cultivar Mauritius Floridian Mean

Type

Mauritius

Floridian

0.9 aa 3.5 b 1.4 a 4.0 b 2.5 Ba

1.9b 0.5 a 1.8 b

1.0 a

1.3 A

aMean separation in columns (lowercase) and rows (uppercase) by Duncan's multiple range test, P = 0.05. Source: Adapted from Stern and Gazit 1998.

432

R. STERN AND S. GAZIT

with the greater attractiveness of M z flowers to the honeybee, make the M z bloom more effective for pollination than the M 1 bloom. Thus, when a pollenizer is needed to increase the productivity of a certain lychee cultivar, we should select a pollenizer whose M z bloom overlaps with the female bloom of that cultivar. Unfortunately, the female bloom of the first cuItivar to bloom is only exposed at the end to its M z bloom (Fig. S.S). The great attractiveness of the lychee bloom to pollinators usually ensures adequate pollination. Indeed, Batten and McConchie (1992) concluded that the pollen transfer process does not limit fruit set, and that the importance of bees has been overstated. 4. The Effect of Self- and Cross-Pollination. The lychee flowering pat-

tern tends to promote cross-pollination. However, the partial overlap between the female bloom and the two male (M 1 and M z) blooms (Fig. S.S) enables close-pollination among flowers on the same tree, or among trees of the same cultivar, thereby providing an opportunity for selffertilization (Joubert 19S6; Stern and Gazit 1996). Lychee is considered to be self-fertile, since monocuItivar lychee plots are capable of bearing good yields, as found for 'Mauritius' in South Africa, 'Brewster' in Florida, 'Hei Ye' in Taiwan and 'Huai Zhi' in China (Campbell and Malo 1965; Joubert 19S6; McConchie and Batten 19S9; Batten and McConchie 1992). Hand-pollination studies in Israel (Stern and Gazit 1995), South Africa (Fivaz and Robbertse 1995), and Australia (Batten and McConchie 1992) confirmed the self-compatibility of several lychee cuItivars. In most cases, the use of self- or foreign pollen had no significant effect on fruit set (Table S.7). However, some pollen donors increased fruit set in certain cuItivars in Australia. Determination of the pollen parent's identity by isozyme analysis again confirmed the self-fertility of 'Mauritius' and 'Floridian' in commercial orchards in Israel (Stern et al. 1993b; Degani et al. 1995b). However, it also demonstrated the importance of cross-pollination in lychee. Almost all fruit on trees close to the second cultivar were hybrids. In addition, there was a pronounced selective abscission in favor of hybrid fruit over seIfed ones. About a month after fruit set, the hybrid rate in a 'Mauritius' plot was 30 percent, increasing to 76 percent at harvest. The survival advantage of the hybrid fruitlet may derive from the fact that due to inbreeding depression selfed progeny often have a less vigorous embryos than outcrossed ones (Sedgeley and Griffin 19S9). 'Mauritius' yield was not correlated with either hybrid percentage or proximity to 'Floridian' trees, indicating that cross-pollination by

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

433

'Floridian' holds no advantage over self-pollination. In contrast, 'Floridian' yield was higher close to 'Mauritius' and a significant positive relationship was observed between its yield and hybrid percentage or proximity to 'Mauritius,' indicating a beneficial effect of cross-pollination by 'Mauritius' (Degani et al. 1995b). V. FRUIT DEVELOPMENT AND ABSCISSION

The mature lychee fruit is a tuberculate berry, oval to ovoid in shape, about 3 to 3.S em in diameter and 3 to 4 em long. The attractive leathery peel (pericarp) is yellow-red to red. A few days after harvest it turns brown and brittle. The juicy flesh (aril) is white and translucent, containing a single glossy dark brown seed. In most cultivars the seed is usually large; only a small percentage may be small or shrunken. In some cultivars most or all of the seeds are degenerate (chicken tongue) (Joubert 1986; Galan-Sauco 1989; Tindall 1994). A. Fruit Growth and Development 1. Morphology. The lychee ovary is bi-Iobed, with one ovule in each

lobe. The two lobes stand at almost right angles to the pedicel and style (Fig. 8.4A). Usually, only one lobe develops into a fruit, while the second lobe remains attached, eventually reduced to a tiny appendix (Plate 8.4). The developing ovule gradually straightens up, until at two to three weeks it forms a straight line with the pedicel; at this stage the style is found at the bottom of the fruit, near the pedicel (Khan 1929). When neither of the two ovules has been successfully fertilized, the two lobes may still enlarge considerably and persist up to four weeks. Inflorescences heavy with two-lobed fruitlets may be a common sight two or three weeks after female anthesis, but these fruitlets will not develop into normal fruits (Plate 8.S). In rare cases, the two ovary lobes develop into twin full-size fruits, which are borne on one pedicel (Khan 1929; Joubert 1967, 1986). The rarity of twin fruits stems from the rare occurrence of two normal ovules in the same flower; for example, only one (0.2%) out of 49S 'Mauritius' and 'Floridian' flowers examined in Israel had two normal ovules (Stern et al. 1996b). 2. Anatomy. The mature fruit is composed of three components: peel (pericarp), flesh (aril) and seed. The anatomical development of the 'Tai So' ('Mauritius,' 'Hong Huay') fruit was described by Joubert (1967, 1986) and Chaitrakulsub et al. (1988).

434

R. STERN AND S. GAZIT

The Peel (Pericarp). About two weeks after fertilization, the ovary wall differentiates into three layers: exocarp (epicarp), mesocarp, and endocarp. Cell divisions continue for about 70 days and then only cell enlargement takes place. The exocarp consists mainly of external protuberances supported by two to three layers of sclereids. The mesocarp consists of parenchymatous cells and vascular bundles. The endocarp consists of two to three layers of small cells on the inner surface of the pericarp. These three layers of the pericarp make up the leathery peel of the fruit. The pressure exerted by the developing aril causes elongation of the pericarp cells to form the thin pericarp of the mature fruit. Peel thickness reaches a maximum value of 3 mm in the fourth week, and then decreases gradually to 0.1 mm in the ninth week. Its weight increases consistently up to the eighth week, and does not change appreciably thereafter. The Aril. The aril develops from the funiculus or the nearby outer integument, just above the obturator (Huang and Qiu 1987; Robbertse et al. 1993; Huang 2001). The assumption that the obturator gives rise to the aril (Banerji and Chaudhuri 1944; Joubert 1986) appears to be erroneous. The aril appears at the beginning as a ring of white tissue around the seed base. Its initial growth is slow; rapid growth starts simultaneously with cotyledon development and continues to fruit maturity. The aril eventually produces a thick fleshy layer, which envelops the seed. The mature aril consists of large, irregular, thin-walled cells. The Seed. The endosperm is the first tissue to divide after fertilization. The young nuclear endosperm is a liquid. It changes into a gelatinous cellular tissue at about three to four weeks. It is absorbed by the developing embryo and disappears with embryo maturation. It persists longer when the developing embryo degenerates. At three weeks the embryo is globular; with the appearance of the cotyledon initials at four weeks, it is at the microscopic "heart" stage. For the first month the tiny embryo is embedded at the micropylar end and cannot be observed with the naked eye. By the fifth week its growing cotyledons emerge and it can be easily observed. Rapid embryo growth starts at this stage; in about two weeks it reaches its final size, filling the seed cavity (Fig. 8.10, Plate 8.6). The cotyledons develop into the seed coat (testa). In the first month, the elongated light green seed coat comprises the main component of the seed. With embryo maturation, the leathery seed coat hardens and changes color to dark brown. 3. Phases in Fruit Development. The development of the 'Mauritius' fruit was studied by Joubert (1967, 1986) in South Africa and by Stern et al. (1995) in Israel (Fig. 8.10). Three distinct phases were distinguished:

435

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

24

---Fruit -o-Aril

22

-atr- Pericarp

20

I -h:-Seed

'S

-; 18 Q)

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~

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.:i ~

0

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0)

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16 14 12 10 8

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>

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4

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o

2

4

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8

10

12

14

Weeks after full female bloom Fig. 8.10. Cumulative growth of 'Mauritius' lychee fruit, seed, pericarp and aril. (Stern et al. 1995)

First Phase. This phase lasts for about five weeks in Israel and seven to eight weeks in South Africa. At the end of this phase, fruit weight is about 2 g, and is composed mainly of peel (pericarp) (2/3) and seed (1/3). The aril is starting to grow from the base of the seed. The tiny embryo is at the "heart" stage, protruding from the seed coat and it can be seen with the naked eye (Plate 8.6). The remnant of the endosperm may still be present in the seed cavity. Second Phase. This phase lasts for about two weeks in Israel and two to three weeks in South Africa. During this period there is rapid growth of the embryo and the endosperm disappears completely. At the end of this phase, the embryo fills the seed cavity with two well-developed cotyledons, the seed coat has hardened and the aril has started its rapid growth (Plates 8.6 and 8.7). Fruit weight is about 7 g, and is composed of peel (1/3), seed (1/3) and aril (1/3).

436

R. STERN AND S. GAZIT

Third Phase. The last phase lasts for about six weeks in Israel and five to six weeks in South Africa. At the end of this phase, the fruit is ripe, ready for harvest. Fruit weight in Israel is 23 to 24 g, and is composed of peel (1/10), seed (1/10), and edible flesh (aril) (8/10). 4. Fruit Growth Rate. Several studies have determined the growth rate of the fruit and its component tissues (Joubert 1967, 1986; Huang and Xu 1983; Paull et al. 1984; Chaitrakulsub et al. 1988; Stern et al. 1995). The cumulative fresh and dry weights of the whole fruit and its components (pericarp, aril, and seed) follow a sigmoidal growth pattern (Fig. 8.10). The pericarp and the seed reach their maximal fresh weight at about seven weeks, whereas the aril reaches its maximal fresh weight at maturity.

B. Pollen Parent Effect on Seed and Fruit Characteristics

The pollen parent supplies one-half of the seed and one-third of the endosperm genome. These tissues comprise the main portion of the seed; hence, the pollen parent may exert a significant xenic effect on seed characteristics. This phenomenon is well-known in nut crops, where the seed constitutes the harvested product (Sedgley and Griffin 1989). However, it apparently occurs in all crops, but is seldom studied. The embryo and endosperm may exert a significant influence on the fruit maternal tissues (metaxenia). This phenomenon was first found in dates (Nixon 1935; Reuveni 1986), but it is potentially widespread and may be discernible in many other fruit crops. In lychee, several studies have determined the effect of the pollen parent on seed and fruit weights and on the incidence of shriveled seeds with aborted embryos. The identity of the pollen parent was ascertained by either hand-pollination (McConchie et al. 1991; Xiang et al. 2001) or isozyme analysis of the fruit embryo (Stern et al. 1993b; Degani et al. 1995b). From plots with only two cultivars ('Mauritius' and 'Floridian'), selfed and outcrossed embryos were identified by isozyme analysis. Outcrossed fruit were heavier and contained heavier seeds than selfed fruit. This effect was most pronounced for seed weights in selfed 'Floridian' fruit. Discounting the seeds (the xenic effect) still left a clear metaxenic effect, pericarp and aril from outcrossed fruit being heavier than from selfed ones (Stern et al. 1993b; Degani et al. 1995b). Hand-pollination in 'Bengal' with self- and four pollenizer pollens resulted in noticeable differences in fruit weight among the five progeny: one was heavier and two were lighter than the selfed progeny (McConchie et al. 1991), indicating that foreign pollen is not always more effective than self-pollen.

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

437

Lychee fruit may contain a shriveled (chicken tongue) seed, which does not contain a live embryo. Such seeds weigh about 0.5 g, compared to the normal 2 to 4 g (Plate 8.8). Their presence is a great advantage to the consumer because of the higher ratio of flesh to seed. There are great differences among cultivars in the incidence of these desirable fruit. In most, it is quite rare. Only a few (e.g., 'Nuo Mi Ci,' 'Gui Wei,' 'Salathiel') usually have almost only shriveled seeds. Their percentage may change appreciably from year to year and according to locality. Stern et al. (1993b) found a significant positive correlation between the percentage of 'Floridian' fruit with shriveled seeds and the distance to the pollenizer ('Mauritius'). They suggested that self-fertilization in 'Floridian' tends to increase the production of shriveled seeds, probably as a result of inbreeding depression which causes embryo degeneration and abortion. Xiang et al. (2001) hand-pollinated two outstanding cultivars, which usually have a high percentage of shriveled seeds ('Nuo Mi Ci' and 'Gui Wei'), with pollen from several cultivars. They found great differences between the effect of the different pollenizers and the responses of the two pollinated cultivars. 'Nuo Mi Ci' carried a very high percentage (80 to 92%) of fruit with shriveled seeds after pollination with seven pollenizers; however, the use of 'Da Zao' ('Mauritius') pollen resulted in a low percentage (8%) of shriveled-seed fruit. The response in 'Gui Wei' was much more variable: it produced 5, 18, 25, 30,47,65, and 75 percent shriveled-seed fruit after pollination by the seven pollenizers. The lowest percentage was again produced by 'Da Zao' pollen. In Australia, 'Salathiel' ('Southern Cross') produced only shriveled-seed fruit after pollination by five pollenizers (McConchie et al. 1991).

c.

Fruit Abscission

1. Abscission Rate and Pattern. The lychee tree produces a large excess of female flowers. We estimate that a medium-size orchard tree (planted at a spacing of 6 x 6 m) carries about 60,000 female flowers (400 inflorescences, each with about 150 female flowers) (Stern et al. 1993a). At a high yield of 50 kg/tree (14 t ha-1 ), the tree carries about 2,500 mature fruit (20 g each) at harvest. Hence, at best the final fruit set will be only about 4 percent. This simple calculation demonstrates the inevitable massive flower and fruit abscission. Indeed, there are many reports and commentaries dealing with this phenomenon (Mustard et al. 1953; Mustard 1960; Prasad and Jauhari 1963; Chadha and Rajpoot 1969; Hoda et al. 1973; Misra et al. 1973; Pivovaro 1974; Veera and Das 1974; Singh and La11980; Yuan and Huang 1988; McConchie and Batten 1989; Stern et al. 1995, 1997c; Stern and Gazit 1997,1999, 2000b).

R. STERN AND S. GAZIT

438

Most of the massive abscission of flowers and fruitlets occurs during the first month after pollination (Mustard et al. 1953; Joubert 1986; Stern et al. 1995, 1997c; Stern and Gazit 1999). In Israel, by the fifth week after pollination, about 90, 96, and 99 percent of all female flowers abscised in 'Mauritius,' 'Floridian,' and 'Kaimana,' respectively (Fig. 8.11). Apparently, most of the abscised flowers are not fertilized, due to inadequate pollination, nonviable pollen, problems in the fertilization process and defective ovules (Mustard et al. 1953; Mustard 1960; Joubert 1986; McConchie and Batten 1989; Stern and Gazit 1996, 1998). It is difficult to determine the effect of each of these factors, but in Israel, the last factor bears the major responsibility: most of the female flowers are abnormal, in that they lack an embryo sac, egg apparatus, and/or polar nuclei (Stern et al. 1996b, 1997a).

100 . - - - - - - - - - - - - - - . . ,

C)

c:

'S:

.~

::s

t/)

::

::s 10

.....s-

..... 0 ..... c: a>

Mauritius

(,)

s-

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Q.

Floridian

--.__--I~I--__...

b

Kaimana

o

2

4

6

8

10

12

14

Weeks after full female bloom Fig. 8.11. A typical fruit-drop pattern in 'Kaimana,' 'Mauritius,' and 'Floridian' lychee. Each value is the average of 12 inflorescence from uniform trees. Mean separation at harvest by Duncan's multiple range test, P = 0.05. (Stern and Gazit 1999)

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

439

Two distinct abscission periods, with minor variations, were found in 'Mauritius' (Fig. 8.11) (Stern et al. 1995). The first period lasts for about a month, at the end of which 5 to 10 percent of the female flowers survive to develop into small fruitlets. The second abscission wave begins one or two weeks later, lasting for about two weeks. Fruitlets that abscise at this stage weigh 2 to 6 g, and contain a well-developed seed coat. Rapid embryo growth consistently coincided with the second abscission period (Fig. 8.12). The abscission subsides when the embryo has reached full size. During the second period, about half the remaining fruitlets abscise. Hence, only 2 to 5 percent of the initial female flowers develop into mature fruit. Fruit-drop intensity varies according to cultivar, environmental conditions, and cultural practices. It is not rare to find that all fruit of a given panicle have abscised before harvest. In Israel, 'Kaimana' tends to shed almost all its fruitlets in the third and fourth week after fruit set (Fig. 8.11). Initial set by the fourth week was only 4 percent, two weeks later reaching the low final set of about 1 percent. No distinct abscission waves could be discerned (Stern and Gazit 1999). 'Floridian,' on the other hand, exhibits two abscission waves like 'Mauritius'; however, this cultivar tends to retain more fruitlets at the end of the first wave (10 to 20%), but to drop most of them during the second wave, with a final set of 1 to 3 percent (Stern 1992; Stern et al. 1997c). 100

-..-.------------.-3

~

ii .~

c:

... ::::s

II)

10

Cl)

E

2 u. II

o

2

4

6

8

10

12

Weeks after full female bloom Fig. 8.12. A typical patterns of 'Mauritius' lychee fruitlet abscission and seed growth in Israel during 1988. Initial numbers offemale flowers per inflorescence was about 160. Each value is an average of 12 replicates. (Stern et al. 1995)

440

R. STERN AND S. GAZIT

In China, abscission patterns were studied in the normal seeded 'Huai Zhi' and the aborted-seeded 'Nuo Mi Ci' (Yuan and Huang 1988; Wang and Qiu 1997; Huang 2001). They usually found multiple waves, up to three in 'Huai Zhi' and up to five in 'Nuo Mi Ci.' However, there were significant differences among years. In both cultivars, most of the abscission occurred during the first month, in one or two waves. In the seeded cultivar an additional wave occurred during the sixth and seventh weeks and then stopped (similar to the results with seeded cultivars in Israel). In contrast, the aborted seed cultivar had an additional pronounced wave much later, in the ninth week (close to harvest). 2. Abscission Reduction with Plant Growth Regulators. In a large number of fruit crops, including citrus, apples, pears, and peaches, fruitlet abscission can be reduced or prevented by auxin application (Leopold 1958; Weaver 1972; Arteca 1996). Fruit abscission is facilitated by the action of the hydrolytic enzymes polygalacturonase and cellulase, which cause degradation of the cell wall and middle lamella in the abscission zone. Ethylene promotes their synthesis and activity, whereas auxin delays these processes (Goren 1993). Liu (1986) found that the endogenous indolacetic acid (IAA) content in lychee fruitlets rises steeply during the first three weeks of fruit development, from 150 to 850 ~g g-1 FW, but decreases after the onset of rapid embryo development, four to five weeks after fertilization, falling to 300 ~g g-1. Many studies on the effect of various growth regulators on lychee initial and final fruit set were reported from India, especially in the 1970s. These started with Prasad and ]auhari's (1963) report on great increases in fruitlet retention and yield on panicles sprayed with naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 35 to 100 mg L-1, with a concurrent increase in fruit size. The following studies were also conducted, on a rather small scale, at the panicle level, or on tree parts. A large assortment of growth regulators was sprayed in addition to NAA and 2,4,5-T. Most were reported to reduce fruit drop, sometimes significantly. Khan et al. (1976) stated that NAA at 20 mg L-1, 2,4,5,-T at 10 mg L-1, gibberellic acid (GA 3 ) at 100 mg L-1 and 2 cloroethyl trimethyl ammonium chloride (CCC) at 250 mg L-1, check fruit drop significantly. Hoda et al. (1973) reported that NAA at 10 mg L-1 and 2,4-dichlorophenoxyacetic acid (2,4-D) at 15 mg L-1, especially in combination with a prior spray of zinc sulfate at 1 percent, have a significant positive effect on fruit retention. Singh and Lal (1980) concluded that GA 3 at 50 mg L-1 increases fruit retention. A number of other studies also produced positive results (Veera and Das 1974; Verma et al. 1980; Yuan and Huang 1991), whereas others failed to reduce

8. THE REPRODUCTIVE BIOLOGY OF THE LYCHEE

441

abscission (Misra et al. 1973; Singh and Dhillon 1981). However, the successful reports were not followed up with larger-scale studies at the tree and orchard levels, in order to arrive at a reliable method that could be recommended for use. The fact that the use of growth regulators for the reduction of fruitlet drop is not recommended in India (Pandey and Sharma 1989) reflects this situation. Several studies conducted in Israel did not verify the ability of NAA, 2,4,5-T or GA 3 to reduce lychee fruit drop (Pivovaro 1974; Shalem-Galon 1980; Stern et al. 1995). The contradiction with the positive reports from India and China may be the result of genetic differences between cultivars, environmental conditions, or the timing of the synthetic auxin application. It is also possible that differences in the formulation of the sprayed substances affected their efficacy. In Israel, Pivovaro (1974) found that the auxin 2,4,5-trichlorophenoxypropionic acid (2,4,5-TP), at 50 to 400 mg L-1, is consistently effective at reducing lychee fruit drop. The commercial product he used (Tipimon) is a liquid solution, containing 6.8 percent 2,4,5-TP, formulated as a triethanolamine salt. However, most of the resulting mature fruit was seedless, very small, with no market value. In another study, Tipimon failed to reduce fruit drop when applied seven to eight weeks after fruit set (Shalem-Galon, 1980). Later, Stern et al. (1995) found that timing is critical: spraying 'Mauritius' is effective only when executed about five weeks after peak female bloom, when fruitlet weight is about 2 g (Plate 8.6). At this stage, the embryo begins its rapid growth, followed, a few days later, by a second wave of fruit drop (Fig. 8.12). Fruitlet drop was consistently and significantly reduced and yield increased by spraying at that stage. The first success at the panicle level was followed by successful spraying at the tree level and then at the orchard level (Stern 1992; Stern et al. 1995). Based on this study, it has been recommended that mature 'Mauritius' orchards be sprayed at the 2 g stage with 100 mg L-1 2,4,5-TP (0.15% Tipimon) at 1000 L ha-1. 'Floridian' responded in a similar manner but the timing was less critical: it could also be sprayed about four weeks after fruit set, when fruitlet weight is about 1 g (Stern et al. 1997c). These recommendations have been adopted by the lychee industry in Israel. It should be noted that this spray might cause some foliar scorching, especially if spraying volume is excessive. 'Floridian' is more susceptible than 'Mauritius,' and 'Kaimana' is even more so. Spraying with 2,4,5-TP caused an increase in fruit with shriveled seeds in 'Mauritius,' and particularly in 'Floridian' (Stern et al. 1995, 1997c). This indicates that this treatment can reduce the abscission of fruitlets with degenerate or aborted embryos.

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Stern and Gazit (1997) tested a large number of synthetic auxins, in search of an alternative to 2,4,5-TP. The synthetic auxin 3,5,6-trichloro2-pyridyl-oxyacetic acid (3,5,6-TPA), as formulated in the commercial product Maxim® (manufactured by Dow AgroSciences, and sold as tablets containing 10% a.i.) was effective at 50 mg L-1 at reducing fruitlet abscission in 'Mauritius.' It was also very effective in 'Kaimana,' but only when sprayed early, about three weeks after fruit set, when fruit weight is about 0.5 g (Stern and Gazit 1999). Almost all this later cultivar's fruitlets are shed during the fourth week after fruit set (Fig. 8.11), hence a later treatment cannot be effective. In contrast, Maxim was found to be ineffective with 'Floridian' (Stern et a1. 2000). Preliminary trials in China found significant increases in yield by spraying young trees of 'Hei Ye' and 'Fei Zi Xiao' with Tipimon and Maxim (Stern et a1. 2001). In none of the trials with Maxim spraying was any foliar scorching observed. Spraying with 3,5,6-TPA resulted in a significant increase (14 to 30%) in fruit weight in 'Mauritius,' 'Floridian,' 'Kaimana,' 'Hei Ye' and 'Fei Zi Xiao' (Stern and Gazit 1999; Stern et a1. 2000, 2001). This increase occurred concomitantly with a much higher increase in yield. Hence, the increase in fruit number was the main factor for the yield increase. Obviously, the increase in fruit size cannot be explained by fruit thinning, as suggested for citrus (Zaragoza et a1. 1992) and apple (Dennis 1986). The treatment probably makes the fruit a stronger sink (Agusti et a1. 1995). The concomitant significant increase in lychee seed weight (Stern et a1. 2000) apparently reflects this effect. The aril's TSS (total soluble solids) level did not decrease with the pronounced yield increase, indicating that the higher crop load had not exhausted the tree's capacity. Recently, Stern et al. (2000) found the highest yield in 'Mauritius' and 'Floridian' after spraying with 2,4,5-TP at 67 mg L-1 (0.1 % Tipimon) and about one week later with Maxim (3,5,6-TPA at 20 mg L-1). The increased yield was apparently the result of a reduction in fruitlet drop (by both auxins) and increased fruit weight (by the second, 3,5,6-TPA treatment). The combined treatment is now used on a commercial scale in Israel. In conclusion, the two synthetic auxins (2,4,5-TP and 3,5,6-TPA), as formulated in two commercial products (Tipimon and Maxim, respectively), were found to greatly reduce lychee fruitlet abscission and increase yield (Stern and Gazit 2000b). Both auxins are now routinely applied in commerciallychee orchards in Israel. However, other lychee cultivars might respond differently; hence, a tailor-made protocol should be determined for each cultivar. The environment might also affect the

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response to auxin sprays. Thus, in other countries, detailed trials should be conducted for each cultivar, in order to realize the potential of auxins to significantly increase lychee yield. VI. CONCLUDING REMARKS

In lychee, as in other crops, we strive to reach the highest sustainable yield of high-quality fruit. The highest sustained yield for which we have reliable data is about 20 t ha-1 (four-year average for 'Mauritius,' at Lavi orchard, Israel). The fact that fruit weight did not decrease in this orchard indicates that we have not yet reached the commercial yield limit. We estimate that under optimal growing conditions, a sustainable yield of 25 t ha- 1 can be achieved. Many factors are responsible for the rather low yield of 5 to 10 t ha-1 in most of the world's lychee orchards. In this chapter, only those factors related to reproductive biology were covered, assuming that the trees are healthy and well-maintained. Our knowledge and understanding of most aspects of lychee fruit formation has greatly expanded over the last 20 years, with most of the work having been conducted in Australia, Israel, and China. Note that most of the Chinese findings have been inaccessible to the English-speaking world. In our concluding remarks, we will try to highlight the significant advances achieved and the subjects that merit more study in order to realize lychee's yield potential. 1. Fruit Bud FOrInation. Inadequate flowering is sometimes the main limiting factor in obtaining a good lychee yield. It is mainly a problem where the winter is not cold enough and the environmental conditions promote late autumnal vegetative flushes. Breeding and selection of high-quality cultivars, under warm winter conditions, is the best longterm solution for the low-elevation tropicallychee industry. Judicious use of water and/or nutritional stress has been able to prevent autumnal vegetative flushes, ensuring adequate blooming. However, these methods are often not feasible. The use of growth retardants might eventually provide an effective solution in those cases.

2. Normal Female and Male Reproductive Organs. There is likely a widespread problem of defective pollen and even more so ovules in lychee. In Israel, where this aspect was studied, most of the flowers were found to be infertile, with ovaries that did not contain even one normal ovule. There are apparently great differences among cultivars and environmental factors that are also involved. Much more research should be conducted into the factors responsible for the impaired functionality of

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the pollen and ovules. A prerequisite for high yield is an adequate number of flowers with at least one normal ovule.

3. Pollination. Though wind may playa role in lychee pollination, effective insect vectors are essential for adequate pollination. The lychee bloom, especially the female one, is attractive to honeybees and other effective pollinators. Thus, during the female lychee bloom there is usually strong pollinator activity. In addition to pollinators, available viable pollen is also needed. There is usually a partial overlap between the female and male blooms. However, the presence of another cultivar, which has a male bloom (preferably the potent M z) during the first cultivar's peak female bloom, may improve pollination by ensuring the presence of available pollen throughout the female bloom. 4. The Fertilization Process. Rain and hot weather have deleterious effects on pollen germination and the fertilization process. No solution has yet been devised to these weather-related problems.

5. Fruit Set. Putrescine spraying at the female bloom had a pronounced positive effect on yield. We do not know how this effect was achieved. It may be the result of prolonging the effective pollination period, through delayed senescence of the pistil. Thus, the extended bloom of fully mature female flowers coincides with the potent M z bloom. However, an entirely different mechanism may be responsible. 6. Fruit Drop. Massive drop of fruitlets that have the capacity to develop into full-size fruits is a typical undesirable phenomenon in lychee. Spraying with two synthetic auxins (2,4,5-TP and 3,5,6-TPA) produced a significant reduction in fruit drop and subsequently, a great increase in yield in several cultivars. The use of these and other auxins will probably also be effective in reducing fruitlet drop in many other cultivars. However, the successful use of this method depends on its calibration for each cultivar. We envision the breeding and selection of parthenocarpic seedless lychee cultivars in this century. Most of the problems mentioned and discussed in this chapter will no longer be relevant when such cultivars are developed. However, until that time, we hope that this chapter will be instrumental in solving pressing problems related to lychee reproductive biology and in increasing lychee yields throughout the world. LITERATURE CITED Acta Hart. Number 558. Proc. 1st Intnl. Symp. litchi and longan. 2001. (H. Huang and C. Menzel, Eds.), Guangzhou, China.

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Agusti, M., M. EI-Otmani, M. Aznar, M. Juan, and V. Almela. 1995. Effect of 3,5,6-trichloro2-pyridyl-oxyacetic acid on clementine early fruitlet development on fruit size at maturity. J. Hort. Sci. 70:955-962. Alexander, M. P. 1969. Differential staining of aborted, and nonaborted pollen. Stain Technol. 44:117-122. Apelbaum, A., A. C. Burgoon, J. D. Anderson, and M. Lieberman. 1981. Polyamines inhibit biosynthesis of ethylene in higher plant tissue, and protoplasts. Plant Physiol. 68:453-456. Argaman, E. 1983. Effect of temperature, and pollen source on fertilization, fruit set, and abscission in avocado (Persea americana Mill.). (in Hebrew). MS Thesis, Hebrew Univ. Jerusalem, Israel. Arteca, R. N. 1996. Plant growth substances: principles and applications. Chapman and Hall Press, New York. Baker, H. G., and I. Baker. 1983. Floral nectar sugar constituents in relation to pollination type, p. 117-141. In: C. E. Jones and K. J. Little (eds.), Handbook of experimental pollination biology. Van Nostrand Reinhold Co., New York. Banerji, I., and K. 1. Chaudhuri. 1944. A contribution to the life-history of Litchi chinensis Sonn. Proc. Indian Acad. Sci. Section B, 19:19-27. Barker, R. J., and Y. Lehner. 1974. Acceptance, and sustenance value of naturally occurring sugars fed to newly emerged adult workers of honeybees (Apis mellifera L.). J. Expt. Zool. 187:277-286. Batten, D. J. 1982. Lychee. In: C. Hacket and J. Carolane (eds.). Edible horticultural crops: a compendium of information on fruits, vegetables, spice, and nut species. Crop number 550. Academic Press, Sydney. Batten, D. J. 1986. Towards an understanding ofreproductive failure in lychee (Litchi chinensis Sonn.). Acta Hort. 175:79-83. Batten D. J., and C. A. McConchie. 1992. Pollination in lychee. Proc. 3rd Natl. Lychee Seminar Bundaberg, Australia, p. 23-28. Batten, D. J., and C. A. McConchie. 1995. Floral induction in growing buds oflychee (Litchi chinensis), and mango (Mangifera indica). Austral. J. Plant Physiol. 22:783-791. Burgos, L., and J. Egea. 1993. Apricot embryo-sac development in relation to fruit set. J. Hort. Sci. 68:203-208. Burgos, L., T. Berenguer, and J. Egea. 1995. Embryo-sac development in pollinated, and non-pollinated flowers of two apricot cultivars. J. Hort. Sci. 70:35-39. Butcher, F. G. 1956. Bees pollinate lychee blooms. Proc. Fla. Lychee Growers Assoc. 3:59-60. Butcher, F. G. 1957. Pollinating insects on lychee blossoms. Proc. Fla. Stat. Hort. Soc. 70:326-328. Campbell, C. W. 1994. Lychee cultivars in Florida: past, present, and future. Proc. Fla. State Hort. Soc. 107:347-348. Campbell, C. W., and S. E. Malo. 1968. The Lychee. Fruit crops fact sheet 6. Florida Coop. Ext. Serv., Gainsville, F1. Campbell, R. J. 1994. Fall pruning induces blooming in young lychee trees. Proc. Fla. State Hort. Soc. 107:348-350. Chadha, K. L., and M. S. Rajpoot. 1969. Studies on floral biology, fruit set, and its retention, and quality of some lychee varieties. Indian J. Hort. 26:124-129. Chaikiattiyos, S., C. M. Menzel, and T. S. Rasmussen. 1994. Floral induction in tropical fruit trees: effects of temperature, and water supply. J. Hort. Sci. 69:397-415. Chaitrakulsub, T., P. Chaidate, and H. Gemma.1988. Study of fruit development of Litchi chinensis Sonn. var. Hong-Huay. Japanese J. Tropical Agr. 32:201-207.

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Menzel, C. M., and D. R. Simpson. 1987. Effect of cincturing on growth, and flowering of lychee over several seasons in subtropical Queensland. Austral. J. Expt. Agr. 27:733-738. Menzel, C. M., and D. R. Simpson. 1988. Effect of temperature on growth, and flowering of litchi (Litchi chinensis Sonn.) cultivars. J. Hort. Sci. 63:347-358. Menzel, C. M., and D. R. Simpson. 1990a. Does paclobutrazol have role in lychee cultivar? Acta Hort. 275:205-210. Menzel, C. M., and D. R. Simpson. 1990b. Performance, and improvement oflychee cultivars: a review. Fruit Var. J. 44:197-215. Menzel, C. M., and D. R. Simpson. 1991. Effect of temperature, and leaf water stress on panicle, and flower development of litchi (Litchi chinensis Sonn.). J. Hort. Sci. 66:335-344. Menzel, C. M., and D. R. Simpson. 1992. Flowering, and fruit set in lychee (Litchi chinensis Sonn.) in subtropical Queensland. Austral. J. Expt. Agr. 32:105-111. Menzel, C. M., and D. R. Simpson. 1994. Lychee. p. 123-145. In: B. Schaffer, and P. C. Anderson (eds.). Handbook of environment physiology of fruit crops. Vol. 2. Subtropical, and tropical crops. CRC Press, Boca Roton, FL. Menzel, C. M., and D. R. Simpson. 1995. Temperatures above 20°C reduce flowering in lychee (Litchi chinensis Sonn.). J. Hort. Sci. 70:981-987. Menzel, C. M, M. L. Carseldine, and D. R. Simpson. 1988b. Crop development, and leaf nitrogen in lychee in subtropical Queensland. Austral. J. Expt. Agr. 28:793-800. Menzel, C. M., M. L. Carseldine, G. F. Haydon, and D. R. Simpson. 1992. A review of existing, and proposed new leaf nutrient standards for lychee. Scientia Hort. 49:33-53. Menzel, C. M., T. Olesen, and C. A. McConchie, 1999. Making a profit from lychee in Australia. Proc. 5th Natl. Lychee Conference, Twin Waters. Australia, p. 5-15. Menzel, C. M., T. S. Rasmussen, and D. R. Simpson. 1989. Effects oftemperature, and leaf water stress on growth, and flowering of litchi (Litchi chinensis Sonn.). J. Hort. Sci. 64:739-752. Menzel, C. M., K R. Chapman, B. F. Paxton, and D. R. Simpson. 1986. Growth, and yield oflychee cultivars in subtropical Queensland. Austral. J. Expt. Agr. 26:261-265. Menzel, C. M., J. H. Oosthuizen, D. J. Roe, and V. J. Doogan. 1995. Water deficit at anthesis reduce CO 2 assimilation, and yield of lychee (Litchi chinensis Sonn.) trees. Tree Physiol. 15:611-617. Menzel, C. M, B. J. Watson, and D. R. Simpson. 1988a. The lychee in Australia. Queensland Agr. J. 114:19-26. Milne, D. L. 1999a. Lychee production, and research in Southern Africa. Proc. 5th Natl. Lychee Conference, Twin waters, Australia, p. 25-31. Milne, D. L. 1999b. Logistic of growing, and marketing lychee in South Africa. Proc. 5th Natl. Lychee Conference, Twin waters, Queensland, Australia, p. 77-81. Misra, S. K, J. P. Nauriyal, and R. P. Awasthi. 1973. Effect of growth regulators on fruit drop in litchi. Punjab Hort. J. 13:122-126. Mitra, S. K, and D. Sanyal. 1990. Effect of putrescine on fruit set, and quality of litchi. Gartenbauwissenschaft, 55:83-84. Moncur, M. W. 1988. Floral development of tropical, and subtropical fruit, and nut species. C.S.I.R.O., Division of Water, and Land Resources, Australia. Moss, G. I. 1969. Influence of temperature, and photoperiod on flower induction, and inflorescence development in sweet orange (Citrus sinensis L. Osbeck). J. Hort. Sci. 44:311-320. Mulcahy, G. B., and D. L. Mulcahy. 1985. Ovarian influence on pollen tube growth, as indicated by the semivivo technique. Am. J. Bot. 72:1078-1080. Mustard, M. J. 1960. Megagametophytes ofthe lychee (Litchi chinensis Sonn.), Proc. Am. Soc. Hort. Sci. 75:292-304.

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Subject Index (Volume 28) A

Abscission, lychee, 437-443 Allium phytonutrients, 156-159 B

Brassica classification, 27-28

c Cactus grafting, 106-109 Classification, 1-6 Brassica, 27-28 lettuce, 25-27 potato, 23-26 tomato, 21-23 Crucifers phytochemicals, 150-156 Cucumber grafting, 91-96

phytochemicals, 125-185 volatiles, pear, 237-324 Fruit crops: lychee, 393-453 peach thinning, 351-392 pear volatiles, 237-324 virus elimination, 187-236 G

Genetics and breeding, grafting use, 109-115 Graft and grafting, herbacious, 61-124

H Health phytochemicals, vegetables, 125-185 L

D

Dedication, Stevens, M.A., xi-xiii E

Eggplant grafting, 103-104 Eggplant phytochemicals, 162-163 F Flower and flowering: Lychee,397-421 strawberry, 325-349 Fruit: lychee, 433-444 pear volatiles, 237-324

Lettuce classification, 25-27 Lychee: fruit abscission, 437-443 reproductive biology, 393-453 flowering, 397-421 fruit development, 433-436 pollination, 422-428 M

Melon grafting, 96-98 N

Nomenclature, 1-60 Taxonomy, 1-60

Horticultural Reviews, Volume 28, Edited by Jules Janick ISBN 0-471-21542-2 © 2003 John Wiley & Sons, Inc. 454

455

SUBJECT INDEX

o

T

Ornamental plants, cactus grafting, 106-109

p Peach thinning, 351-392 Pear, fruit volatiles, 237-324 Pepper: grafting, 104-105 phytochemicals, 161-162 Physiology: grafting, 78-84 lychee reproduction, 393--453 strawberry flowering, 325-349 Plant: classification, 1-60 systematics, 1-60 Pollination, lychee, 422-428 Potato: classification, 23-26 phytochemicals, 160-161

s Strawberry, flowering, 325-349 Systematics, 1-60

Thinning, peach and Prunus, 351-392 Tomato: classification, 21-23 grafting, 98-103 phytochemicals, 160

v Vegetable crops: Allium phytochemicals, 156-159 cucumber grafting, 91-96 crucifer phytochemicals, 150-156 eggplant grafting, 103-104 eggplant phytochemicals, 162-163 grafting, 61-124 melon grafting 96-98 pepper phytochemicals, 161-162 potato phytochemicals, 160-161 tomato phytochemicals, 160 phytochemicals, 125-185 watermelon grafting, 86-91 Virus elimination, 187-236 Volatiles, pear, 237-324

w Watermelon grafting, 86-91

455

Cumulative Subject Index (Volumes 1-28)

A

Abscisic acid: chilling injury, 15:78-79 cold hardiness, 11 :65 dormancy, 7:275-277 genetic regulation, 16:9-14, 20-21 lychee, 28:437-443 mechanical stress, 17:20 rose senescence, 9:66 stress, 4:249-250 Abscission: anatomy and histochemistry, 1:172-203 citrus, 15:145-182, 163-166 flower and petals, 3:104-107 regulation, 7:415-416 rose, 9:63-64 Acclimatization: foliage plants, 6:119-154 herbaceous plants, 6:379-395 micropropagation, 9:278-281, 316-317 Actinidia,6:4-12 Adzuki bean, genetics, 2:373 Agapanthus, 25:56-57 Agaricus, 6:85-118 Agrobacterium tumefaciens, 3:34 Air pollution, 8:1-42 Alkaloids, steroidal, 25:171-196 Allium phytonutrients, 28:156-159 Almond: bloom delay, 15:100-101 in vitro culture, 9:313 postharvest technology and utilization, 20:267-311

Alocasia, 8:46, 57, see also Aroids Alternate bearinu' chemical thin~ing, 1:285-289 fruit crops, 4:128-173 pistachio, 3:387-388 Aluminum: deficiency and toxicity symptoms in fruits and nuts, 2:154 Ericaceae, 10:195-196 Amarcrinum, 25: 57 Amaryllidaceae, growth, development, flowering, 25:1-70 Amaryllis, 25:4-15 Amorphophallus, 8:46, 57, see also Aroids Anatomy and morphology: apple flower and fruit, 10:273-308 apple tree, 12:265-305 asparagus, 12:71 cassava, 13:106-112 citrus, abscission, 15:147-156 embryogenesis, 1:4-21,35-40 fig, 12:420-424 fruit abscission, 1:172-203 fruit storage, 1:314 ginseng, 9:198-201 grape flower, 13:315-337 grape seedlessness, 11:160-164 heliconia, 14:5-13 kiwifruit, 6:13-50 magnetic resonance imaging, 20:78-86, 225-266 orchid,5:281-283 navel orange, 8:132-133

Horticultural Reviews, Volume 28, Edited by Jules Janick ISBN 0-471-21542-2 © 2003 John Wiley & Sons, Inc. 456

457

CUMULATIVE SUBJECT INDEX

pecan flower, 8:217-255 petal senescence, 1:212-216 pollution injury, 8:15 waxes, 23:1-68 Androgenesis, woody species, 10:171-173

Angiosperms, embryogenesis, 1:1-78 Anthurium: see also Aroids, ornamental fertilization, 5:334-335 Antitranspirants, 7:334 cold hardiness, 11:65 Apical meristem, cryopreservation, 6:357-372

Apple: alternate bearing, 4:136-137 anatomy and morphology of flower and fruit, 10:273-309 bioregulation, 10:309--401 bitter pit, 11 :289-355 bloom delay, 15:102-104 CA storage, 1:303-306 chemical thinning, 1:270-300 fertilization, 1 :105 fire blight control, 1 :423--4 74 flavor, 16:197-234 flower induction, 4:174-203 fruiting, 11:229-287 fruit cracking and splitting, 19:217-262

functional phytonutrients, 27:304 in vitro, 5:241-243; 9:319-321 light, 2:240-248 maturity indices, 13:407--432 mealiness, 20:200 nitrogen metabolism, 4:204-246 replant disease, 2:3 root distribution, 2:453-456 scald, 27:227-267 stock-scion relationships, 3:315-375 summer pruning, 9:351-375 tree morphology and anatomy, 12:265-305

vegetative growth, 11:229-287 watercore, 6:189-251 weight loss, 25:197-234 yield, 1:397--424 Apricot: bloom delay, 15:101-102 CA storage, 1:309 origin and dissemination, 22:225-266

Arabidopsis: molecular biology of flowering, 27:1-39,41-77

Aroids: edible, 8:43-99; 12:166-170 ornamental, 10:1-33 Arsenic, deficiency and toxicity symptoms in fruits and nuts, 2:154

Artemisia, 19:319-371 Artemisinin, 19:346-359 Artichoke, CA storage, 1:349-350 Asexual embryogenesis, 1:1-78; 2:268-310; 3:214-314; 7:163-168, 171-173, 176-177, 184, 185-187, 187-188,189; 10:153-181; 14:258-259, 337-339; 24:6-7; 26:105-110

Asparagus: CA storage, 1:350-351 fluid drilling of seed, 3:21 postharvest biology, 12:69-155 Auxin: abscission, citrus, 15:161, 168-176 bloom delay, 15:114-115 citrus abscission, 15:161, 168-176 dormancy, 7:273-274 flowering, 15:290-291, 315 genetic regulation, 16:5-6, 14, 21-22 geotropism, 15:246-267 mechanical stress, 17:18-19 petal senescence, 11:31 Avocado: CA and MA, 22:135-141 flowering, 8:257-289 fruit development, 10:230-238 fruit ripening, 10:238-259 rootstocks, 17:381-429 Azalea, fertilization, 5:335-337 B

Babaco, in vitro culture, 7:178 Bacteria: diseases of fig, 12:447--451 ice nucleating, 7:210-212; 11:69-71 pathogens of bean, 3:28-58 tree short life, 2:46-47 wilt of bean, 3:46-47 Bacteriocides, fire blight, 1:450--459

CUMULATIVE SUBJECT INDEX

458

Bacteriophage, fire blight control, 1:449-450

Banana: CA and MA, 22:141-146 CA storage, 1:311-312 fertilization, 1:105 in vitro culture, 7:178-180 Banksia, 22:1-25 Bean: CA storage, 1:352-353 fluid drilling of seed, 3 :21 resistance to bacterial pathogens, 3:28-58

Bedding plants, fertilization, 1:99-100; 5:337-341

Beet: CA storage, 1:353 fluid drilling of seed, 3:18-19 Begonia (Rieger), fertilization, 1:104 Biennial bearing. See Alternate bearing Biochemistry, petal senescence, 11:15-43 Bioreactor technology, 24:1-30 Bioregulation: See also Growth substances apple and pear, 10:309-401 Bird damage, 6:277-278 Bitter pit in apple, 11:289-355 Blackberry, harvesting, 16:282-298 Black currant, bloom delay, 15:104 Bloom delay, deciduous fruits, 15:97 Blueberry: developmental physiology, 13:339-405 harvesting, 16:257-282 nutrition, 10:183-227 Boron: deficiency and toxicity symptoms in fruits and nuts, 2:151-152 foliar application, 6:328 nutrition, 5:327-328 pine bark media, 9:119-122 Botanic gardens, 15:1-62 Bramble, harvesting, 16:282-298 Branching, lateral: apple, 10:328-330 pear, 10:328-330 Brassica classification, 28:27-28 Brassicaceae, in vitro, 5:232-235 Breeding. See Genetics and breeding Broccoli, CA storage, 1:354-355 Brussels sprouts, CA storage, 1:355

Bulb crops: See also Tulip development, 25:1-70 flowering, 25:1-70 genetics and breeding, 18:119-123 growth, 25: 1-70 in vitro, 18:87-169 micropropagation, 18:89-113 root physiology, 14:57-88 virus elimination, 18:113-123

c CA storage. See Controlled-atmosphere storage Cabbage: CA storage, 1:355-359 fertilization, 1:117-118 Cactus: crops, 18:291-320 grafting, 28:106-109 reproductive biology, 18:321-346 Caladium. See Aroids, ornamental Calciole, nutrition, 10:183-227 Calcifuge, nutrition, 10:183-227 Calcium: bitter pit, 11:289-355 cell wall, 5:203-205 container growing, 9:84-85 deficiency and toxicity symptoms in fruits and nuts, 2:148-149 Ericaceae nutrition, 10:196-197 foliar application, 6:328-329 fruit softening, 10:107-152 nutrition, 5:322-323 pine bark media, 9:116-117 tipburn, disorder, 4:50-57 Calmodulin, 10:132-134, 137-138 Caparis, See Caper bush Caper bush, 27:125-188 Carbohydrate: fig, 12:436-437 kiwifruit partitioning, 12:318-324 metabolism, 7:69-108 partitioning, 7:69-108 petal senescence, 11:19-20 reserves in deciduous fruit trees, 10:403-430

Carbon dioxide, enrichment, 7:345-398, 544-545

Carnation, fertilization, 1:100; 5:341-345

CUMULATIVE SUBJECT INDEX

Carrot: CA storage, 1:362-366 fluid drilling of seed, 3:13-14 Caryophyllaceae, in vitro, 5:237-239 Cassava, 12:158-166; 13:105-129; 26:85-159 Cauliflower, CA storage, 1:359-362 Celeriac, CA storage, 1:366-367 Celery: CA storage, 1:366-367 fluid drilling of seed, 3:14 Cell culture, 3:214-314 woody legumes, 14:265-332 Cell membrane: calcium, 10:126-140 petal senescence, 11:20-26 Cellular mechanisms, salt tolerance, 16:33-69 Cell wall: calcium, 10:109-122 hydrolases,5:169-219 ice spread, 13:245-246 tomato, 13:70-71 Chelates, 9:169-171 Cherimoya, CA and MA, 22:146-147 Cherry: bloom delay, 15:105 CA storage, 1:308 origin, 19:263-317 Chestnut: blight, 8:281-336 in vitro culture, 9:311-312 Chicory, CA storage, 1:379 Chilling: injury, 4:260-261; 15:63-95 injury, chlorophyll fluorescence, 23:79-84 pistachio, 3:388-389 Chlorine: deficiency and toxicity symptoms in fruits and nuts, 2:153 nutrition, 5:239 Chlorophyll fluorescence, 23:69-107 Chlorosis, iron deficiency induced, 9:133-186 Chrysanthemum fertilization, 1:100-101; 5:345-352 Citrus: abscission, 15:145-182 alternate bearing, 4:141-144

459 asexual embryogenesis, 7:163-168 CA storage, 1:312-313 chlorosis, 9:166-168 cold hardiness, 7:201-238 fertilization, 1:105 flowering, 12:349-408 functional phytochemicals, fruit, 27:269-315 honey bee pollination, 9:247-248 in vitro culture, 7:161-170 juice loss, 20:200-201 navel orange, 8:129-179 nitrogen metabolism, 8:181 practices for young trees, 24:319-372 rootstock, 1:237-269 viroid dwarfing, 24:277-317 Classification:

Brassica, 28:27-28 lettuce, 28:25-27 potato, 28:23-26 tomato, 28:21-23 Clivia, 25:57 Cloche (tunnel), 7:356-357 Coconut palm: asexual embryogenesis, 7:184 in vitro culture, 7:183-185 Cold hardiness, 2:33-34 apple and pear bioregulation, 10:374-375 citrus, 7:201-238 factors affecting, 11:55-56 herbaceous plants, 6:373-417 injury, 2:26-27 nutrition, 3:144-171 pruning, 8:356-357 Co10casia, 8:45, 55-56, see also Aroids Common blight of bean, 3:45-46 Compositae, in vitro, 5:235-237 Container production, nursery crops, 9:75-101 Controlled-atmosphere (CA) storage: asparagus, 12:76-77, 127-130 chilling injury, 15:74-77 flowers, 3:98; 10:52-55 fruit quality, 8:101-127 fruits, 1:301-336; 4:259-260 pathogens, 3:412-461 seeds, 2:134-135 tropical fruit, 22:123-183 tulip, 5:105

CUMULATIVE SUBJECT INDEX

460 Controlled-atmosphere (CA) storage (cont.) vegetable quality, 8:101-127 vegetables, 1:337-394; 4:259-260 Controlled environment agriculture, 7:534-545, see also Greenhouse and greenhouse crops; hydroponic culture; protected culture Copper: deficiency and toxicity symptoms in fruits and nuts, 2:153 foliar application, 6:329-330 nutrition, 5:326-327 pine bark media, 9:122-123 Corynebacterium flaccumfaciens, 3:33,46 Cowpea: genetics, 2:317-348 U.S. production, 12:197-222 Cranberry: botany and horticulture, 21:215-249 fertilization, 1:106 harvesting, 16:298-311 Crinum, 25:58 Crucifers phytochemicals, 28:150-156 Cryopreservation: apical meristems, 6:357-372 cold hardiness, 11:65-66 Cryphonectria parasitica. See Endothia parasitica Crytosperma, 8:47, 58, see also Aroids Cucumber: CA storage, 1:367-368 grafting, 28:91-96 Cucurbita pepo, cultivar groups history, 25:71-170 Currant, harvesting, 16:311-327 Custard apple, CA and MA, 22:164 Cyrtanthus, 25:15-19 Cytokinin: cold hardiness, 11 :65 dormancy, 7:272-273 floral promoter, 4:112-113 flowering, 15:294-295, 318 genetic regulation, 16:4-5, 14,22-23 grape root, 5:150, 153-156 lettuce tipburn, 4:57-58 petal senescence, 11:30-31 rose senescence, 9:66

D

Date palm: asexual embryogenesis, 7:185-187 in vitro culture, 7:185-187 Daylength. See Photoperiod Dedication: Bailey, L.H., 1:v-viii Beach, S.A., l:v-viii Bukovac, M.J., 6:x-xii Campbell, C.W., 19:xiii Cummins, J.N., 15:xii-xv Dennis, F.G., 22:xi-xii De Hertogh, A.A., 26:xi-xii Faust, Miklos, 5:vi-x Hackett, W.P., 12:x-xiii Halevy, A.H., 8:x-xii Hess, c.E., 13:x-xii Kader, A.A., 16:xii-xv Kamemoto, H., 24:x-xiii Looney, N.E., 18:xiii Magness, J.R., 2:vi-viii Moore, J.N., 14:xii-xv Possingham, J.V., 27:xi-xiii Pratt, c., 20:ix-xi Proebsting, Jr., E.L., 9:x-xiv Rick, Jr., C.M., 4:vi-ix Ryugo, K., 25:x-xii Sansavini, S., 17:xii-xiv Sherman, W.B., 21:xi-xiii Smock, RM., 7:x-xiii Stevens, M.A., 28:xi-xiii Weiser, c.J., l1:x-xiii Whitaker, T.W., 3:vi-x Wittwer, S.H., 10:x-xiii Yang, S.F., 23:xi Deficit irrigation, 21:105-131 Deficiency symptoms, in fruit and nut crops, 2:145-154 Defoliation, apple and pear bioregulation, 10:326-328 'Delicious' apple, 1:397-424 Desiccation tolerance, 18:171-213 Dieffenbachia. See Aroids, ornamental Dioscorea. See Yam Disease: and air pollution, 8:25 aroids, 8:67-69; 10:18; 12:168-169 bacterial, of bean, 3:28-58

CUMULATIVE SUBJECT INDEX cassava, 12:163-164 control by virus, 3:399-403 controlled-atmosphere storage,

461

Environment: air pollution, 8:20-22 controlled for agriculture, 7:534-545

3:412-461

cowpea, 12:210-213 fig, 12:447-479 flooding, 13:288-299 hydroponic crops, 7:530-534 lettuce, 2:187-197 mycorrhizal fungi, 3:182-185 ornamental aroids, 10:18 resistance, acquired, 18:247-289 root, 5:29-31 stress, 4:261-262 sweet potato, 12:173-175 tulip, 5:63, 92 turnip moasic virus, 14:199-238 waxes, 23:1-68 yam (Dioscorea), 12:181-183 Disorder: see also Postharvest physiology: bitterpit, 11:289-355 fig, 12:477-479 pear fruit, 11:357-411 watercore, 6:189-251; 11:385-387 Dormancy, 2:27-30 blueberry, 13:362-370 release in fruit trees, 7:239-300 tulip, 5:93 Drip irrigation, 4:1-48 Drought resistance, 4:250-251 cassava, 13:114-115 Durian, CA and MA, 22:147-148 Dwarfing: apple, 3:315-375 apple mutants, 12:297-298 by virus, 3:404-405

controlled for energy efficiency, 1:141-171; 9:1-52

embryogenesis, 1:22,43-44 fruit set, 1:411-412 ginseng, 9:211-226 greenhouse management, 9:32-38 navel orange, 8:138-140 nutrient film technique, 5:13-26 Epipremnum. See Aroids, ornamental Eriobotrya japonica. See Loquat Erwinia: amylovora, 1:423-474 lathyri, 3:34 Essential elements: foliar nutrition, 6:287-355 pine bark media, 9:103-131 plant nutrition, 5:318-330 soil testing, 7:1-68 Ethylene: abscission, citrus, 15:158-161, 168-176

apple bioregulation, 10:366-369 avocado, 10:239-241 bloom delay, 15:107-111 CA storage, 1:317-319, 348 chilling injury, 15:80 citrus abscission, 15:158-161, 168-176

cut flower storage, 10:44-46 dormancy, 7:277-279 flowering, 15:295-296, 319 flower longevity, 3:66-75 genetic regulation, 16:6-7, 14-15, 19-20

E

Easter lily, fertilization, 5:352-355 Eggplant: grafting, 28:103-104 phytochemicals, 28:162-163 Embryogenesis. See Asexual embryogenesis Endothia parasitica, 8:291-336 Energy efficiency, in greenhouses, 1:141-171; 9:1-52

kiwifruit respiration, 6:47-48 mechanical stress, 17:16-17 petal senescence, 11:16-19, 27-30 rose senescence, 9:65-66 Eucharis, 25:19-22 Eucrosia, 25:58 F

Feed crops, cactus, 18:298-300 Feijoa, CA and MA, 22:148

462 Fertilization and fertilizer: anthurium, 5:334-335 azalea, 5:335-337 bedding plants, 5:337-341 blueberry, 10:183-227 carnation, 5:341-345 chrysanthemum, 5:345-352 controlled release, 1:79-139; 5:347-348 Easter lily, 5:352-355 Ericaceae, 10:183-227 foliage plants, 5:367-380 foliar, 6:287-355 geranium, 5:355-357 greenhouse crops, 5:317-403 lettuce, 2:175 nitrogen, 2:401-404 orchid, 5:357-358 poinsettia, 5:358-360 rose, 5:361-363 snapdragon, 5:363-364 soil testing, 7:1-68 trickle irrigation, 4:28-31 tulip, 5:364-366 Vaccinium, 10:183-227 zinc nutrition, 23:109-128 Fig: industry, 12:409-490 ripening, 4:258-259 Filbert, in vitro culture, 9:313-314 Fire blight, 1:423-474 Flooding, fruit crops, 13:257-313 Floral scents, 24:31-53 Floricultural crops: see also individual crops: Amaryllidaceae, 25:1-70 Banksia, 22:1-25 fertilization, 1:98-104 growth regulation, 7:399-481 heliconia, 14:1-55

Leucospermum, 22:27-90 postharvest physiology and senescence, 1:204-236; 3:59-143; 10:35-62; 11:15-43 Pratea, 26:1-48 Florigen, 4:94-98 Flower and flowering: Amaryllidaceae, 25:1-70 apple anatomy and morphology, 10:277-283

CUMULATIVE SUBJECT INDEX

apple bioregulation, 10:344-348 Arabidopsis, 27:1-39, 41-77 aroids, ornamental, 10:19-24 avocado, 8:257-289 Banksia, 22:1-25 blueberry development, 13:354-378 cactus, 18:325-335 citrus, 12:349-408 control, 4:159-160; 15:279-334 development (postpollination), 19:1-58 fig, 12:424-429 grape anatomy and morphology, 13:354-378 homeotic gene regulation, 27:41-77 honey bee pollination, 9:239-243 induction, 4:174-203,254-256 initiation, 4:152-153 in vitro, 4:106-127 kiwifruit, 6:21-35; 12:316-318

Leucospermum, 22:27-90 lychee, 28:397-421 orchid, 5:297-300 pear bioregulation, 10:344-348 pecan, 8:217-255 perennial fruit crops, 12:223-264 phase change, 7:109-155 photoperiod,4:66-105 pistachio, 3:378-387 postharvest physiology, 1:204-236; 3:59-143; 10:35-62; 11:15-43 postpollination development, 19:1-58 protea leaf blackening, 17:173-201 pruning, 8:359-362 raspberry, 11:187-188 regulation in floriculture, 7:416-424 rhododendron, 12:1-42 rose, 9:60-66 scents, 24:31-53 senescence, 1:204-236; 3:59-143; 10:35-62; 11:15-43; 18:1-85 strawberry, 28:325-349 sugars, 4:114 thin cell layer morphogenesis, 14:239-256 tulip, 5:57-59 water relations, 18:1-85 Fluid drilling, 3:1-58 Foliage plants: acclimatization, 6:119-154

463

CUMULATIVE SUBJECT INDEX

fertilization, 1:102-103; 5:367-380 Foliar nutrition, 6:287-355 Freeze protection. See Frost protection Frost: apple fruit set, 1:407-408 citrus, 7:201-238 protection, 11 :45-109 Fruit: abscission, 1:172-203 abscission, citrus, 15:145-182 apple anatomy and morphology, 10:283-297

apple bioregulation, 10:348-374 apple bitter pit, 11:289-355 apple flavor, 16:197-234 apple maturity indices, 13:407-432 apple ripening and quality, 10:361-374 apple scald, 27:227-267 apple weight loss, 25:197-234 avocado development and ripening, 10:229-271

bloom delay, 15:97-144 blueberry development, 13:378-390 cactus physiology, 18:335-341 CA storage and quality, 8:101-127 chilling injury, 15:63-95 coating physiology, 26:161-238 cracking, 19:217-262 diseases in CA storage, 3:412-461 drop, apple functional phytochemicals, 27:269-315 growth measurement, 24:373-431 kiwifruit, 6:35-48; 12:316-318 loquat, 23:233-276 lychee, 28:433-444 maturity indices, 13:407-432 navel orange, 8:129-179 nectarine, postharvest, 11:413-452 nondestructive postharvest quality evaluation, 20:1-119 olive processing, 25:235-260 peach, postharvest, 11:413-452 pear, bioregulation, 10:348-374 pear, fruit disorders, 11:357-411 pear maturity indices, 13:407-432 pear ripening and quality, 10:361-374 pear scald, 27:227-267 pear volatiles, 28:237-324 pistachio, 3:382-391 phytochemicals, 28:125-185

plum, 23:179-231 quality and pruning, 8:365-367 ripening, 5:190-205 set, 1:397-424; 4:153-154 set in navel oranges, 8:140-142 size and thinning, 1:293-294; 4:161 softening, 5:109-219; 10:107-152 splitting, 19:217-262 strawberry growth and ripening, 17:267-297

texture, 20:121-224 thinning, apple and pear, 10:353-359 tomato parthenocarpy, 6:65-84 tomato ripening, 13:67-103 volatiles, pear, 28:237-324 Fruit crops: see also individual crop alternate bearing, 4:128-173 apple bitter pit, 11:289-355 apple flavor, 16:197-234 apple fruit splitting and cracking, 19:217-262

apple growth, 11:229-287 apple maturity indices, 13:407-432 apple scald, 27:227-267 apricot, origin and dissemination, 22:225-266

avocado flowering, 8:257-289 avocado rootstocks, 17:381-429 berry crop harvesting, 16:255-382 bloom delay, 15:97-144 blueberry developmental physiology, 13:339-405

blueberry harvesting, 16:257-282 blueberry nutrition, 10:183-227 bramble harvesting, 16:282-298 cactus, 18:302-309 carbohydrate reserves, 10:403-430 CA and MA for tropicals, 22:123-183 CA storage, 1:301-336 CA storage diseases, 3:412-461 cherry origin, 19:263-317 chilling injury, 15:145-182 chlorosis, 9:161-165 citrus abscission, 15:145-182 citrus cold hardiness, 7:201-238 citrus, culture of young trees, 24:319-372

citrus dwarfing by viroids, 24:277-317 citrus flowering, 12:349-408 cranberry, 21:215-249

464

CUMULATIVE SUBJECT INDEX

Fruit crops (cant.) cranberry harvesting, 16:298-311 currant harvesting, 16:311-327 deficit irrigation, 21:105-131 dormancy release, 7:239-300 Ericaceae nutrition, 10:183-227 fertilization, 1 :104-106 fig, industry, 12:409-490 fireblight, 11 :423-474 flowering, 12:223-264 foliar nutrition, 6:287-355 frost control, 11:45-109 grape flower anatomy and morphology, 13:315-337

grape harvesting, 16:327-348 grape irrigation, 27:189-225 grape nitrogen metabolism,

pecan flowering, 8:217-255 photosynthesis, 11 :111-15 7 Phytophthora control, 17:299-330 plum origin, 23:179-231 pruning, 8:339-380 rambutan, 16:143-196 raspberry, 11:185-228 roots, 2:453-457 sapindaceous fruits, 16:143-196 short life and replant problem, 2:1-116 strawberry fruit growth, 17:267-297 strawberry harvesting, 16:348-365 summer pruning, 9:351-375 Vaccinium nutrition, 10:183-227 virus elimination, 28:187-236 water status, 7:301-344 Functional phytochemicals, fruit,

14:407-452

grape pnming, 16:235-254, 336-340 grape root, 5:127-168 grape seedlessness, 11 :164-176 grapevine pruning, 16:235-254, 336-340

27:269-315

Fungi: fig, 12:451-474 mushroom, 6:85-118 mycorrhiza, 3:172-213; 10:211-212 pathogens in postharvest storage,

honey bee pollination, 9:244-250, 254-256 jojoba, 17:233-266 in vitro culture, 7:157-200; 9:273-349 irrigation, deficit, 21:105-131 kiwifruit, 6:1-64; 12:307-347 lingonberry, 27:79-123 longan, 16:143-196 loquat, 23:233-276 lychee, 16:143-196, 28:393-453 muscadine grape breeding, 14:357-405 navel orange, 8:129-179 nectarine postharvest, 11 :413-452

nondestructive postharvest quality evaluation, 20:1-119 nutritional ranges, 2:143-164 olive salinity tolerance, 21:177-214 orange, navel, 8:129-179 orchard floor management, 9:377-430 peach origin, 17:331-379 peach postharvest, 11:413-452 peach thinning, 28:351-392 pear fruit disorders, 11:357-411; 27:227-267

pear maturity indices, 13:407-432 pear scald, 27:227-267 pear volatiles, 28:237-324

3:412-461

truffle cultivation, 16:71-107 Fungicide, and apple fruit set, 1:416 G

Galanthus, 25:22-25 Garlic, CA storage, 1:375 Genetic variation: alternate bearing, 4:146-150 photoperiodic response, 4:82 pollution injury, 8:16-19 ternperature-photoperiod interaction, 17:73-123

Genetics and breeding: aroids (edible), 8:72-75; 12:169 aroids (ornamental), 10:18-25 bean, bacterial resistance, 3:28-58 bloom delay in fruits, 15:98-107 bulbs, flowering, 18:119-123 cassava, 12:164 chestnut blight resistance, 8:313-321 citrus cold hardiness, 7:221-223 cranberry, 21:236-239 embryogenesis, 1:23 fig, 12:432-433 fire blight resistance, 1:435-436

CUMULATIVE SUBJECT INDEX flowering, 15:287-290, 303-305, 306-309,314-315;27:1-39,41-77 flower longevity, 1:208-209 ginseng, 9:197-198 grafting use, 28:109-115 in vitro techniques, 9:318-324; 18:119-123 lettuce, 2:185-187 lingonberry, 27:108-111 loquat, 23:252-257 muscadine grapes, 14:357-405 mushroom, 6:100-111 navel orange, 8:150-156 nitrogen nutrition, 2:410-411 pineapple, 21:138-164 plant regeneration, 3:278-283 pollution insensitivity, 8:18-19 potato tuberization, 14:121-124 rhododendron, 12:54-59 sweet potato, 12:175 sweet sorghum, 21:87-90 tomato parthenocarpy, 6:69-70 tomato ripening, 13:77-98 tree short life, 2:66-70 Vigna, 2:311-394 waxes, 23:50-53 woody legume tissue and cell culture, 14:311-314 yam (Dioscorea), 12:183 Geophyte. See Bulb, tuber Geranium, fertilization, 5:355-357 Germination, seed, 2:117-141, 173-174; 24:229-275 Germplasm: cryopreservation,6:357-372 in vitro, 5:261-264; 9:324-325 pineapple, 21:133-175 Gibberellin: abscission, citrus, 15:166-167 bloom delay, 15:111-114 citrus, abscission, 15:166-167 cold hardiness, 11 :63 dormancy, 7:270-271 floral promoter, 4:114 flowering, 15:219-293, 315-318 genetic regulation, 16:15 grape root, 5:150-151 mechanical stress, 17:19-20 Ginseng, 9:187-236 Girdling, 4:251-252

465 Glucosinolates, 19:99-215 Gourd, history, 25:71-171 Graft and grafting: herbaceous, 28:61-124 incompatibility, 15:183-232 phase change, 7:136-137, 141-142 rose, 9:56-57 Grape: CA storage, 1:308 chlorosis, 9:165-166 flower anatomy and morphology, 13:315-337 functional phytochemicals, 27:291-298 irrigation, 27:189-225 harvesting, 16:327-348 muscadine breeding, 14:357-405 nitrogen metabolism, 14:407-452 pollen morphology, 13:331-332 pruning, 16:235-254, 336-340 root, 5:127-168 seedlessness, 11:159-187 sex determination, 13:329-331 Gravitropism, 15:233-278 Greenhouse and greenhouse crops: carbon dioxide, 7:357-360, 544-545 energy efficiency, 1:141-171; 9:1-52 growth substances, 7:399-481 nutrition and fertilization, 5:317-403 pest management, 13:1-66 vegetables, 21:1-39 Growth regulators. See Growth substances Growth substances, 2:60-66; 24:55-138, see also Abscisic acid, Auxin, Cytokinins, Ethylene, Gibberellins abscission, citrus, 15:157-176 apple bioregulation, 10:309-401 apple dwarfing, 3:315-375 apple fruit set, 1:417 apple thinning, 1:270-300 aroids, ornamental, 10:14-18 avocado fruit development, 10:229-243 bloom delay, 15:107-119 CA storage in vegetables, 1:346-348 cell cultures, 3:214-314 chilling injury, 15:77-83 citrus abscission, 15:157-176 cold hardiness, 7:223-225; 11:58-66 dormancy, 7:270-279

CUMULATIVE SUBJECT INDEX

466

Growth substances (cont.) embryogenesis, 1:41-43; 2:277-281 floriculture, 7:399-481 flower induction, 4:190-195 flowering, 15:290-296 flower storage, 10:46-51 genetic regulation, 16:1-32 ginseng, 9:226 grape seedlessness, 11:177-180 hormone reception, 26:49-84 in vitro flowering, 4:112-115 mechanical stress, 17:16-21 meristem and shoot-tip culture, 5:221-227

navel oranges, 8:146-147 pear bioregulation, 10:309-401 petal senescence, 3:76-78 phase change, 7:137-138, 142-143 raspberry, 11 :196-197 regulation, 11:1-14 rose, 9:53-73 seedlessness in grape, 11:177-180 triazole, 10:63-105 H

Haemanthus, 25:25-28 Halo blight of beans, 3:44-45 Hardiness, 4:250-251 Harvest: flower stage, 1:211-212 index, 7:72-74 lettuce, 2:176-181 mechanical of berry crops, 16:255-382 Hazelnut. See Filbert Health phytochemicals: fruit, 27:269-315 vegetables, 28:125-185 Heat treatment (postharvest), 22:91-121 Heliconia, 14:1-55 Herbaceous plants, subzero stress, 6:373-417

Hippeastrum, 25:29-34 Histochemistry: flower induction, 4:177-179 fruit abscission, 1:172-203 Histology, flower induction, 4:179-184, see also Anatomy and morphology Honey bee, 9:237-272

Horseradish, CA storage, 1:368 Hydrolases, 5:169-219 Hydroponic culture, 5:1-44; 7:483-558 HymenocaJJis, 25:59 Hypovirulence, in Endothia parasitica, 8:299-310

Ismene, 25:59 Ice, formation and spread in tissues, 13:215-255

Ice-nucleating bacteria, 7:210-212; 13:230-235

Industrial crops, cactus, 18:309-312 Insects and mites: aroids, 8:65-66 avocado pollination, 8:275-277 fig, 12:442-447

hydroponic crops, 7:530-534 integrated pest management, 13:1-66 lettuce, 2:197-198 ornamental aroids, 10:18 tree short life, 2:52 tulip, 5:63, 92 waxes, 23:1-68 Integrated pest management: greenhouse crops, 13:1-66 In vitro: abscission, 15:156-157 apple propagation, 10:325-326 aroids, ornamental, 10:13-14 artemisia, 19:342-345 bioreactor technology, 24:1-30 bulbs, flowering, 18:87-169 cassava propagation, 13:121-123; 26:99-119

cellular salinity tolerance, 16:33-69 cold acclimation, 6:382 cryopreservation, 6:357-372 embryogenesis, 1:1-78; 2:268-310; 7:157-200; 10:153-181

environmental control, 17:123-170 flowering bulbs, 18:87-169 flowering, 4:106-127 pear propagation, 10:325-326 phase change, 7:144-145 propagation, 3:214-314; 5:221-277; 7:157-200; 9:57-58, 273-349; 17:125-172

467

CUMULATIVE SUBJECT INDEX thin cell layer morphogenesis, 14:239-264 woody legume culture, 14:265-332 Iron: deficiency and toxicity symptoms in fruits and nuts, 2:150 deficiency chlorosis, 9:133-186 Ericaceae nutrition, 10:193-195 foliar application, 6:330 nutrition, 5:324-325 pine bark media, 9:123 Irrigation: deficit, deciduous orchards, 21:105-131 drip or trickle, 4:1-48 frost control, 11:76-82 fruit trees, 7:331-332 grape, 27:189-225 grape root growth, 5:140-141 lettuce industry, 2:175 navel orange, 8:161-162 root growth, 2:464-465

Jojoba, 17:233-266 Juvenility, 4:111-112 pecan, 8:245-247 tulip, 5:62-63 woody plants, 7:109-155 K

Kale, fluid drilling of seed, 3:21 Kiwifruit: botany, 6:1-64 vine growth, 12:307-347 L

Lamps, for plant growth, 2:514-531 Lanzon, CA and MA, 22:149 Leaves: apple morphology, 12:283-288 flower induction, 4:188-189 Leek: CA storage, 1:375 fertilization, 1:118 Leguminosae, in vitro, 5:227-229; 14:265-332

Lemon, rootstock, 1:244-246, see also Citrus Lettuce: CA storage, 1:369-371 classification, 28:25-27 fertilization, 1:118 fluid drilling of seed, 3:14-17 industry, 2:164-207 seed germination, 24:229-275 tipburn, 4:49-65 Leucojum, 25:34-39 Leucospermum, 22:27-90 Light: fertilization, greenhouse crops, 5:330-331 flowering, 15:282-287, 310-312 fruit set, 1:412-413 lamps, 2:514-531 nitrogen nutrition, 2:406-407 orchards, 2:208-267 ornamental aroids, 10:4-6 photoperiod,4:66-105 photosynthesis, 11:117-121 plant growth, 2:491-537 tolerance, 18:215-246 Lingonberry, 27:79-123 Longan: See also Sapindaceous fruits CA and MA, 22:150 Loquat: botany and horticulture, 23:233-276 CA and MA, 22:149-150 Lychee: See also Sapindaceous fruits CA and MA, 22:150 flowering, 28:397-421 fruit abscission, 28-437-443 fruit development, 28:433-436 pollination, 28:422-428 reproductive biology, 28:393-453 Lycoris, 25:39-43 M Magnesium: container growing, 9:84-85 deficiency and toxicity symptoms in fruits and nuts, 2:148 Ericaceae nutrition, 10:196-198 foliar application, 6:331 nutrition, 5:323 pine bark media, 9:117-119

CUMULATIVE SUBJECT INDEX

468 Magnetic resonance imaging, 20:78-86, 225-266 Male sterility, temperature-photoperiod induction, 17:103-106 Mandarin, rootstock, 1:250-252 Manganese: deficiency and toxicity symptoms in fruits and nuts, 2:150-151 Ericaceae nutrition, 10:189-193 foliar application, 6:331 nutrition, 5:235-326 pine bark media, 9:123-124 Mango: alternate bearing, 4:145-146 asexual embryogenesis, 7:171-173 CA and MA, 22:151-157 CA storage, 1:313 in vitro culture, 7:171-173 Mangosteen, CA and MA, 22:157 Mechanical harvest, berry crops, 16:255-382 Mechanical stress regulation, 17:1-42 Media: fertilization, greenhouse crops, 5:333 pine bark, 9:103-131 Medicinal crops: artemisia, 19:319-371 poppy, 19:373-408 Melon grafting, 28:96-98 Meristem culture, 5:221-277 Metabolism: flower, 1:219-223 nitrogen in citrus, 8:181-215 seed,2:117-141 Micronutrients: container growing, 9:85-87 pine bark media, 9:119-124 Micropropagation, see also In vitro; propagation: bulbs, flowering, 18:89-113 environmental control, 17:125-172 nuts, 9:273-349 rose, 9:57-58 temperate fruits, 9:273-349 tropical fruits and palms, 7:157-200 Microtus. See Vole Modified atmosphere (MA) for tropical fruits, 22:123-183 Moisture, and seed storage, 2:125-132

Molecular biology: cassava, 26:85-159 floral induction, 27:3-20 flowering, 27:1-39;41-77 hormone reception, 26:49-84 Molybdenum nutrition, 5:328-329 Monocot, in vitro, 5:253-257 Monstera. See Aroids, ornamental Morphology: navel orange, 8:132-133 orchid,5:283-286 pecan flowering, 8:217-243 Moth bean, genetics, 2:373-374 Mung bean, genetics, 2:348-364 Mushroom: CA storage, 1:371-372 cultivation, 19:59-97 spawn, 6:85-118 Muskmelon, fertilization, 1:118-119 Mycoplasma-like organisms, tree short life, 2:50-51 Mycorrhizae: container growing, 9:93 Ericaceae, 10:211-212 fungi, 3:172-213 grape root, 5:145-146 N

Narcissus, 25:43-48 Navel orange, 8:129-179 Nectarine: bloom delay, 15:105-106 CA storage, 1:309-310 postharvest physiology, 11:413-452 Nematodes: aroids, 8:66 fig, 12:475-477 lettuce, 2:197-198 tree short life, 2:49-50 Nerine, 25:48-56 NFT (nutrient film technique), 5:1-44 Nitrogen: CA storage, 8:116-117 container growing, 9:80-82 deficiency and toxicity symptoms in fruits and nuts, 2:146 Ericaceae nutrition, 10:198-202 fixation in woody legumes, 14:322-323

CUMULATIVE SUBJECT INDEX foliar application, 6:332 in embryogenesis, 2:273-275 metabolism in apple, 4:204-246 metabolism in citrus, 8:181-215 metabolism in grapevine, 14:407-452 nutrition, 2:395,423; 5:319-320 pine bark media, 9:108-112 trickle irrigation, 4:29-30 vegetable crops, 22:185-223 Nomenclature, 28:1-60 Nondestructive quality evaluation of fruits and vegetables, 20:1-119 Nursery crops: fertilization, 1:106-112 nutrition, 9:75-101 Nut crops: see also individual crop almond postharvest technology and utilization, 20:267-311 chestnut blight, 8:291-336 fertilization, 1:106 honey bee pollination, 9:250-251 in vitro culture, 9:273-349 nutritional ranges, 2:143-164 pistachio culture, 3:376-396 Nutrient: concentration in fruit and nut crops, 2:154-162

film technique, 5:1-44 foliar-applied, 6:287-355 media, for asexual embryogenesis, 2:273-281

media, for organogenesis, 3:214-314 plant and tissue analysis, 7:30-56 solutions, 7:524-530 uptake, in trickle irrigation, 4:30-31 Nutrition (human): aroids, 8:79-84 CA storage, 8:101-127 phytochemicals in fruit, 27:269-315 phytochemicals in vegetables, 28:125-185

steroidal alkaloids, 25:171-196 Nutrition (plant): air pollution, 8:22-23, 26 blueberry, 10:183-227 calcifuge, 10:183-227 cold hardiness, 3:144-171 container nursery crops, 9:75-101 cranberry, 21:234-235

469

ecologically based, 24:156-172 embryogenesis, 1 :40-41 Ericaceae, 10:183-227 fire blight, 1:438-441 foliar, 6:287-355 fruit and nut crops, 2:143-164 ginseng, 9:209-211 greenhouse crops, 5:317-403 kiwifruit, 12:325-332 mycorrhizal fungi, 3:185-191 navel orange, 8:162-166 nitrogen in apple, 4:204-246 nitrogen in vegetable crops, 22:185-223

nutrient film techniques, 5:18-21, 31-53

ornamental aroids, 10:7-14 pine bark media, 9:103-131 raspberry, 11:194-195 slow-release fertilizers, 1:79-139

o Oil palm: asexual embryogenesis, 7:187-188 in vitro culture, 7:187-188 Okra: botany and horticulture, 21:41-72 CA storage, 1:372-373 Olive: alternate bearing, 4:140-141 salinity tolerance, 21:177-214 processing technology, 25:235-260 Onion: CA storage, 1:373-375 fluid drilling of seed, 3:17-18 Opium poppy, 19:373-408 Orange: see also Citrus alternate bearing, 4:143-144 sour, rootstock, 1:242-244 sweet, rootstock, 1:252-253 trifoliate, rootstock, 1:247-250 Orchard and orchard systems: floor management, 9:377-430 light, 2:208-267 root growth, 2:469-470 water, 7:301-344 Orchid: fertilization, 5:357-358

CUMULATIVE SUBJECT INDEX

470 Orchid (cont.) pollination regulation of flower development, 19:28-38 physiology, 5:279-315 Organogenesis, 3:214-314, see also In vitro; tissue culture Ornamental plants: see also individual plant Amaryllidaceae Banksia, 22:1-25 cactus grafting, 28-106-109 chlorosis, 9:168-169 fertilization, 1:98-104, 106-116 flowering bulb roots, 14:57-88 flowering bulbs in vitro, 18:87-169 foliage acclimatization, 6:119-154 heliconia, 14:1-55 Leucospermum, 22:27-90 orchid pollination regulation, 19:28-38 poppy, 19:373-408 protea leaf blackening, 17:173-201 rhododendron, 12:1-42 p

Paclobutrazol. See Triazole Papaya: asexual embryogenesis, 7:176-177 CA and MA, 22:157-160 CA storage, 1:314 in vitro culture, 7:175-178 Parsley: CA storage, 1:375 drilling of seed, 3:13-14 Parsnip, fluid drilling of seed, 3:13-14 Parthenocarpy, tomato, 6:65-84 Passion fruit: in vitro culture, 7:180-181 CA and MA, 22:160-161 Pathogen elimination, in vitro, 5:257-261 Peach: bloom delay, 15:105-106 CA storage, 1:309-310 origin, 17:333-379 postharvest physiology, 11:413-452 short life, 2:4 summer pruning, 9:351-375 thinning, 28:351-392 wooliness, 20:198-199

Peach palm (Pejibaye): in vitro culture, 7:187-188 Pear: bioregulation, 10:309-401 bloom delay, 15:106-107 CA storage, 1:306-308 decline, 2:11 fire blight control, 1:423-474 fruit disorders, 11:357-411; 27:227-267 fruit volatiles, 28:237-324 in vitro, 9:321 maturity indices, 13:407-432 root distribution, 2:456 scald, 27:227-267 short life, 2:6 Pecan: alternate bearing, 4:139-140 fertilization, 1:106 flowering, 8:217-255 in vitro culture, 9:314-315 Pejibaye, in vitro culture, 7:189 Pepper (Capsicum): CA storage, 1:375-376 fertilization, 1:119 fluid drilling in seed, 3:20 grafting, 28:104-105 phytochemicals, 28:161-162 Persimmon: CA storage, 1:314 quality, 4:259 Pest control: aroids (edible), 12:168-169 aroids (ornamental), 10:18 cassava, 12:163-164 cowpea, 12:210-213 ecologically based, 24:172-201 fig, 12:442-477 fire blight, 1:423-474 ginseng, 9:227-229 greenhouse management, 13:1-66 hydroponics, 7:530-534 sweet potato, 12:173-175 vertebrate, 6:253-285 yam (Dioscorea), 12:181-183 Petal senescence, 11:15-43 pH:

container growing, 9:87-88 fertilization greenhouse crops, 5:332-333

CUMULATIVE SUBJECT INDEX

pine bark media, 9:114-117 soil testing, 7:8-12, 19-23 Phase change, 7:109-155 Phenology: apple, 11:231-237 raspberry, 11:186-190 Philodendron. See Aroids, ornamental Phosphonates, Phytophthora control, 17:299-330 Phosphorus: container growing, 9:82-84 deficiency and toxicity symptoms in fruits and nuts, 2:146-147 nutrition, 5:320-321 pine bark media, 9:112-113 trickle irrigation, 4:30 Photoautotrophic micropropagation, 17:125-172 Photoperiod, 4:66-105,116-117; 17:73-123 flowering, 15:282-284, 310-312 Photosynthesis: cassava, 13:112-114 efficiency, 7:71-72; 10:378 fruit crops, 11:111-157 ginseng, 9:223-226 light, 2:237-238 Physiology: see also Postharvest physiology bitter pit, 11 :289-355 blueberry development, 13:339-405 cactus reproductive biology, 18:321-346 calcium, 10:107-152 carbohydrate metabolism, 7:69-108 cassava, 13:105-129 citrus cold hardiness, 7:201-238 conditioning 13:131-181 cut flower, 1:204-236; 3:59-143; 10:35-62 desiccation tolerance, 18:171-213 disease resistance, 18:247-289 dormancy, 7:239-300 embryogenesis, 1:21-23; 2:268-310 floral scents, 24:31-53 flower development, 19:1-58 flowering, 4:106-127 fruit ripening, 13:67-103 fruit softening, 10:107-152 ginseng, 9:211-213

471 glucosinolates, 19:99-215 grafting, 28:78-84 heliconia, 14:5-13 hormone reception, 26:49-84 juvenility, 7:109-155 lettuce seed germination, 24:229-275 light tolerance, 18:215-246 loquat, 23:242-252 lychee reproduction, 28:393-453 male sterility, 17:103-106 mechanical stress, 17:1-42 nitrogen metabolism in grapevine, 14:407-452 nutritional quality and CA storage, 8:118-120 olive salinity tolerance, 21:177-214 orchid,5:279-315 petal senescence, 11:15-43 photoperiodism, 17:73-123 pollution injury, 8:12-16 polyamines, 14:333-356 potato tuberization, 14:89-188 pruning, 8:339-380 raspberry, 11:190-199 regulation, 11:1-14 root pruning, 6:158-171 roots of flowering bulbs, 14:57-88 rose, 9:3-53 salinity hormone action, 16:1-32 salinity tolerance, 16:33-69 seed,2:117-141 seed priming, 16:109-141 strawberry flowering, 28:28:325-349 subzero stress, 6:373-417 summer pruning, 9:351-375 sweet potato, 23:277-338 thin cell layer morphogenesis, 14:239-264 tomato fruit ripening, 13:67-103 tomato parthenocarpy, 6:71-74 triazoles, 10:63-105; 24:55-138 tulip, 5:45-125 vernalization, 17:73-123 volatiles, 17:43-72 watercore, 6:189-251 water relations cut flowers, 18:1-85 waxes, 23:1-68 Phytochemicals, functional: fruits, 27:269-315 vegetables, 28:125-185

472

Phytohormones. See Growth substances Phytophthora control, 17:299-330 Phytotoxins, 2:53-56 Pigmentation: flower, 1:216-219 rose, 9:64-65 Pinching, by chemicals, 7:453-461 Pineapple: CA and MA, 22:161-162 CA storage, 1:314 genetic resources, 21:138-141 in vitro culture, 7:181-182 Pine bark, potting media, 9:103-131 Pistachio: alternate bearing, 4:137-139 culture, 3:376-393 in vitro culture, 9:315 Plantain: CA and MA, 22:141-146 in vitro culture, 7:178-180 Plant: classification, 28: 1-60 protection, short life, 2:79-84 systematics, 28:1-60 Plastic cover, sad production, 27:317-351 Plum: CA storage, 1:309 origin, 23:179-231 Poinsettia, fertilization, 1:103-104; 5:358-360 Pollen, desiccation tolerance, 18:195 Pollination: apple, 1:402-404 avocado, 8:272-283 cactus, 18:331-335 embryogenesis, 1:21-22 fig, 12:426-429 floral scents, 24:31-53 flower regulation, 19:1-58 fruit crops, 12:223-264 fruit set, 4:153-154 ginseng, 9:201-202 grape, 13:331-332 heliconia, 14:13-15 honey bee, 9:237-272 kiwifruit, 6:32-35 lychee, 28:422-428 navel orange, 8:145-146

CUMULATIVE SUBJECT INDEX orchid, 5:300-302 petal senescence, 11:33-35 protection, 7:463-464 rhododendron, 12:1-67 Pollution, 8:1-42 Polyamines, 14:333-356 chilling injury, 15:80 Polygalacturonase, 13:67-103 Poppy, opium, 19:373-408 Postharvest physiology: almond, 20:267-311 apple bitter pit, 11 :289-355 apple maturity indices, 13:407-432 apple scald, 27:227-257 apple weight loss, 25:197-234 aroids, 8:84-86 asparagus, 12:69-155 CA for tropical fruit, 22:123-183 CA storage and quality, 8:101-127 chlorophyll fluorescence, 23:69-107 coated fruits & vegetables, 26:161-238 cut flower, 1:204-236; 3:59-143; 10:35-62 foliage plants, 6:119-154 fruit, 1:301-336 fruit softening, 10:107-152 heat treatment, 22:91-121 lettuce, 2:181-185 low-temperature sweetening, 17:203-231 MA for tropical fruit, 22:123-183 navel orange, 8:166-172 nectarine, 11:413-452 nondestructive quality evaluation, 20:1-119 pathogens, 3:412-461 peach,11:413-452 pear disorders, 11:357-411; 27:227-267 pear maturity indices, 13:407-432 pear scald, 27:227-257 petal senescence, 11:15-43 protea leaf blackening, 17:173-201 quality evaluation, 20:1-119 scald, 27:227-267 seed,2:117-141 texture in fresh fruit, 20:121-244 tomato fruit ripening, 13:67-103 vegetables, 1:337-394

473

CUMULATIVE SUBJECT INDEX watercore, 6:189-251; 11:385-387 Potassium: container growing, 9:84 deficiency and toxicity symptoms in fruits and nuts, 2:147-148 foliar application, 6:331-332 nutrition, 5:321-322 pine bark media, 9:113-114 trickle irrigation, 4:29 Potato: CA storage, 1:376-378 classification, 28:23-26 fertilization, 1:120-121 low temperature sweetening, 17:203-231 phytochemicals, 28:160-161 tuberization, 14:89-198 Processing, table olives, 25:235-260 Propagation: see also In vitro apple, 10:324-326; 12:288-295 aroids, ornamental, 10:12-13 bioreactor technology, 24:1-30 cassava, 13:120-123 floricultural crops, 7:461-462 ginseng, 9:206-209 orchid,5:291-297 pear, 10:324-326 rose, 9:54-58 tropical fruit, palms 7:157-200 woody legumes in vitro, 14:265-332 Protaceous flower crop: see also Protea Banksia, 22:1-25

Leucospermum, 22:27-90 Protea, leaf blackening, 17:173-201 Protected crops, carbon dioxide, 7:345-398 Protoplast culture, woody species, 10:173-201 Pruning, 4:161; 8:339-380 apple, 9:351-375 apple training, 1:414 chemical, 7:453-461 cold hardiness, 11:56 fire blight, 1:441-442 grapevines, 16:235-254 light interception, 2:250-251 peach,9:351-375 phase change, 7:143-144 root, 6:155-188

Prunus: see also Almond; Cherry; Nectarine; Peach; Plum in vitro, 5:243-244; 9:322 root distribution, 2:456

Pseudomonas: phaseo1icola, 3:32-33, 39,44-45 solanacearum, 3:33 syringae, 3:33, 40; 7:210-212 Pumpkin, history, 25:71-170 Q

Quality evaluation: fruits and vegetables, 20:1-119, 121-224 nondestructive, 20:1-119 texture in fresh fruit, 20:121-224 R

Rabbit, 6:275-276 Radish, fertilization, 1:121 Rambutan: See Sapindaceous fruits CA and MA, 22:163 Raspberry: harvesting, 16:282-298 productivity, 11:185-228 Rejuvenation: rose, 9:59-60 woody plants, 7:109-155 Replant problem, deciduous fruit trees, 2:1-116 Respiration: asparagus postharvest, 12:72-77 fruit in CA storage, 1:315-316 kiwifruit, 6:47-48 vegetables in CA storage, 1:341-346 Rhizobium, 3:34,41 Rhododendron, 12:1-67 Rice bean, genetics, 2:375-376 Root: apple, 12:269-272 cactus, 18:297-298 diseases, 5:29-31 environment, nutrient film technique, 5:13-26 Ericaceae, 10:202-209 grape, 5:127-168 kiwifruit, 12:310-313

CUMULATIVE SUBJECT INDEX

474

Root (cant.) physiology of bulbs, 14:57-88 pruning, 6:155-188 raspberry, 11 :190 rose, 9:57 tree crops, 2:424-490 Root and tuber crops: Amaryllidaceae, 25:1-79 aroids, 8:43-99; 12:166-170 cassava, 12:158-166; 26:85-159 low-temperature sweetening, 17:203-231

minor crops, 12:184-188 potato tuberization, 14:89-188 sweet potato, 12:170-176 sweet potato physiology, 23:277-338 yam (Dioscorea), 12:177-184 Rootstocks: alternate bearing, 4:148 apple, 1:405-407; 12:295-297 avocado, 17:381-429 citrus, 1:237-269 cold hardiness, 11:57-58 fire blight, 1:432-435 light interception, 2:249-250 navel orange, 8:156-161 root systems, 2:471-474 stress, 4:253-254 tree short life, 2:70-75 Rosaceae, in vitro, 5:239-248 Rose: fertilization, 1:104; 5:361-363 growth substances, 9:3-53 in vitro, 5:244-248

s Salinity: air pollution, 8:25-26 olive, 21:177-214 soils, 4:22-27 tolerance, 16:33-69 Sapindaceous fruits, 16:143-196 Sapodilla, CA and MA, 22:164 Scadoxus, 25:25-28

Scald, apple and pear, 27:227-265 Scoring, and fruit set, 1:416-417 Seed: abortion, 1:293-294

apple anatomy and morphology, 10:285-286

conditioning, 13:131-181 desiccation tolerance, 18:196-203 environmental influences on size and composition, 13:183-213 flower induction, 4:190-195 fluid drilling, 3:1-58 grape seedlessness, 11:159-184 kiwifruit, 6:48-50 lettuce, 2:166-174 lettuce germination, 24:229-275 priming, 16:109-141 rose propagation, 9:54-55 vegetable, 3:1-58 viability and storage, 2:117-141 Secondary metabolites, woody legumes, 14:314-322

Senescence: chlorophyll senescence, 23:88-93 cut flower, 1:204-236; 3:59-143; 10:35-62; 18:1-85

petal, 11:15-43 pollination-induced, 19:4-25 rose, 9:65-66 whole plant, 15:335-370 Sensory quality: CA storage, 8:101-127 Shoot-tip culture, 5:221-277, see also Micropropagation Short life problem, fruit crops, 2:1-116 Signal transduction, 26:49-84 Small fruit, CA storage, 1:308 Snapdragon fertilization, 5:363-364 Sod production, 27:317-351 Sodium, deficiency and toxicity symptoms in fruits and nuts, 2:153-154

Soil: grape root growth, 5:141-144 management and root growth, 2:465-469

orchard floor management, 9:377-430 plant relations, trickle irrigation, 4:18-21

stress, 4:151-152 testing, 7:1-68; 9:88-90 zinc, 23:109-178 Soilless culture, 5:1-44

475

CUMULATIVE SUBJECT INDEX Solanaceae: in vitro, 5:229-232 steroidal alkaloids, 25:171-196 Somatic embryogenesis. See Asexual embryogenesis Sorghum, sweet, 21:73-104 Spathiphyllum. See Aroids, ornamental Squash, history, 25:71-170 Stem, apple morphology, 12:272-283 Sternbergia, 25:59 Steroidal alkaloids, solanaceous, 25:171-196 Storage: see also Postharvest physiology, Controlled-atmosphere (CA) storage cut flower, 3:96-100; 10:35-62 rose plants, 9:58-59 seed,2:117-141 Strawberry: fertilization, 1:106 flowering, 28:325-349 fruit growth and ripening, 17:267-297 functional phytonutrients, 27: 303-304 harvesting, 16:348-365 in vitro, 5:239-241 Stress: benefits of, 4:247-271 chlorophyll fluorescence, 23:69-107 climatic,4:150-151 flooding, 13:257-313 mechanical, 17:1-42 petal, 11:32-33 plant, 2:34-37 protectants (triazoles), 24:55-138 protection, 7:463-466 salinity tolerance in olive, 21:177-214 subzero temperature, 6:373-417 waxes, 23:1-68 Sugar: see also Carbohydrate allocation, 7:74-94 flowering, 4:114 Sugar apple, CA and MA, 22:164 Sugar beet, fluid drilling of seed, 3:18-19 Sulfur: deficiency and toxicity symptoms in fruits and nuts, 2:154 nutrition, 5:323-324 Sweet potato: culture, 12:170-176

fertilization, 1:121 physiology, 23:277-338 Sweet sop, CA and MA, 22:164 Symptoms, deficiency and toxicity symptoms in fruits and nuts, 2:145-154 Syngonium. See Aroids, ornamental Systematics, 28:1-60 T Taro. See Aroids, edible Taxonomy, 28:1-60 Tea, botany and horticulture, 22:267-295 Temperature: apple fruit set, 1:408-411 bloom delay, 15:119-128 CA storage of vegetables, 1:340-341 chilling injury, 15:67-74 cryopreservation, 6:357-372 cut flower storage, 10:40-43 fertilization, greenhouse crops, 5:331-332 fire blight forecasting, 1:456-459 flowering, 15:284-287, 312-313 interaction with photoperiod, 4:80-81 low temperature sweetening, 17:203-231 navel orange, 8:142 nutrient film technique, 5:21-24 photoperiod interaction, 17:73-123 photosynthesis, 11:121-124 plant growth, 2:36-37 seed storage, 2:132-133 subzero stress, 6:373-417 Texture in fresh fruit, 20:121-224 Thinning: apple, 1:270-300 peach and Prunus, 28:351-392 Tipburn, in lettuce, 4:49-65 Tissue: see also In vitro culture 1:1-78; 2:268-310; 3:214-314; 4:106-127; 5:221-277; 6:357-372; 7:157-200; 8:75-78; 9:273-349; 10:153-181, 24:1-30 cassava, 26:85-159 dwarfing, 3:347-348 nutrient analysis, 7:52-56; 9:90

476 Tomato: CA storage, 1:380-386 classification, 28:21-23 chilling injury, 20:199-200 fertilization, 1:121-123 fluid drilling of seed, 3:19-20 fruit ripening, 13:67-103 galacturonase, 13:67-103 grafting,28:98-103 greenhouse quality, 26:239 parthenocarpy, 6:65-84 phytochemicals, 28:160 Toxicity symptoms in fruit and nut crops, 2:145-154 Transport, cut flowers, 3:100-104 Tree decline, 2:1-116 Triazoles, 10:63-105; 24:55-138 chilling injury, 15:79-80 Trickle irrigation, 4:1-48 Truffle cultivation, 16:71-107 Tuber, potato, 14:89-188 Tuber and root crops. See Root and tuber crops Tulip: See also Bulb fertilization, 5:364-366 in vitro, 18:144-145 physiology, 5:45-125 Tunnel (cloche), 7:356-357 Turfgrass, fertilization, 1:112-117 Turnip, fertilization, 1:123-124 Turnip Mosaic Virus, 14:199-238 U

Urd bean, genetics, 2:364-373 Urea, foliar application, 6:332 V

Vaccinium, 10:185-187, see also Blueberry; Cranberry; Lingonberry functional phytonutrients, 27:303 Vase solutions, 3:82-95; 10:46-51 Vegetable crops: see also Specific crop Allium phytochemicals, 28:156-159 aroids, 8:43-99; 12:166-170 asparagus postharvest, 12:69-155 cactus, 18:300-302

CUMULATIVE SUBJECT INDEX cassava, 12:158-166; 13:105-129; 26:85-159 CA storage, 1:337-394 CA storage and quality, 8:101-127 CA storage diseases, 3:412-461 Caper bush, 27:125-188 chilling injury, 15:63-95 coating physiology, 26:161-238 crucifer phytochemicals, 28:150-156 cucumber grafting, 28:91-96 ecologically based, 24:139-228 eggplant grafting, 28:103-104 eggplant phytochemicals, 28:162-163 fertilization, 1:117-124 fluid drilling of seeds, 3:1-58 gourd history, 25:71-170 grafting, 28:61-124 greenhouse management, 21:1-39 greenhouse pest management, 13:1-66 honey bee pollination, 9:251-254 hydroponics, 7:483-558 lettuce seed germination, 24:229-275 low-temperature sweetening, 17:203-231 melon grafting, 28:96-98 minor root and tubers, 12:184-188 mushroom cultivation, 19:59-97 mushroom spawn, 6:85-118 N nutrition, 22:185-223 nondestructive postharvest quality evaluation, 20:1-119 okra, 21:41-72 pepper phytochemicals, 28:161-162 phytochemicals, 28:125-185 potato phytochemicals, 28:160-161 potato tuberization, 14:89-188 pumpkin history, 25:71-170 seed conditioing, 13:131-181 seed priming, 16:109-141 squash history, 25:71-170 steroidal alkaloids, Solanaceae, 25:171-196 sweet potato, 12:170-176 sweet potato physiology, 23:277-338 tomato fruit ripening, 13:67-103 tomato (greenhouse) quality: 26:239-319 tomato parthenocarpy, 6:65-84

477

CUMULATIVE SUBJECT INDEX tomato phytochemicals, 28:160 tropical production, 24:139-228 truffle cultivation, 16:71-107 watermelon grafting, 28:86-91 yam (Dioscorea), 12:177-184 Vegetative tissue, desiccation tolerance, 18:176-195 Vernalization, 4:117; 15:284-287; 17:73-123 Vertebrate pests, 6:253-285 Vigna: see also Cowpea genetics, 2:311-394 U.S. production, 12:197-222 Viroid, dwarfing for citrus, 24:277-317 Virus: benefits in horticulture, 3:394-411 dwarfing for citrus, 24:277-317 elimination, 7:157-200; 9:318; 18:113-123; 28:187-236 fig, 12:474-475 tree short life, 2:50-51 turnip mosaic, 14:199-238 Volatiles, 17:43-72; 24:31-53; 28:237-324 Vole, 6:254-274

w Walnut, in vitro culture, 9:312 Water relations: cut flower, 3:61-66; 18:1-85 deciduous orchards, 21:105-131 desiccation tolerance, 18:171-213 fertilization, grape and grapevine, 27:189-225 kiwifruit, 12:332-339 light in orchards, 2:248-249 photosynthesis, 11:124-131 trickle irrigation, 4:1-48 Watercore, 6:189-251 apple, 6:189-251 pear, 11:385-387

Watermelon: fertilization, 1:124 grafting, 28:86-91 Wax apple, CA and MA, 22:164 Waxes, 23:1-68 Weed control, ginseng, 9:228-229 Weeds: lettuce research, 2: 198 virus, 3:403 Woodchuck,6:276-277 Woody species, somatic embryogenesis, 10:153-181

x Xanthomonas phaseoli, 3:29-32, 41, 45-46 Xanthophyll cycle, 18:226-239 Xanthosoma, 8:45-46, 56-57, see also Aroids Sugar: see also Carbohydrate allocation, 7:74-94 flowering, 4:114 y Yam (Dioscorea), 12:177-184 Yield: determinants, 7:70-74; 97-99 limiting factors, 15:413-452

z Zantedeschia. See Aroids, ornamental Zephyranthes,25:60-61 Zinc: deficiency and toxicity symptoms in fruits and nuts, 2:151 foliar application, 6:332, 336 nutrition, 5:326; 23:109-178 pine bark media, 9:124

Cumulative Contributor Index (Volumes 1-28)

Abbott, J.A., 20:1 Adams III, W.W., 18:215 Aldwinckle, H.S., 1:423; 15:xiii Amarante, e., 28:161 Anderson, I.e., 21:73 Anderson, J.L., 15:97 Anderson, P.e., 13:257 Andrews, P.K., 15:183 Ashworth, E.N., 13:215; 23:1 Asokan, M.P., 8:43 Atkinson, D., 2:424 Aung, L.H., 5:45

Bower, J.P., 10:229 Bradley, G.A., 14:xiii Brandenburg, W., 28:1 Brennan, R, 16:255 Broadbent, P., 24:277 Broschat, T.K., 14:1 Brown, S. 15:xiii Buban, T., 4:174 Bukovac, M.J., 11:1 Burke, M.J., 11:xiii Buwalda, J.G., 12:307 Byers, RE., 6:253; 28:351

Bailey, W.G., 9:187 Baird, L.A.M., 1:172 Banks, N.H., 19:217; 25:197; 26:161 Barden, J.A., 9:351 Barker, A.V., 2:411 Bass, L.N., 2:117 Bassett, e. L., 26:49 Becker, J.S., 18:247 Beer, S.V., 1:423 Behboudian, M.H., 21:105; 27:189 Bennett, AB., 13:67 Benschop, M., 5:45 Ben-Ya'acov, A., 17:381 Benzioni, A, 17:233 Bevington, K.B., 24:277 Bewley, J.D., 18:171 Binzel, M.L., 16:33 Blanpied, G.D., 7:xi Bliss, F.A, 16:xiii; 28:xi Boardman, K. 27 xi Borochov, A, 11:15

Caldas, L.S., 2:568 Campbell, L.E., 2:524 Cantliffe, D.J., 16:109; 17:43; 24:229; 28:325 Carter, G., 20:121 Carter, J.V., 3:144 Cathey, H.M., 2:524 Chambers, RJ., 13:1 Chandler, e.K. 28:325 Charron, e.S., 17:43 Chen, Z., 25:171 Chin, e.K., 5:221 Clarke, N.D., 21:1 Coetzee, J. H., 26:1 Cohen, M., 3:394 Collier, G.F., 4:49 Collins, G., 25:235 Collins, W.L., 7:483 Colmagro, S., 25:235 Compton, M.E., 14:239 Conover, e.A., 5:317; 6:119

Horticultural Reviews, Volume 28, Edited by Jules Janick ISBN 0-471-21542-2 © 2003 John Wiley & Sons, Inc. 478

479

CUMULATIVE CONTRIBUTOR INDEX Coppens d'Eeckenbrugge, G., 21:133 Costa, G. 28:351 Coyne, D.P., 3:28 Crane, J.e., 3:376 Criley, RA., 14:1; 22:27; 24:x Crawly, W., 15:1 Cutting, J.G., 10:229 Daie, J., 7:69 Dale, A., 11:185; 16:255 Darnell, RL., 13:339, 28:325 Davenport, T.L., 8:257; 12:349 Davies, F.S., 8:129; 24:319 Davies, P.J., 15:335 Davis, T.D., 10:63; 24:55 Decker, H.F., 27:317 DeEll, J.R, 23:69 DeGrandi-Hoffman, G., 9:237 De Hertogh, A.A., 5:45; 14:57; 18:87; 25:1 Deikman, J., 16:1 DellaPenna, D., 13:67 Demmig-Adams, B., 18:215 Dennis, F.G., Jr., 1:395 Dorais, M., 26:239 Doud, S.L., 2:1 Dudareva, N., 24:31 Duke, S.O., 15:371 Dunavent, M.G., 9:103 Duval, M.-F., 21:133 Diizyaman, E., 21:41 Dyer, W.E., 15:371 Early, J.D., 13:339 Eastman, K., 28:125 Elfving, D.e., 4:1; 11:229 EI-Goorani, M.A., 3:412 Esan, E.B., 1:1 Evans, D.A., 3:214 Ewing, E.E., 14:89 Faust, M., 2:vii, 142; 4:174; 6:287; 14:333; 17:331; 19:263; 22:225; 23:179 Fenner, M., 13:183 Fenwick, G.R, 19:99 Ferguson, A.R, 6:1 Ferguson, I.B., 11:289 Ferguson, J.J., 24:277 Ferguson, L., 12:409 Ferree, D.e., 6:155 Ferreira, J.F.S., 19:319

Fery, RL., 2:311; 12:157 Fischer, RL., 13:67 Fletcher, RA., 24:53 Flick, e.E., 3:214 Flore, J.A., 11:111 Forshey, e.G., 11:229 Franks, R G., 27:41 Fujiwara, K., 17:125 Gazit, S., 28:393 Geisler, D., 6:155 Geneve, RL., 14:265 George, W.L., Jr., 6:25 Gerrath, J.M., 13:315 Gilley, A., 24:55 Giovannetti, G., 16:71 Giovannoni, J.J., 13:67 Glenn, G.M., 10:107 Goffinet, M.e., 20:ix Goldschmidt, E.E., 4:128 Goldy, RG., 14:357 Goren, R, 15:145 Gosselin, A., 26:239 Goszczynska, D.M., 10:35 Grace, S.e., 18:215 Graves, CJ., 5:1 Gray, D., 3:1 Grierson, W., 4:247 Griffen, G.J., 8:291 Grodzinski, B., 7:345 Gucci, R, 21:177 Guest, D.I., 17:299 Guiltinan, M.J., 16:1 Hackett, W.P., 7:109 Halevy, A.H., 1:204; 3:59 Hallett, I.C, 20:121 Hammerschmidt, R, 18:247 Hanson, E.J., 16:255 Harker, F.R, 20:121 Heaney, RK., 19:99 Heath, RR, 17:43 Helzer, N.L., 13:1 Hendrix, J.W., 3:172 Henny, RJ., 10:1 Hergert, G.B., 16:255 Hess, F.D., 15:371 Hetterscheid, W.L.A., 28:1 Heywood, V., 15:1 Hjalmarsson, I., 27:79-123

480 Hogue, E.]., 9:377 Holt, J.S., 15:371 Huber, D.J., 5:169 Hunter, E.1., 21:73 Hutchinson, J.F., 9:273 Hutton, RJ., 24:277 Indira, P., 23:277 Ingle, M. 27:227 Isenberg, F.M.R, 1:337 Iwakiri, B.T., 3:376 Jackson, J.E., 2:208 Janick, J., 1:ix; 8:xi; 17:xiii; 19:319; 21:xi; 23:233 Jarvis, W.R, 21:1 Jenks, M.A., 23:1 Jensen, M.H., 7:483 Jeong, B.R, 17:125 Jewett, T.J., 21:1 Joiner, J.N., 5:317 Jones, H.G., 7:301 Jones, J.B., Jr., 7:1 Jones, RB., 17:173 Kagan-Zur, V., 16:71 Kalt, W. 27:269; 28:125 Kang, S.-M., 4:204 Kato, T., 8:181 Kawa, L., 14:57 Kawada, K., 4:247 Kelly, J.F., 10:ix; 22:xi Kester, D.E., 25:xii Khan, A.A., 13:131 Kierman, J., 3:172 Kim, K.-W., 18:87 Kinet, J.-M., 15:279 King, G.A., 11:413 Kingston, CM., 13:407-432 Kirschbaum, D.S. 28:325 Kliewer, W.M., 14:407 Knight, R.J., 19:xiii Knox, RB., 12:1 Kofranek, A.M., 8:xi Korcak, RF., 9:133; 10:183 Kozai, T., 17:125 Krezdorn, A.H., l:vii Kushad, M.M., 28:125

CUMULATIVE CONTRIBUTOR INDEX Lakso, A.N., 7:301; 11:111 Laimer, M., 28:187 Lamb, RC, 15:xiii Lang, G.A., 13:339 Larsen, RP., 9:xi Larson, RA., 7:399 Leal, F., 21:133 Ledbetter, CA., 11:159 Lee, J.-M., 28:61 Li, P.H., 6:373 Lill, RE., 11:413 Lin, S., 23:233 Liu, Z., 27:41 Lipton, W.J., 12:69 Littlejohn, G.M., 26:1 Litz, RE., 7:157 Lockard, RG., 3:315 Loescher, W.H., 6:198 Lorenz, O.A., 1:79 Lu, R, 20:1 Lurie, S., 22:91-121 Lyrene, P., 21:xi Maguire, K.M., 25:197 Manivel, 1., 22:267 Maraffa, S.B., 2:268 Marangoni, A.G., 17:203 Marini, RP., 9:351 Marlow, G.C, 6:189 Maronek, D.M., 3:172 Martin, G.G., 13:339 Masiunas, J., 28:125 Mayak, S., 1:204; 3:59 Maynard, D.N., 1:79 McConchie, R, 17:173 McNicol, RJ., 16:255 Merkle, S.A., 14:265 Michailides, T.J., 12:409 Michelson, E., 17:381 Mika, A., 8:339 Miller, A.R, 25:171 Miller, S.S., 10:309 Mills, H.A., 2:411; 9:103 Mills, T.M., 21:105 Mitchell, CA., 17:1 Mizrahi, Y., 18:291,321 Molnar, ].M., 9:1 Monk, G.]., 9:1

CUMULATIVE CONTRIBUTOR INDEX Monselise, S.P., 4:128 Moore, G.A, 7:157 Mor, Y., 9:53 Morris, J.R, 16:255 Murashige, T., 1:1 Murr, D.P., 23:69 Murray, S.H., 20:121 Myers, P.N., 17:1 Nadeau, J.A., 19:1 Nascimento, W.M., 24:229 Neilsen, G.H., 9:377 Nelson, P.V., 26:xi Nerd, A., 18:291, 321 Niemiera, AX., 9:75 Nobel, P.S., 18:291 Nyujto, F., 22:225 Oda, M., 28:61 O'Donoghue, E.M., 11:413 Ogden, RJ., 9:103 O'Hair, S.K, 8:43; 12:157 Oliveira, CM., 10:403 Oliver, M.J., 18:171 O'Neill, S.D., 19:1 Opara, L.U., 19:217; 24:373; 25:197 Ormrod, D.P., 8:1 Ortiz, R, 27:79 Palser, B.F., 12:1 Papadopoulos, A.P., 21:1; 26:239 Pararajasingham, S., 21:1 Parera, CA., 16:109 Paris, H.S., 25:71 Pegg, KG., 17:299 Pellett, H.M., 3:144 Perkins-Veazil, P., 17:267 Pichersky, E., 24:31 Piechulla, B., 24:31 Ploetz, RC, 13:257 Pokorny, F.A., 9:103 Poole, RT., 5:317; 6:119 Poovaiah, B.W., 10:107 Portas, CAM., 19:99 Porter, M.A, 7:345 Possingham, J.V., 16:235 Prange, RK, 23:69 Pratt, C, 10:273; 12:265

481 Predieri, S., 28:237 Preece, J.K, 14:265 Priestley, CA, 10:403 Proctor, J.T.A, 9:187 Puonti-Kaerlas, J., 26:85 Quamme, H., 18:xiii Raese, J.T., 11:357 Ramming, D.W., 11:159 Rapparini, F., 28:237 Ravi, V., 23:277 Reddy, A.S.N., 10:107 Redgwell, RJ., 20:121 Reid, M., 12:xiii; 17:123 Reuveni, M., 16:33 Richards, D., 5:127 Rieger, M., 11:45 Roper, T.R, 21:215 Rosa, E.A.S., 19:99 Roth-Bejerano, N., 16:71 Roubelakis-Angelakis, KA., 14:407 Rouse, J.1., 12:1 Royse, D.J., 19:59 Rudnicki, RM., 10:35 Ryder, KJ., 2:164; 3:vii Sachs, R, 12:xiii Sakai, A., 6:357 Salisbury, F.B., 4:66; 15:233 Saltveit, M.E., 23:x San Antonio, J.P., 6:85 Sankhla, N., 10:63; 24:55 Saure, M.e, 7:239 Schaffer, B., 13:257 Schenk, M.K., 22:185 Schneider, G.W., 3:315 Schuster, M.1., 3:28 Scorza, R, 4:106 Scott, J.W., 6:25 Sedgley, M., 12:223; 22:1; 25:235 Seeley, S.S., 15:97 Serrano Marquez, C, 15:183 Sharp, W.R, 2:268; 3:214 Sharpe, RH., 23:233 Shattuck, V.I., 14:199 Shear, C.B., 2:142 Sheehan, T.J., 5:279

482 Shipp, J.L., 21:1 Shirra, M., 20:267 Shorey, RH., 12:409 Simon, J.E., 19:319 Singh, Z. 27:189 Sklensky, D.E., 15:335 Smith, A.H., Jr., 28:351 Smith, M.A.L., 28:125 Smith, G.S., 12:307 Smock, RM., 1:301 Sommer, N.F., 3:412 Sondahl, M.R, 2:268 Sopp, P.I., 13:1 Soule, J., 4:247 Sozzi, G. 0., 27:125 Sparks, D., 8:217 Splittstoesser, W.E., 6:25; 13:105 Spooner, D.M., 28:1 Srinivasan, c., 7:157 Stang, E.J., 16:255 Steffens, G.L., 10:63 Stern, RA., 28:393 Stevens, M.A., 4:vii Stroshine, RL., 20:1 Struik, P.e., 14:89 Studman, c.J., 19:217 Stutte, G.W., 13:339 Styer, D.J., 5:221 Sunderland, K.D., 13:1 Sung, Y., 24:229 Suninyi, D., 19:263; 22:225; 23:179 Swanson, B., 12:xiii Swietlik, D., 6:287; 23:109 Syvertsen, J.P., 7:301 Tattini, M., 21:177 Tetenyi, P., 19:373 Theron, K.I., 25:1 Tibbitts, T.W., 4:49 Timon, B., 17:331 Tindall, H.D., 16:143 Tisserat, B., 1:1 Titus, J.S., 4:204 Trigiano, RN., 14:265 Tunya, G.O., 13:105

CUMULATIVE CONTRIBUTOR INDEX Upchurch, B.L., 20:1 Valenzuela, H.R, 24:139 van Doorn, W.G., 17:173; 18:1 van den Berg, W.L.A., 28:1 van Kooten, 0., 23:69 van Nocker, S. 27:1 Veilleux, RE., 14:239 Vorsa, N., 21:215 Vizzotto, G., 28: 351 Wallace, A., 15:413 Wallace, D.H., 17:73 Wallace, G.A., 15:413 Wang, c.Y., 15:63 Wang, S.Y., 14:333 Wann, S.R, 10:153 Watkins, C.B., 11:289 Watson, G.W., 15:1 Webster, B.D., 1:172; 13:xi Weichmann, J., 8:101 Wetzstein, H.Y., 8:217 Whiley, A.W., 17:299 Whitaker, T.W., 2:164 White, J.W., 1:141 Williams, E.G., 12:1 Williams, M.W., 1:270 Wismer, W.V., 17:203 Wittwer, S.H., 6:xi Woodson, W.R, 11:15 Wright, R.D., 9:75 Wutscher, H.K., 1:237 Yada, RY., 17:203 Yadava, U.L., 2:1 Yahia, E.M., 16:197; 22:123 Yan, W., 17:73 Yarborough, D.E., 16:255 Yelenosky, G., 7:201 Zanini, E., 16:71 Zieslin, N., 9:53 Zimmerman, RH., 5:vii; 9:273 Ziv, M., 24:1 Zucconi, F., 11:1