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:
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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
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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)
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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
Hawkes 1990
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et
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Chomatophylla ex"",ool'O 1989 '-+--!---!-+{ 1 I - f-I-' Ingifolia Ochoo ex C>rr<1I 1%1 • (S 21+-~-+-H61+--l-+-r-tf-------+<2 ::M:!!.aglia 2:=·=Bi.::..'rer=191:::.2=:::..:.::=---t--.::::+++-I-----.-:::,"4-+-+-87~R=f-++------t-< 1 Tuberosa CRydb)H.wI<.... I944
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~
Subacaulia Bu!<,n<>mnuJ VaviIoviana BU~~:e::ki~9 Acaulia JU:K::=::'1~59 Longipedicellata JU:K:'::~~ ~59
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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."
1. PLANT NOMENCLATURE AND TAXONOMY
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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LEE AND M. QDA
(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.
84
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
J.
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)
90
]. 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
2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS
91
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
J. LEE AND M. aDA
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.
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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
2. GRAFTING OF HERBACEOUS VEGETABLE AND ORNAMENTAL CROPS
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.
J. LEE AND M. aDA
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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
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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.
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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]
172
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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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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.
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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
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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 observati