Plant Breeding Reviews, Volume 20

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WILEYe BOOK WILEY JOSSEY-BASS PFEIFFER J.K.LASSER CAPSTONE WILEY-LISS WILEY-VCH WILEY-INTERSCIENCE

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PLANT BREEDING REVIEWS Volume 20

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Plant Breeding Reviews is sponsored by: American Society for Horticultural Science Crop Science Society of America Society of American Foresters National Council of Commercial Plant Breeders

Editorial Board, Volume 20 G. R. Askew F. A. Bliss M. Gilbert

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PLANT BREEDING REVIEWS Volume 20

edited by

Jules Janick Purdue University

NEW YORK

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

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This book is printed on acid-free paper. Copyright © 2001 by John Wiley & Sons, Inc. 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: PERMREQ @ WILEY.COM. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold with the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional person should be sought. Library of Congress Catalog Card Number: 83-641963 ISBN 0-471-38788-6 ISSN 0730-2207 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

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Contents

List of Contributors 1. Dedication: Norman Willison Simmonds; Plant Breeder, Teacher, Administrator

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William Spoor and Frederick England

2. The Origin of Maize: Evidence for Tripsacum Ancestry Mary W. Eubanks I. Introduction II. Theoretical Framework III. Taxonomic Classification IV. The Nature of the Evidence V. Synthesis and Future Directions Literature Cited

3. History of Public Onion Breeding Programs in the United States

15 16 18 22 31 55 57

67

Irwin L. Goldman, Geoffrey Schroeck, and Michael J. Harvey I. Introduction 68 II. History of Onion in the Americas 71 III. State Experiment Station Programs 95 IV. Future Prospects for Public Onion Breeding in the United States 99 Literature Cited 101 v

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CONTENTS

4. Current and Future Issues in Lettuce Breeding

105

Edward J. Ryder I. Introduction II. History of Lettuce Breeding III. Breeding Efforts IV. Lettuce Breeding for the Future Literature Cited

105 106 107 117 130

5. Cactus Pear Domestication and Breeding

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Candelario Mondragon Jacobo I. Introduction II. Origin and Early Development III. Genetic Resources IV. Breeding Objectives V. Breeding Techniques VI. Breeding Systems VII. Summary and Future Prospects Literature Cited

136 138 141 145 152 158 161 162

6. Somatic Hybridization and Applications in Plant Breeding

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Alexander A. T. Johnson and Richard E. Veilleux I. Introduction II. Recent Efforts in Somatic Hybridization III. Conclusion Literature Cited

168 173 209 212

Subject Index

227

Cumulative Subject Index

229

Cumulative Contributor Index

245

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Contributors Frederick England, Formerly of Scottish Plant Breeding Station, Pentlandfield, Midlothian, Scotland Mary W. Eubanks, Department of Botany, Duke University, Durham, North Carolina 27708-0338, [email protected] Irwin L. Goldman, Department of Horticulture, 1575 Linden Drive, University of Wisconsin–Madison, Madison, Wisconsin 53706, [email protected] Michael J. Harvey, USDA—Agricultural Research Service and Department of Horticulture, 1575 Linden Drive, University of Wisconsin–Madison, Madison, Wisconsin 53706 Candelario Mondragon Jacobo, Instituto Nacional de Investigaciones Forestales y Agropecuarias, Guanajuato, Mexico Alexander A. T. Johnson, Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0327 Edward J. Ryder, U.S. Agricultural Research Station, U.S. Department of Agriculture, Agricultural Research Service, 1636 E. Alisal Street, Salinas, California 93905, [email protected] Geoffrey Schroek, Department of Horticulture, 1575 Linden Drive, University of Wisconsin–Madison, Madison, Wisconsin 53706 William Spoor, Department of Biotechnology, Plant Science Division, Scottish Agricultural College, West Mains Road, Edinburgh, EH9 3JG, Scotland [email protected] Richard E. Veilleux, Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0327 Masahiko Yamada, Persimmon and Grape Research Center, National Institute of Fruit Tree Science, Akitsu, Hiroshima 729-2494, Japan Keizo Yonemori, Laboratory of Pomology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan, [email protected] Zhao-Bang Zeng, Department of Statistics, North Carolina State University, Raleigh, NC 27695-1932

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Norman W. Simmonds

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1 Dedication: Norman Willison Simmonds Plant Breeder, Teacher, Administrator William Spoor Department of Biotechnology, Plant Science Division, Scottish Agricultural College, West Mains Road, Edinburgh, EH9 3JG, Scotland Frederick England Formerly of Scottish Plant Breeding Station, Pentlandfield, Midlothian, Scotland Norman W. Simmonds’s long and distinguished professional career has centered around three major crops, namely, bananas, potatoes, and sugar cane. Along the way, he has made additional substantial contributions in other crops, to plant breeding in general, to general taxonomy, economic botany, and tropical farming systems. In all these fields he has contributed creatively and provocatively. His output has been enormous, diverse, and profound.

CAREER Norman Willison Simmonds was born in Bedford (England) on December 15, 1922. His father was a civil servant and his mother of Scottish (Perthshire) farming stock, the Willisons, from whom he takes his middle name. Norman attended Whitgift School in Croydon from 1934 to 1940 and from there won an Open Exhibition (scholarship) to Downing College, Cambridge. At school he was stimulated, as were so many budding botanists, by Cecil T. Prime, that notable teacher. While at Cambridge, he was much influenced by Professor D. G. Catcheside, who

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encouraged his interest in genetics and cytogenetics. After a distinguished undergraduate career, he was awarded a First class degree in the Natural Sciences Tripos part II (Botany, with a strong bent to genetics and plant breeding). In 1943 he was granted a Colonial Agricultural Scholarship and studied at Cambridge and the Imperial College of Tropical Agriculture (ICTA) in Trinidad and in 1944 obtained his B.A. from the University of Cambridge. This was followed by an M.A. in 1948 and later, in 1966, a Sc.D. The introduction to tropical agriculture afforded by the time spent in Trinidad was to be the start of a life-time interest in tropical agriculture, the crops and the demands, following his employment as a Botany lecturer at ICTA in 1945. His stay in the West Indies was to last for 15 years, ending in 1959 as Senior Cytogeneticist, Banana Research Scheme. During this period he established himself as a vigorous researcher, initially with K. S. Dodds, developing a banana breeding strategy through constructed diploids crossed to triploids, thence to tetraploids. It was also during this time that he started to develop ideas on genetic resources, conservation, and utilization following two major collecting trips to East Africa (1948) and the Pacific, Malaysia, Thailand, and North India (1954/55). Material from these trips is now proving of value as later generation parents, but at the time led to enhanced evolutionary understanding of the group. This extensive experience led to two key books, the standard monograph Bananas (1959, 1966, with R. H. Stover in 1987), long regarded as the banana researcher’s bible, and Evolution of the Bananas (1962) in addition to 40 papers published during his period in Trinidad. In 1959, Norman Simmonds returned to the United Kingdom as Head of the Potato Genetics Department at the John Innes Institute at Hertford, rejoining K. S. Dodds, the then Director. He characteristically threw himself into research in this new crop challenge, publishing an array of papers ranging over tuber dormancy, seed germination, polyploidy, callus differentation, virus transmission, chimeral and other mutants, linkage studies, and disease resistance. It was during this period that he developed the concept of genetic base broadening, now found to be fundamental and effective for potatoes. This has proved to be a valuable recurring theme in much of his later work as it became obvious that such an approach had general applicability to a very wide range of crop species. It is one of Norman’s regrets that, while his ideas on base broadening have received wide theoretical acceptance, they have been relatively little applied in practice, although enjoying considerable success in sugar cane improvement and to a lesser extent in oil palm and rubber development. While at the John Innes Institute, he started two very

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important consultancies: on West Indies sugar breeding in Barbados and on rubber in Malaysia. This broadening of his crop interests allowed him to bring experiences in other clonal crops to the benefit of these two economically important tropical plantation crops. These consultancies among others allowed him to build up a wide circle of friends and acquaintances. He realized that breeders of particular crops have much to gain from crosslinking with the experience of others. This developed over the years and could be said to have culminated in the book Evolution of Crop Plants (1976), edited by Norman Simmonds but consisting of the authoritative contributions of almost 86 authors, experts in their field, in addition to six chapters contributed by himself. This extremely popular book has done much to provoke interest in crop plants and the systematic approach required by the editor (introduction, cytotaxonomic background, early history, recent history, and prospects) has proved to be particularly valuable to educators wherever crop evolution and breeding is taught. The original book has now been replaced by a second edition (Smartt and Simmonds, 1995) which, in addition to the necessary updating, has retained the much valued presentation formula. In 1965, Norman Simmonds moved northwards nearer to his family’s origin, when he took on the post of Director of the Scottish Plant Breeding Station, then at Pentlandfield on the outskirts of Edinburgh. This demanding role, at a time of increasing pressure for new varieties suited to Scottish conditions left little time for personal research and many of the ideas initiated at the John Innes Institute, had to be left to others to pick up. However, he did make time to re-establish his education links, developing teaching initiatives with the Botany Department of the University of Edinburgh and this thread would be carried through to the final phase of his professional career when he joined the staff of the Edinburgh School of Agriculture in 1976, then under the enlightened direction of Professor Noel Robertson. This return to academia allowed Norman to bring his knowledge and experience to bear in a wide range of areas. One of his first activities was to produce a new and much needed textbook on plant breeding (Principles of Crop Improvement, 1979). This provided students and practitioners with a synthesis of current breeding approaches along with a bibliography that would allow the motivated into the current literature in any particular area. This has proved to be a very worthy and highly regarded addition to the texts currently available on plant breeding and, having recently undergone some modifications, has appeared as a 2nd Edition (Simmonds and Smartt, 1999). During this period and continuing well after his formal retirement in 1982 at the age of 60, he developed consultancies and took ever wider

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interests in tropical crops at large. He travelled widely throughout the tropics, to some 20 countries, partly as Chairman of the Quinquennial Review of the International Board for Plant Genetic Resources and later on behalf of FAO and the World Bank. For the last he prepared a review (1983/84) which was assimilated into Bank policy though not without sociological controversy. In addition to the above he continued to make valuable contributions to consultancies carried out for Sugar Cane Breeding, in the West Indies; Rubber Research Institute of Malaysia; BookerDaehnfeldt, Denmark; and Copersucar, Brazil, to name but a few. Although well into his retirement now, Professor Simmonds remains no less active academically and continues to review and publish on a wide range of topics, not only within his broad subject area, but also in relation to his hobbies, most notably on trout fishing. Major recent reviews by him have covered horizontal resistance to diseases on crops, an interest originally fired by John Niederhauser almost 30 years before in Mexico; potato propagation by seed as distinct from clonal propagation by tubers (a recently fashionable topic); tropical crops and their improvement; and, most recently, an informal history of statistics for Plant Breeding Reviews, Volume 17. His interest in statistics goes back to the start of his academic training at University and has been demonstrated throughout much of his published work, indicating both his liking for the subject and his growing appreciation of its importance for biology and agriculture. Although largely self-taught, he has always been able to focus on the principles and build upon these to produce a clearer understanding of the importance and applicability of statistics in plant genetics breeding and agriculture for the benefit of students and practitioners.

THE MAN Norman Simmonds is a unique and stimulating individual, with diverse interests. He is an iconoclast, a gifted scientist and profound thinker, a stimulating teacher, a loyal colleague and friend, and a kind and generous man who has been ably supported in all his endeavors by his loving wife, Christa. As a result of his teaching duties he has profoundly affected hundreds of students and has directly influenced many people now in senior positions in agricultural research and administration. His many travels and consultancies have made him a well known and well regarded figure in tropical agriculture and in many developing countries of the world. He is a world authority in many areas and crops. As a result

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of his skill as a writer and editor he has become one of the most respected names in plant breeding, stimulating interest and endeavor in others. Over the years he has received many accolades. Recognition by the University of Edinburgh for his contributions in research and education resulted in a well deserved Honorary Professorship in 1975, and similarly he was elected to be a fellow of the Royal Society of Edinburgh in 1970. One international award which gave him considerable pleasure was that of Distinguished Economic Botanist by the American Society of Economic Botany in 1991, the only non-American then to be so awarded. Norman Simmonds’s career, which has demonstrated clear commitment to improving knowledge, has been an inspiration to us all and we proudly dedicate this volume of Plant Breeding Reviews to him.

PUBLICATIONS OF NORMAN SIMMONDS Books Simmonds, N. W. 1959. Bananas. Longman, London. Simmonds, N. W. 1962. The evolution of the bananas. Longman, London. Simmonds, N. W. 1966. Bananas, 2nd edition. Longman, London. Simmonds, N. W. 1976. The evolution of crop plants (ed.). Longman. London. Simmonds, N. W. 1979. Principles of crop improvement. Longman, London. Simmonds, N. W., and R. H. Stover. 1987. Bananas, 3rd edition. Longman, London. Smartt, J., and N. W. Simmonds. 1995. Evolution of crop plants, 2nd edition. Longman, London. Simmonds, N. W., and J. Smartt. 1999. Principles of crop improvement, 2nd edition. Blackwell Science, Oxford.

Papers Bananas Dodds, K. S., and N. W. Simmonds. 1946. Genetical and cytological studies of Musa VIII. The formation of polyploid spores. J Genet. 47:233–241. Simmonds, N. W. 1946. The relative yields of bananas and potatoes. Trop. Agr. (Trinidad) 23:226–228. Simmonds, N. W., and K. S. Dodds. 1947. Persistence of a nucleolar remnant during meiosis in a diploid banana. Ann. Bot. (London) 11:370–374. Simmonds, N. W., and K. S. Dodds. 1948. Genetical and cytological studies of Musa IX. The origin of an edible diploid and the significance of interspecific hybridisation in the banana complex. J. Genet. 48:285–296. Simmonds, N. W. 1948. Genetical and cytological studies of Musa X. Stomatal size and plant vigour in relation to polyploidy. J. Genet. 49:57–68. Simmonds, N. W., and K. S. Dodds. 1948. Sterility and parthenocarpy in diploid hybrids of Musa. Heredity 2:101–117.

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Simmonds, N. W. 1948. The effects of ploidy upon the leaf of Musa. Ann. Bot. (London) 12:441–453. Simmonds, N. W., and K. S. Dodds. 1949. Meiosis in seeded diploids of Musa. J. Genet. 49:221–251. Simmonds, N. W. 1950. Polyploidy in bananas. Proc. VII Int. Bot. Congr. (Stockholm), 335. Baker, R. E. D., and N. W. Simmonds. 1951. Bananas in East Africa I. Emp. J. Expl. Agr. 19:283–290. Baker, R. E. D., and N. W. Simmonds. 1951. Banana research: Changes in outlook. Trop. Agr. (Trinidad) 28:43–45. Simmonds, N. W. 1952. Experiments on the pollination of seeded diploid bananas. J. Genet. 51:32–40. Simmonds, N. W. 1952. The strength of banana petioles in relation to ploidy. Ann. Bot. (London) 16:341–347. Simmonds, N. W. 1952. The germination of banana seeds. Trop. Agr. (Trinidad) 29:2–16. Simmonds, N. W., and K. Shepherd. 1952. An Asian banana (Musa acuminata) in Pemba, Zanzibar Protectorate. Nature (London) 169:507. Simmonds, N. W., and R. E. D. Baker. 1952. Bananas in East Africa II. Emp. J. Expl. Agr. 20:66–76. Simmonds, N. W. 1952. La banane à la Guadeloupe et à la Martinique. Fruits d’Outre Mer 7:67–69. Simmonds, N. W. 1953. Segregations in some diploid bananas. J. Genet. 51:458–469. Simmonds, N. W. 1953. The development of the banana fruit. J. Expt. Bot. 4:87–105. Baker, R. E. D., and N. W. Simmonds. 1953. The genus Ensete in Africa. Kew Bul. 1953:405–416. Simmonds, N. W. 1953. Classification of the bananas. II. T. Musa erecta U. Musa angustigemma. Kew Bul. 1953:571–574. Simmonds, N. W. 1953. Notes on the banana bunch. Trop. Agr. (Trinidad) 30:54–59. Simmonds, N. W., and F. J. Simmonds. 1953. Experiments on the banana borer, Cosmopolites sordidus, in Trinidad. B.W.I. Trop. Agr. (Trinidad) 30:216–223. Simmonds, N. W. 1954. Isolation in Musa section Eumusa and Rhodochlamys. Evolution 8:65–74. Simmonds, N. W. 1954. Mutations in the Cavendish banana group. Trop. Agr. (Trinidad) 31:131–132. Simmonds, N. W. 1954. Anthocyanins in bananas. Nature (London) 173:402. Simmonds, N. W. 1954. Anthocyanins in bananas. Ann. Bot. 18:471–82. Steward, F. C., and N. W. Simmonds. 1954. Growth promoting substances in the ovary and immature fruit of the banana. Nature (London) 43:1083–1084. Simmonds, N. W. 1954. A survey of the Cavendish group of bananas. Trop. Agr. (Trinidad) 31:126–130. Simmonds, N. W. 1954. Varietal identification in the Cavendish group of bananas. J. Hort. Sci. 29:81–88. Simmonds, N. W. 1954. Notes on banana varieties in Hawaii. Pacific Sci. 8:226–229. Simmonds, N. W. 1955. Wild bananas in Malaya. Malayan Nat. J. 10:1–8. Simmonds, N. W., and K. Shepherd. 1955. The taxonomy and origins of the cultivated bananas. J. Linn. Soc. Bot. 55:302–312. Simmonds, N. W. 1956. Botanical results of the banana collecting expedition 1954–5. Kew Bul. 1956:463–489. Simmonds, N. W. 1956. A banana collecting expedition to South East Asia and the Pacific. Trop. Agr. (Trinidad) 33:251–271. Simmonds, N. W. 1958. Ensete cultivation in the southern Highland of Ethiopia: A review. Trop. Agr. (Trinidad) 35:302–307.

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Simmonds, N. W. 1959. Experiments on the germination of banana seeds. Trop. Agr. (Trinidad) 36:259–273. Simmonds, N. W. 1960. Megasporogenesis and female fertility in three edible triploid bananas. J. Genet. 57:269–278. Simmonds, N. W. 1960. Notes on banana taxonomy. Kew Bul. 1960:212. Simmonds, N. W. 1960. Experiments on banana fruit development. Ann. Bot. (London) 24:212–222. Simmonds, N. W. 1960. The growth of post-war West Indian banana trades. Trop. Agr. (Trinidad) 37:279–283. Simmonds, N. W. 1962. The classification and nomenclature of the bananas and potatoes: Some implications. Proc. Linn. Soc. (London) 173:11–13. Simmonds, N. W. 1985. Classification and breeding of the bananas. p. 62–73. In: IRAZ/IDRC, Workshop on Banana Production in Eastern and Central Africa, Burundi. IDRC, Ottawa, Canada. Simmonds, N. W. 1985. Bananas, Musa cvs. p. 17–24. In: FAO Workshop on Disease Resistance in Tropical Perennial Crops. FAO, Rome. Simmonds, N. W. 1987. Classification and breeding of bananas. p. 69–73. In: G. J. Persley, and E. A. DeLanghe, Banana and plantain breeding strategies, ACIAR/INIBAP, Canberra. Simmonds, N. W., and S. T. C. Weatherup. 1990. Numerical taxonomy of the wild bananas. New Phytol. 115:567–571. Simmonds, N. W., and S. T. C. Weatherup. 1990. Numerical taxonomy of cultivated bananas. Trop. Agr. (Trinidad) 67, 90–92.

Potatoes Simmonds, N. W. 1963. Correlated seed and tuber dormancy in potatoes. Nature (London) 19:720–721. Simmonds, N. W. 1963. Experiments on the germination of potato seeds. Eur. Potato J. 6:45–60, 69–76. Simmonds, N. W. 1963. Abbreviations of potato names. Eur. Potato J. 6:186–190. Simmonds, N. W. 1963. Studies of the tetraploid potatoes. I. J. Linn. Soc. Bot. 58:461–474. Simmonds, N. W. 1964. Studies of the tetraploid potatoes. II. J. Linn. Soc. Bot. 59:43–56. Simmonds, N. W. 1964. The genetics of seed and tuber dormancy in the cultivated potatoes. Heredity 19:489–504. Simmonds, N. W. 1964. Observations on potato callus and adventitious shoot formation. Am. Potato J. 41:129–136. Simmonds, N. W. 1965. Some experimental techniques with potatoes. Eur. Potato J. 8:125–132. Simmonds, N. W. 1965. Attempted graft-transmission of potato tuber dormancy. Eur. Potato J. 8:197–199. Simmonds, N. W. 1965. Seed size in the cultivated potatoes. Euphytica 14:143–152. Simmonds, N. W. 1965. Chimeral potato mutants. J. Hered. 56:139–142. Simmonds, N. W., and J. B. Harborne. 1965. Control of malvidin synthesis in the cultivated potatoes. Heredity 20:315–318. Simmonds, N. W. 1965. Somatic segregation of the spectacle pattern on potato tubers. Heredity 20:277–288. Simmonds, N. W. 1965. Mutant expression in diploid potatoes. Heredity 20:65–72. Simmonds, N. W. 1966. Studies of the tetraploid potatoes III. J. Linn. Soc. Bot. 59:279–288. Simmonds, N. W. 1966. Linkage to the S-locus in diploid potatoes. Heredity 21:473–479. Simmonds, N. W., and P. E. Waggoner. 1966. Stomata and transpiration of droopy potatoes. Plant Physiol. 41:1268–1271.

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Simmonds, N. W., and J. F. Malcolmson. 1967. Resistance to late blight in Andigena potatoes. Eur. Potato J. 10:161–166. Simmonds, N. W. 1968. Prolonged storage of potato seeds. Eur. Potato J. 11:150–156. Simmonds, N. W. 1968. Change of leaf size in the evolution of the Tuberosum potatoes. Euphytica 17:504–506. Simmonds, N. W. 1969. Variegated mutant plastid chimeras of potatoes. Heredity 24: 303–306. Simmonds, N. W. 1969. The genetics of spectacle in diploid potatoes. Heredity 24:487–490. Simmonds, N. W. 1971. The potential of potatoes in the tropics. Trop. Agr. (Trinidad) 48:291–299. Simmonds, N. W. 1973. A note on somatic segregation of the spectacle pattern in potatoes. Heredity 31:405–407. Simmonds, N. W., and R. J. Killick. 1974. Specific gravity of potato tubers as a character showing small genotype environment interactions. Heredity 32:109–112. Simmonds, N. W. 1974. Dry matter content of potatoes in relation to country of origin. Potato Res. 17:178–186. Simmonds, N. W. 1976. Neotuberosum and the genetic base in potato breeding. ARC Res. Rev. 2:9–11. Simmonds, N. W. 1977. Relations between specific gravity, dry matter content and starch content in potatoes. Potato Res. 20:137–140. Simmonds, N. W. 1980. Comparisons of the yields of four potato varieties in trials and in agriculture. Expl. Agr. 16:393–398. Simmonds, N. W., and R. L. Wastie. 1987. Assessment of horizontal resistance to late blight of potatoes. Ann. Appl. Biol. 11:213–221. Simmonds, N. W. 1998. A review of potato propagation by seed as distinct from clonal propagation by tubers. Potato Res. 40:191–214.

Sugarcane Simmonds, N. W. 1967. Potato breeding ideas applied to sugarcane. SCBNL 19:1–8. Simmonds, N. W. 1967. Botanical nomenclature of sugarcane. SCBNL 9–11. Simmonds, N. W. 1969. Radiation-induced non-flowering mutants. SCBNL 24:4–7. Simmonds, N. W. 1971. More generations? SCBNL 26:4–6. Simmonds, N. W., and R. J. Killick. 1972. More generations? SCBNL 29:4–6. Simmonds, N. W. 1972. Profitability selection in relation to trials economy. SCBNL 29:20–23. Simmonds, N. W., and D. I. T. Walker. 1972. Rate of turnover of cane varieties. SCBNL 30:8–10. Simmonds, N. W. 1973. Optimal replanting time for sugarcane. Int. Sugar J. 75:107–108. Simmonds, N. W. 1976. Towards a strategy for sugarcane smut control in the West Indies. Int. Sugar J. 78:329–330. Simmonds, N. W. 1976. A slide rule for sequential Brix sampling. SCBNL 38:701. Simmonds, N. W. 1976. Two points about selection intensity. SCBNL 38:73–74. Simmonds, N. W. 1976. Progress in sugarcane breeding. SCBNL 38:75–78. Simmonds, N. W. 1979. The impact of plant breeding on sugarcane yields in Barbados. Trop. Agr. (Trinidad) 56:289–300. Simmonds, N. W. 1981. Comparisons of the performance of sugarcane varieties in trials and in agriculture. Expl. Agr. 17:137–144. Simmonds, N. W. 1983. Contingency and correlation: Applications to sugarcane breeding. SCBNL 45:20–25.

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Simmonds, N. W. 1983. Variety trials. SCBNL 45:26–29. Simmonds, N. W., and D. I. T. Walker. 1984. Breeding, selection and trials. p. 124–135. In: F. H Blackburn, Sugar cane. Longman, London. Simmonds, N. W. 1984. A note on the strategy of breeding clonal crops. Heredity 53: 397–410. Simmonds, N. W. 1984. Decentralised selection. Sugar Cane 6:8–10. Simmonds, N. W., and D. I. T. Walker. 1986. An economic selection index for sugar cane breeding. Euphytica 35:311–317. Simmonds, N. W. 1986. Sugarcane, Saccharum. p. 115–124. In: FAO Workshop on Disease Resistance in Tropical Perennial Crops. FAO, Rome. Simmonds, N. W. 1987. The leading features of cane breeding. Sugar Cane 1987 (Suppl.):8–11. Simmonds, N. W., and D. A. Elston. 1988. Models of sugar cane smut disease and their implications for testing variety resistance. J. Appl. Ecol. 23:319–329. Simmonds, N. W. 1994. Some speculative calculations on the dispersal of sugar cane smut disease. Sugar Cane 1994/1:2–5. Simmonds, N. W. 1995. Reflections on sugar cane. Int. Sugar J. 97:24–26.

Other Plants Simmonds, N. W. 1945. Meiosis in tropical Rhoeo discolor. Nature (London) 155:731. Simmonds, N. W. 1945. Biological flora of the British Isles. Polygonum, P. persicaria, P. lapathifolium, P. petecticale. J. Ecol. 33:117–143. Simmonds, N. W., and K. S. Dodds. 1946. A cytological basis of sterility in Tripsacum laxum. Ann. Bot. (London) 9:109–116. Simmonds, N. W. 1946. Biological flora of the British Isles. Gentiana pneumonanthe. J. Ecol., 33:295–307. Simmonds, N. W. 1949. Notes on the biology of the Aracaea of Trinidad. J. Ecol. 38:277–291. Simmonds, N. W. 1950. The Aracaea of Trinidad and Tobago, B.W.I. Kew Bul. 1950:391–406. Baker, R. E. D., and N. W. Simmonds. 1953. Solanaceae. Flora Trin. Tob. 2(4):241–272. Simmonds, N. W. 1954. Chromosome behaviours in some tropical plants. Heredity 8:139–146. Simmonds, N. W., and E. Harrison. 1959. Genetics of reaction to pepper vein-banding virus. Genetics 44:1281–1289. Simmonds, N. W. 1960. Flower colour in Lochnera rosea. Heredity 14:253–261. Simmonds, N. W. 1964. Nyctaginaceae—Batidaceae. Flora Trin. Tob. 2(7):439–480. Simmonds, N. W. 1966. Plant and seed colours in Chenopodium pallidicaule. Heredity 21:316–317. Simmonds, N. W. 1967. Hydrocharitaceae—Burmanniaceae. Flora Trin. Tob. 3(1):4–12. Simmonds, N. W. 1967. Marantaceae. Flora Trin. Tob. 3(2):4–34. Simmonds, N. W. 1971. The breeding system of Chenopodium quinoa. I. Male sterility. Heredity 27:73–82.

Plant Breeding Simmonds, N. W. 1961. Mating systems and the breeding of perennial crops. Advancem. Sci. (London) 18:183–186. Simmonds, N. W. 1962. Variability in crop plants, its use and conservation. Biol. Rev. 37:442–465.

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Simmonds, N. W. 1969. Genetical bases of plant breeding. J. Rubb. Res. Inst. Malaya 21:1–10. Simmonds, N. W. 1973. Plant breeding. Phil. Trans. Royal Soc. Lond., B. 267:145–156. Simmonds, N. W. 1974. Costs and benefits of an agricultural research institute. R&D Managem. 5:23–28. Simmonds, N. W. 1977. Breeding perennial crops. TAA Newslett. 17(4):27–29. Simmonds, N. W. 1980. Polyploidy in plant breeding. SPAN 23:73–75. Simmonds, N. W. 1981. Genotype (G), Environment (E) and GE components of crop yields. Expl. Agr. 17:355–362. Simmonds, N. W. 1983. Plant breeding: The state of the art. p. 5–25. In: T. Kosuge, C. P. Meredith and A. Hollaender (eds.), Genetic engineering of plants. Plenum Press, New York. Simmonds, N. W. 1983. Strategy of disease resistance breeding. p. 637–654. In: J. P. Gustafson (ed.), Gene manipulation and crop improvement, Columbia, MO (16th Stadler Symp.). Simmonds, N. W. 1985. A plant breeder’s perspective of durable resistance. FAO Plant. Prot. Bul. 33:13–17. Simmonds, N. W. 1985. Perspectives on the evolutionary history of tree crops. p. 3–12. In: M. G. R. Cannell and J. E. Jackson (eds.), Attributes of trees as crop plants, ITE. Simmonds, N. W. 1985. Two stage selection strategy in plant breeding. Heredity 55:393–399. Simmonds, N. W. 1986. Strategies for disease resistance breeding in tropical perennial crops. p. 3–16. In: FAO Workshop on Disease Resistance in Tropical Perennial Crops. FAO, Rome. Simmonds, N. W. 1986. The strategy of rubber breeding. Int. Rubber Conf., Kuala Lumpur, 1985 3:115–126. Simmonds, N. W. 1986. Theoretical aspects of synthetic/polycross populations of rubber seedlings. J. Nat. Rubb. Res. 1:1–15. Simmonds, N. W. 1986. Oil palm, Elaeis guineensis. p. 79–84. In: Workshop on Disease Resistance in Tropical Perennial Crops. FAO, Rome. Kennedy, A. J., G. Lockwood, G. Mossu, N. W. Simmonds, and G. Y. Tan. 1987. Cocoa breeding, past, present and future. Cocoa Growers Bul. 38:5–22. Simmonds, N. W. and S. Rajaram. (eds.) 1988. Breeding strategies for resistance to the rusts of wheat. CIMMYT, Mexico. Simmonds, N. W. 1988. Synthesis: The strategy of rust resistance breeding. p. 119–136. In: N. W. Simmonds, and S. Rajaram (eds.), Breeding strategies for resistance to the rusts of wheat, CIMMYT, Mexico. Simmonds, N. W. 1988. Environmental features of plant breeding. p. 3–10. In: AAB, Environmental aspects of applied biology, AAB, Warwick. Simmonds, N. W. 1989. Rubber breeding. p. 85–124. In: C. C. Webster and W. J. Baulkwill (eds.), Rubber, Longman, London. Simmonds, N. W. 1989. Economic aspects of plant breeding, with special reference to economic index selection. Res. and Dev. in Agr. 6:53–62. Patterson, H. D., and N. W. Simmonds. 1989. Tables to calculate means in a doubly truncated bivariate normal population. Euphytica 42:241–249. Simmonds, N. W. 1989. A statistical vade-mecum for the itinerant agriculture/researcher. Trop. Agr, (Trinidad) 67:9–15. Simmonds, N. W. 1989. How frequent are superior genotypes in plant breeding populations? Biol. Rev. 64: 341–365.

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Simmonds, N. W. 1990. The social context of plant breeding. Plant Breed. Abstr. 60:337–341. Simmonds, N. W. 1990. Breeding horizontal resistance to South Americal leaf blight of rubber. J. Nat. Rubb. Res. 5:102–113. Simmonds, N. W. 1990. Plant breeding, seed technology and beyond. Proc. ISTA Conf., Edinburgh 1989, p. 81–88. Simmonds, N. W. 1991. Genetics of horizontal resistance to diseases of crops. Biol. Rev. Camb. Phil. Soc. 66:189–241. Simmonds, N. W. 1991. Selection for local adaptation in a plant breeding programme. Theor. Appl. Genet. 82:363–367. Simmonds, N. W. 1991. Uniformity and yield in hybrid crop cultivars. Trop. Agr. (Trinidad) 68: 198–199. Simmonds, N. W., and M. Talbot. 1992. Analysis of on-farm rice yield data from India. Expl. Agr. 28:325–329. Simmonds, N. W. 1992. Cocoa breeding in Sabah. Trop. Agr. Assoc. Newslett. 12(2):9. Spoor, W., and N. W. Simmonds. 1993. Pot trials as an adjunct to cereal breeding and evaluation of genetic resources. Food Crops Res. 35:205–213. Simmonds, N. W. 1993. Introgression and incorporation. Strategies for the use of crop genetic resources. Biol. Rev. Camb. Phil. Soc., 68:539–562. Simmonds, N. W. 1993. Tropical plant breeding: success or failure or a bit of each? Trop. Agr. Assoc. Newslett. 13:3–5. Simmonds, N. W. 1994. The breeding of perennial crops. Int. Workshop on Conservation Characterization and Utilization of Cocoa Genetic Resources into the 21st Century. CRU/UWI, Trinidad, 1992, p. 156–162. Simmonds, N. W. 1994. Horizontal resistance to cocoa diseases. Cocoa Growers’ Bul. 47:42–52. Simmonds, N. W. 1994. Yield and sugar content in sugar beet. Int. Sugar J. 96:414–416. Simmonds, N. W. 1995. Plant breeding. Biol. Sci. Rev., Jan. 1995:32–34. Simmonds, N. W. 1996. Family selection in plant breeding. Euphytica 90:201–208. Simmonds, N. W. 1996. Bias in the estimation of horizontal resistance to airborne fungi in plant breeding. Bot. J. Scotl. 48:275–284. Simmonds, N. W. 1996. Profits, projects and plant breeding. Trop. Agr. Assoc. Newslett. 16(1):31–32. Simmonds, N. W. 1997. Gough and Chittenden, two rubber pioneers. Planter, Kuala Lumpur. Simmonds, N. W. 1997. Tropical crops and their improvement. p. 257–293. In: C. C. Webster and P. N. Wilson. Agriculture in the tropics, 3rd ed. Blackwell, Oxford.

Phenolics Forsyth, W. G. C., and N. W. Simmonds. 1954. A survey of the anthocyanins of some tropical plants. Proc. Roy. Soc. B. 142:549–564. Simmonds, N. W., and R. Stevens. 1956. Occurrence of the methylene-dioxy bridge in the phenolic components of plants. Nature (London) 178:752–753. Forsyth, W. G. C., and N. W. Simmonds. 1957. Anthocyanidins of Lochnera rosea. Nature (London) 180:247. Harborne, J. B., and N. W. Simmonds. 1964. The natural distribution of the phenolic aglycones. Ch. 3. In: J. B. Harborne (ed.), Biochemistry of phenolic compounds. Academic Press, London.

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Miscellanea Simmonds, N. W. 1951. Notes on field management at the Botany Department of the I.C.T.A., Trinidad. Trop. Agr. (Trinidad) 28:70–75. Simmonds, N. W. 1956. Fishing rod botany: A review. Key Bul. 1956:135–140. Simmonds, N. W. 1957. Botanical problems for the amateur naturalist. J. Trinidad. Food. Nat. Club, p. 14–20. Kirkpatrick, T. W., and N. W. Simmonds. 1958. Bamboo borers and the moon. Trop. Agr. (Trinidad) 35:299–301. Simmonds, N. W. 1959. Notes on archery in Southeast Asia and the Pacific. J. Roy, Asiatic Soc. (Mal. Br.) 23:107–164. Simmonds, N. W. 1962. Questions about crops. Biol. Hum. Affairs 27:1–6. Simmonds, N. W. 1965. The grain Chenopods of the tropical American highlands. Econ. Bot. 19:223–235. Simmonds, N. W. 1966. Tissue cultures and the study of plant morphogenesis. Advancem. Sci. 22:178–183. Simmonds, N. W. 1968. The Scottish plant breeding station. Scott. Agr. 47:79–83. Simmonds, N. W. 1971. The Scottish plant breeding station. Scott. Agr. 50:113–118. Simmonds, N. W. 1977. Approximations for i, intensity of selection. Heredity 38:413–414. Simmonds, N. W. 1978. Genetic conservation: An introductory discussion of needs and principles. Seed Technology for Genebanks, IBPGR, Rome, p. 1–9. Simmonds, N. W. 1980. Monocarpy, calendars and flowering cycles in Angiosperms. Key Bul. 35:235–245. Simmonds, N. W. 1982. Some ideas on botanical research on rubber. Trop. Agr. (Trinidad) 59:1–8. Simmonds, N. W. 1982. Genetic conservation: The context of the workshop. p. 1–3. In: J. Withers and J. T. Williams (eds.), Crop genetic resources: The conservation of difficult material, IUBS/IBPGR. Simmonds, N. W. 1983. Exploited plants. Rubber, Biol. 30:153–157. Simmonds, N. W. 1983. Sowing the seeds of change. Times Higher Educ. Suppl. June 24., p. 13 Simmonds, N. W. 1984. Duplication of research: A good or a bad thing? J. Opl. Res. Soc. 36:55–59. Simmonds, N. W. 1985. Farming systems research: A review. World Bank, Washington DC. Simmonds, N. W. 1986. A short review of farming systems research in the tropics. Expl. Agr. 22:1–13. Simmonds, N. W. 1988. A re-examination of some grafting experiments with rubber. J. Nat. Rubber Res. 3:30–41. Simmonds, N. W. 1988. Observations on induced diffusion of innovations as a component of tropical agricultural extension systems. Agr. Admin Ext. 28:207–216. Simmonds, N. W. 1988. A morning in the market. Biologist 35:131–133. Simmonds, N. W. 1990. My week as an ageing applied geneticist. Biologist 37:115–116. Simmonds, N. W. 1991. The earlier British contribution to tropical agricultural research. Trop. Agr. Assoc. Newslett. 11(2):2–7. Simmonds, N. W. 1992. Bandwagons I have known. Trop. Agr. Assoc. Newslett. 11(4):7–10. Simmonds, N. W. 1992. Reflections on five crops. Econ. Bot. 46:4–9. Simmonds, N. W. 1992. An international college of tropical agriculture. Trop. Agr. Assoc. Newslett. 12(4):30. Simmonds, N. W. 1993. Visit to Syria. Trop. Agr. Assoc. Newslett. 31(3):27–28.

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Simmonds, N. W. 1994. Diseases of tropical crops: problems and controls. Bot. J. Scotl. 47:129–137. Simmonds, N. W. 1995. Tree biology, forestry and agriculture, especially in the tropics: A personal viewpoint. Bot. J. Scotl. 47:211–227. Simmonds, N. W. 1995. Food crops: 500 years of travels. CSSA Spec. Publ. 23:31–45. Simmonds, N. W. 1995. The relation between yields and protein in cereal grain. J. Sci. Food Agr. 67:309–315. Simmonds, N. W. 1996. Yields of cereal grain and protein. Expl. Agr. 32:351–356. Simmonds, N. W. 1996. Two iconoclasts. Agricultural research and over-population. TAA Newslett. 16(6):5–6. Simmonds, N. W. 1997. Pie in the sky. TAA Newslett. 17/2:1–5 and The Planter 73:615–633. Simmonds, N. W. 1998. Cocoa confusion. TAA Newslett. 18(3):26–27. Simmonds, N. W. 1999. Agricultural research revolutionized. TAA Newslett. 19(2):36–39. Simmonds, N. W. 1999. An informal history of statistics. Plant Breed. Rev. 17:259–315.

Essays in SPBS Annual Report Simmonds, N. W. 1967. What’s wrong with plant breeding? Annu. Rep. SPBS 46:13–20. Simmonds, N. W. 1969. Prospects of potato improvement. Annu. Rep. SPBS 48:18–38. Simmonds, N. W. 1971. Recent developments and future trends. Annu. Rep. SPBS 50:61–67. Simmonds, N. W. 1972. Research, development and exploitation in agriculture. Annu. Rep. SPBS 51:29–36. Simmonds, N. W. 1973. The old order changeth. Annu. Rep. SPBS 52:38–45. Simmonds, N. W. 1974. Plant genetics and crop involvement. Annu. Rep. SPBS 53:45–53. Simmonds, N. W. 1975. The place of economics. Annu. Rep. SPBS 54:44–53. Simmonds, N. W. 1978. The cereal breeding situation. Annu. Rep. SPBS 47: 16–34.

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2 The Origin of Maize: Evidence for Tripsacum Ancestry Mary W. Eubanks* Department of Botany, Duke University, Durham, North Carolina 27708-0338

I. INTRODUCTION II. THEORETICAL FRAMEWORK III. TAXONOMIC CLASSIFICATION A. Zea B. Tripsacum IV. THE NATURE OF THE EVIDENCE A. Morphology B. Cytology C. Molecular Biology D. Archaeology E. Plant Breeding and Genetics V. SYNTHESIS AND FUTURE DIRECTIONS A. Paleoethnobotany B. Crop Improvement LITERATURE CITED

To find among wild plants, living or extinct, the prototype of a domesticated form is a peculiar problem, and it usually offers more difficulty than would be expected. The improvement of a wild plant through domestication is at best a slow process which involves so little of the spectacular that it is not likely to be recorded in detail; the human part of the process may, in fact, be done unconsciously, and its significance may be *A special thank you to W. C. Galinat, H. G. Wilkes, anonymous reviewers, and J. Janick for critical comments that improved the manuscript and helped me address key questions about the hybrids between Tripsacum and Zea diploperennis. I gratefully acknowledge research support under National Science Foundation grant nos. 9660146, 9801386, and DEB-94-15541. Plant Breeding Reviews, Volume 20, Edited by Jules Janick ISBN 0-471-38788-6 © 2001 John Wiley & Sons, Inc. 15

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realized only long after the fundamental parts of it have been accomplished and its beginnings have been forgotten. If, as is evident in the domestication of maize, the process began long ago, and through the agency of a people taking only its first step toward civilization, the difficulty of finding out anything about it by direct methods is greatly increased. (Paul Weatherwax 1935)

I. INTRODUCTION Of the 13 crop plants that stand between humans and starvation, maize (Zea mays L.), or corn as it is called in the United States, is the only one for which there is no conclusive evidence concerning its origin except that it is native to the Americas. Although it is widely grown throughout the world today, Wilkes and Goodman characterize maize as having “a passport without a birth certificate” (1996) because its original parentage is still shrouded in mystery. The oldest archaeological remains of maize, excavated in the Valley of Tehuacán in southern Mexico, suddenly appear in the archaeological record around 5000 B.C. (Mangelsdorf et al. 1964, 1967a; Flannery and MacNeish 1997). These ancient maize cobs show all the morphological traits that distinguish it from its wild relatives, but specimens showing the intermediate steps of this transformation have not been recovered (Galinat 1985), and there is disagreement among scientists about how those key morphological mutations occurred and which ancestor(s) were involved. We still do not know if maize descended from a single ancestor (Beadle 1939, 1980; Galinat 1971; Doebley 1990), or if it evolved in a reticulate pattern of hybridization and introgression with its wild relatives (Mangelsdorf and Reeves 1939; Wilkes 1979; Mangelsdorf, Roberts, and Rogers 1981; Eubanks 1995, 1997a). Since the 1930s, different theoretical positions have often been hotly debated, and the corn wars still rage today with vitriolic barbs often directed at researchers who challenge popular dogma (Dold 1997). Egos aside, elucidating the question of the origin of maize could have significant consequences for maize improvement. A better understanding of the genetic affinities between maize and other grasses may enhance plant breeders’ ability to identify new sources of genetic variation that could be valuable for crop improvement. Accurate knowledge of the phylogenetic history of maize may aid characterization of the genetic mechanisms governing crossability of germplasm in breeding programs and crop plant evolution. With the advent of molecular mapping, it can be inferred from comparative genomics that all grasses contain essentially the same genes in similar chromosome posi-

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tions and orientation (Moore et al. 1995). Basically what differentiates grass genomes are the amount of repetitive DNA and chromosomal rearrangements in large-scale units. This synteny and collinearity promises to be a valuable tool in bioengineering because the search for a gene in a species with high levels of repetitive DNA, such as wheat and maize, can be accelerated by locating the same gene in a grass with a smaller genome like sorghum or rice (Bennetzen and Freeling 1997). However, utility of this phenomenon for genomics is confounded by the fact that collinearity is not conserved at the small-scale level of genomic organization (Bennetzen et al. 1998). Approximately 20–40% of markers on recombinational maps do not exhibit collinearity or synteny (Bennetzen and Freeling 1997; Gale and Devos 1998). Therefore, characterization of patterns of speciation among closely related taxa is necessary for detection of small-scale rearrangements that perturb local gene composition and order (i.e. microcolinearity) not revealed by most comparative genetic maps, as well as delineation of other exceptions to collinearity and synteny that have largely been ignored in plant genomics research to date. In one exception, the chromosome 4 complex controlling the female spikelet-rachis relationship in maize versus teosinte was compared to the segment of wheat that controls several separate floral characters (Galinat 1970). As genetic engineering replaces traditional plant breeding programs, understanding both taxonomic and evolutionary relationships is essential for agroecological prediction of potential transfer of alien genes between wild or weedy taxa and their domesticated counterparts, which is the critical component of environmental risk assessment posed by dispersals of genetically modified organisms (Arriola and Ellstrand 1996; Paoletti and Pimentel 1996; Kendall et al. 1997; Snow and Palma 1997). In regard to the question of the origin of maize, Weatherwax (1935) remarked: “When taken all together the fragments of information we have do not fit together into a picture which all will accept as expressing a fact. We have had, rather, a series of growing theories, necessarily recast from time to time in the light of new discoveries or new interpretations.” The goal of this review is to refocus perspective on the origin of maize debate in view of recent developments in plant breeding, genetics, and molecular biology. I begin by describing various hypotheses about the origin of maize with emphasis on ideas that have had the greatest impact on the debate. This is followed by review of taxonomic schemes characterizing the closest wild relatives of maize and the evidence from morphology, cytology, molecular biology, archaeology, and experimental crossing studies. The concluding remarks will synthesize what is known about the phylogenetic history of maize in light of the

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latest findings, and outline some directions for future exploration in the continuing odyssey of maize evolution. For other reviews on this subject, see Mangelsdorf (1974), Randolph (1976), Galinat (1977), Goodman (1988), Doebley (1990), Wilkes and Goodman (1996).

II. THEORETICAL FRAMEWORK There are four general schools of thought regarding the origin of maize: (1) maize evolved from an extinct or as yet undiscovered wild grass (Kempton 1937; Mangelsdorf and Reeves 1939; Mangelsdorf 1974, 1986; Wilkes 1979; Goodman 1988); (2) the ancestor of maize is annual teosinte, a weedy grass that grows in Mexico, Guatemala, and Honduras, and is the closest wild relative of maize (Ascherson 1877; Vavilov 1931; Beadle 1939; Galinat 1971; de Wet and Harlan 1972; Iltis 1972, 1983; Kato 1976, 1984; Doebley 1983, 1990); (3) maize and its closest relatives, teosinte and Tripsacum, arose from an unidentified ancestor (Montgomery 1906; Weatherwax 1918); (4) maize derived from hybridization between teosinte and another wild grass (Harshberger 1896; Collins 1912; Eubanks 1995, 1997a). The most widely known version of the first school of thought was the Mangelsdorf and Reeves (1939) tripartite theory. In this scheme, an extinct wild maize hybridized with Tripsacum, a wild related grass, giving rise to annual teosintes, the closet relatives of maize. Introgression between domesticated maize and the teosintes gave rise to the explosive genetic diversity of maize evidenced by the approximately 250 extant Latin American land races. This hypothesis was modified by Mangelsdorf after a new diploid perennial teosinte (Zea diploperennis Iltis, Doebley and Guzmán) was reported (Iltis et al. 1979). This new teosinte inspired Garrison Wilkes (1979) to postulate that rather than deriving from hybridization between an extinct wild ancestral maize and Tripsacum, the annual teosintes arose from hybridization between maize in the early stages of cultivation with perennial teosinte. Mangelsdorf tested the Wilkes hypothesis in crosses between Z. diploperennis and Palomero Toluqueño, a primitive Mexican popcorn (Cámara-Hernández and Mangelsdorf 1981; Mangelsdorf, Roberts, and Rogers 1981), and recovered numerous phenotypes with all of the characteristics of races of annual teosinte among the segregants. He interpreted this finding as experimental confirmation of Wilkes’s idea about the origin of annual teosinte. Consequently, he replaced Tripsacum in the original tripartite hypothesis with perennial teosinte (1983, 1986).

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The second school of thought, that teosinte is the ancestor of maize, was proposed by Ascherson (1877) years before the birth of modern genetics. Subsequent advocates of this idea have offered various explanations of how it happened. Beadle (1939, 1980) favored the hypothesis that maize arose from teosinte through human selection for mutations in the teosinte spike because it was the simplest explanation, and it accounted for the close relationship and crossability between maize and teosinte. Although Beadle (1939) acknowledged the possibility that teosinte was secondarily derived, he was not willing to accept the assumption of an extinct or as yet undiscovered wild ancestor. Beadle (1939) stated: “Any hypothesis involving an extinct or unknown ancestor must remain unsatisfactory until tangible evidence for the existence of such a plant is forthcoming.” Discovery of a block of genes on chromosome 4 that govern key traits of the maize ear that was evidently not in Tripsacum enhanced feasibility of the teosinte hypothesis (Galinat 1971). This was one reason why Galinat, a former associate of Mangelsdorf, became an advocate of the teosinte theory. Galinat, who has conducted extensive morphological and genetic studies on the pistillate inflorescences of maize and teosinte (1971), has proposed two independent origins of maize by human selection (1992). According to Galinat (1992, 1994), one of the ancestors is the small-grained Balsas teosinte (Zea mays ssp. parviglumis) from Guerrero, and the other is the large-seeded annual teosinte (Z. mays ssp. mexicana) from the Chalco area in central Mexico. He believes that genetic recombination resulting from hybridization between individuals of the two derivative pathways accounts for the rich biodiversity of maize. Another advocate of the teosinte hypothesis favors a single origin in which natural selection acted on annual teosinte (Zea mays ssp. parviglumis) growing in the Río Balsas drainage (Doebley 1990). Doebley’s (1990) conclusions are based on interpretation of two lines of molecular systematic evidence. In the early 1980s, isozyme studies of thirteen enzyme systems in maize and teosinte (Mastenbroek et al. 1981; Goodman and Stuber 1983; Stuber and Goodman 1983; Doebley et al. 1984, 1985, 1987; Smith et al. 1984, 1985) indicated that Balsas teosinte is most closely related to maize. The second line of molecular evidence, chloroplast (cp) DNA analysis (Doebley et al. 1987), partially complemented results of isozyme studies, but did not divide Chalco teosinte (Z. m. spp. mexicana) and Balsas teosinte into separate subspecies. Therefore, based on cpDNA findings, if teosinte is the progenitor, highland teosinte from the Valley of Mexico, as proposed in Galinat’s double origin scheme, is as likely to be ancestral to maize as Balsas teosinte. The

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discrepancy in cpDNA data was explained as due either to reticulate evolution (introgression via hybridization) or lineage sorting (retention of polymorphisms from a common ancestor) (Doebley et al. 1987; Doebley 1990). Another variation on the theme of transformation of the teosinte spike into the maize ear is the catastrophic sexual transmutation theory or CSTT (Iltis 1983). CSTT proposes that the development of a key trait distinguishing maize from the wild relatives, two kernels per cupule in maize in contrast to a single kernel per cupule in teosinte, was due to a sex change in the paired staminate flowers that gave rise to the paired female flowers of maize. This putative hormonal switch converting the male flowers to female florets was caused by pathogens or environmental change. Although the third school of thought that maize, teosinte, and Tripsacum all diverged from an unidentified common ancestor never received popular acclaim, it deserves consideration. Weatherwax, a grass systematist par excellence who viewed the origin question from the wholistic perspective of grass taxonomy, morphology, anatomy, genetics, cytology, and evolution (1935), believed that wild maize might still exist in an as yet undiscovered, isolated location in tropical America where it is protected from grazing animals by thorny vegetation or other isolating landscape features. Based on careful study of maize and related wild grasses, he projected what the wild plant would look like as follows: it would be perennial by means of basal offshoots and rhizomes; it would have variable inflorescences, some entirely staminate, some pistillate, and some a mixture of male and female flowers; it would have a tall stem, and some branches would be better developed than others; it would have grains protruding beyond the bracts that are easily removed from the fruiting structure, and it would have a high degree of mutability. The fourth school of thought is that maize derived from hybridization between two wild grasses. This links with Mangelsdorf and Reeves (1939) in the first school of thought, because although they believed maize derived from an extinct wild maize ancestor, they invoked hybridization and introgression as central to the evolution of domesticated maize and the annual teosintes. The distinction here is no presumption of an extinct wild maize; the parents of maize can be found among extant taxa. The best evidence indicating hybridization may have played a key role in the origin of maize (Eubanks 1995, 1997a) are experimental hybrids between Tripsacum and diploid perennial teosinte. Fertile hybrid plants (Fig. 2.1) look a lot like the hypothetical wild maize described by Weatherwax (1935), and the ears produced by segregating intercrosses among hybrid lineages in a Tripsacum cytoplasm

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Fig. 2.1.

Tripsacum × Zea diploperennis F2 plant.

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(Fig. 2.2) have features of the oldest maize cobs found in dry caves in the Valley of Tehuacán, Mexico (Mangelsdorf et al. 1967a; MacNeish and Eubanks 2000), as well as “hybrid” specimens (Fig. 2.3) from Valenzuela Cave in Tamaulipas, Mexico (Mangelsdorf et al. 1967b).

III. TAXONOMIC CLASSIFICATION Maize belongs to the grass family, Poaceae (syn. Gramineae), subfamily, Panicoideae, which includes the majority of grasses in tropical and subtropical regions throughout the world. Whereas most grasses have perfect flowers, maize and its wild relatives, teosinte and Tripsacum, are monoecious, i.e. they have separate staminate and pistillate flowers on the same plant, and this was the reason maize and its relatives were once grouped into a separate taxonomic tribe the Maydeae (syn. Tripsaceae). Stebbins and Crampton (1961) revised that classification and placed Zea and Tripsacum in the Andropogoneae. Other members of the Andropogoneae endemic to the New World include Manisuris, Coelorachis, and Elyonurus. Old World Andropogoneae include Coix, Trilobachne, Polytoca, Schleracne, Chionacne, as well as the important economic grasses sugar cane (Saccharum) and sorghum (Sorghum) (Kellogg 1998). A. Zea Annual teosinte is the closest relative of maize (Reeves and Mangelsdorf 1942; Wilkes 1967). It has the same chromosome number as maize (2n = 20), and hybridizes easily with it. Like maize, teosintes bear their staminate flowers in tassels at the summit of the main stems, and pistillate flowers are borne laterally in leaf axils. The spike of teosinte is a single row of rachid segments in an alternate or two-ranked arrangement (Fig. 2.4). The grain is encased in hard fruitcases formed by the endurated outer glumes and the dark brown fruits shatter upon maturity for natural seed dispersal. Originally classified in the genus Euchlaena (Schrader 1833), teosinte was subsequently moved to Zea, the same genus as maize (Reeves and Mangelsdorf 1942). Classification based on phenetic characters of male spikelet morphology delineated four annual and two perennial teosinte species in Mexico and Guatemala (Doebley and Iltis 1980; Iltis and Doebley 1980; Doebley 1990). This classification divided Zea into two sections. Section Zea includes two Mexican annual teosintes (Z. mays ssp. mexicana and Z. mays ssp. parviglumis), an annual teosinte from western Guatemala (Z. mays ssp. huehuetenangensis), and domesticated maize (Z. mays ssp. mays). Section Luxuriantes includes another

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Fig. 2.2. Four-rowed Tripsacum × Zea diploperennis F3 ear that represents an experimentally reconstructed prototype of primitive maize.

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Fig. 2.3. A “hybrid” pistillate inflorescence excavated in Valenzuela Cave, Tamaulipas, Mexico. The specimen labelled “possible F1 corn × teosinte closely resembles an inflorescence of Tripsacum × Z. diploperennis. From the collections at the Harvard University Herbaria.

Guatemalan teosinte (Z. luxurians) and the two perennial species (Z. perennis and Z. diploperennis). Table 2.1 compares the two taxonomic classifications and aligns them with the geographic races of annual teosinte described by Wilkes (1967, 1972). The teosintes today extend from northern Mexico to southern Guatemala and northern Nicaragua. All populations are found west of the Sierra mountain range that extends through central Mexico (Fig. 2.5). However, prehistoric remains of teosinte dating to circa 2200 B.C. have been recovered in archaeological context in Tamaulipas (Mangelsdorf et al. 1967b), which is east of

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Classification schemes for maize and teosinte.

Early classificationz

Revised schemey

Geographic racesx

ZEA (formerly EUCHLAENA) Z. (or E.) mexicana Z. (or E.) mexicana Z. (or E.) mexicana Z. (or E.) mexicana Z. (or E.) mexicana Z. mays

Z. mays ssp. mexicana Z. mays ssp. mexicana Z. mays spp. mexicana Z. mays ssp. parviglumis Z. mays ssp. huehuetenangensis Z. mays ssp. mays

Nobogame Central Plateau Chalco Balsas Huehuetenango

LUXURIANTES Z. (or E.) luxurians Z. (or E.) perennis Z. diploperennis

Z. luxurians Z. perennis Z. diploperennis

Guatemala Perennial (2n = 40) Perennial (2n = 20)

z

Mangelsdorf and Reeves 1942. Iltis and Doebley 1980. x Wilkes 1967. y

the escarpment and beyond its natural geography today. These plant materials could have been brought to Tamaulipas by humans moving into the region from the west, or the natural range of teosinte might have extended to the eastern slopes of the Sierras in prehistoric times. Although current popular opinion is that annual teosinte from the Río Balsas River drainage in Guerrero, Mexico is the progenitor of maize (Doebley 1990), the evidence does not clearly resolve whether the role of this teosinte is ancestral or sister to maize. There is evidence to support the hypothesis that introgression of teosinte germplasm contributed to the rapid evolution of diverse maize land races in prehistoric times (Wellhausen et al. 1952; Wilkes 1977, 1979, 1989), and recent experimental evidence (Eubanks 1995, 1997a; MacNeish and Eubanks 2000) signals the possibility that the mutant phenotypes of the pistillate inflorescence selected by humans were generated by intergeneric hybridization between teosinte and Tripsacum rather than by spontaneous mutations affecting the teosinte spike. These eventually evolved and became fixed via artificial selection and the domestication process. B. Tripsacum An important but more distantly related grass is Tripsacum, a rhizomatous perennial, widely distributed throughout North and South America (Fig. 2.6). In contrast to maize and teosinte, Tripsacum usually bears both

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A teosinte pistillate spike.

Map showing the biogeographic distribution of teosinte species.

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Fig. 2.5.

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Fig. 2.6.

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Map showing the biogeographic distribution of Tripsacum species.

staminate and pistillate flowers on the same spike, with the staminate flowers directly above the pistillate flowers (Fig. 2.7). Cutler and Anderson (1941) recognized seven species, and two more were added a few

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Fig. 2.7.

A Tripsacum spike showing female flowers subtending the male flowers.

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years later (Randolph 1950; Randolph and Hernández-X 1950). Subsequent surveys of Tripsacum throughout Latin American expanded the genus to include as many as 16 species (de Wet et al. 1976, 1983; Brink and de Wet 1983), each with a gametic chromosome number of n = 18 and ploidy levels ranging from 2n to 6n. The Tripsacum species have been divided into two sections: Tripsacum and Fasciculata (see Table 2.2). Section Tripsacum is distinguished by stiff inflorescences in which both of the paired staminate flowers are sessile; whereas in section Fasciculata the inflorescences are pendulous and the upper member of the staminate floret pair is pedicellate. The gametophytic (n) chromosome number of Tripsacum is 18 (Berthaud et al. 1997), but Stebbins (1950) suggested × = 9 because Tripsacum is polyploid. Evidence in support of this comes from Manisuris, a closely related New World tropical grass that has often been placed in the same genus as Tripsacum (Hitchcock 1935; Mangelsdorf 1974). Its haploid number is nine (Reeves and Mangelsdorf 1935), and Edgar Anderson (1944) proposed that Tripsacum was an allopolyploid comprised of two

Table 2.2.

Classification of the genus Tripsacumz.

Classification

Geographic distribution

TRIPSACUM T. dactyloides T. bravo T. australe T. andersonii T. floridanum T. latifolium T. zopilotense T. cundinamarce T. intermedium T. manisurioides T. peruvianum

North to South America Valle de Bravo, Mexico South America Mesoamerica and South America Southern Florida and Cuba Mexico and Central America Guerrero, Mexico Venezuela Mexico to Honduras Chiapas, Mexico Ecuador and Peru

FASICULATA T. laxum (synonym T. fasiculatum) T. lanceolatum T. maizar T. pilosum T. jalapensey

Veracruz, Mexico Arizona, Chihuahua, Durango, Sinaloa Nayarit, Veracruz, Oaxaca Central Mexico to Honduras Guatemala

z

Brink and de Wet 1983. de Wet et al. 1983.

y

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separate genomes XXYY, with the X genome represented by Manisuris. On the basis of intergenomic mapping experiments, Galinat et al. (1964; Galinat 1973) proposed that Tripsacum is an ancient amphidiploid derived from hybridization between Manisuris and wild maize. Although there have been literally thousands of attempts to cross annual teosinte and Tripsacum, they did not produce viable hybrids (Tantravahi 1968; Mangelsdorf 1974). Mangelsdorf and Reeves (1931) were the first to succeed in crossing Tripsacum with maize, which led to their hypothesis (1939) that a hybridization event between Tripsacum and an extinct wild maize gave rise to domesticated maize and annual teosintes. Maize-Tripsacum hybrids, however, usually require special techniques in pollination and embryo germination, and they are sterile. Partial fertility can be restored in the pistillate florets by treatment with colchicine to induce chromosome doubling. Some of the initial objections to the Mangelsdorf and Reeves hypothesis were based on the difficulty of making intergeneric crosses and hybrid sterility, plus lack of evidence for hybrids between Tripsacum and Zea in nature. Since then, however, evidence for at least one natural hybrid (Tripsacum andersonii) between Zea and Tripsacum has been forthcoming (de Wet et al. 1983; Talbert et al. 1990). Although the once renowned theory of Mangelsdorf and Reeves is no longer popular, Tripsacum continues to be investigated as a source of beneficial traits for maize improvement (Eubanks 1989, 1992, 1994, 1995, 1996, 1997a, 1998; Kindiger and Beckett 1990; Burkhart et al. 1994; Leblanc et al. 1995; Berthaud et al. 1996, 1997; Savidan et al. 1996; Kindiger and Sokolov 1998), and new evidence from crossing studies discussed below suggests the hybridization hypothesis involving Tripsacum in the origin of maize warrants rethinking (Eubanks 1995, 1997a).

IV. THE NATURE OF THE EVIDENCE Interpretations of evidence from cytology, morphology, taxonomy, classical and molecular genetics, experimental breeding, and archaeology have led to divergent conclusions about the origin of maize. The reason why the origin of maize is still a conundrum may be that the undiscovered missing link is yet to be found, or experts have failed to arrange available pieces of the puzzle into proper order and arrive at the correct synthesis. A clearer understanding of the origin and evolutionary trajectory of maize will: (1) aid development of new cultivars better adapted to diverse habitats and shifting environmental parameters accompanying global climate change; (2) enhance improvement in productivity of

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this important cereal crop in an era of burgeoning population growth and diminishing area of arable land for agriculture. A. Morphology The ear of maize is a morphological structure unparalleled in the botanical kingdom. Whereas the pistillate inflorescences of its wild relatives are distichously arranged spikes of 5 to 12 caryopses encased in hard fruitcases, alternately stacked one on top of the other, that shatter upon maturity for natural seed dispersal, the many-rowed maize ear bears hundreds of exposed kernels that occur in pairs in cupules fused to form a rigid, nonshattering rachis (the cob). The entire structure is enveloped by a multiple of condensed leaf sheaths (the husks). Maize has been so radically modified by artificial selection that the plant is no longer capable of naturally reproducing itself. It would ultimately go extinct without human intervention to plant and harvest the seed. Although maize geneticists have identified and mapped five genes primarily responsible for this transformation using crosses between teosinte and maize (Langham 1940; Mangelsdorf 1947; Rogers 1950; Galinat 1977, 1985; Doebley 1990), how this assemblage originated is still not completely resolved. The key genes for morphological traits that distinguish domesticated maize from its wild relatives include: (1) two ranked (tr1) versus many ranked on the short arm of chromosome 2 (Langham 1940; Rogers 1950); (2) rind and pith abscission (ri1/ph1) on the short arm of chromosome 4, responsible for the rigid rachis or cob (Galinat 1975, 1978); (3) a single female spikelet (pd1) versus two kernels per cupule in maize on the long arm of chromosome 3 (Langham 1940; Rogers 1950); (4) teosinte glume architecture (tga1) on the short arm of chromosome 4 involved in cupule reduction causing exposure of the maize grains compared to a kernel that is completely enclosed in a highly endurated fruitcase (Galinat 1970; Dorweiler et al. 1993); (5) teosinte branched (tb1) on the long arm of chromosome 1, which controls the reduction of the multiple branched culms of teosinte to a single stalk in maize (Burnham 1961; Doebley et al. 1995). Other loci that have important effects are: tunicate (tu1) on the long arm of chromosome 4 (Collins 1917a,b; Mangelsdorf and Galinat 1964), which is responsible for converting the hard, erect outer glumes of teosinte and Tripsacum to the softer reflexed glumes subtending maize kernels; terminal ear from lateral branch (te1) on the long arm of chromosome 3 (Matthews et al. 1974), which is responsible for the suppression of apical meristem growth to form a single large ear enclosed in modified leaf sheaths that form the protective husk and position the ear proximal to the main culm; a gene for four-

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ranked arrangement that contributes to polystichy on the long arm of chromosome 9, and a gene or genes that have multiple effects on the short arm of chromosome 5. If wild maize plants were perennial, as Weatherwax proposed (1935), another important locus is the one for perennialism (pe*-d) on the long arm of chromosome 4 (Mangelsdorf and Dunn 1984a,b) that is responsible for the switch from the perennial habit to the annual, in which more energy goes into grain production as opposed to root development. Table 2.3 summarizes the key genes involved in maize domestication. Through artificial selection of these traits, the anomolous, domesticated form that produces large quantities of nutritious grain, and can be easily grown, harvested, stored, and processed, evolved within a span of a few thousand years. Annual teosinte is often a weedy mimic of maize in fields in Mexico and Central America and is difficult to distinguish from maize until the plants flower. The principal difference is that teosinte produces small spikes of about 7 to 12 kernels enclosed in hard fruitcases that disarticulate upon maturity, whereas maize has large, multiple-rowed ears with hundreds of kernels on a firm cob enclosed by husks that can be easily harvested and stored. There are more subtle differences in plant architecture related to branching habit, but overall resemblance between Mexican annual teosintes and maize and their fertile hybrids is why

Table 2.3. maizeZ.

Genes involved in the transformation from the wild form to domesticated

Trait

Symbol

Teosinte branched Two-ranked ear Paired spikelets Terminal ear to lateral branch

tb1 tr1 pd1 te1

1L 2S 3L 3L

Teosinte glume architecture Rind and pith abscission Tunicate Perennialism Multiple effects

tga1 ri1/ph1 tu1 pe*-d

4S 4S 4L 4L 5S

Four-ranked ear Z

Chromosome

9L

Molecular marker loci UMC107, adh1 UMC53, UMC34 UMC63 UMC50, bnl5.37, npi296 UMC42 php20725, UMC87 UMC66 UMC15, UMC52 npi409, UMC90, UMC27, UMC40 UMC114, UMC95

An allele from the Tripsacum parent that is not found in the Zea diploperennis parent is present in Tripsacum-diploperennis hybrids at all of the above molecular marker loci except bnl5.37.

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some scientists have postulated that teosinte is the wild progenitor of maize. Various ideas ranging from selection for mutations affecting the pistillate structure (Beadle 1939; Galinat 1988; Doebley 1996) to developmental transmutation that converted the male panicle into the female spike (Iltis 1983) have been put forward to explain the transformation of the teosinte ear into the complex structure of the maize ear. However, no one has practically demonstrated how such a change could have occurred (Kellogg and Birchler 1993). Intermediate gradations have been reconstructed experimentally in crosses between maize and teosinte (Langham 1940; Mangelsdorf 1947; Rogers 1950; Galinat 1985), but such breeding experiments have failed to recover pure segregating parental phenotypes as would generally be expected if annual teosinte is the maize progenitor. Therefore, an alternative explanation some scientists have proposed is that the progenitor of domesticated maize is either an unidentified ancestor or an extinct plant. Although the plant habit and reproductive spikes of maize and teosinte differ from Tripsacum, particular features of the Tripsacum inflorescence warrant consideration. As mentioned earlier, in Tripsacum the staminate and pistillate flowers are borne on the same spike. This trait, considered primitive (Sundberg and Orr 1986), is present in the most ancient prehistoric maize found in dry caves in the Tehuacán Valley, Mexico (Mangelsdorf et al. 1967a), and it occurs in a few extant South America maize races like Coroico (Galinat 1971). It is also seen in teosintes that flower late in the season, indicating some evidence for daylength control (Wilkes 1967). Mangelsdorf (1968) recovered Tripsacum inflorescence phenotypes in Maíz Amargo plants, indicating the presence of Tripsacum germplasm in this race from Argentina. Furthermore, although highly productive races of maize were available during the pre-Columbian Classic period (circa 250–900 A.D.), depictions of maize on Zapotec ceremonial urns from the Valley of Oaxaca, Mexico (Fig. 2.8), consistently portray primitive races with staminate tips (Fig. 2.9), indicating this feature was common and had important symbolic significance (Eubanks 1999a). The paradox of retention of this primitive feature and its stylized depiction above naturalistic ears impressed from molds made from real maize ears is unclear, but for some reason the staminate tip is importantly linked to native American beliefs and rituals associated with maize. Another key trait of maize, two kernels paired in a single cupule, is not found in teosinte but apparently has a genetic precursor in Tripsacum. Some Tripsacum plants develop two to four embryos in a single seed, with as many as 50% bearing pairs of kernels in a cupulate

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Fig. 2.8. Zapotec urn depicting the God of Glyph “L” excavated from Tomb 124 at Monte Albán in the Valley of Oaxaca. Museo Nacional de Antropología Mexico, D.F. Accession no. 6-6223.

fruitcase (Farquharson 1954; Dewald et al. 1987). Ears of F1 Tripsacumteosinte hybrids have pairs of exposed kernels in fused cupules (Eubanks 1995). They resemble the oldest archaeological maize remains from Tehuacán, Mexico, and appear to approximate a reconstructed prototype of early maize (MacNeish and Eubanks 2000). The distinctive ear morphology led Eubanks (1995, 1997a) to reintroduce the idea that transformation of the ear leading to domesticated maize may have been initiated from hybridization between Tripsacum and Zea.

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Fig. 2.9. Ceramic model close-up of mold-made maize with stylized staminate tip depicted on Zapotec urn from the Valley of Oaxaca, Mexico.

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B. Cytology The haploid chromosome number of maize and annual teosinte is n = 10. The ten chromosomes, designated one to ten from longest to shortest, respectively, can be identified cytologically under a light microscope by their relative lengths, distinctive chromomere patterns, position of centromeres, and distinctive dark-staining regions that appear like beads on a string and are referred to as knobs. Maize and the annual teosintes share approximately 20 of the same knob positions, and teosinte has some additional knobs not found in maize (Longley 1941a,b). In contrast to maize and the annual teosintes, Zea luxurians from Guatemala, the perennial teosintes, and Tripsacum have small terminal knobs at the ends of their chromosomes (Longley 1941a; Kato 1976, 1984; Ting et al. 1981; Pasupuleti and Galinat 1982). The karyograms in Fig. 2.10 illustrate the knob configurations in Zea and Tripsacum. As would be expected from the similarity in chromosome number and architecture, hybrids between maize and annual teosinte occur naturally and are usually fertile. Kato (1976, 1984) and Doebley (1984) view the fact that annual teosinte has the same knobs as maize, plus additional ones, as evidence that maize arose from teosinte. Since a study of knob patterns in 15 species from four unrelated plant families (Lima-de-Faria 1976, 1983) showed that knobs in perennial species are found at the ends of chromosomes but in cultivated species tend to move to more internal positions, such logic may be untenable. A possible alternative explanation is that the interstitial knobs of the annual teosintes, as well as maize, may somehow be derived from the terminal knobs of more primitive perennial relatives. A characteristic feature of knobs in maize is that they occupy a fixed position on a particular chromosome when visible and their specific size and shape is stably inherited within lines (McClintock 1929; McClintock et al. 1981). The assumption has been made that knob inheritance is as precise as that of a mutant gene, and knobs have been used as markers in the same sense that mutant genes have been (Rhoades 1955). Evidence from cytogenetic study of inheritance of knobs in maize-teosinte hybrids, however, revealed that knob expression in interspecific hybrids is a complex pattern in which knobs may be lost or the number amplified beyond the expected number, and they can transpose from terminal to internal chromosomal positions (Eubanks 1987a, 1987b, 1987c, 1988). Until the function and origin of knob heterochromatin in plants is better understood, assumptions correlating knobs with evolutionary relationships are premature. Interfertility among species is an important indicator signaling relatedness. Maize crosses naturally with teosinte and there is a range in

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Fig. 2.10. Karyogram depicting the knob constitutions of maize, teosinte, Tripsacum, and Tripsacum-diploperennis hybrids. A large open circle represents the nucleolus; the small open circle is the centromere; large and small black circles indicate knob positions.

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Fig. 2.10.

(continued)

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degree of hybrid fertility depending on which taxa are crossed. Tripsacum (n = 18) and maize (n = 10) can be crossed, but resulting progeny (2n = 28 for maize cross diploid Tripsacum and 2n = 46 for tetraploid Tripsacum) are male sterile and almost always female sterile. Although efforts to artificially cross Tripsacum with annual teosinte have failed to produce viable embryos (Randolph 1950; Tantravahi 1968; Mangelsdorf 1974), there is evidence of at least one Zea-Tripsacum hybrid in nature. Talbert et al. (1990) showed that Tripsacum andersonii (2n = 64) is a natural hybrid in which a Tripsacum female parent crossed with Zea, most likely the Guatemalan teosinte Z. luxurians. A new development in recent years has been the successful crossing of Tripsacum dactyloides and diploid perennial teosinte Z. diploperennis (n = 10), which produces fully fertile hybrid plants (2n = 20) (Eubanks 1987a, 1989, 1992, 1994, 1995, 1996, 1997a, 1998). Reduction in number of terminal knobs and appearance of internal knobs in these hybrids (Eubanks 1987a) demonstrates that the internal knobs of maize and annual teosinte could be derived from hybridization between these perennial relatives. C. Molecular Biology The molecular evidence for maize evolution includes isozyme analyses and DNA studies of nuclear and cytoplasmic genes. There are incongruities in different data sets, and interpretations about their significance for maize evolution vary. Although it is now possible to sequence DNA and sophisticated computer programs generate cladograms from sequence data, different methods yield different results and different interpretations are often obtained from different data sets (Moritz and Hillis 1996). There may not be consensus about what a particular gene sequence signifies for evolutionary relationships, how or what forces of selection are operating on a specific segment of the genome, and what it reveals about relationships among groups of organisms. In isozyme analysis, banding patterns from different species are compared and the observed variation observed subjected to statistical analysis to predict relationships among species. Studies have been conducted to analyze variation in 13 isozyme systems in maize and teosinte races (Mastenbroek et al. 1981; Goodman and Stuber 1983; Stuber and Goodman 1983; Doebley et al. 1984, 1985, 1987a; Smith et al. 1984, 1985). The isozyme data delineated two taxonomic groups: section Zea, composed of the Mexican annual teosintes (Z. m. ssp. mexicana, Z. m. ssp. parviglumis) and maize (Z. m. ssp. mays), and section Luxuriantes composed of the Guatemalan teosintes (Z. m. ssp. huehuetenangensis and Z. lux-

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urians) and the perennials (Z. diploperennis and Z. perennis). From these data, it can be inferred that Z. m. ssp. parviglumis (i.e. Balsas teosinte) is most closely related to maize. Doebley (1990) interprets this as evidence that maize is descended directly from Balsas teosinte. Comparative isozyme data for Tripsacum have not been published. Chloroplast (cp) DNA restriction analysis (Doebley et al. 1987; Doebley 1990) complemented results of isozyme studies with the exception that it placed Z. m. ssp. huehuetenangensis in the Zea group. It did not indicate that Z. m. ssp. mexicana and Z. m. ssp. parviglumis are two separate subspecies. Doebley (1990) suggested the explanation for this cpDNA discrepancy is reticulate evolution of Zea or retention of cpDNA polymorphisms from a common ancestor. Chloroplast DNA analysis comparing 14 Tripsacum species to Z. m. ssp. parviglumis and Z. diploperennis showed few differences between Zea and Tripsacum, and confirmed “a very close relationship between these genera” (Larson and Doebley 1994). The results were interpreted as indicating that neither genus was anciently derived. The same study examined restriction site variation in nuclear ribosomal genes (rDNA) and found such a high degree of variation within individual plants that it could not be employed for phylogenetic analysis. The rDNA data, however, did confirm a Zea-Tripsacum hybrid origin for T. andersonii, which shared three mutated restriction sites with Z. m. ssp. parviglumis and Z. diploperennis, and a fourth site with just Z. diploperennis. Examination of the cpDNA data reported also indicates possible Zea-Tripsacum hybridization events. Mutated cpDNA polymorphisms were shared among Z. diploperennis, T. bravum, and T. pilosum, and among Z. diploperennis, T. dactyloides, T. floridanum, and T. zopilotense (Larson and Doebley 1994). Alternatively, these shared polymorphisms among Zea and Tripsacum may be alleles retained as a result of lineage sorting. Molecular data for maize were not reported in this study. Using restriction enzyme analyses of nuclear ribosomal (rDNA) genes in Zea and Tripsacum, Zimmer et al. (1988) did not obtain enough variable characters to produce a phylogeny. There was high similarity between Z. m. ssp. mays and Z. m. ssp. mexicana in the intergenic spacer (IGS) region of rDNA, and both species have a restriction site not present in any of the other relatives. The 5S DNA data, however, revealed differences in every single taxa except Z. diploperennis and Z. perennis, which matched each other. An intriguing finding of this study was loss of restriction sites in maize-teosinte hybrids that would normally have been inherited from the teosinte parent. In contrast, restriction analysis of ribosomal DNA in maize-Tripsacum hybrids showed new restriction sites generated between the 26S and 17S genes (Lin et

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al. 1985). Eubanks (1995) has observed similar loss and gain of restriction sites in teosinte-Tripsacum hybrids. These findings indicate that hybridizations between Zea and Tripsacum spark mutation events. Novel forms generated by such wide crosses could provide the diversity for the rapid evolution that occurred in domesticated maize around 7,000 years ago. A phylogenetic study of the ribosomal internal transcribed spacer (ITS) sequences of Zea and Tripsacum confirmed these genera are closely related and form a single clade (Buckler and Holtsford 1996). The ITS data presented a different picture of Zea phylogeny. The authors produced gene trees using four different statistical programs. With Tripsacum as the outgroup for rooting the trees, results consistently (1) failed to support division of Zea into separate Zea and Luxuriantes clades; (2) placed Z. diploperennis and Z. perennis together; (3) placed the perennial teosintes and Z. luxurians basal to the other Zeas; (4) placed Z. m. ssp. huehuetenangensis basal to Z. m. ssp. mays; and (5) placed Z. m. ssp. mays basal to Z. m. ssp. parviglumis and Z. m. ssp. mexicana with one exception. In some cladograms the teosinte race Nobogame was basal to Z. m. ssp. mays. Because the basic assumption of the cladistic methods employed for analyzing the ITS data set is that descent is monophyletic from a single ancestor, if evolution is reticulate involving hybridization and introgression among the taxa being investigated (McDade 1992), this approach is inadequate for detecting the correct evolutionary pathway. Characterization of the nuclear gene Adh1, which codes for the enzyme alcohol dehydrogenase (Gaut and Clegg 1993), revealed six different alleles of the gene in maize, perennial teosinte, and the Guatemalan teosintes that were not detected in the isozyme assays. Since this showed that isozyme data does not reveal all the alleles at a locus, its usefulness for characterization of the evolutionary history of maize appears limited. Computation of genetic distance for Adh1 polymorphisms revealed that in some cases there is less distance between maize and teosinte than within maize and teosinte lines. This suggests there has either been extensive introgression between maize and teosinte and/or the Adh1 locus is an ancient polymorphism that predates divergence. Similar results were reported from analysis of a segment of the Adh2 gene in modern maize, prehistoric maize from Peru, the teosintes, and Tripsacum (Goloubinoff et al. 1993). Sequence diversity in prehistoric maize was comparable to that of modern maize and one of the alleles in ancient maize was identical to perennial teosinte. The authors interpreted these findings as evidence that maize arose from multiple ancestral populations.

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As can be seen from the above, the molecular evidence does not provide a robust picture of maize evolution; different methods and different genes reveal different things that cannot be deciphered at the species level to provide a comprehensive picture. Most molecular studies have not included many, if any, Tripsacum taxa; consequently most results reported are restricted by the prevailing assumption that teosinte is the ancestor of maize and do not provide necessary information to evaluate critically the relative merits of a monophyletic origin versus a hybrid origin of maize. D. Archaeology In addition to preserved plant materials in archaeological contexts that provide direct evidence of plant use and domestication, evidence from microfossils (phytoliths and pollen) have been employed to supplement reconstruction of the evolutionary history of maize in areas where conditions are not amenable to preservation of macro remains. Although there are limitations in discriminating maize, teosinte, and Tripsacum using these microfossils (Dunn 1983; Lippi et al. 1984; Piperno and Pearsall 1993; Eubanks 1997b; Rovner 1999), the data provide corroborative support for the presence of Zea and Tripsacum when they converge with other lines of evidence in the archaeological record like macro plant remains and cultural artifacts. Parameters that have been used to distinguish pollen of maize from teosinte and Tripsacum are diameter and axis/pore ratio. However, size inflation due to chemical preparation and different mounting media can increase measurements by as much as 35% (Christensen 1946; Andersen 1960; Cushing 1961), and environmental conditions can have significant effect on pollen size (Kurtz et al. 1960). When maize was identified from pollen in cores taken from approximately 70 m below the surface in Mexico City, it was thought to be clear evidence of ancient maize, but was subsequently challenged and remains unresolved because of ambiguities in distinguishing maize pollen from teosinte and Tripsacum (Mangelsdorf et al. 1978). Recent systematic study of Zea and Tripsacum pollen spinule patterns using scanning electron microscopy underscores how problematic pollen identification is (Eubanks 1997b). Although it is sometimes possible to clearly identify Tripsacum by sculpturing of spinules on the grain surface, it is not possible with available techniques to make a definite identification of maize from teosinte or hybrids between Zea and Tripsacum. Phytoliths (literally “plant stones”) are formed by deposits of silica in certain plant cells. Upon plant decomposition, these microscopic silica

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bodies may be deposited in the soil locally or wind borne and deposited at great distances from the site of origin (Baker 1959; Smith et al. 1970; Dunn 1983). They can be separated from soil samples by density gradient centrifugation, then examined microscopically, and sometimes identified to genus (Rovner 1971; Pearsall 1978; Piperno 1988a). Identifications of maize are based on frequency of size and class of crossshaped phytoliths. Because there is considerable overlap in size between teosinte and maize, and conditions of deposition may influence frequencies, there is ambiguity in reports of presence of maize based solely on phytolith data (Dunn 1983; Roosevelt 1984). Piperno’s (1988a) careful, systematic study of maize and teosinte phytoliths has led her to suggest that some of the identification ambiguity may be explained by hybridization between maize and teosinte. Pearsall (1978) identified cross-shaped phytoliths as maize in Valdivia-phase deposits radiocarbon dated from 3000 to 2300 B.C. in southwest Ecuador. Identification of these phytoliths has been revised to indicate they fall within the “maize/wild” size overlap with the claim maize was probably present (Piperno 1988). Cross-shaped phytoliths have been identified in soils from other sites in central Panama and Ecuador ranging from 7000 B.C. to 500 A.D. (Pearsall 1978; Piperno 1984, 1988b; Bush et al. 1989). Pollen identified as maize was present with phytoliths in preceramic contexts deposits dating circa 4900 B.C. at the Cueva de los Ladrones site in Panama (Piperno et al. 1985), and in sediments from Lake Ayauch in Amazonian Ecuador radiocarbon dated approximately 4000 B.C. (Bush et al. 1989). Although phytolith evidence signals the possibility that maize was present very early in Central and South America, such a scenario is not firmly established because of uncertainties about maize pollen and phytolith identification and deposition. Unequivocal evidence for the earliest maize is still the archaeological remains of cobs excavated from dry caves in the Valley of Tehuacán in south central Mexico, originally radiocarbon dated to 5200 b.c. (Mangelsdorf et al. 1964, 1967a). Although the original 14C dates have been challenged by recent accelerator mass spectrometry (AMS) to 3600 B.C. (Long et al. 1989), the cobs used in the redating had been treated with bedacryl, a chemical preservative (Flannery and MacNeish 1997). Since the specimens were not treated with a solvent to remove the bedacryl prior to dating, the original 14C dates are probably the more reliable. Therefore, the date of first appearance of maize around 7,000 years ago is still considered accurate here. These early cobs were remarkably uniform in size, ranged from 19 mm to 25 mm long and had four to eight rows of kernels surrounded by very long glumes. The well preserved specimens from Tehuacán provide a complete evolutionary sequence of

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maize evolution up to 1500 A.D., beginning with the earliest, tiny 8rowed ears that were transformed into early cultivated maize, then graded into early tripsacoid maize, and ultimately changed to the Nal Tel-Chapalote complex, late tripsacoid, and slender popcorn of later phases (Mangelsdorf et al. 1967a). Archaeological research may ultimately reveal whether there was a single origin or multiple origins of domesticated maize, as well as where, how, and when it originated and diffused from its Mesoamerican cradle into North and South America. E. Plant Breeding and Genetics As stated earlier, maize and annual teosinte have the same chromosome number and hybridize easily. A number of crossing studies have been conducted to map the genetic differences between teosinte and maize. As early as 1896, Harshberger’s experiments crossing maize and teosinte led him to conclude that maize was the offspring of teosinte and another grass. Emerson and Beadle (1932) studied crosses between Z. m. ssp. mexicana (races Chalco and Durango), Z. luxurians (Florida teosinte), and perennial teosinte (2n = 40), and a maize line with two marker loci c and Wx on chromosome 9. They determined that crossing-over was approximately the same in teosinte as in maize. Durango teosinte, which has an inversion in chromosome 9 that causes reduction in crossing over, was the sole exception. Mangelsdorf and Reeves (1939) examined crosses between Florida teosinte and a maize line with marker loci on chromosomes 2, 4, 6, and 9; Langham (1940) studied maize × Durango teosinte hybrids; Rogers (1950) mapped linkage relations in hybrids between maize and Z. m. ssp. mexicana (races Chalco, Nobogame, Central Plateau, and Durango), Z. m. ssp. huehuetenangensis and Z. luxurians; and Beadle produced hybrids between Chalco teosinte and the primitive Mexican popcorn Chapalote (1972) in a large-scale crossing experiment. All of these experiments, though differing on some specifics, showed that certain loci are inherited together in clusters and, except when crossing over occurs, these linked blocks of genes act like one supergene. Linkage groups on chromosomes 1, 3, 4, and 9 were found to be largely responsible for the characteristics that distinguish maize from teosinte. More recently, Doebley et al. (1990) extended mapping of Chalco teosinte hybridized with Chapalote maize to characterize inheritance of quantitative trait loci (QTLs) or compound loci using restriction fragment length polymorphism (RFLP) molecular marker loci. QTL mapping is a technique that scores continuous variation and provides greater flexibility in determining relationships if traits do not segregate in accordance with strict Mendelian ratios, probably because

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of within-locus crossing-over. This work indicated there are five linkage blocks on chromosomes 1, 2, 3, 4, and 5 that distinguish teosinte from maize. Because the phenotypic effects of these blocks of genes in the teosintemaize mapping studies were similar to effects of Tripsacum chromosomes in maize-Tripsacum hybrids, Mangelsdorf (1974) regarded “teosinte as essentially maize with a relatively few blocks of genes from Tripsacum.” When asked, as he often was: “If teosinte is a hybrid between maize and Tripsacum, why do you not synthesize it by crossing the putative parents?” Mangelsdorf’s answer, at the time before the collections of Latin American maize races and Tripsacums had been completed, was that since they did not suspect they were dealing with an allopolyploid hybrid that might be easily synthesized by crossing its putative parents and so little was known about the Tripsacum species and types of maize, it would be extremely difficult to identify the correct parental species involved. The discovery of Zea diploperennis was significant because for the first time we had a perennial teosinte with the same chromosome number as maize. However, its chromosome architecture was quite distinct from maize and the Mexican teosintes. The perennial has terminal instead of internal knobs. A study of the inheritance of knob heterochromatin in hybrids between the primitive popcorns Palomero Toluqueño and Chapalote revealed these incompatibilities in chromosome architecture were accompanied by cytological anomalies as well as bizarre mutations in hybrid plants that only produced staminate flowers or ears in which the kernel orientation on the cob was reversed and the silks that clumped at the base of the ear never emerged for pollination (Eubanks 1987a). Eubanks (1987a) observed that the terminal knobs of diploperennis resembled those of Tripsacum (see Fig. 2.10), and in spite of their different chromosome numbers, the total length (501.6 µm) of the ten diploperennis chromosomes (Pasupuleti and Galinat 1982) is virtually the same as the total length (496.8 µm) of the 18 haploid chromosomes of Tripsacum (Chandravadana et al. 1971). Thus, she surmised that the similarities in chromosome architecture might enhance pairing and recovery of viable hybrids. Controlled cross-pollinations of Z. diploperennis (2n = 20) and Tripsacum (2n = 36 and 2n = 72), in which both taxa have served as pollen donor and pollen recipient, yielded fertile hybrid plants (Eubanks 1989, 1992, 1994, 1995, 1996, 1997a, 1998). Three hybrid plants obtained from controlled cross pollinations between Tripsacum dactyloides and Zea diploperennis have been described (Eubanks 1989, 1992, 1996) and are referred to by their culti-

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var names: ‘Sun Dance’, ‘Tripsacorn’, and ‘Sun Star’. The Zea diploperennis parents used to make these crosses were plants grown from seed provided by Professor Hugh Iltis of the University of Wisconsin at Madison. The seed was collected from two populations growing in Jalisco, Mexico. One was labelled Jalisco R. Guzmán M. #777, and the other Upper las Joyas Iltis et al. #1250. Two clones of Tripsacum dactyloides growing at the University of Indiana at Bloomington Experiment Station were the Tripsacum parents in the crosses. One that was growing next to a door of the greenhouse is a tetraploid (2n = 72). The other, which was growing adjacent to the fence, is a diploid (2n = 36). ‘Sun Dance’ was derived from applying tetraploid Tripsacum pollen to the styles of a diploperennis plant grown from Iltis et al. #1250 seed. ‘Sun Star’ was derived from applying diploid Tripsacum pollen to a plant grown from Guzmán #777. ‘Tripsacorn’ was produced by applying pollen from a diploperennis plant grown from Iltis et al. #1250 seed to the styles of an emasculated tetraploid Tripsacum plant. Two other hybrid plants were obtained from pollinating diploperennis Guzmán with the diploid Tripsacum pollen (an illustration of one of these hybrids can be seen in Eubanks 1995, Fig. 1). Chromosomes of all of these hybrids have been examined in standard cytological preparations of microsporocytes stained with acetocarmine (Eubanks 1987a). Based on numbers of chromosomes observed at diakinesis (Eubanks 1995, Fig. 7), 2n = 20 in the hybrids. Degree of pairing observed in cells ranges from 10 bivalents to 9 bivalents and 2 univalents or 8 bivalents and 4 univalents. Approximately one quarter of the cells observed contain one or more nuclear bodies that are as large as the nucleolus in some cells. No irregularities have been observed in metaphase or anaphase, but indicators for recombinant chromosomal events can be observed in pachytene. These include translocations, inversions, and irregular alignment. The plants of Tripsacum-Z. diploperennis hybrids, like their parents, are perennial and send out shoots from underground rhizomes. They are fully fertile and have the same chromosome number as Zea (2n = 20). They grow up to 2 m in height and produce as many as 50 primary culms. Plants can be propagated asexually by rhizome divisions and rooting cuttings, as well as sexually by seed. There is a wide range of variation in the inflorescences on a single plant, including forms resembling both parents as well as novel forms that have characteristics of a wild maize prototype. Staminate flowers are produced in tassels at the tips of the culms or they are borne on a lateral spike above the pistillate flowers. One type of pistillate inflorescence is a single rowed spike of four to six distichously arranged caryopses in hard, shell-like fruitcases that disarticulate upon maturity for natural seed dispersal; whereas

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another form has paired kernels partially enclosed in glumes with fused rachid segments approaching a cob-like structure (Fig. 2.11). Types that have many of the same characteristics of the oldest maize remains in the archaeological record have been recovered in segregating phenotypes from hybrid intercrosses in which Tripsacum is the female parent throughout the lineage (MacNeish and Eubanks 2000) (Fig. 2.12). The phenotypic similarity to ancient maize, the same chromosome number as maize, and the fertility of the hybrids led Eubanks (1995) to

Fig. 2.11. Z. diploperennis × Tripsacum F1 pistillate spike with paired, slightly exposed kernels in each cupule representing an intermediate form between teosinte and maize that may simulate “wild” maize.

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Fig. 2.12. Ears resembling archaeological specimens of early cultivated maize from the Valley of Tehuacán, Mexico that were derived experimentally from intercrosses of Tripsacum × Z. diploperennis hybrid progeny with Tripsacum as the maternal parent throughout the descent line.

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propose that these hybrids represented a reconstructed prototype of wild maize. However, these same characteristics were so unexpected that critics contested the validity of the hybrids (Dold 1997) and have claimed the plants were self or sib pollinated by Zea diploperennis, or the Zea diploperennis plants used to make the crosses were contaminated with maize, or Eubanks’s original crosses were contaminated by stray maize pollen. In order to determine the parentage of the putative hybrids and test her hypothesis that maize had two ancestors instead of one, Eubanks initiated DNA fingerprinting employing a number of molecular marker loci that Doebley et al. (1990) had demonstrated were linked to traits involved in the transformation to maize. Because the hybrids contained bands that were present in the Tripsacum parents and were not present in the diploperennis parents, maize, or annual teosintes, Eubanks concluded that the plants were true hybrids between Tripsacum and Zea diploperennis (1995, 1997a, 1999b). Furthermore, molecular analysis showed that the hybrids have alleles inherited from the Tripsacum parent not found in Z. diploperennis at most of the loci for the key traits distinguishing maize from its wild relatives (see Table 2.3), and these same alleles are inherited in subsequent generations of hybrids (Eubanks 1997a). Marker loci with unique Tripsacum contributions on chromosomes 1, 2, 3, 4, 6, and 9 corresponded well with results of previous investigators (Mangelsdorf 1947; Rogers 1950; Doebley et al. 1990; Doebley and Stec 1991; Dorweiler et al. 1993; Doebley et al. 1995). The findings also correlated well with Galinat’s (1973) intergenomic mapping data for maize, teosinte and Tripsacum, and Blakey’s (1993) molecular map of Tripsacum that showed homeology between certain Tripsacum linkage groups and maize chromosomes. To also test the hypothesis that maize arose via hybridization between Tripsacum and a primitive Zea, Eubanks (1999b) extended DNA fingerprinting to include populations of five or more individuals of three annual teosintes (Z. m. ssp. parviglumis, Z. m. ssp. mexicana, Z. luxurians), and three ancient indigenous primitive popcorns (Nal Tel and Chapalote from Mexico, and Pollo from South America). Results revealed there are Tripsacum alleles in the hybrids and primitive maize races that are not found in any of the teosintes at 21 out of 74 molecular marker loci (see Fig. 2.13). Although these results might possibly be explained by lineage sorting because Tripsacum is a distant relative of maize, if teosinte is the sole ancestor of maize such a high frequency of Tripsacum alleles in maize that are not present in teosinte would not be expected and the more probable explanation is that maize is a segmental allopolyploid (Galinat et al. 1964; Galinat 1973; Gaut and Doebley 1997) with Zea and Tripsacum contributing to the maize genome. Fig. 2.14 shows a putative evolutionary trajectory for maize via

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Fig. 2.13. Schematic representation of the ten linkage groups of maize showing loci for 74 molecular markers used in DNA fingerprinting of Zea and Tripsacum. The marker loci in bold are associated with genes involved in the transformation to domesticated maize where alleles are present in the Tripsacum parents, but not in Zea diploperennis parents, and are in the Tripsacum-Z. diploperennis hybrids. This molecular evidence supports the hypothesis that maize derives from intergeneric hybridization between Tripsacum and Zea diploperennis.

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Fig. 2.14. Putative evolutionary trajectory of Zea mays based on experimentally reconstructed prototypes derived from cross pollinating Tripsacum dactyloides and Zea diploperennis. F1 hybrid progeny are designated “wild” maize and subsequent segregating intercrosses that trace descent through Tripsacum maternal inheritance may represent “early cultivated” maize.

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hybridization based on Eubanks’s experimental reconstruction of prototypic forms. It is not easy to obtain hybrids between Tripsacum and Zea diploperennis. It takes hundreds of pollinations to obtain a viable seed and many of the seedlings die within two weeks of germination. The rare successful hybrids all involve a change in chromosome number from the expected 2n = 28 (10 chromosomes from diploperennis and 18 from diploid Tripsacum) or 2n = 46 (10 chromosomes from diploperennis and 36 from tetraploid Tripsacum) to 2n = 20. There is precedent for such change in chromosome number in other wide cross hybrids that may be explained by the processes of chromosome elimination, fusion, and/or rearrangements involving breakage, translocations, and inversions (John and Freeman 1975; McClintock 1984; Singh 1993; Wagner et al. 1993). Cytological examination of chromosomes of the Tripsacumdiploperennis hybrids indicates there has been change in chromsome structure. For example, there is a knob at end of the long arm of Z. diploperennis chromosome 6 (Fig. 2.10D). In the hybrids, the long arm of chromosome 6 has been shortened and the knob has either been eliminated or translocated (Fig. 2.10F). The phenomenon of genomic rearrangement and elimination of targeted chromosomes in interspecific hybrids has been reported in Hordeum (Linde-Laursen and von Bothmer 1988). There is a well characterized mechanism for eliminating multivalents formed during meiotic prophase in favor of bivalents between homoeologous chromosomes (Jenkins et al. 1988; White et al. 1988; Davies et al. 1990). Furthermore, it has been demonstrated in a Lolium hybrid that differences in length of homoeologues is accommodated for by the structure of the synaptonemal complex (Jenkins and White 1990), and diploid hybrids instead of expected polyploids are formed. Such interspecific diploids have been reported for Hypochoeris maculata × H. grandiflora (Compositae), Festuca drymeja × F. scariosa (Gramineae), and Lolium temulentum × L. perenne (Gramineae) (Jenkins and White 1990). Diploids have also been recovered from the intergeneric cross Solanum lycopersicoides × Lycopersicon esculentum (Chetelat et al. 1989). In crosses between oilseed rape Brassica napus (2n = 38) with wild radish B. raphanistrum (2n = 18), plants with 2n = 18 are recovered in the F4 generation (Chèvre et al. 1997). When B. napus (2n = 38) is crossed with its wild relative B. campestris (2n = 20), hybrid plants with 20 chromosomes and pollen fertility greater than 90% are recovered in F1 progeny (Mikklesen et a. 1996). Backcrosses of amphidiploids to diploid parental species are also known to occur in nature. For example, hybrids between the diploid spotted orchid Dactylorhiza fuchsii and

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the tetraploid marsh orchid D. purpurella in Durham County, England produce diploid progeny with the same chromosome number as D. fuchsii (Lord and Richards 1977). A possible explanation for the unexpected success of the crosses between Tripsacum and diploid perennial teosinte may be that Tripsacum in an amphidiploid (i.e. allopolypolid comprised of two different genomes) as proposed by Stebbins (1950) and supported by experimental intergeneric mapping by Galinat et al. (1964). Allopolyploids carry large numbers of duplicated genes. Results of studies employing genetic (Rhoades 1951), biochemical (Wendell et al. 1986), and molecular (Helentjaris et al. 1988) methods have confirmed that maize has many duplicated genes in its genome. Therefore, maize appears to be a cryptic or veiled polyploid (Wagner et al. 1993). If Tripsacum derived from a cross between an ancestral Zea diploperennis and another grass, the Tripsacum-diploperennis hybrids may represent backcrosses to an ancient progenitor. Allopolyploidy has played a role in the evolution of such crops as wheat, cotton, tobacco, and sugarcane. An experimental method of genome analysis to characterize ancestral species is backcrossing known or synthesized allopolyploids to putative diploid progenitors. Relationships between species are determined by the degree of chromosome pairing and level of fertility in offspring derived from those crosses (Jensen 1989; Singh 1993). Hybrids from such experiments are sometimes instrumental in overcoming reproductive barriers and serve as bridge species for transferring desired genes into crops (Hadley and Openshaw 1980). The high fertility and excellent chromosome pairing of the Tripsacum-diploperennis hybrids, plus their ability to bridge reproductive barriers for transferring Tripsacum genes to maize, signals a close phylogenetic relationship between Tripsacum and Z. diploperennis that is supported by the molecular data (Eubanks 1995, 1997a, 1999b; Buckler and Holtsford 1996). Since diploperennis and Tripsacum are wind pollinated, the greenhouse pollinations did not require any special techniques, and the caryopses germinated naturally, there is the possibility that natural hybrids could have formed at the margins of sympatric populations of Tripsacum and Zea diploperennis. Although Zea diploperennis is an endangered species with only four populations in Jalisco today, it was presumably more widespread at one time. The geographical range of Tripsacum dactyloides includes Jalisco and neighboring states of western Mexico (Figs. 2.5 and 2.6); therefore, it seems reasonable to conjecture that crosses between these genera could have occurred in nature during their evolution.

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V. SYNTHESIS AND FUTURE DIRECTIONS The question of the origin and ancestry of cultivated crop plants has important implications for archaeology and agronomy (Bates and Deyoe 1973; Zohary 1973; Harlan 1976). Walton C. Galinat, a plant breeder whose career has encompassed the study of maize evolution for half a century, advised: “An efficient exploitation of the variation that lingers from the past requires an understanding of the evolutionary pathways . . . Insights of what came ‘in the beginning’ provide an understanding of how corn evolved to its present state and, thereby, indicate possible extrapolation for a future evolution in symbiotic harmony with [hu]man[s]” (1977). A. Paleoethnobotany There are two archaeological models, Río Balsas and Tehuacán, that explain the origin of maize agriculture (MacNeish and Eubanks 2000). The Río Balsas or lowland model is centered around the teosinte hypothesis. It contends that maize originated from natural mutations in annual teosinte growing in the lowlands of the Río Balsas drainage in Guerrero, Mexico, where it was harvested and possibly cultivated by humans before 7,500 years ago. The paleoecological evidence presented in support of the Río Balsas model includes pollen and phytolith profiles from lowland Mesoamerica. As pointed out earlier, these methods are handicapped by ambiguity in microfossil identification and deposition. Thus, presently there is no rigorous evidence to support the Río Balsas model. Molecular evidence viewed as supporting this model includes isozyme, cpDNA, and more recently ITS studies (Piperno and Pearsall 1998). The focus of these studies was almost exclusively on Zea and comparable data for Tripsacum species were not reported. Because there are now known limitations to application of the above molecular methods for deriving accurate phylogenetic relationships of closely related taxa, particularly when there has been introgression as between teosinte, maize, and Tripsacum, the Río Balsas model is noticeably weakened. The Tehuacán or highland model, on the other hand, is based on the direct evidence of maize macro remains excavated in dry caves in the Valley of Tehuacán, Mexico, and experimental evidence from plant breeding in which a prototype “wild” maize that bears striking similarities to the oldest archaeological maize remains has been reconstructed by crossing Tripsacum and perennial teosinte. Additional support for the Tehuacán model is the molecular evidence from DNA fingerprinting that

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shows that the maize prototype hybrids carry alleles from Tripsacum that are also found in ancient indigenous maize races but not in any of the teosintes. Although the two models differ in how and which teosinte species was transformed to become maize, both are in agreement that teosinte is an ancestor of maize. Genomics research now underway promises to provide a wealth of new molecular data to guide our understanding of maize diversity and make it possible to relate the findings to the archaeological record in a way that will bring new insights into the evolutionary history of maize, thus broadening our understanding of how it interfaces with New World culture history during the transition from hunting and foraging to the development of agriculture at the end of the Pleistocene. B. Crop Improvement During the 1990s the agricultural landscape has changed rapidly as seed companies have been bought by chemical companies incorporating transgene technologies to develop genetically modified organisms (GMOs) with new traits such as herbicide tolerance and pest resistance. Approximately 45% of all American corn growers are now planting transgenic corn that carries the Bt gene for resistance to European corn borer and genes for tolerance to particular brands of herbicides. One rationale driving advances in transgene technology is concern about how agriculture will be able to produce enough food for an ever-growing human population and concerns about the environment (Kendall et al. 1997). This technology allows transfer of genes from different organisms such as animals and bacteria, or new genes synthesized in the laboratory, into crop plants, and theoretically it will enable production of crops with capabilities unprecedented in nature. There are still hurdles in development of transgenic technology, particularly in maize. Organisms have naturally evolved mechanisms to protect themselves from invasion of foreign DNA, and these same mechanisms often prevent successful incorporation or desired expression of transgenes. Better knowledge about the evolution of maize will provide molecular biologists with greater understanding of DNA architecture and genetic mechanisms involved in gene expression that will enhance their ability to overcome these innate biological protections and improve ability to insert, manipulate, and express transgenes in maize. On the other hand, there are concerns engendering violent opposition to GMOs. These include indications that deployment of transgenic crops may impair natural ecosystems and have harmful effects on beneficial organisms (NABC 1988, 1991; Lewis and Palevitz 1999). It is suggested

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that the movement of transgenes into wild or weedy crop relatives via cross pollination could lead to weeds no longer susceptible to herbicides or species with new traits that could disrupt the balance among species in nature and impact natural habitats in ways similar to the introduction of exotic plants that in some cases have virtually eliminated native species (Goldburg 1992; Raybould and Gray 1994; Mikkelsen et al. 1996; Chèvre et al. 1997). Other concerns are potential harm to beneficial insects, e.g. lethal toxicity of maize pollen carrying the Bt gene when ingested by Monarch butterfly larvae (Losey et al. 1999). There are also indications that target insects such as the European corn borer develop resistance to the Bt gene (Huang et al. 1999), and allergic reactions to transgenic crops have been reported in humans (Nordlee et al. 1996). The scientific controversies surrounding transgene technology have been exploited by advocacy groups and reported in the popular press, resulting in growing consumer resistance to agricultural biotechnology in the European Union, Japan, other countries, and recently the United States (Williams 1998; Lewis and Palevitz 1999). Whatever the outcome, it seems clear that better characterization of the evolutionary relationships of maize is important. Accurate knowledge of its phylogenetic history will also enhance opportunities for improving maize using either traditional breeding methods to utilize beneficial genes from wild relatives that have evolved natural survival mechanisms for coping with pests and abiotic stress and which are harmless to humans or via transgene technology. In these changing times in the history of agriculture, Galinat’s (1977) insightful observation that knowledge of the origin of maize will guide its future evolution still holds true.

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Smith, J. S. C., M. M. Goodman, and C. W. Stuber. 1984. Variation within teosinte: II. Numerical analysis of allozyme data. Econ. Bot. 38:97–113. Smith, J. S. C., M. M. Goodman, and C. W. Stuber. 1985. Relationships between maize and teosinte of Mexico and Guatemala: Numerical analysis of allozyme data. Econ. Bot. 39:12–24. Smith, R. M., P. C. Twiss, R. K. Krauss, and M. J. Brown. 1970. Dust deposition in relation to site, season, and climatic variables. Soil Sci. Soc. Am. Proc. 34:112–117. Snow, A. A., and P. M. Palma. 1997. Commercialization of transgenic plants: Potential ecological risks. BioScience 47:86–96. Stebbins, G. L. 1950. Variation and evolution in plants. Columbia University Press, New York. Stebbins, G. L., and B. Crampton. 1961. A suggested revision of the grass genera of temperate North America. Recent Adv. Bot. 1:133–145. Stuber, C. W., and M. M. Goodman. 1983. Allozyme genotypes for popular and historically important inbred lines of corn, Zea mays L. USDA Agri. Res. Results, Southern Series 16, New Orleans, LA. Sundberg, M. D., and A. R. Orr. 1986. Early inflorescence and floral development in Zea diploperennis, diploperennial teosinte. Am. J. Bot. 73:1699–1712. Talbert, L. E., J. F. Doebley, S. Larson, and V. L. Chandler. 1990. Tripsacum andersonii is a natural hybrid involving Zea and Tripsacum: Molecular evidence. Am. J. Bot. 77:722–726. Tantravahi, R. V. 1968. Cytology and crossability relationships of Tripsacum. Harvard Univ. Bussey Inst., Cambridge, MA. Ting, Y. C., M. G. Gu, and M. Yu. 1981. Meiotic observations of diploid perennial teosinte. Maize Gen. Coop. Newslett. 55:21–22. Vavilov, N. I. 1931. Mexico and Central America as the principal centre of origin of cultivated plants of the New World. Bul. Appl. Bot., Gen. Plant Breeding 16:1–248. Wagner, R. P., M. P. Maguire, and R. L. Stallings. 1993. Chromosomes: A synthesis. WileyLiss, New York. Weatherwax, P. 1918. The evolution of maize. Bull. Torrey Bot. Club 45:309–342. Weatherwax, P. 1935. The phylogeny of Zea mays. Am. Midland Naturalist 16:1–71. Wellhausen, E. J., L. M. Roberts, E. Hernandez X. in collaboration with P. C. Mangelsdorf. 1952. Races of maize in Mexico: Their origin, characteristics and distribution. Harvard Univ. Bussey Inst., Cambridge, MA. Wendell, J. F., C. W. Stuber, M. D. Edwards, and M. M. Goodman. 1986. Duplicated chromosome segments in Zea mays. I. Further evidence from hexokinase enzymes. Theor. Appl. Gen. 72:178–185. White, J., G. Jenkins, and J. S. Parker. 1988. Elimination of multivalents during meiotic prophase in Scilla autumnalis. I. Diploid and triploid. Genome 30:930–939. Wilkes, H. G. 1967. Teosinte: The closest relative of maize. Harvard Univ. Bussey Inst., Cambridge, MA. Wilkes, H. G. 1972. Maize and its wild relatives. Science 177:1071–1077. Wilkes, H. G. 1977. The origin of corn—studies of the last hundred years. p. 211–223. In: D. Seigler (ed.), Crop resources. Academic Press, New York. Wilkes, H. G. 1979. Mexico and Central America as a center for the origin of maize. Crop Improv. (India) 6:1–18. Wilkes, H. G. 1989. Maize: Domestication, racial evolution, and spread. p. 441–455. In: D. R. Harris and G. C. Hillman (eds.), Foraging and farming. Unwin Hyman Ltd., London.

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3 History of Public Onion Breeding Programs in the United States Irwin L. Goldman* and Geoffrey Schroeck Department of Horticulture, 1575 Linden Drive, University of Wisconsin–Madison, Madison, WI 53706 Michael J. Havey USDA Agricultural Research Service and Department of Horticulture, 1575 Linden Drive, University of Wisconsin–Madison, Madison, WI 53706

I. INTRODUCTION A. Origin of Onion B. Onion as a World Crop C. Onion Biology and Horticulture II. HISTORY OF ONION IN THE AMERICAS A. Origin of Onion and Settlement of the New World B. Adaptation of Storage Onion to Shorter Days C. Variability and Cultivar Distinctions D. Founding Populations III. STATE EXPERIMENT STATION PROGRAMS A. California B. Colorado C. Idaho D. Iowa E. Louisiana F. Michigan G. New Mexico H. New York I. Texas

*Corresponding author, [email protected], FAX 608-262-4743

Plant Breeding Reviews, Volume 20, Edited by Jules Janick ISBN 0-471-38788-6 © 2001 John Wiley & Sons, Inc. 67

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J. Utah K. Wisconsin IV. FUTURE PROSPECTS FOR PUBLIC ONION BREEDING IN THE UNITED STATES LITERATURE CITED

I. INTRODUCTION A. Origin of Onion The bulb onion (Allium cepa L., Alliaceae) originated in the region comprising Afghanistan, Iran, and the southern portion of the former Soviet Union (Havey 1995). The genus Allium is highly diverse and contains more than 600 species, among them the edible species A. fistulosum (bunching onion, Japanese bunching onion, or Welsh onion), A. sativum (garlic), A. ampeloprasum (leek), A. schoenoprasum (chive), and A. tuberosum (chinese chives). The bulb onion, A. cepa, is the most widely cultivated of these species. Vegetable Alliums are distributed throughout the temperate zones of the Northern Hemisphere and have been cultivated for thousands of years. Bulb onion has been cultivated for more than 5,000 years and is not known to exist in the wild. The closest wild relatives are A. galanthum and A. vavilovii (Hanelt 1990), both of which can be found in the area comprising Turkmenistan, Tajikistan, Uzbekistan, and northern Afghanistan. Allium cepa has been divided into two broad horticultural groups, the common onion and aggregatum groups (Hanelt 1990; Brewster 1994). The common onion group comprises the majority of bulb onions grown throughout the world. The majority of onions in this group are grown from seed to produce large bulbs. The common onion group also contains potato or multiplier onion and the ever-ready onion (Pike 1986). Potato or multiplier onions have smaller bulbs than the typical bulb onion and often do not flower and produce seed. They are typically propagated asexually from divided bulbs. The ever-ready onion possesses smaller bulbs than the multiplier onion and does not typically flower. Bulbs of the aggregatum group are smaller and are usually divided into clusters of small bulbs. This group includes the multiplier onions and shallots, each of which can form up to 20 bulbs in a cluster. Shallots form a single bulb and exhibit foliar maturity by prostrate tops, both of which are similar to the typical bulb onion. Unlike the bulb onion, however, shallots are typically propagated asexually through bulb division. Most of the onions from the aggregatum group are vegetatively reproduced, although seed production is a viable alternative. Populations of bulb onion spread from Asia to Europe and were introduced to the United States by European immigrants and by trade with

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European countries. Farmers maintained these introductions as openpollinated populations for approximately three centuries before dedicated scientific programs to improve onion were established. Although the modern U.S. marketplace contains a variety of onion market classes, it is possible to simplify these classes into three primary types of onion: storage, Grano or Valencia, and Spanish. The latter two categories comprise onion cultivars typically grown under a daylength of less than 13 h, and stored briefly in cold temperatures or consumed immediately. Storage onions are typically grown under daylengths greater than 14 h and can be held in cold temperature storage for a considerable length of time. Virtually all of the onion germplasm developed in the United States has its origin in Europe. Introduction of onion germplasm to this country occurred during two primary periods: the early 17th century arrival of English Puritans, and the 19th century arrival of immigrants from southern European countries such as Spain and Italy. Storage onion germplasm was introduced during the former period, while Grano and Spanish germplasm were introduced during the latter. Although some information about the founding onion populations in the United States has been collected (Magruder et al. 1941), no systematic study of their relationships has been published. A proliferation of cultivar names during the 19th century during the early growth of the vegetable seed industry (Tracy 1903) led to much confusion about the genetic relationships among open-pollinated populations. One of the primary objectives of this review is to clarify relationships among onion populations and germplasm accessions used in U.S. onion breeding during the 20th century. B. Onion as a World Crop Worldwide, onion ranks among the most important vegetable crops, with a total production of 37 million tonnes in 1998. Onion typically follows only tomato, potato, and lettuce in value among all vegetable crops in the United States, which is the world’s third largest onion producer, with 3 million tonnes produced on 64,751 ha in 1998, valued in excess of $830 million dollars. Onion is produced from Scandanavia to the humid tropics, although the great majority of production occurs in temperate and sub-tropical regions (Brewster 1994). Important exporters of onion include Spain, India, Mexico, Turkey, U.S.A., Poland, and The Netherlands. International trade in onion bulbs exceeds 2 million tonnes per year (Brewster 1994). Much of the European bulb onion production is spring-sown and fall-harvested. Even though much of this crop can be stored as bulbs and sold during the fall, winter, and spring months, there is short supply of onion during the late spring and early summer. Onion bulbs produced

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in the Southern hemisphere in South America, New Zealand, and Australia are sold during the spring and summer in Europe to fill this niche. The onion market in the United States is supplied by crops from various regions throughout the country. Bulbs from fall-sown crops in the western United States (California, Colorado, Idaho) are harvested beginning in the summer and are sold throughout the country during the summer and fall. Pungent, long-storing bulbs from spring-sown crops in the northern states (Michigan, New York, Wisconsin, among others) are sold throughout the fall, winter, and spring months. Mild bulbs from fallsown crops in Texas, New Mexico, and Arizona are harvested between January and June, thus assuring for continuous availability of onion bulbs. While pungent, long-storing onions grown in the northern states are typically stored at ambient conditions during the fall and winter, much of the trade in mild bulb onion is facilitated by controlledatmosphere storage facilities, which make use of elevated CO2 levels and precision control of humidity to increase bulb storage life. C. Onion Biology and Horticulture The bulb onion exhibits a large range of variability in many horticulturally important characteristics. A sample of these includes bulb shape, which can range from a flattened globe to cylindrical; bulb color, which can be white, yellow, brown, red, purple, or chartreuse; foliage color; presence of bulbils in the inflorescence; resistance to bolting; and daylength required for bulb formation. This latter characteristic is of primary importance for adaptation of onion populations to various world regions, as both daylength and temperature influence bulb formation. The interaction between onion bulb formation and daylength was first noted by Garner and Allard (1920) and has been a subject of great importance for onion producers as onion cultivars have spread throughout the world. In brief, daylengths in excess of 14 h (actually night lengths less than 10 h) induce bulb formation in long-day onion, while daylengths less than 13 h (night lengths greater than 11 h) induce bulb formation in short-day onion. Bulb formation in intermediate types is induced at daylengths between 13 and 14 h. Because bulb formation is due to an interaction between temperature and daylength, the exact daylength required to form adequate bulbs varies with environment and latitude. In general, short-day onions are used in latitudes with photoperiods from just slightly above 12 to approximately 14 h. Intermediate-day onions are grown as a fall-sown crop in middle latitudes where they are overwintered and harvested in late spring and early summer. Longday onions are typically spring-sown at northern latitudes and har-

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vested in late summer or early fall. Daylengths ideal for bulb formation occur during mid-summer, resulting in adequate bulb formation prior to frost. Onion bulbing in a particular photoperiod is hastened by increased temperatures. Onion cultivars adapted to long days will not form bulbs at photoperiods of 12–13 h, whereas short-day cultivars planted at greater than 14 h daylengths will begin to form bulbs immediately upon emergence, resulting in very small bulbs. All of the widely cultivated Alliums have a basic chromosome number of eight (Havey 1993). Bulb onion is diploid with 2n = 2x = 16. Onion is an excellent model for cytological investigation due to its relatively large chromosomes. Relative to other important crop plants, few genes have been identified in bulb onion. The biennial nature of the onion plant and the time necessary to complete segregation analysis may be partly responsible for the lack of genetic information on this important crop. In addition, onion possesses one of the largest genomes of all crop plants (King et al. 1998; Arumuganathan and Earle 1991), making molecular investigation more challenging. Genes for restoration of male fertility; resistance to Fusarium basal rot, downy mildew, ozone, and pink root; and anther, foliage, seed, and bulb colors have been characterized (reviewed in Havey 1993; Cramer and Havey 1999). Rabinowitch (1988) and Cramer and Havey (1999) reviewed 24 isozyme markers developed for onion. Recently, more than 125 DNA markers were identified and characterized in a segregating population and the first molecular map of onion was developed (King et al. 1998; Cramer and Havey 1999). The goals of this paper are: (1) to present a brief history and pedigree of the founding onion populations in the United States; (2) to trace the development of inbred lines, hybrids, and open-pollinated populations following the establishment of the national onion breeding program in 1936 by Dr. Henry A. Jones; and (3) to describe the pedigree relationships among all public onion germplasm releases in the United States since the first releases from the national onion breeding program in 1931.

II. HISTORY OF ONION IN THE AMERICAS A. Origin of Onion and Settlement of the New World As stated previously, the center of origin for the bulb onion is a region comprised by Afghanistan, Iran, and the southern portion of the former Soviet Union (Jones and Mann 1963; Havey 1995). The bulb onion is only known in cultivation. Little information exists regarding spread of the bulb onion from Asia, although physicians commented on the use

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of onions as curatives by the sixth century B.C.E. in India (Havey 1995) and by the first century in Greece (Jones and Mann 1963). Widespread cultivation of onion was noted in Europe by the Middle Ages. Popularity of the bulb onion to cultures throughout Asia and Europe was due to its threefold use as a succulent vegetable, a flavorant for numerous cuisines, and as an herbal remedy for a wide variety of human ailments (Block 1992). Although the bulb onion is an old world crop, this historical treatment deals exclusively with the introduction to and spread of bulb onion in the New World from a plant breeding point of view. The European encursion in the Americas, referred to by the Europeans as the “discovery of the New World,” encountered various civilizations that proved to be unaware or only vaguely aware of each other; although the common cultivation of maize and beans suggested more ancient contacts. By the beginning of the 17th century along the eastern coast of North America, Native American tribes cultivated not only maize and beans but a number of edible root crops that were not native to North America, such as turnip, parsnip, carrot, and onion. These plants were introduced by trade just prior to, or during the period of, early settlement by Europeans (Arrington 1922; Fuess 1935). The presence of native Allium species, such as A. canadense in eastern North America has been well-documented, and some of these species were and continue to be routinely used for similar vegetable and medicinal purposes as bulb onion. However, despite the fact that Native Americans on the eastern coast of the United States cultivated NewWorld crops such as beans and maize, there is no evidence that they cultivated the bulb onion prior to the arrival of European traders during the late 16th and early 17th centuries. Cultivation of a bulb onion crop in Europe was well established by the time of the European encursion into eastern North America. The bulb onion crop in northern Europe was typically spring sown and harvested in the fall, whereupon bulbs were cured, stored, and consumed during the winter months. Thus, the bulb onion from northern Europe is considered a storage onion. By contrast, certain populations of bulb onion produced in southern Europe did not possess sufficient storability and were considered separately from the storage onion. The story of storage onions in the New World likely begins with the Puritans, the 17th-century English dissenters who were escaping religious persecution from the Church of England. The Puritans fled first to Holland and, after a short stay, arrived in the New World in 1620, landing off the coast of what is now the Commonwealth of Massachusetts. The first immigrants were from the area around Dorchester, located in the southern part of England. Within several years these immigrants,

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referred to as Pilgrims after 1793, established the Massachusetts Bay Company and more settlers began to arrive. The beginnings of Europeanstyle agriculture in the New World can be traced to this period because the first settlers brought seeds of European crops with them (Fuess 1935). It is likely that they also brought onion seed that became the foundation populations for storage onion production in the New World. Many references point to non-native crops grown by the Pilgrims during the early years of their settlements (Fuess 1935; Adams and Stiles 1974). Agricultural records indicate that grains such as wheat, rye, barley, and oats were carried by early settlers from England to the New World (Fuess 1935). Hedrick (1919) pointed out that onion was likely among the garden plants sown by Columbus at Isabela Island in 1494, although they are not specifically mentioned. Sir John Endicott arrived in Naumkeag (coastal Massachusetts) in 1628 and obtained a large land grant to establish a settlement in the area that became known as Salem (Danvers Mirror 1899). During the winter of 1628, the Massachusetts Bay Company shipped “a hogshead each, in the ear, of wheat, rye barley, and oats, beans, peas, woad seed, madder roots and seeds, potatoes, and hemps and flax seed” from England to Salem (cited in Fuess 1935). This shipment was clearly to be used for planting the following spring. B. Adaptation of Storage Onion to Shorter Days The area near Dorchester, England, the original home of the “Pilgrims,” is located near 51° latitude, while Salem, Massachusetts, their first settlement in the New World, is located near 41° latitude. This 10° difference in latitude is significant for bulb onion production because of daylength sensitivity. Long-day onions are grown throughout the middle and northern parts of the United States, whereas short-day onions are grown in more southern latitudes. Despite the mild, maritime climate in southern England, onion seed introduced by Puritans from Dorchester in southern England would be long-day because, on July 1, the daylength at Dorchester, England is 16 h and 27 min, whereas the daylength at Salem, Massachusetts is 15 h and 11 min; a difference of 1.35 h. Because daylength is a primary factor influencing onion bulb development, this difference is enough to affect bulb formation in long-day onion (Garner and Allard 1923; Magruder and Allard 1937). July 1 daylengths more similar to the Salem, Massachusetts area can be found in Mid-Southern Italy and Spain (Table 3.1). Italy and Spain possess daylengths slightly shorter than that found in Boston on July 1. An example of the effect of daylength on onion bulb formation occurred during a period of onion seed shortages in the United States

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Table 3.1. Daylength and foliar maturity in three onion cultivars adapted to Northern European latitudes. Source: Magruder and Allard (1937). Percentage of plants with prostrate dry foliage Cultivar Wolska Yellow Rijnsburg Yellow Zittau Round Maderia Sweet Spanish

Native latitude

12h

14h

16h

18h

52° 52° 51° 41° 39°

0 0 0 41 39

0 0 12 100 93

25 53 33 95 100

60 55 42 100 100

in the early part of the 20th century. During this period, seed was imported from Northern Europe to the United States. However, in many cases, this seed failed to produce normal bulbs (Magruder and Allard 1937). Although this failure was attributed to many causes, it is likely that the primary one was photoperiod. Northern European countries (parts of Belgium, Germany, Poland, and the Netherlands) are located at latitudes exceeding 51°, and onion populations adapted to these latitudes would necessarily be adapted to daylengths exceeding that found in nearly all of the northern United States. A landmark study by Magruder and Allard (1937) examined 10 onion cultivars commonly grown at a range of latitudes in Europe for foliar and bulb maturity traits when grown in the United States under a range of daylengths. Foliar maturity in onion is manifest by both prostrate leaves (known as “tops down”) and dry foliage. Magruder and Allard (1937) evaluated three cultivars adapted to 52° latitude: ‘Wolska’ (Poland), Yellow Rijnsburg (Germany), and Yellow Zittau (Germany) at 12, 14, 16, and 18 h photoperiods. Only 12% of the plants in plantings of one of these cultivars (Yellow Zittau) exhibited foliar maturity under a 14 h daylength, whereas no foliar maturity was observed for the other two cultivars (Table 3.1). Only 25%–50% of the plants exhibited foliar maturity under a 16 hour daylength. Bulbing was similarly inhibited. These three cultivars exhibited approximately 40% bulbing under a 14 h daylength and 100% bulbing under a 16 h daylength. Therefore, at a daylength similar to that found in Salem, Massachusetts, complete foliar and bulb maturity would not have been attained. By contrast, the onion cultivars Round Madeira and Sweet Spanish, both adapted to lower latitudes (41° and 30°, respectively) exhibited 100% and 93% foliar maturity, respectively, when grown under a 14 h daylength (Table 3.1). These data suggest that short-day onion cultivars adapted to latitudes

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similar to the Boston area would likely have exhibited some bulb formation (despite the slightly longer daylength in the Boston area) in contrast to the long-day cultivars introduced by the Puritans, which likely did not form bulbs with a frequency greater than 50%. The lack of adequate bulbing is of great importance to onion production from both a consumption and seed-supply standpoint. Poorly formed or small bulbs will obviously result in less crop for human consumption. In addition, poorly formed bulbs, particularly those that are not yet mature, will not store well during the vernalization period and therefore will produce poor seed yield the following season. Since onion is a biennial plant, seed production depends upon a supply of goodquality bulbs following the vernalization phase. Foliar maturity is closely tied to storability in that dry, mature foliage results in closure of the “neck” of the onion plant. A tight closure of this neck junction between the foliage and storage leaves aids in the prevention of neck rot pathogens and facilitates bulb storage (Hawthorn and Pollard 1954). During the 17th century in the New World, it is likely that the vernalization requirement was accomplished by storing onion bulbs during the winter in piles covered with hay or in cellars designed to store roots. Under these conditions, foliar maturity would be an important criterion for ensuring a supply of good-quality bulbs for storage and subsequent seed production. No mention of onion as a crop during the early period of settlement may reflect the challenge early settlers had in modifying the response of introduced onion populations to the New England photoperiod. Onion is a biennial plant and therefore requires two years to complete its life cycle under natural conditions. Selection for adaptation to shorter daylength might have taken a number of generations before adequate foliar and bulb maturity was obtained. Natural selection under daylengths such as those reported for Salem would have accomplished this goal during regular onion production. However, the lack of information on selection response for modification of daylength response in long-day onion makes this speculation a working hypothesis at best. It is possible that the warmer growing season in Massachusetts compared to Dorchester, England, would increase bulbing, since the bulbing response is due in part to temperature and in part to daylength. Furthermore, mention of bulb onion as a crop in Massachusetts occurred as early as 1629 (Hedrick 1919), when William Wood described their cultivation in the New World, suggesting that at least some onion crop production was underway during the first decade of New World settlement. Despite this reference, it does not appear that widespread cultivation of onion took place until later in the 17th century. More research is needed to deter-

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mine if a bottleneck in onion adaptation due to photoperiod was indeed present during the early 17th century in the New World. C. Variability and Cultivar Distinctions During the 18th and 19th centuries, it was not common to refer to certain cultivated vegetable crops by cultivar names. Cultivar distinctions did exist for certain ornamental plants and fruit trees; however, many vegetables were typically known by very broad names. Wieder (1966) reproduced a page from the James L. Belden Seed Catalog (circa 1821) in her book on the history of Wethersfield, Connecticut. Belden offers onion seed for sale, and the three types of onion seeds listed are simply named white, yellow, and red. Similar distinctions are made for other vegetables, such as cucumber, winter squash, and carrot (Wieder 1966). The lack of cultivar distinction for crops such as onion suggests onion populations were maintained by color type but not necessarily by bulb type. Thus it is possible that a single color population contained a diversity of bulb phenotypes and, possibly, a wealth of genetic diversity for various traits. The ‘Danvers Yellow Globe’ onion, perhaps the most important storage onion population ever developed in the United States, has as its parentage a population known simply as “common yellow” (Magruder et al. 1941). A population known as “common red” was also in existence in New England during the early period of settlement. ‘Wethersfield Large Red’, which was the dominant red onion on the East Coast (Wieder 1966; Magruder et al. 1941) may also have been selected from a common pool of red germplasm. This important population contained a multiplicity of bulb types. ‘Wethersfield Large Red’ exhibited a flattened bulb. ‘Southport Red Globe’, a population developed in Southport, Connecticut, exhibited a globe-shaped bulb and yet was selected from ‘Wethersfield Large Red’ (Magruder et al. 1941), suggesting that a large amount of phenotypic variability was present in early open-pollinated populations. D. Founding Populations Four founding populations make up much of the modern onion germplasm used in the United States (Plate 1). These are ‘Danvers Yellow Globe’ (storage), ‘Valencia’ (synonymous with ‘Grano’), ‘White Bermuda’, and ‘Spanish’. Each of these will be discussed in the following section. A pedigree diagram (Plate 1) describing the relationships among all U.S. public onion germplasm released during the period

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1931–1997 is presented as a guide to the narrative in Sections 4 and 5. Additional references used to create the pedigree diagram in Plate 1 include Beaumont et al. (1935), Coulter and Willard (1949), Erwin and Harter (1924–1926), Flint (1865), Mally (1915), Perry and Jones (1957), Peterson and Haber (1941), and S. Nagai (unpublished). 1. Storage Germplasm. ‘Danvers Yellow Globe’ was developed in Danvers, Massachusetts, and perfected by Daniel Buxton (Anon. 1877). Danvers was among the first settlements in Essex County, Massachusetts, and among the first where agricultural expertise was developed. The ‘Danvers Yellow Globe’ onion was also known as ‘Yellow Globe Danvers’, ‘Yellow Danvers’, ‘Round Yellow Danvers’, ‘Yellow Danvers Flat’, and other similar names. The importance of this onion as a progenitor of most, if not all, of the United States storage onion germplasm cannot be overestimated. In fact, it is likely that much of the storage onion germplasm developed during the 20th century in the United States is derived originally from this important population. In addition, ‘Danvers Yellow Globe’ was introduced to France around 1850 and became an important cultivar in Europe during the same period when it was achieving prominence in the United States (Vilmorin-Andreiux 1885). Testifying to the prominence of this cultivar, Lester Morse, founder of C.C. Morse Seed Growers of California, stated that “if the world could have but one variety, it should choose Yellow Danvers Flat of California grown seed” (Morse 1923). The exact origin of ‘Danvers Yellow Globe’ is unknown; however Magruder et al. (1941) suggest it is derived from both ‘Silverskin’ and a population known as “common yellow.” Since, as mentioned earlier, onion populations prior to the 19th century were typically named for their color, it is impossible to determine the origin of “common yellow.” On the other side of the pedigree, ‘Silverskin’ is mentioned as a popular onion cultivar by the European seedsman Vilmorin-Andrieux, published in translation in 1885 (Vilmorin-Andrieux 1885). Vilmorin-Andrieux, a scion of the famous Vilmorin family who helped establish the modern seed industry in Europe, published descriptions and illustrations of prominent vegetable varieties cultivated in Europe during the latter part of the 19th century. Three onion cultivars with descriptions similar to the ‘Silverskin’ found in European seed catalogs are the ‘Early Paris SilverSkinned’, ‘Globe Silver-Skinned’, and the ‘White Portugal’ onions. Although the ‘White Portugal’ and ‘Globe Silver-Skinned’ exhibit a globeshaped bulb while the ‘Paris Silver-Skinned’ exhibits a flattened shape, these cultivars possess the name ‘Silverskin’ among their common synonyms in different languages (Vilmorin-Andrieux 1885). ‘Paris Silver-

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I. GOLDMAN, G. SCHROECK, AND M. HAVEY

Skinned’ is said to have its origin in the southern Italian onions imported to France (Vilmorin-Andrieux 1885). Selection was practiced within the open-pollinated ‘Yellow Globe Danvers’ to develop a number of important second-generation openpollinated populations. Populations such as ‘Mountain Danvers’, ‘Early Yellow Globe’, ‘Rochester Bronze’, and others were derived from ‘Danvers Yellow Globe’ during the latter part of the 19th century and the early part of the 20th century. Selection from an open-pollinated population such as ‘Danvers Yellow Globe’ does not preclude introgression of other germplasm during their development; however, in most cases we have little if any information on the kinds of germplasm used during the selection process. An exception is ‘Downing Yellow Globe’, which was selected by Ken Trapp of Michigan (unpublished notes, ca. 1950) using ‘Southport Yellow Globe’, ‘Valencia’, and ‘Siberian Red.’ ‘Extra Early Yellow’ was selected into ‘Early Yellow Globe’ and incorporated into many of the early “B” series inbreds such as B2108B (B is for Beltsville, Maryland, home of the Agricultural Research Service of the United States Department of Agriculture and formerly of the National Onion Breeding Program), which ultimately figured into a number of the early hybrid releases from the national program (Jones 1946–1956; Davis 1957–1965) as shown in Table 3.2. ‘Downing Yellow Globe’ was selected into ‘Rochester Bronze’, which formed the foundation population for most of the “W” (for University of Wisconsin) inbred releases developed by W. H. Gabelman during the 1950s to the 1990s (Goldman 1996), as shown in Table 3.3. Since ‘Danvers Yellow Globe’ was the dominant storage onion population during the early days of onion breeding in the United States, it is likely that it can be counted as the foundation population for storage onion germplasm. Another important storage population was ‘Southport Yellow Globe’, developed in Southport, Connecticut, and used as a parent for a number of important populations including ‘Brigham Yellow Globe’. Although the origin of ‘Southport Yellow Globe’ is not precisely known, it is likely that it traces back, at least partially, to ‘Danvers Yellow Globe’. Descriptions of the two cultivars are very similar and differ primarily in terms of depth of color (Greiner 1893; Jones and Rosa 1928; Magruder et al. 1941). A survey of RFLP-based genetic distance among storage onion open-pollinated populations revealed few differences among these populations (Havey and Bark 1994), suggesting that the storage onion germplasm pool is quite narrow. ‘Southport Yellow Globe’ was selected into the early MSU inbreds (for Michigan State University) by C. E. Peterson. ‘Brigham Yellow Globe’ was selected into a series of “B” inbreds such as B2215C and used in early hybrids (Table 3.2). ‘Brigham

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Table 3.2. Hybrids and open-pollinated populations released from public onion breeding programs in the United States, 1931–1997.

Year

Population or hybrid

? ? ? ?

Stockton G-36 Lord Howe Island White Persian Crystal Grano

1931 1935

New Mexico Early Grano Red 21

1935

Brown 5

1939

Spanish No. 2

1943 1945

California Hybrid Red #1 Excel

1946

San Joaquin

1947

Calred

1947

Texas Early Grano 502 Crystal Wax L. 690 Beltsville Bunching

1949 1950

Origin or parents Stockton Yellow Globe Introduced from Australia PI 86279 from Persia Recessive white bulb from field of Early Grano Babosa California Early Red (Same as California Early Red UC #1) Australian Brown (Same as Australian Brown UC #5) Sweet Spanish selected under field pink root pressure Ital. Red 13-53 × Lord Howe Island Yellow Bermuda S1 Family (986) G-36 (S1 of Stockton Yellow Globe) × Early Grano (Ital. Red 13-53 × L. Howe Island) × Lord Howe Island New Mexico Early Grano

Cooperating experiment station or entity California California California

New Mexico

California California

California and USDA Texas, California, USDA California, USDA California, USDA Texas

S1 Family

Texas, USDA California, USDA

1952

Crystal Hybrid

1952 1953 1953 1953 1953

Granex Abundance Aristocrat Bonanza Champion

Natural amphidiploid of White Portugal 3-203-113-5 × Nebuka L690A × CC163 (L=Lubbock, CC=Crystal City, TX) YB986A × TEG951C B2108A × B2215C B2218A × B2215C B2190A × B2215C B2146A × B2215C

1953 1953 1953

Contender Early Harvest Elite

B2133A × B2215C B2108A × TEG951C B5546A × B2215C

Texas, USDA Texas, USDA Iowa, Idaho, USDA Ohio, Idaho, Iowa, USDA Idaho, Iowa, USDA New York, Idaho, Iowa, USDA Idaho, Iowa, USDA USDA Indiana, Idaho, Iowa, USDA

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Table 3.2.

(continued)

Year

Population or hybrid

1953 1953 1953 1953 1954 1954

Encore Epoch Pioneer Surprise Fiesta Eclipse

1955 1956 1956 1957 1957 1957 1957 1957 1959 1959 1962 1962 1962 1963 1963 1963 1964 1965 1966 1972 1972 1972 1972 1974

Early Crystal 281 White Granex Pilot Premier Snow White Bronze Perfection Spartan Golden Beauty Empire Treasure Nugget Hickory Spartan Era Spartan Gem El Capitan Chieftain Sunburst Spartan Banner Spartan Bounty Fusario 12 Fusario 24 Fusario 142 Fusario 245 Spartan Sleeper

1979

Spartan Banner 80

1981

NuMex BR1

1982

Sweet Sandwich

Origin or parents B2129A × B2215C B2264A × B2215C B15-108A × B2215C B2207A × B2215C B2190A × B12115C (Crystal Wax × Excel) × Excel Selfed; white bulbs selected Crystal Wax × Excel (986) L303A × B1410C B2264A × B1340B B2117A × B2215C CW15-8-4-1A × WSS34-89-1-4 B12132A × B2215C IA467-8A × B2215C B2133A × B12132B B2267 × B2215C B2267 × B 12115C IA36A × W101B IA163A × W101B IA736A × MSU611C MSU728A × MSU611C P54-371A × P54-306C U16-3-1A × B12115-2C W4A × W101B MSU2399A × MSU611C MSU1411A × MSU611C W101A × W202B W202A × W404B (W101A × W404B) × W202B Fusario 24 × W205B (MSU2935A × MSU1459B) × MSU4535B (MSU611-1A × MSU611B) × MSU2399B Texas Early Grano 502 PRR (MSU5718A × MSU8155B) × MSU826B

Cooperating experiment station or entity Iowa, Idaho, USDA Iowa, Idaho, USDA Colorado, Idaho, USDA USDA Idaho, Iowa, USDA Texas, USDA Texas, USDA Texas, USDA Colorado, USDA New York, USDA USDA Wisconsin, Idaho, USDA Michigan, Iowa, USDA Idaho, Wisconsin, USDA New York, Idaho, USDA USDA Iowa, Wisconsin, USDA Iowa, Wisconsin, USDA Michigan, USDA Michigan, USDA Idaho, USDA Utah, Idaho, USDA Wisconsin Michigan Michigan Wisconsin Wisconsin Wisconsin Wisconsin Michigan, USDA Michigan, USDA New Mexico (Corgan 1984) Michigan, New York, USDA (Peterson et al. 1986)

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3. HISTORY OF PUBLIC ONION BREEDING PROGRAMS IN THE U.S. Table 3.2.

Year 1983

81

(continued)

Population or hybrid

Origin or parents

Cooperating experiment station or entity

TEG951C

Texas (Pike et al. 1988a)

Texas Early Grano 502 × Ben Shemen Texas Early Grano 502 × Ben Shemen Texas Early Grano 502 × Ben Shemen Ben Shemen

Texas (Pike et al. 1988b)

1986

Texas Grano 1015Y Texas Grano 1025Y Texas Grano 1030Y Texas Grano 1105Y NuMex Sundial

1986

NuMex Suntop

Ben Shemen

1986

NuMex Sunlight

Texas Early Grano 502 PRR

1987 1990 1991 1992

Blitz NuMex Starlite Perla NuMex Casper

1992

Texas Grano 438

1992 1992

Dorada NM899

1994

NuMex Jose Fernandez

1994

NuMex Bolo

1995 1995

NuMex Mesa NuMex Dulce

(B6693A × B2923B) × B7728B Texas Early Grano PRR Ben Sheman Temprana, Snow White, New Mexico White Grano, and white selection from TEG502PRR Texas Early Grano 502 × Ben Shemen Ben Sheman Early Supreme, Southport White Globe, New Mexico White Grano PRR, White Creole, and Ringmaster NuMex BR1, Buffalo, Ben Shemen, Ben Shemen × Sweet Spanish, Peckham YSS, Tucker YSS × PI249538, El Capitan × PI 249538 Spartan hybrids, Tucker YSS, Utah YSS, El Capitan, Ring King, Inca, Peckham YSS, Ben Shemen, Colorado #6, Texas Grano 502 PRR NuMex BR1 × Buffalo Low pungency strain of NuMex Starlite

1983 1983 1983

Texas (Pike et al. 1988c) Texas (Pike et al. 1988d) New Mexico (Corgan 1988a) New Mexico (Corgan 1988b) New Mexico (Corgan 1988b) USDA New Mexico Texas New Mexico

Texas Texas New Mexico

New Mexico

New Mexico

New Mexico New Mexico (Wall and Corgan 1998)

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Table 3.2.

(continued)

Year

Population or hybrid

1995

NuMex Crispy

1995 1995 1995 1995 1996

NuMex Vado NuMex Luna NuMex Centric Texas Early White Spartan Supreme

1997

NuMex Sweetpack

1997

New York Sweet Blush

Origin or parents Temprana × (YB986A × Temprana) Ben Sheman × NuMex BR1 Ben Sheman × NuMex BR1 Sweet Spanish Ringold × unknown white (MSU2091A × MSU2086B) × MSU2076 Texas Early Grano, TG1015Y, Excel, and TEG502 PRR (MSU5718A × MSU8155B) × Kelsae Sweet Giant

Cooperating experiment station or entity New Mexico New Mexico New Mexico New Mexico Texas Michigan New Mexico New York

Yellow Globe’ was also selected into ‘Iowa Yellow Globe’ and then into inbred lines such as IA736 by C. E. Peterson (Table 3.3). 2. Grano and Bermuda Germplasm. The short-day populations known as ‘Grano’ and ‘Bermuda’ originated from two key open-pollinated populations: ‘Valencia’ and ‘White Bermuda’ (Magruder et al. 1941; Onion World 1998a,b). ‘Valencia’ was introduced to the United States from Spain in 1925, and ‘White Bermuda’ was introduced to the United States in 1883 from Bermuda, although the latter cultivar originated in Italy (Magruder et al. 1941). By the time of their introduction, both of these populations had long histories in onion production in southern Europe. A detailed history of the introduction of Bermuda onion into Texas has been published (Onion World 1998a,b). Culture of ‘Grano’ onions was studied in detail and perfected in New Mexico during the early part of the 20th century. Much of the credit for this effort goes to Fabian Garcia who, through his position with the New Mexico Agricultural Experiment Station, helped to establish ‘Grano’ onion culture in New Mexico (Garcia 1904, 1910, 1912; Garcia and Fite 1931). Two distinct types of open-pollinated onion populations were and are still grown in the Valencia area of Spain. These are the ‘Babosa’ and the ‘Grano’ populations. The ‘Babosa’ types are short-day, mild types while the ‘Grano’ are long-day types with significant storage potential. It may be that the name Grano, which means grain in Spanish, was used by onion farmers in Spain to refer to an onion cultivar that could be stored,

Inbred

L690A&B CC163 YB986A&B

TEG951C

B2215C

B12115C

L36

L365

B2108A&B B2218A&B B2190A&B B2146A&B B2133A&B B5546A&B B2129A&B

1952 1952 1952

1952

1952

1953

1953

1953

1953 1953 1953 1953 1953 1953 1953

Abundance, Early Harvest Aristocrat Bonanza, Fiesta Champion Contender, Golden Beauty Elite Encore

Abundance, Aristocrat, Bonanza, Bronze Perfection, Champion, Contender, Elite, Empire, Encore, Epoch, Fiesta, Pioneer, Premier, Spartan, Surprise Fiesta, Treasure

Granex, Early Harvest

Crystal Hybrid Crystal Hybrid Granex

Used in public hybrid

Iowa, Idaho, USDA Ohio, Idaho, Iowa, USDA Idaho, Iowa, USDA New York, Idaho, Iowa, USDA Idaho, Iowa, USDA Indiana, Idaho, Iowa, USDA Iowa, Idaho, USDA

Texas, USDA

Texas, USDA

Idaho, USDA

Iowa, Idaho, USDA

Texas, USDA

Texas, USDA Texas, USDA Texas, USDA

Cooperating experiment station or entity

9/26/2000 1:14 PM

Utah Sweet Spanish from Ferry Morse [(YB × White Persion) × YB] × YB selfed; selected yellows (Crystal Wax × Excel) × Excel selfed; selected whites Early Yellow Globe Brigham Yellow Globe Brigham Yellow Globe Early Yellow Globe Early Yellow Globe Early Yellow Globe Early Yellow Globe

L=Laredo, TX CC=Crystal City, TX Self of single bulb of Yellow Bermuda (Excel 986) Self of single bulb of Texas Grano Early Grano 502 S2 from Brigham Yellow Globe

Origin

Inbred populations released from public onion breeding programs in the United States, 1952–1997.

Year

Table 3.3.

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83

84

Inbred

B2264A&B B15-108A&B B2207A&B L303A&B

B1410C

B1340B B2217A&B B2228A&B B2117A&B CW15-8-4-1A&B

WSS 34-89-1-4

B12132A&B IA467-8A&B

IA736A&B B5546A&B B2264B PRR U16-3-11A&B U16-3-10-2A&B P54-9C

1953 1953 1953 1956

1956

1956 1956 1956 1957 1957

1957

1957 1957

1958 1958 1958 1959 1959 1959

(continued)

Year

Table 3.3.

Brigham Yellow Globe Yellow Globe Danvers Brigham Yellow Globe (Crystal Wax × Excel) × Excel selfed; selected whites (TEG951C × Crystal Grano) S2 of white bulb Currier Sweet Spanish S1 Brigham Yellow Globe Brigham Yellow Globe Early Yellow Globe Cochise White from Arizona (dominant white) White Sweet Spanish from Utah Sweet Spanish Utah Sweet Spanish Mass of several S1 from bulb of Melzer’s Yellow Globe Iowa Yellow Globe 44 White Perianth Brigham Yellow Globe Cochise Brown Cochise Brown Yellow Sweet Spanish— Peckham strain

Origin

Nugget, Spartan Era Early Yellow Globe

Bronze Perfection, Golden Beauty Spartan

Snow White

Pilot USDA USDA Premier Snow White

Iowa, Michigan, USDA USDA Texas, USDA Utah, USDA Utah, USDA Idaho, USDA

Wisconsin, Idaho, USDA Michigan, Iowa, USDA

USDA

New York, USDA USDA

Colorado, USDA

Texas, USDA

Iowa, Idaho, USDA Colorado, Idaho, USDA USDA Texas, USDA

Cooperating experiment station or entity

9/26/2000 1:14 PM

White Granex

Epoch, Pilot Pioneer Surprise White Granex

Used in public hybrid

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P1015C

B2267A&B MSU611C

B2190A&B PRR IA1718A&B IA2997A&B B12115-2C W101A&B

IA163A&B MSU728A&B W4A&B P54-371A&B

P54-306A&B

CO6-7-1

MSU611C M-100 MSU2399A&B MSU1411A&B W52C WD7C B2246B

1959

1959 1960

1960 1960 1960 1961 1962

1962 1963 1963 1963

1963

1963

1963 1964 1965 1966 1966 1966 1967

Spartan Banner Spartan Bounty

Colorado

El Capitan

Chieftain Nugget, Hickory, Sunburst, Fusario 12, Fusario 142 Hickory Spartan Gem Sunburst El Capitan

Empire, Treasure Spartan Banner, Spartan Bounty, Spartan Era, Spartan Gem

USDA California Michigan Michigan Wisconsin Wisconsin Idaho, Iowa, USDA

Idaho, USDA

Iowa, Wisconsin, USDA Michigan, USDA Wisconsin, USDA Idaho, USDA

Iowa, Texas, USDA Iowa, USDA Iowa, Texas, USDA Idaho, USDA Iowa, Wisconsin, USDA

New York, Idaho, USDA Michigan

Idaho, USDA

9/26/2000 1:14 PM

Iowa Yellow Globe 44 Iowa Yellow Globe 44 S5 from B15-108-1B × B2267B Yellow Sweet Spanish— Peckham strain Yellow Sweet Spanish— Utah strain Yellow Sweet Spanish— Colorado #6 Improved strain Early White Sweet Spanish Early Yellow Globe ? Subtich Yellow Globe Brown Beauty Brown seeded segregate from single BYG bulb

Brigham Yellow Globe Iowa Yellow Globe 44 Iowa Yellow Globe 44 Utah Sweet Spanish Rochester Bronze

Yellow Sweet Spanish— Peckham strain Brigham Yellow Globe Downing Yellow Globe

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85

86

Inbred

B58-614B

IA2997A&B PRR W202A&B

W205A&B W206C W207C W404A&B

MSU2935A&B MSU1459B MSU4535B

MSU611-1A&B MSU611A&B Ga-C 76

MSU826A&B

MSU5718A&B

MSU8155B

1967

1967 1967

1967 1967 1967 1972

1974 1974 1974

1979 1979 1980

1982

1982

1982

(continued)

Year

Table 3.3.

Iowa Yellow Globe 53 Single plant from IYG 53 Downing Yellow Globe— Trapp’s strain Downing Yellow Globe Downing Yellow Globe Amphidiploid population from A. galanthum PI 280091 × B2215C S3 Massed of Downing Yellow Globe—Trapp’s strain Single plants from Iowa Yellow Globe 53 MSU728B × B5546B

Fusario 24, Fusario 142, Fusario 245 Spartan Sleeper Spartan Sleeper Spartan Sleeper

Fusario 12, Fusario 24, Fusario 142, Fusario 245 Fusario 245

Used in public hybrid

USDA

USDA USDA

USDA USDA USDA

Michigan, USDA Michigan, USDA Michigan, USDA

Wisconsin Wisconsin Wisconsin Wisconsin

USDA Wisconsin

USDA

Cooperating experiment station or entity

9/26/2000 1:14 PM

C72 × B2215C B2190 B2264B × B2215C Rochester Bronze

Brown seed from B2246B × B2264B (black seed) Iowa Yellow Globe Rochester Bronze

Origin

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B2923A&B

B6693A&B

B9885A&B

B9897A&B

B11278B

B11377B

W407A&B W419A&B W420A&B

W426A&B B7728A&B

B1731A&B

B1750A&B B1794A&B

B2371C

1983

1983

1983

1983

1983

1983

1983 1983 1983

1983 1984

1987

1987 1987

1987

Single plant from Iowa Yellow Globe 53 Single plants from Iowa Yellow Globe 53 Glossy Inbred = White Persian (FPI 86279) × Long-day material Glossy Inbred = White Persian (FPI 86279) × Long-day material Glossy Inbred = White Persian (FPI 86279) × Long-day material Glossy Inbred = White Persian (FPI 86279) × Long-day material W202B × W4B W101B × W205B W404B × Buckskin Rochester Bronze W202B × W205B F3M5 from B2108B × C566 (A. vavilovii PI281727) F3M4 from MSU5785B × MSU826B F2M4 from B2923B × B1803B F3M5 from MSU8155B × MSU826B F1M4 from MSU611-30B × Sapporo-Ki USDA

USDA USDA

USDA

Wisconsin USDA

Wisconsin Wisconsin Wisconsin

Iowa, Michigan, Wisconsin, USDA

9/26/2000 1:14 PM

Iowa, Michigan, Wisconsin, USDA

Iowa, Michigan, Wisconsin, USDA

Michigan, Wisconsin, USDA Iowa, Michigan, Wisconsin, USDA

Michigan, Wisconsin, USDA

3499 P-03 Page 87

87

88

B9161A&B

CMS-ga 614, 2215, 8111, 8152, 8154, 8155, 8364, 8540, 8543 B8103

B8156

B8157

B8158

B8159

B8348

f-c 8434

1987

1988

1988

1988

1988

1988

1988

1988

Tetraploid from A. fistulosum Evergreen Bunching (Asgrow) Tetraploid from A. fistulosum Hakushyu Giant (Takii) Tetraploid from A. fistulosum Ishikura Long White (Takii) Tetraploid from A. fistulosum Kincho Long White (Takii) Tetraploid from A. fistulosum Tsukuba Long White (Takii) Tetraploid from A. fistulosum Tokyo Long White Bunching (Harris) A. fistulosum (PI 223853) × A. cepa (PI 239633 × Penn. Dutch Long Bottle) amphidiploid

F3M6 from MSU611-1B × MSU2399B BC4 male-sterile lines possessing the cytoplasm of A. galanthum PI 280091

Origin

Used in public hybrid

USDA

USDA

USDA

USDA

USDA

USDA

USDA

USDA

USDA

Cooperating experiment station or entity

9/26/2000 1:14 PM

1988

Inbred

(continued)

Year

Table 3.3.

3499 P-03 Page 88

f-c 8492

f-c 8497

f-c 8615

f-c 8407

f-c 8432

223853 ms

6007 ms

W417A&B

W434A&B W435A&B W438A&B W439A&B

W440A&B W441A&B

1988

1988

1988

1988

1988

1989

1989

1990

1990 1990 1990 1990

1990 1990

A. fistulosum (PI 223853) × A. cepa (P52-364A × PI 246322) amphidiploid A. fistulosum (PI 223853) × A. cepa (Crystal Wax) amphidiploid A. fistulosum (PI 433631) × A. cepa (Southport White Globe) amphidiploid A. fistulosum (PI 223853) × A. cepa (Southport White Globe) amphidiploid A. fistulosum (PI 223853) × A. cepa (White Knight) amphidiploid Genic male-sterile of A. fistulosum Kujyu-Negi (PI 223853) Genic male-sterile of top-set onion (A. × proliferum) × A. fistulosum W4B × Buckskin Rochester Bronze Synthetic population Synthetic population Synthetic population (Buckskin Rochester Bronze × W4B) × Sapporo-Ki Synthetic population Synthetic population Wisconsin Wisconsin Wisconsin

Wisconsin Wisconsin Wisconsin

Wisconsin

USDA

USDA

USDA

9/26/2000 1:14 PM

USDA

USDA

USDA

USDA

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89

90

Inbred

W445C W446A&B W447A&B W449C

B3350A&B

W454A&B W455A&B W456A&B W457A&B W458A&B W459A&B W460A&B W461A&B CMS-ga 614, 8111, 8152,

B1717A&B

B1828A&B

B2354A&B

1990 1990 1990 1990

1990

1993 1993 1993 1993 1993 1993 1993 1993 1998

1998

1998

1998

(continued)

Year

Table 3.3.

Sapporo-Ki × W202B Synthetic population Synthetic population Sapporo-Ki × (Buckskin Rochester Bronze × W404B) F2M2SM2 from MSU4535B × MSU611B W404B × PI 264650 W404B × PI 264650 W404B × PI 264650 W404B × PI 264650 PI 264650 × W404B PI 264650 × W404B W404B × PI 264650 PI 264650 × W404B BC7 male-sterile lines possessing the cytoplasm of A. galanthum PI 280091 F2MSM4 from MSU826B × B2737B F2M2SM2 from MSU826B × MSU611-1B S2M5 from {[(4929 × 5147) × (2737 × 4921)] S1} × {[(MSU826 × 2737) × (MSU611-1 × K62)] S1}

Origin

Used in public hybrid

USDA, Wisconsin

USDA, Wisconsin

USDA, Wisconsin

Wisconsin Wisconsin Wisconsin Wisconsin Wisconsin Wisconsin Wisconsin Wisconsin USDA, Wisconsin

9/26/2000 1:14 PM

USDA

Wisconsin Wisconsin Wisconsin Wisconsin

Cooperating experiment station or entity

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like grain, during the winter months. Both the ‘Babosa’ and ‘Grano’ types were introduced from Spain into Texas; however, many of the early selections mentioned below list only ‘Valencia’ as a source population. ‘Valencia’ was originally selected into ‘New Mexico Early Grano’, which was the progenitor of ‘Texas Early Grano’. The Early Grano populations were selected into three key populations: ‘Texas Grano’, ‘Texas Early Grano 502’, and ‘Crystal Grano’. It is likely that the source of these populations was a ‘Babosa’ type. A self-pollination of a single plant of ‘Texas Early Grano 502’ produced ‘Texas Early Grano 951C’, which was used to produce the popular cultivar ‘Texas 1015Y’ by L. Pike at Texas A&M University. ‘Texas Early Grano 502’ was also selected into many of the NuMex cultivars developed at New Mexico State University by J. Corgan. ‘Texas Early Grano 951C’ was also used as a parent of the very popular hybrid known as ‘Granex’. The other parent was YB986, a male sterile derivative of ‘Excel’, which was derived from ‘Yellow Bermuda’. ‘White Bermuda’ was also selected into the popular ‘Crystal Wax’, which served as a parent of L690 and ultimately to ‘Crystal Hybrid’ (Jones 1946–1956; Davis 1957–1965). ‘White Bermuda’ first appeared in an 1888 listing by Peter Henderson and Co. According to the description given by D. Landreth and Co. for 1890, ‘White Bermuda’ “has quite a yellowish color” (Magruder et al. 1941). ‘White Bermuda’ may be simply a pale derivative of ‘Yellow Bermuda’ (Magruder et al. 1941). 3. Spanish Germplasm. Some accounts suggest ‘Sweet Spanish’ or the Spanish onion germplasm was first officially introduced into the United States in 1916 (Magruder et al. 1941; Havey and Bark 1994), although there is evidence that introductions were made much earlier. A population known as ‘Sweet Spanish’ was introduced in 1916 from Spain and may have served as the progenitor for the populations ‘Yellow Sweet Spanish’, and specific strains such as ‘Utah Sweet Spanish’, ‘Yellow Sweet Spanish Peckham’, ‘Yellow Sweet Spanish Utah’, and ‘Currier Sweet Spanish’ in the Western United States. However, T. T. Greiner’s 1893 treatise on onion growing, Onions for Profit, published by the W. Atlee Burpee Co. of Philadelphia, Pennsylvania (Greiner 1893), mentions both ‘Spanish’ and ‘Prizetaker’ (a Spanish cultivar grown in the United States; see below) as leading cultivars in the 1890s. Thus, it is likely Spanish germplasm was already important in the United States prior to the turn of the 20th century. An interesting account of Spanish onion germplasm introduction was recently brought to our attention by J. F. Watson II of Sunseeds, Brooks, Oregon. Lester Morse of the C. C. Morse Seed Company described the introduction of Spanish germplasm to California in 1886 in his 1923

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publication Field Notes on Onions (Morse 1923). According to Morse, large yellow onions of the cultivar ‘Spanish King’ were shipped to many parts of the world from Spain. Mr. C. C. Morse noted the fine appearance of a shipment of these onions at the port in San Francisco and purchased 50 crates to be shipped to Santa Clara, California. These onion bulbs were planted and seed was produced during 1887. The 35 kg of seed this crop produced were purchased by Mr. William Henry Maule, who subsequently listed this cultivar as ‘Prizetaker’ in his 1888 catalog (Morse 1923). ‘Prizetaker’ is listed as an important onion in many early reports (Jones and Rosa 1928; Greiner 1893). Interestingly, Morse (1923) mentions a separate cultivar designation known as ‘Denia’, which refers to the Spanish port city from which the original ‘Spanish King’ onions were shipped. Under California conditions, the ‘Prizetaker’ population drifted somewhat and was refreshed every several years with seed obtained from ‘Denia’. ‘Yellow Sweet Spanish’ was the progenitor of many of the important “P” series inbreds developed by D. Franklin at Idaho (“P” refers to Parma, Idaho, the location of the experiment station). ‘Utah Sweet Spanish’ was the progenitor of “B” series inbreds such as B12115C and of ‘White Sweet Spanish’. ‘Currier Sweet Spanish’ was used to select “B” series inbreds such as B1340B. 4. Male Sterility. Fundamental to the development of modern onion hybrids was the discovery of sterile cytoplasm and its nuclear restorer by Jones and Clarke (1943). The source of sterility for the entire national program was a single plant from ‘Italian Red’, known as Italian Red 1353. Originally, ‘Italian Red’ was known as ‘California Early Red’ or ‘California Early Red UC#1’, the latter of which was released in 1935. These populations arose from ‘Red Italian Tripoli’, a popular Italian onion cultivar. ‘California Early Red’ and ‘Italian Red’ were common cultivars grown by market gardeners in the San Francisco Bay area in the early part of the 20th century. Italian Red 13-53 was observed in 1925, propagated asexually using top sets, and went on to become the female parent of the first released hybrid, ‘Calred’, in 1947 (Jones 1946–1956; Davis 1957–1965). 5. National Onion Breeding Program. Up through the mid 1900s, onion seed was produced by individual growers and commercially by groups or individuals. This seed production was based on mass selection of bulbs, maintaining strains adapted to a specific growing region. In the 1920s, breeding programs were established in the public sector to improve the quality of onion populations available to growers and consumers. The first onion-breeding program in the United States was sup-

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ported by the California Agricultural Experiment Station. In 1922, Henry A. Jones joined the faculty of the University of California–Davis and initiated research on the breeding and genetics of onion. The contributions of Jones epitomize the early years of public onion breeding in the United States. His laudable accomplishments have been documented elsewhere (Jones 1944; Simon et al. 1991; Whitaker 1983). While at the UC–Davis, Jones and colleagues established many of the techniques still widely used by onion breeders of today, such as self or small mass pollinations using flies (Jones 1923; Jones and Emsweller 1934). Jones and Davis (1944) observed that inbreeding onion for one to two generations often produced uniform lines. Vigor was restored by crossing between inbreds and a few hybrid combinations were higher yielding than the parental populations. Jones and Davis (1944) concluded that uniform, high-yielding hybrids could be generated after inbreeding and selection for two to five generations. Most publicly released onion inbreds were developed by selfing individual plants from open-pollinated cultivars or recombinations of previously developed inbreds. The advantages of hybrid onion could not be realized without a method to ensure cross pollination. The onion umbel consists of hundreds of small, perfect flowers making emasculation impractical. The production of hybrid onion seed became economically feasible with the discovery of cytoplasmic male sterility (CMS). Although maize researchers had described a maternal effect for CMS, the report by Jones and Clarke (1943) was the first to recognize the interaction between the cytoplasm and nucleus in conditioning male sterility. Jones and Clarke (1943) also described the use of CMS to produce hybrid seed. This discovery must be considered as Jones’s most significant contribution to plant breeding. After the discovery and characterization of CMS, onionbreeding efforts in the public and private sector have concentrated on the development of hybrids. However, the development of hybrids from some open-pollinated populations, e.g. ‘Texas Early Grano’ types, is limited because of a high frequency of the nuclear male-fertility restoration allele (Little et al. 1944; Davis 1957) or the predominance of sterile (S) cytoplasm (Havey 1993). In 1936, Jones left UC–Davis to establish the onion breeding program of the United States Department of Agriculture (USDA) at Beltsville, Maryland. The truly unique attribute of Jones’s USDA program was the development of an onion-breeding network in the United States. The USDA provided Jones with a modest budget and he used these funds to support cooperators at up to 22 state agricultural experiment stations. The early cooperators with Jones included G. Davis and L. Mann of

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California, D. Franklin of Idaho, A. Kehr of Iowa, E. Tims of Louisiana, C. Peterson of Michigan, B. Perry of Texas, H. Munger of New York, and W. Gabelman of Wisconsin. These cooperators worked with Jones to develop onion inbreds, test experimental hybrids, and identify superior combinations. From about 1952 to 1960, Jones grew his experimental material at the USDA field station at Greeley, Colorado. He appointed D. Franklin, W. Gabelman, and H. Munger as USDA collaborators. These three men along with C. Peterson, a USDA employee until he moved to Michigan State, met every February for a week in Greeley and at other locations to evaluate inbreds and hybrids and, most importantly, openly discuss ideas, germplasm, and techniques to successfully expand cooperation between the federal and state programs. Jones also invited representatives from commercial seed companies to these evaluations. During the 21-year tenure of Jones, the USDA and cooperating state agricultural experiment stations released 29 populations or hybrids (Table 3.2) and 28 inbreds (Table 3.3). Jones’s tenure as the leader of onion breeding in the United States was extremely successful. He and his cooperators established the genetic basis of cytoplasmic male sterility in onion, successfully applied this system to the development of hybrid onion, and worked to transfer this technology to the private sector. Jones developed the first onion hybrid, ‘California Hybrid #1’, using clonal propagules of the single plant source of cytoplasmic male sterility (Italian Red 13-53) as the seed parent in crosses with ‘Lord Howe Island’. The first seed-propagated hybrid (‘Calred’) was released in 1947. Having established the technology to use cytoplasmic male sterility to produce hybrid onion seed, Jones and colleagues developed a series of hybrids of each major market class in the United States. In 1952, the USDA and six state agricultural experiment stations released 11 hybrids of long-day storage onions (Table 3.2). Cooperation between the USDA and the Idaho and Texas Agricultural Experiment Stations developed the first hybrids from ‘Spanish’ (‘El Capitan’ in 1963) and short-day onions (‘Crystal Hybrid’ and ‘Granex’ in 1952), respectively. YB986A, the seed parent of ‘Granex’, is still used in short-day hybrids; to date, more hybrid onion seed has probably been produced on this single sterile line than any other line. Today, hybrid onions are commonly grown throughout the world, many generated from inbreds developed by Jones and his cooperators and almost all using the cytoplasmic-genic male sterility system discovered by Jones and Clarke. In 1957, Jones left the USDA to assume a position with Dessert Seed Company, El Centro, California. First, A. Kehr moved from Iowa to Beltsville. Later, E. Davis assumed direction of the USDA program from 1957 to 1968. During this period, the USDA and cooperating experiment stations released 21 inbreds and 9 hybrids (Table 3.2 and Table 3.3). In

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1966, G. McCollum moved from Parma, Idaho, to Beltsville and established a program concentrating on the crossing relationships among onion and closely related wild Allium species and introgression of diverse germplasm in the bulb onion. Prior to his retirement in 1988, McCollum released nine cytoplasmic-male-sterile lines developed from backcrossing of A. galanthum to A. cepa, six populations of tetraploid A. fistulosum, six amphidiploid populations derived from A. fistulosum × A. cepa hybrids, and genic-male-sterile lines of A. fistulosum derived from an A. cepa × A. fistulosum hybrid (Table 3.3). In 1968, the onion-breeding efforts of the USDA program moved from Beltsville to Madison, Wisconsin, and C. E. Peterson assumed responsibilities. Peterson primarily developed elite inbreds for production of disease-resistant, well-storing, long-day hybrids grown in the eastern half of the United States. Twenty-one inbreds were developed by Peterson and jointly released by the USDA and the Agricultural Experiment Stations of Iowa, Michigan, New York, and Wisconsin (Table 3.3); publicly named hybrids from these inbreds include ‘Spartan’, ‘Spartan Era’, ‘Spartan Gem’, ‘Spartan Sleeper’, ‘Spartan Banner 80’, ‘Sweet Sandwich’ (Peterson et al. 1986), and ‘Blitz’ (Table 3.2). Many state programs cooperated closely with the USDA National Onion Program in the evaluation of populations or hybrids. Some states supported breeding efforts independent of the USDA, of which the most successful are or were located in California, Idaho, Texas, Michigan, New Mexico, and Wisconsin.

III. STATE EXPERIMENT STATION PROGRAMS A. California After Jones’s departure in 1936, D. R. Porter carried on the onion breeding work until 1938, when G. N. Davis took over. By 1945, the California Agricultural Experiment Station had released nine improved cultivars from indigenous or introduced populations, including selections from ‘California Early Red’, ‘Stockton Early Yellow’, ‘Sweet Spanish’, ‘Early Grano’, ‘Lord Howe Island’, and ‘Australian Brown’. ‘San Joaquin’ was the first release of a new cultivar derived from hybridization between two existing populations, ‘Stockton G-36’ and ‘Early Grano’. For a short period of time during the 1950s and 1960s, Louis Mann directed onion breeding efforts in California until his death in the early 1960s. Unfortunately, California has not supported an onion breeding program since the death of Mann, even though this state is one of the world’s primary production areas.

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B. Colorado Two contrasting cultivars of onion were grown in Colorado: ‘Mountain Danvers’ is an early, pungent, well-storing population originally cultivated on the western slopes of the Rocky Mountains, and ‘Yellow Sweet Spanish’ is grown in the Arkansas Valley and Northern Colorado. ‘Colorado No. 6’ is a Spanish population released for production in the Arkansas Valley. C. Idaho At Parma, Delance Franklin worked on the development and evaluation of inbreds of the ‘Sweet Spanish’ onion from 1942 until his retirement in 1974. Franklin was involved in the development of 17 inbreds and the first storage × Spanish hybrid (‘Fiesta’) and the first true Spanish hybrid, ‘El Capitan’ (Tables 3.1 and 3.2). The Parma inbreds are still widely used and highly prized in the private sector for the production of numerous Spanish hybrids. Franklin also completed definitive experiments establishing the best methods to produce hybrid-onion seed. After Franklin’s retirement, the onion breeding program at Parma was discontinued. In recognition of his superior contributions to Idaho agriculture, Franklin was awarded an honorary doctorate from the University of Idaho. D. Iowa Onion breeding efforts in Iowa were initiated by E. S. Haber in 1940 and continued by C. E. Peterson, at that time a USDA employee. Work concentrated on two strains grown in Iowa, ‘Brigham Yellow Globe’ grown in Northern Iowa and ‘Scott County Globe’ grown from sets in Eastern Iowa, although ‘Melzer’s Yellow Globe’ was also used. Peterson developed synthetics by recombining lines that had been self-pollinated for one generation, e.g. ‘Iowa Yellow Globe 44’ developed from Iowa-grown ‘Brigham Yellow Globe’ and released in 1948 as a storage type. Inbreds selected from ‘Iowa Yellow Globe 44’ and ‘Iowa Yellow Globe 53’ are listed in Table 3.3. E. Louisiana J. C. Miller and Mr. Cochran developed a strain of ‘Red Creole (C-5)’, selecting for a deep pink color. E. C. Tims, a plant pathologist, joined the onion breeding efforts in 1947 and A. Kehr directed the Louisiana onion breeding from 1949 until he joined the USDA and moved to Iowa in

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1954. Crosses between ‘Calred’ (Italian Red 13-53 as seed parent) and ‘Creole’ were initiated to develop mildew resistant ‘Creole’ lines. Amphidiploids from interspecific hybrids between shallot and A. fistulosum were backcrossed to onion to develop shallots resistant to pink root and onion yellow dwarf virus (triploid ‘Delta Giant’ shallot). F. Michigan P. M. Harmer cooperated with the USDA in testing experimental hybrids in Michigan. Onion breeding efforts were initiated when C. E. Peterson moved to Michigan State University (MSU) from Iowa State University in 1954. Peterson developed numerous inbreds and hybrids during his 14-year tenure at MSU (Tables 3.2 and 3.3). In 1968, Peterson moved to Wisconsin to direct the USDA onion breeding program. Many of the inbreds released by Peterson while employed by the USDA had MSU numbers because their development was initiated while he was in Michigan. After Peterson left MSU, the onion breeding program was directed by Grant Vest (1970 to 1976). From 1983 to 1998, Lowell Ewart directed the MSU program and released ‘Spartan Supreme’ in 1996. G. New Mexico The New Mexico State Agricultural Experiment Station has played a pivotal role in the development of short-day onion cultivars. In 1927 a population from Spain, ‘Valencia Grano 9452’, was introduced into the United States, selected by F. Garcia for earliness and bolting resistance, and released as ‘New Mexico Early Grano’ in 1931. Numerous important short-day populations were subsequently selected from this population, such as ‘Texas Early Grano (TEG) 502’ (1947), ‘TEG951C’ (1952), ‘Temprana’ (1979), and others (Tables 3.2 and 3.3). J. N. Corgan directed the New Mexican onion breeding program from 1976 to his retirement in 1996. Corgan developed a series of synthetic populations with excellent bolting resistance for New Mexican production, such as ‘NuMex BR1’ (Corgan 1984) and ‘NuMex Sundial’, ‘NuMex Suntop’ (Corgan 1988a), ‘NuMex Sunlite’ (Corgan 1988b), among others (Table 3.2). The New Mexico program is presently directed by C. Cramer. H. New York Cooperative onion breeding efforts between Cornell University and the USDA were initiated in 1938. Early efforts concentrated on selection of disease resistant onion. A. G. Newhall provided bulbs of New York

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grown ‘Early Yellow Globe’ and ‘Brigham Yellow Globe’ strains to Jones for development of many of the first publicly developed inbreds. Henry Munger was the primary onion breeder in New York until his retirement in 1988. Munger inbred bulbs of ‘Brigham Yellow Globe’, ‘Early Yellow Globe’, and ‘Southport Yellow Globe’ with simultaneous crossing to a sterile line. The selfed progeny were evaluated for vigor, bulb appearance, uniformity, and the ability to maintain male sterility. Tom Walters directed the New York onion breeding program from Munger’s retirement until 1997. Walters developed and released ‘New York Sweet Blush’ (Table 3.2). I. Texas The Texas Agricultural Experiment Station (TAES) and the USDA established cooperative onion breeding efforts in 1940. Bruce Perry initially directed the Texas onion breeding efforts, followed by Paul Leeper. The TAES released ‘Texas Early Grano’ (an early selection out of Grano) and ‘TEG502’, a slightly earlier selection out of ‘TEG’ with more upright foliage. The experiment station was involved in the development of ‘Excel’ (YB986), a pink-root resistant early selection that quickly replaced it parental cultivar ‘Yellow Bermuda’. A CMS-sterile derivative of ‘Excel’ (YB986A) was crossed with ‘TEG951C’ (originated from the self-pollination of a single bulb of ‘TEG 502’) to produce ‘Granex’, one of the first shortday hybrid onion cultivars. ‘Granex’ is still widely grown in the United States and throughout the tropics. ‘Laredo (L) 690’, a selection out of ‘Crystal Wax’, was developed cooperatively with the USDA and released in 1949. ‘Crystal Hybrid’ (L-690 × CC-163), is a white hybrid and the only other publicly developed short-day hybrid. The superiority of the ‘Texas Grano’ derivatives is evidenced by their complete substitution of the ‘Yellow Bermuda’ types. L. Pike has directed the Texas onion breeding since Leeper’s retirement. His releases are widely grown and commercially successful, especially ‘Texas Grano 1015Y’ (Pike et al. 1988). Additional releases from Pike’s program are listed in Table 3.2. J. Utah A. E. Clarke, a USDA employee, and L. H. Pollard developed a series of sterile and maintainer lines from ‘Utah Yellow Sweet Spanish’, ‘Utah White Sweet Spanish’, ‘Cochise Brown’, and ‘Cochise White’. Dr. Clarke transferred to Parma, Idaho, in February 1952, but died in November of that same year. A series of inbreds were developed from ‘Cochise Brown’ (Table 3.3).

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K. Wisconsin Initial onion research at the University of Wisconsin was directed by R. H. Larson, a USDA employee, and J. C. Walker, concentrating on selection of lines resistant to smut, pink root, and smudge. In the early 1950s, Wisconsin researchers evaluated long- by short-day hybrids (e.g. ‘Early Harvest’) to identify uniform early-maturing materials to replace production of bulbs grown from sets. W. H. Gabelman directed the onion breeding at Wisconsin from 1949 until his retirement in 1992. Openpollinated populations used by Gabelman included ‘Rochester Bronze’ and ‘Buckskin Rochester Bronze’, derivatives of ‘Downing Yellow Globe’. The significant and numerous releases from Gabelman’s program were recently reviewed (Goldman 1996) and are included in Tables 3.2 and 3.3 for convenience.

IV. FUTURE PROSPECTS FOR PUBLIC ONION BREEDING IN THE UNITED STATES In this review, we attempted to document the significant contributions of public-sector researchers to the breeding and genetics of onion. In the first half of the 20th century, the USDA and many state agricultural experiment stations supported onion research. Technologies developed by these public programs established the foundation for the development of superior open-pollinated and hybrid-onion cultivars. Inbreds and elite populations developed by the public sector were used by private seed companies to produce hybrid-onion seed for the United States and the world. At the end of the 20th century, public programs are still supported by the USDA and the agricultural experiment stations of Georgia, New Mexico, New York, Texas, and Wisconsin. Unfortunately, support has waned and public programs that once had a significant effect on onion breeding and genetics have disappeared from California, Colorado, Idaho, Iowa, Louisiana, Michigan, and Utah. This loss is significant because four of these states—California, Colorado, Idaho, and Michigan—contribute more than 60% of the U.S. spring-planted, fallharvested onion crop (including dehydrator onions). Other states with significant production, such as Oregon or Washington (28% of the springplanted, fall-harvested onion crop in the United States), have never supported major onion-breeding programs (http://www.nass.usda.gov/). There have also been significant consolidations in the private sector; many smaller seed companies producing hybrids for specific market niches have been purchased or merged together.

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Presently, the main challenge facing public-sector researchers of relatively minor crops, such as onion, is funding. Breeding programs in individual states have historically received funding from indigenous grower groups through check-offs or marketing orders. These sources of funds still exist in many production areas, such as in New York, New Mexico, Georgia, Wisconsin, or Idaho, but levels of funding have not kept up with the increased costs of research. Federal sources of funding, such as through granting agencies of the USDA or the National Science Foundation, have become extremely competitive and are dominated by research on model organisms or major crop species. In spite of these reductions in number of public sector projects and limited financial resources, the challenges and opportunities in onion research are enormous. Onion consumption steadily increases each year as U.S. consumers eat more fresh or lightly processed vegetables. Onion possesses unique health-enhancing thiosulfinates that possess antioxidant activities (Yin and Cheng 1998), reduce serum cholesterol (Sainani et al. 1976), and enhance in vitro antiplatelet activity (Goldman et al. 1995; Bordia et al. 1996). Quercitin, the main flavonoid in onion (Price and Rhodes 1997), may also have significant anticarcinogenic and antithrombotic properties (Leighton et al. 1992). These health-enhancing attributes can be exploited to produce new value-added cultivars for consumers. However, the relatively long generation time of onion slows genetic progress (Cramer and Havey 1999). The huge nuclear genome of onion makes cloning and manipulation of genes difficult (King et al. 1998). Although haploid extraction (Muren 1989; Campion et al. 1992; Javornik et al. 1998) and transformation (Eady et al. 1996) of onion have been reported, these technologies are far from routine. Each of these difficulties make public-sector research ever more important. Publicsector programs should be able to make long-term commitments to develop new technologies for onion improvement and produce new value-added onion cultivars for the consumer. Private investment in onion research should be stimulated by the huge value of the onion crop, but companies must be willing to make long-term investments to reap any reward. The public and private sectors have supported, and continue to support, research on onion genetics and breeding. The development of new elite onion cultivars is dependent on the continued financial support of local growers’ groups, state and federal formula funding, corporate gifts and grants, and competitive federal grants. With this support, publicsector researchers can commit to long-term experiments aimed at genetic improvement of onion, application of biotechnological approaches, and development of new value-added cultivars for consumers world-wide.

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LITERATURE CITED Adams, S. W., and H. R. Stiles. 1974. The history of ancient Wethersfield. A facsimile of the 1904 Edition. Volume 1. New Hampshire Publ. Co., Somersworth. Anon. 1877. 24th Annual Report of the Secretary of the Massachusetts Board of Agriculture for 1876. Albert J. Wright, State Printer, Boston. Arrington, B. F. 1922. Municipal history of Essex County in Massachusetts. Vol. 1. Lewis Historical Publ. Co., New York. Arumugunathan, K., and E. Earle. 1991. Nuclear DNA content of some important plant species. Plant Mol. Biol. Rep. 9:208–218. Beaumont, A. B., M. E. Snell, W. L. Doran, and A. I. Bourne. 1935. Onions in the Connecticut Valley. Massachusetts Agr. Expt. Sta. Bul. 318. Massachusetts State College, Amherst. Block, E. 1992. The organosulfur chemistry of the genus Allium—Implications for the organic chemistry of sulfur. Agnew. Chem. Int. Ed. Engl. 31:1135–1178. Bordia, T., N. Mohammed, M. Thomson, and M. Ali. 1996. An evaluation of garlic and onion as antithrombotic agents. Prostaglandins, Leukotrienes and Essential Fatty Acids 54:183–186. Brewster, J. L. 1994. Onions and other vegetable alliums. CAB Int., Wallingford, UK. Campion, B., E. Vicini, M. Schiavi, and A. M. Falavigna. 1992. Advances in haploid plant induction in onion (Allium cepa L.) through in vitro gynogenesis. Plant Sci. 86:97–104. Corgan, J. N. 1984. ‘NuMex BR1’ onion. HortScience 19:593. Corgan, J. N. 1988a. ‘NuMex Sundial’ and ‘NuMex Suntop’ onion. HortScience 23:421–422. Corgan, J. N. 1988b. ‘NuMex Sunlite’ onion. HortScience 23:423–424. Coulter, F., and J. Willard. 1949. Wethersfield, Large Red. Seed World. Jan. 21. p. 20–21. Cramer, C. S., and M. J. Havey. 1999. Morphological, biochemical, and molecular markers in onion. HortScience. 34:589–593. Danvers Mirror, 1899. Danvers, Massachusetts. Davis, E. 1957–1965. Annual reports to cooperators from the national onion breeding program. Plant Industry Station, Beltsville, MD. Davis, E. W. 1957. The distribution of the male-sterility gene in onion. J. Am. Soc. Hort. Sci. 70:316–318. Eady, C. C., C. E. Lister, Y. Suo, and D. Schaper. 1996. Transient expression of uidA constructs in in vitro onion (Allium cepa L.) cultures following particle bombardment and Agrobacterium-mediated DNA delivery. Plant Cell Rep. 15:958–962. Erwin, A. T., and W. L. Harter. 1926. Onion industry in Pleasant Valley, Iowa. Iowa Agr. Expt. Sta. Bul. 1924–1926. 264–286. Flint, C. L. 1865. Agriculture of Massachusetts. Second Series. 12th Annual Report of the Secretary of the Massachusetts Board of Agriculture for 1864. Wright and Potter, State Printer, Boston. Fuess, C. M. 1935. The story of Essex County. Vol. 1. Am. Hist. Soc. Inc., New York. Garcia, F. 1904. Onion culture. Bul. 52, New Mexico College of Agriculture and Mechanic Arts, Agr. Expt. Sta., Santa Fe, NM. Garcia, F. 1910. Onion tests, 1905–1909. Bul. 74, New Mexico College of Agriculture and Mechanic Arts, Agr. Expt. Sta., Santa Fe, NM. Garcia, F. 1912. Growing Denia onion seed. Bul. 82, New Mexico College of Agriculture and Mechanic Arts, Agr. Expt. Sta., Santa Fe, NM. Garcia, F., and A. B. Fite. 1931. Early Grano onion culture. Bul. 193, New Mexico College of Agriculture and Mechanic Arts, Agr. Expt. Sta., Santa Fe, NM. Garner, W. W., and H. A. Allard. 1920. Effect of the relative length of day and night and

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other factors of the environment on growth and reproduction in plants. J. Agr. Res. 18:553–606. Garner, W. W., and H. A. Allard. 1923. Further studies in photoperiodism, the response of the plant to relative length of day and night. J. Agr. Res. 23:871–920. Goldman, I. L. 1996. A list of germplasm releases from the University of Wisconsin onion breeding program, 1957–1993. HortScience 31:878–879. Goldman, I. L., B. S. Schwartz, and M. Kopelberg. 1995. Variability in blood platelet inhibitory activity of Allium (Alliaceae) species accessions. Am. J. Bot. 82:827–832. Greiner, T. 1893. Onions for profit. W. Atlee Burpee Co., Philadelphia. Hanelt, P. 1990. Taxonomy, evolution, and history. p. 1–26. In: H. Rabinowitch and J. Brewster (eds.), Onions and allied crops. CRC Press, Boca Raton, FL. Havey, M. J. 1991. History of releases from the onion breeding program of the United States Department of Agriculture. Allium Improv. Newsl. 1:50–54. Havey, M. J. 1993. A putative donor of S-cytoplasm and its distribution among openpollinated populations of onion. Theor. Appl. Genet. 86:128–134. Havey, M. J. 1995. Onion and other cultivated alliums. In: J. Smartt and N. W. Simmonds (eds.), Evolution of crop plants. Wiley, New York. Havey, M. J., and O. H. Bark. 1994. Molecular confirmation that sterile cytoplasm has been introduced into open-pollinated Grano onion cultivars. J. Am. Soc. Hort. Sci. 119:90–93. Hawthorn, L. R., and L. H. Pollard. 1954. Vegetable and flower seed production. Blakiston Co., New York. Hedrick, U. P. 1919. Sturtevant’s notes on edible plants. J.B. Lyon Co., Albany, NY. Javornik, B., B. Bohanec, and B. Campion. 1998. Second cycle gynogenesis in onion, Allium cepa L., and genetic analysis of the plants. Plant Breed. 117:275–278. Jones, H. A. 1923. Pollination and self-fertility in the onion. Proc. Am. Soc. Hort. Sci. 20:191–197. Jones, H. A. 1944. Problems and progress in onion breeding. Herbertia 11:175–294. Jones, H. A. 1946–1956. Annual Reports to Cooperators from the National Onion Breeding Program. Plant Industry Station, Beltsville, MD. Jones, H. A., and A. Clarke. 1943. Inheritance of male sterility in the onion and the production of hybrid seed. Proc. Am. Soc. Hort. Sci. 43:189–194. Jones, H. A., and G. Davis. 1944. Inbreeding and heterosis and their relation to the development of new varieties of onions. USDA Tech. Bul. 874. Jones, H. A., and S. L. Emsweller. 1934. The use of flies as onion pollinators. Proc. Am. Soc. Hort. Sci. 31:160–164. Jones, H. A., and L. K. Mann. 1963. Onions and their allies. Interscience Publ., New York. Jones, H. A., and J. T. Rosa. 1928. Truck crop plants. McGraw-Hill, New York. King, J. J., J. M. Bradeen, O. Bark, J. A. McCallum, and M. J. Havey. 1998. A low-density genetic map of onion reveals a role for tandem duplication in the evolution of an extremely large diploid genome. Theor. Appl. Genet. 96:52–62. Leighton, T., C. Ginther, L. Fluss, W. K. Harter, J. Cansado, and V. Notario. 1992. Molecular characterization of quercetin and quercetin glycosides in Allium vegetables. In: Phenolic compounds and their effects on human health II. Amer. Chem. Society. p. 220–238. Little, T., H. Jones, and A. Clarke. 1944. The distribution of the male-sterility gene in varieties of onion. Hebertia 11:310–312. Magruder, R., and H. A. Allard. 1937. Bulb formation in some American and European varieties of onions as affected by length of day. J. Agr. Res. 54:719–752. Magruder, R., R. Webster, H. Jones, T. Randall, G. Snyder, H. Brown, L. Hawthorn, and A.

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L. Wilson. 1941. Descriptions of types of principal American varieties of onions. USDA Misc. Pub. 435. Washington, DC. Mally, F. W. 1915. The Bermuda onion. Texas Dept Agr. Bul. 40. A. C. Baldwin and Sons, Austin. Morse, L. L. 1923. Field notes on onions. C.C. Morse and Co., San Francisco, CA. Muren, R. C. 1989. Haploid plant induction from unpollinated ovaries in onion. HortScience 24:833–834. Onion World. 1998a. Onion history, Part 1. The onion that came to Texas but never left the same. May–June. Columbia Publ., Yakima, WA. 20–23. Onion World. 1998b. Onion history, Part 2. The onion that came to Texas but never left the same. July–August. Columbia Publ., Yakima, WA. 8–14. Perry, B. A., and H. A. Jones. 1957. Onion varieties in Texas. Texas Agr. Expt. Sta. Bul. 854. Peterson, C. E., and E. S. Haber. 1941. Iowa Yellow Globe 44. Seed World. Feb. 21. 14–15. Peterson, C., P. Simon, and L. Ellerbrock. 1986. ‘Sweet Sandwich’ onion. HortScience 21: 1466–1468. Pike, L. 1986. Onion breeding. p. 357–394. In: M. Bassett (ed.), Breeding vegetable crops. AVI Publishing Co., Westport, CT. Pike, L. M., R. S. Horn, C. R. Andersen, P. W. Leeper, and M. E. Miller. 1988a. ‘Texas Grano 1015Y’: A mild pungency, sweet, short-day onion. HortScience 23:634–635. Pike, L. M., R. S. Horn, C. R. Andersen, P. W. Leeper, and M. E. Miller. 1988b. ‘Texas Grano 1025Y’: A medium length storage shortday onion. HortScience 23:635–636. Pike, L. M., R. S. Horn, C. R. Andersen, P. W. Leeper, and M. E. Miller. 1988c. ‘Texas Grano 1030Y’: A late maturing, mild pungency short-day onion. HortScience 23:636–637. Pike, L. M., R. S. Horn, C. R. Andersen, P. W. Leeper, and M. E. Miller. 1988d. ‘Texas Grano 1105Y’: A late maturing medium length storage short-day onion. HortScience 23: 638–639. Price, K. R., and M. J. C. Rhodes. 1997. Analysis of the major flavonol glycosides present in four varieties of onion and changes in composition resulting from autolysis. J. Sci. Food Agr. 74:331–339. Rabinowitch, H. D. 1988. Genetics and breeding: State of the art or too slow but not too late. Proc. EUCARPIA 4th Allium Symp. Inst. Hort. Res., Wellsbourne, Warwick, UK. p. 57–70. Sainani, G. S., D. B. Desai, K. N. Moore. 1976. Onion, garlic, and atherosclerosis. Lancet. 575–576. Simon, P. W., W. H. Gabelman, and D. F. Franklin. 1991. Henry A. Jones (1889–1981). HortScience 26:1115–1118. Tracy, W. W. 1903. List of American varieties of vegetables for the years 1901 and 1902. United States Department of Agriculture Bureau of Plant Industry Bul. 21. Government Printing Office, Washington, D.C. Vilmorin-Andrieux, M. M. 1885. The vegetable garden. English edition, originally published under the direction of W. Robinson. John Murray, London. Wall, M., and J. Corgan. 1998. NuMex Dulce onion. HortScience 33:762–763. Weider, L. M. 1966. The Wethersfield Story. Pequot Press, Stonington, CT. Whitaker, T. W. 1983. Dedication: Henry A. Jones (1889–1981) plant breeder extraordinaire. Plant Breed. Rev. 1:1–10. Yin, M. C., and W. S. Cheng. 1998. Antioxidant activity of several Allium members. J. Agric. Food Chem. 46:4097–4101.

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4 Current and Future Issues in Lettuce Breeding Edward J. Ryder US Agricultural Research Station, U.S. Department of Agriculture, Agricultural Research Service, 1636 E. Alisal Street, Salinas, CA 93905

I. INTRODUCTION II. HISTORY OF LETTUCE BREEDING III. BREEDING EFFORTS A. Head and Rosette Type B. Resistance to Diseases, Insects, and Disorders C. Use of Interspecific Crosses D. Development of Breeding Tools E. Human Health Aspects IV. LETTUCE BREEDING FOR THE FUTURE A. The Tools of Biotechnology B. Public and Private Breeding Responsibilities C. Pesticides and other Chemicals D. Lettuce in Nutrition and Health E. Food Safety Concerns F. Value-added and other New Products G. Lettuce as a Research Organism LITERATURE CITED

I. INTRODUCTION Lettuce is virtually synonymous with salad. No other vegetable, leafy or otherwise, occurs as frequently as lettuce in the salads of the world. Especially in the United States, it has become a staple crop, comparable to tomatoes, potatoes, onions, cabbage, and beans. Lettuce is grown commercially in many countries, particularly in the temperate zones, and is grown in home gardens nearly everywhere. The

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United States is by far the largest commercial producer of lettuce; in 1997, lettuce of all types was grown on 113,000 ha, which produced more than 3.9 million tonnes, worth over $1.6 billion. About 90% of the production is in two states, California (72–75%) and Arizona (15–18%) (Ryder 1999). Lettuce (Lactuca sativa L.) is in the Asteraceae, subfamily Cichorioideae, tribe Lactuceae. It is closely related to common wild lettuce or prickly lettuce, L. serriola L. It forms a taproot and numerous feeder roots. Above ground, leaves are arranged spirally on a shortened stem, forming a rosette. At a stage controlled by temperature and daylength, the stem elongates forming a corymbose panicle of numerous capitula of 12–20 perfect florets. The structure of the flower is such that it is obligately self-pollinated, and some manipulation is required to make crosses. Fertilized florets mature to form achenes, which are small, elongated fruits that are treated like seeds (Ryder 1999).

II. HISTORY OF LETTUCE BREEDING In 1922, Ivan C. Jagger arrived in San Diego, California to begin a lettuce breeding program for the U.S. Department of Agriculture. His assignment was to defeat a disease of lettuce called brown blight that was devastating lettuce fields in the California desert and coastal districts, thus threatening the survival of the burgeoning California lettuce industry. That was the beginning of the first public lettuce breeding program in the United States and probably in the world. Public lettuce breeding was therefore 75 years old in 1997. This number has the sound of a milestone, a time to assess the progress so far and to look ahead and consider the prospects for the future. In subsequent years, lettuce breeding programs were established in several state experiment stations, notably California, New York, Florida, Texas, and Michigan. Public programs also were established in several other countries, such as England, the Netherlands, France, and Israel. In addition, seed companies in the Netherlands, England, France, Japan, Australia, the United States, and other countries established programs with increasing reliance on genetics and other scientific tools. Early genetics studies, primarily in the 1930s and 1940s, established many of the parameters that defined both the scope and the limitations on lettuce breeding activity. One group of studies identified other species that could be crossed with lettuce using essentially standard methods. There are about 100 species of Lactuca; authors differ in the number they list.

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Babcock et al. (1937) published the chromosome numbers for 25 species of Lactuca. There were three groups with haploid numbers n = 8, 9, and 17. The species with n = 17 are considered to be amphidiploids of species with n = 8 and n = 9. Thompson et al. (1941) showed that lettuce (L. sativa L., n = 9) formed a compatibility group with three other nine chromosome species, L. serriola L. (prickly lettuce), L. saligna L., and L. virosa L. These species could not be crossed, using standard methods, with any of the other species they tested. In more recent years, various authors showed that several forms with distinct species names were probably forms of L. serriola or primitive forms of L. sativa. These include L. altaica Fisch. et Mey., L. augustana All., L. quercina L., L. dregeana D.C., and L. aculeata Boiss. and Ky. Zohary (1991), grouped L. serriola, L. aculeata, L. altaica, L. dregeana, L. scarioloides Boiss., L. azerbaijanica Rech., and L. georgica Grossh. as the species most closely related taxonomically to lettuce. The pioneering inheritance study in lettuce was conducted by C. E. Durst (1930) for his Ph.D. thesis. He studied the inheritance of anthocyanin content, seed color, spines, and leaf lobing, all identified as single gene traits. He also, as did Lewis (1931), studied various leaf, bolting, and head characters, all of which were quantitatively inherited, and would require a different approach to breeding technique. However, single genes for specific aspects of bolting and head development were identified by Bremer and Grana (1935). In addition, two genes for disease resistance were identified in those early years, for downy mildew (Jagger and Whitaker 1940) and for powdery mildew (Whitaker and Pryor 1941). This paper presents a view of the future of lettuce breeding and genetics, in the light of its breeding history as described, present activities, availability of new tools, and some of the author’s prejudices as a public sector scientist. The interested reader may wish to explore the lettuce plant further in the author’s recently published book, Lettuce, Endive and Chicory (Ryder 1999).

III. BREEDING EFFORTS As breeding programs developed through the ensuing years to the present time, the breeding efforts worldwide can be grouped into five categories of research: 1. Breeding for type, stressing traits for head and rosette formation, uniformity, earliness, color, size, and bolting resistance. Breeding

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3.

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pathways were dictated by the needs of the subtypes of lettuce: crisphead, butterhead, romaine, leaf, and Latin. Crispheads and butterheads form heads with overlapping leaves and fairly precise requirements for acceptable conformation and shape. The other subtypes form modified leaf rosettes, with less demanding size and shape requirements. Breeding for resistances to diseases, insects, and disorders. Early breeding efforts focused on downy mildew resistance, followed later by work with lettuce mosaic and big vein. In the last 25 years, resistance studies have proliferated, to include virus diseases (lettuce infectious yellows, lettuce chlorosis, beet western yellows, cucumber mosaic), fungal diseases (sclerotinia drop, verticillium wilt, stemphylium, anthracnose), bacterial diseases (corky root, bacterial leaf spot), insects (lettuce root aphid, green peach aphid, lettuce aphid, sweetpotato whitefly, pea leafminer), and physiological disorders (tipburn, rib discoloration). Use of interspecific crosses. In addition to crosses with L. serriola and primitive forms of L. sativa, some use has been made of L. saligna and L. virosa as parents. Development of breeding tools. These have included improved crossing techniques and use of early flowering and male sterility traits. In recent years, breeders have been making use of biotechnology techniques. Breeding for improvement of traits affecting human health or wellbeing. This is another emerging area. At this time the most notable specific effort involves the reduction of nitrate content. In a more general sense, breeding for disease and insect resistance leads to cultivars requiring fewer chemical control treatments and therefore is important to human health.

Before discussing these research goals, it might be useful and interesting to discuss one plant breeding goal conspicuous by its absence: yield per se. Yield is not a stated goal of lettuce breeding because there is a fixed maximum yield based upon the number of plants in a unit area. One plant is one unit of yield and the maximum number of plants on one land unit is always the same. For example, lettuce in California is planted on 1 m (40 inch) beds with two rows on each bed at a fixed spacing. Seed is space planted and thinned to a fixed distance between plants, usually 25 cm (10 in) for head lettuce. This means there are about 76,600 plants per ha, which is also the maximum yield. This translates to 3,200 cartons of 24 heads each per ha; therefore, the number of cartons packed is the usual harvest and packing criterion. The

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weight and dimensions of the head are also variables, as the number of heads and weight per carton affect market price. Maximization of yield is based on factors like plant survival and harvestability under stress of disease or insect attack rather than the effects of genetics, nutrients, and water inputs on the number and size of fruit or seeds per plant. Other rosette forming crop species also are under a similar constraint. A. Head and Rosette Type Breeding for type emphasizes heading or rosetting characteristics, as well as uniformity, earliness, and leaf shape, color, and size. Lettuce is grown in several forms, each with its own requirements. These include, in most production areas, the following: two types of crisphead (iceberg and Batavia), butterhead, romaine, leaf, and Latin. Iceberg lettuce is the most demanding for the grower and therefore for the breeder. Iceberg forms a rosette of leaves in its early growth stage. At a certain point a new leaf is curved rather than flat and thus begins the enclosure of the subsequently forming leaves. The head fills internally and becomes larger and firmer until it reaches a stage where the size and firmness are considered desirable. It should be harvested at this point, because it continues to produce new leaves, which exert severe pressure, eventually causing the outer petioles to crack. At the same time, bitter substances increase, making the leaves less desirable for eating. The time between optimum size, firmness, and quality and overmaturity may be only a few days. Therefore it behooves the producer to harvest during this interval or risk loss of quality and a reduced price. The breeder’s responsibility therefore includes several factors of improvement. A cultivar should have a spherical head for ease of packing in a carton. In most production areas, it should be dark green. The head should be well filled and firm and not loose and puffy. It should reach this optimum shape and firmness at a size to allow packing 24 heads in a carton of standard dimensions. Smaller or larger heads are less desirable. These traits should converge for all the heads in a field at the same time, i.e., development should be uniform. These factors are all judged in a subjective manner, by sight and touch. In addition, the plant should not begin the seed stalk elongation (bolting) stage prematurely. This characteristic is dependent upon temperature and daylength in a genetic-environmental interaction. High temperature and long days induce bolting in sensitive cultivars (Rappaport and Wittwer 1956). Early seed stalk formation may prevent heading completely or result in an elongated stem inside the head, which increases bitterness and may affect shape adversely.

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Batavia lettuce is similar to iceberg lettuce in general appearance. However, the head is smaller and less firm at optimum maturity. Developmental criteria are therefore less stringent. Batavia type lettuces are more common in Europe than in the United States, and are rarely found in other lettuce producing countries. Butterhead lettuce also forms heads, but appearance, size, and conformation are substantially different from the crisphead type. Most cultivars are light to medium green. The heads at optimum development are smaller, earlier maturing, and slightly less dense than crisphead cultivars. The leaves have a soft oily texture. Butterhead cultivars are grown in protected structures as well as in the field. Protected cultivars are grown during the winter months and are smaller and much looser headed than the summer field cultivars. Butterhead cultivars are more uniform in development than crisphead cultivars, so that the convergence of heading traits is more easily achieved. Within the butterhead subtype, the Boston group is light green with relatively large heads, while the Bibb group is smaller and darker green and has reddish leaf margins. Romaine (cos) lettuce forms an upright loaf-shaped rosette of relatively long, spatulate leaves. The top may be open, or the leaves may bend inwards slightly near the apex. There is no true head formed, so the producer can choose, within limits, when to harvest. Variation in leaf length, texture, and color may occur. Leaf lettuces vary substantially in several traits. Cultivars vary in shades of green. Cultivars with anthocyanin occur in various shades and patterns of red color. Leaves may be lobed or entire. They may be highly frilled. They may be broad and crisp similar to crisphead cultivars, but they remain flat and in a rosette rather than forming a head. Finally, the Latin type has romaine-like leaves, but they are shorter than romaine. The texture is crisp and succulent, although the appearance of the leaf is similar to the butterhead. Thus, each subtype of lettuce has its own unique breeding requirements for appearance and shape that the breeder must include among the goals for development of new cultivars. B. Resistance to Diseases, Insects, and Disorders Nearly all lettuce breeding programs include development of resistant cultivars. The earliest work was initiated by Jagger in 1922, with the development of brown blight resistant cultivars. Jagger’s first three releases, ‘Imperial 2’, ‘Imperial 3’, and ‘Imperial 6’, were essentially selections from surviving plants in fields of susceptible ‘New York Special’ (Jagger et al. 1941). The New York type was the mainstay of the

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developing western lettuce industry, which had been threatened by the brown blight disease. Subsequent releases were derived from crosses with ‘White Chavigne’, a brown blight resistant butterhead, and ‘Blonde Lente a Monter’, a downy mildew resistant romaine. Breeding for downy mildew resistance became a major activity in most American and European lettuce breeding programs; downy mildew was a widespread problem, and it was difficult to deal with because new virulent strains of the causal organism (Bremia lactucae Regel) appeared periodically. Breeders were repeatedly required to develop new resistant cultivars from new sources of resistance. The basis for the relationship between the fungus and the host was worked out in the pioneering studies of I. R. Crute and A. G. Johnson, who developed a gene-for-gene hypothesis (Crute and Johnson 1976; Johnson et al. 1977). Single major effect genes conferred complete resistance in lettuce cultivars. These genes were matched by dominant avirulence genes in B. lactucae: the dominant allele of a numbered Dm gene confers resistance against all isolates expressing the matching avirulence gene. All other combinations result in disease. For example, Dm1 confers resistance to all isolates expressing Avr-1, but not isolates expressing avr-1, Dm-2 to all expressing Avr-2, but not isolates expressing avr-2, etc. The fungus can change in an asexual manner, or by crosses between opposite mating types (Michelmore and Ingram 1980, 1981; Hulbert et al. 1988; Hulbert and Michelmore 1988). Because of the rapid changes in the fungus, and the consequent repeated loss of resistant cultivars, downy mildew continues to be a major disease problem of lettuce. Breeders are looking for alternate and supplemental methods of control. At the U.S. Agricultural Research Station in Salinas, we are working with field resistance to this fungus. This is a mature plant resistance based upon quantitative genetics, which may be more durable than single gene resistance (Crute and Norwood 1981; Norwood et al. 1985). This type of resistance is found in the cultivar ‘Iceberg’ and other cultivars, and is manifested by smaller and fewer lesions, rather than complete resistance. Another approach is to use several new Dm genes at once, either dispersed in several cultivars released simultaneously or accumulated in one cultivar (R. W. Michelmore, pers. commun.). A third approach is to combine complete resistance from a Dm gene with quantitative field resistance, so as to provide high levels of resistance, backed up by the field resistance when the Dm gene is overcome. Also, genetic resistance of any type can be combined with fungicidal treatment (Crute 1984). In virus disease resistance work, the genes for lettuce mosaic virus (LMV) resistance have been used in breeding programs. The original

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work reported resistance alleles g (Bannerot et al. 1969) and mo (Ryder 1970), which for many years were believed to be identical. However, they reacted differentially to a pathotype of LMV derived from endive (Dinant and Lot 1992). They are now considered to be alleles of the same locus, mo-11, derived from Gallega and mo-12, derived from PI 251245. (See Irwin et al. 1999 for a discussion of gene nomenclature.) The former has been used in breeding programs in Europe, and the latter in the United States. Since mo-12 may have a greater range of resistance (including resistance to the endive pathotype), it is likely to be used more frequently in future breeding programs. Plants with these alleles react to the virus with a restricted symptom, as compared to the mottling effect in susceptible plants. Recently, a gene has been identified in an Egyptian stem lettuce, which is partially dominant and produces a systemic reaction, but with mild symptoms (Ryder 1996). When combined with mo-12, it confers a high level of resistance and should be useful in breeding programs. Virulent isolates of LMV that may cause severe necrosis have been found in various parts of the world. Most of these have not persisted, but some have, and the search for new resistance genes continues. Genetics of resistance is known for several other problems. Resistance to corky root, a bacterial disease (Rhizomonas suberifaciens van Bruggen, Jochimsem et Brown), is controlled by a single recessive allele (Brown and Michelmore 1988). Resistance to anthracnose, a fungus disease (Microdochium panattoniana (Berl.) Sutton et al.), is controlled by 2–4 dominant loci (Ochoa et al. 1987). Beet western yellows is a virus disease, and a greater problem in Europe than in the United States. Resistance has been identified in two lettuce cultivars and is conferred by a single recessive allele (Pink et al. 1991). Another source was found in a line of L. virosa and is conferred by a single dominant allele (Maisonneuve et al. 1991). Resistance to root aphid (Pemphigus bursarius L.) is controlled by a single dominant allele (Ellis et al. 1994). Lettuce aphid (Nasonovia ribis nigri Mosley) resistance is controlled by a single partially dominant allele, which also gives partial resistance to the green peach aphid (Myzus persicae Sulzer) (Eenink et al. 1982). Breeding for resistance to each of these problems is in progress. Cultivars resistant to LMV, downy mildew, corky root, root aphid, and/or lettuce aphid have been released. Breeding programs for several other diseases and insects are active and in various stages of development, despite the fact that the genetic basis is at present not known. Big vein, caused by a virus-like entity, was at one time only a problem in the western United States, but is now a worldwide problem. Several breeding programs exist; we have released

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three cultivars with moderate resistance, and other cultivars with similar resistance are in existence. We have screened many cultivars and have identified over 25 with resistance (Ryder and Robinson 1995 and unpublished). Tipburn is a physiological disorder associated with low calcium in leaf marginal tissues. Most breeding programs screen for resistance to tipburn, but there has been no useful genetic information developed. We have breeding programs for resistance to Sclerotinia minor Jagger; Verticillium dahliae Kleb, a new lettuce disease (Davis et al. 1997); lettuce infectious yellows/lettuce chlorosis viruses; and pea leafminer (Liriomyza huidobrensis Blanchard). C. Use of Interspecific Crosses Crossing of cultivated lettuce with other forms and species has been practiced increasingly by breeders. The term “other forms” refers to forms with species names, as described above, and to primitive forms of L. sativa. The other species are: L. serriola, L. virosa, and L. saligna. A primitive form of L. sativa, obtained through the Plant Introduction Service of the U.S. Department of Agriculture, has been used in breeding and genetics studies. PI 251245 is resistant to LMV and was crossed with cultivated lettuce to produce ‘Vanguard 75’, the first crisphead lettuce with resistance to LMV (Ryder 1979). This accession flowers very early; the inheritance of this trait is being studied in this line and in several other accessions. The close relationship of L. serriola to L. sativa induces some taxonomists to consider that together they comprise a pan-species. Various accessions of L. serriola appear in the pedigrees of lettuce cultivars and breeding populations as sources of resistance to downy mildew (Bohn and Whitaker 1951) and corky root (Sequeira 1970; Brown and Michelmore 1988). L. serriola has served as a bridge species in crosses with L. virosa (Thompson and Ryder 1961; Eenink et al. 1982). L. virosa is less closely related and is difficult to cross with L. sativa. Nevertheless, it has contributed to at least two notable breeding accomplishments. R. C. Thompson made the cross in 1938, in a complex cross with accessions of L. serriola and a crisphead cultivar of lettuce, ‘Great Lakes’ (Thompson and Ryder 1961). The F1 was sterile, but produced seed when treated with colchicine. The plants were tetraploid. A cross with a butterhead cultivar, ‘Red Besson’, followed by selection for diploidy, produced a line that was crossed with ‘Climax’. Continued selection yielded a line that was used in further crosses, which eventually led to the cultivar ‘Vanguard’. ‘Vanguard’ was released in 1958 as the first cultivar developed from a cross with L. virosa. Most crisphead

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cultivars in use today derive from the materials associated with this and a few other crosses (Fig. 4.1). Two accessions of L. virosa were found to have a dominant allele for resistance to the lettuce aphid, Nasonovia ribis nigri (Eenink et al. 1982). The lettuce aphid has been considered to be the most serious aphid pest in Europe. (It was recently identified in California, and has become a serious pest there as well.) Again, L. serriola was used as a bridge species in successful crosses with cultivated lettuce. Both butterhead and crisphead cultivars were derived from this material in Dutch breeding programs.

Imperial 152 × Brittle Ice L. virosa × (L. serriola × Great Lakes) F1

USDA 1237 colchicine Red Besson × USDA 3486 (4n) BC to 2n USDA 5504 (2n) × Climax Great Lakes 6238 × USDA 45325 DMR Selection PI 251245 × Vanguard LMVR

USDA 8830 × Calmar

6 BC to Vanguard

Vanguard 75

Salinas

Fig. 4.1. Condensed pedigrees for development of landmark iceberg type cultivars, from which all iceberg cultivars used today are derived. DMR = downy mildew resistant; LMVR = lettuce mosaic virus resistant.

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L. saligna is a small, slow growing wild species native to southern Europe. Crosses with L. sativa are easier to make than L. virosa-L. sativa crosses. L. saligna has been used in several breeding programs. It contributed resistance to cucumber mosaic virus in the development of the crisphead cultivar ‘Salad Crisp’ by R. W. Robinson at Cornell University (Pers. commun.). L. saligna is also a potential source of resistance to downy mildew and lettuce infectious yellows. D. Development of Breeding Tools Two genetic breeding tools have been identified in lettuce, but have had limited use in breeding programs. Six traits for male sterility have been identified. Four are due to a recessive allele, one to a dominant allele, and one to a dominant-recessive pair (Robinson et al. 1983). Male sterility has potential use for creating F1 hybrids, although for lettuce the potential usefulness of hybrids is uncertain. There is indication of hybrid vigor in some crosses, although in self-pollinated species, protection of the identity of the hybrid is likely to be more important for the creator of the hybrid. However, lettuce pollen is sticky and not windblown, and there are no insects that work flowers efficiently. Pollination by hand yields about 10 seeds per pollination and would therefore be highly inefficient and expensive. The most likely means of creating hybrid cultivars is multiplication of a single hybrid plant by cell culture. The other trait is early flowering. Two genes control this trait (Ryder 1983, 1988). In the homozygous series, the double dominant reduces flowering time to about 45 days in specific conditions; one single dominant reduces flowering time to 65 days; the other single dominant reduces flowering time to 90 days; and the double recessive flowers in 140 days. This trait can be used to accelerate a backcrossing program, reducing by half the time required to transfer an allele to the recurrent parent (Fig. 4.2). E. Human Health Aspects Although any breeding development has social implications by virtue of maintaining the food supply and by reducing the use of chemicals through development of resistant cultivars, the only one to date with a readily identifiable human health goal is breeding for reduced nitrate content. Two health problems have been identified as associated with excess nitrate content. One is the blue baby syndrome, methaemoglobinaemia, in which nitrate reduces to nitrite, which acts to prevent proper

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Ef-1Ef-1 Ef-2Ef-2 × ef-1ef-1 ef-2ef-2 Early Late Ef-1ef-1Ef-2ef-2 Intermediate

Ef-1Ef-1 Ef-2ef-2

(Discard)

Ef-1ef-1 Ef-2Ef-2

(Discard)

ef-1ef-1 ef-2ef-2

(Discard)

Ef-1ef-1 Ef-2ef-2 Intermediate

×

×

ef-1ef-1 ef-2ef-2 Late

ef-1ef-1 ef-2ef-2 Late

BC1

BC2

BC3 etc.

Ef-1ef-1 Ef-2ef-2 Ef-1Ef-1 Ef-2ef-2 Ef-1ef-1 ef-2ef-2 ef-1ef-1 ef-2ef-2

Select late genotype

Fig. 4.2. A backcross sequence utilizing early flowering genes to accelerate the process. Early and late parents planted at different times to insure flowering at same time. A useful allele (not shown) desired for transfer may be selected in each generation. Number of days to first flower: Ef-1Ef-1Ef-2Ef-2: 45 days; Ef-1ef-1Ef-2ef-2: 65 days; and ef-1ef-1ef-2ef2: 140 days, during long-day season in the greenhouse.

oxygen assimilation, especially in developing fetuses. The other is a possible association of ingestion of nitrates with gastric cancers. This involves conversion of nitrate to nitrite, followed by formation of nitrosamines. Increased nitrate content in lettuce is particularly a concern in the production of butterhead lettuce in greenhouses under low light conditions. The genetic basis for nitrate content has been explored by Reinink (1991) in Holland. He reported on the results of a diallel analysis, which showed that additive genetic effects contributed most to variation among lettuce cultivars, thus offering the opportunity to select for lower nitrate content in a breeding program.

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IV. LETTUCE BREEDING FOR THE FUTURE With this summary of the various aspects of lettuce breeding up to the present, let us now examine the “bridge to the 21st Century” and beyond for the science and art of lettuce breeding. It must be realized that all the avenues of research pursued up to now should be continued, as the need will continue for disease and pest resistance, product quality, and exploration of related species. Two areas that may be expected to expand in scope and emphasis will be: (1) the development of biotechnological tools and (2) dealing with societal obligations in breeding programs. I propose to examine seven issues that one may expect to draw attention in the coming generations: (1) the use of biotechnology, (2) public and private breeding responsibilities, (3) the use of pesticides and other chemicals, (4) the contribution of lettuce to the human diet, (5) food safety aspects, (6) value-added and other new products, and (7) lettuce as a research organism. A. The Tools of Biotechnology Biotechnology is an issue that is both new and significant in crop research, whether in breeding, genetics, plant pathology, or other fields, and offers a wealth of techniques to the study of many crops. In lettuce breeding, the techniques that are in various stages of application now are: cell and tissue culture, creation of genetic maps, map-based cloning, marker-assisted selection, and transformation. These techniques are already in use in many laboratories. It may be confidently anticipated that this use will increase in the coming years. Tissue culture of lettuce was first achieved by Doerschug and Miller (1967), who regenerated shoots from various tissues. Cell or protoplast culture for regeneration of plants was described by Engler and Grogan (1984). These asexual techniques have become routine in lettuce. They have several possible applications in plant breeding. Lettuce is an obligately self-fertilizing species. The structure of the composite flower and the low number of achenes per flower make it quite difficult to produce F1 seed in volume, should the production of F1 hybrids ever become desirable for either biological or security reasons. Reproduction of a desirable hybrid can be achieved by multiplication through cell culture, if the creation of somatic variation can be eliminated or minimized. A more pedestrian use of asexual reproduction would be the rescue of a desired, selected plant in danger of dying from disease or physical damage. Plants can be reproduced quite easily by removal of various

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tissues from the original plant and tissue culturing them or rooting them directly. The greatest contribution to plant breeding is likely to come from the combination of molecular techniques in genetic mapping, markerassisted selection, and transformation. Creation of a complete and thorough genetic map made up of multiple molecular markers, combined with standard genetic markers, and genes of direct value in breeding, will provide the breeder with the means to facilitate breeding procedures. Close linkage of genetic or molecular markers with desirable genes for disease resistance or other economic traits enables the selection of plants with the desired trait in a greenhouse, and perhaps even more efficiently and quickly, in the laboratory. A detailed genetic map has been described by Kesseli et al. (1994). It is composed of 319 loci, consisting of 282 RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) loci, as well as seven isozyme, 19 disease resistance, and 11 morphological loci. An estimation of the size of the genome suggests that the average distance between loci is 6.1 cM. However, gaps of up to 27 cM still exist, indicating an uneven distribution of known loci on the map. Additional technologies for generating map data are becoming available. RFLP technology was the first means beyond standard genetic techniques and izozyme analysis for genetic analysis and marker generation (O’Brien 1990). There are disadvantages in the use of RFLPs; radioactive materials may be required for detection, large amounts of plant tissue are needed, and the work is laborious and time-consuming. Subsequently, PCR (polymerase chain reaction) amplified genetic markers became available. These include RAPD markers (Williams et al. 1990), SCAR (sequence characterized amplified regions) markers (Paran and Michelmore 1993), AFLP (amplified fragment length polymorphism) markers (Vos et al. 1995), and SAMPL (selectively amplified microsatellite polymorphic locus) markers (Witsenboer et al. 1997). These techniques are being used for map development, location of disease resistance and other important loci, and characterization of intra- and inter-specific relationships. An integrated genetic map for lettuce consisting of over 1,000 markers and nine chromosomal linkage groups will be released in the near future (R. W. Michelmore, pers. commun.). The information on a genetic map can be used for several types of research. It should be noted that a genetic map composed only of molecular markers has limited usefulness. Its usefulness greatly increases with the addition of phenotypic loci, which opens the opportunities for map-based breeding and marker-assisted selection practices.

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The molecular and genetic markers from which the map is made can be useful in the differentiation among lettuce accessions, including cultivars, landraces, and wild species. Several phylogenies have been created. Kesseli and Michelmore (1986) separated 17 cultivars and accessions of L. sativa and 15 Plant Introduction accessions, including wild species, using 42 isozyme systems. Variation of banding patterns at RFLP loci disclosed relationships among 67 accessions of L. sativa and five related wild species (Kesseli et al. 1991). Waycott and Fort (1994) compared RAPD loci of similar butterhead lettuce cultivars, and showed good correlation between morphological variation and molecular variation. Witsenboer et al. (1997) analyzed SAMPL loci of 58 accessions, including both cultivars and related species. Relationships shown by biological and chromosome analysis compare well with those obtained by molecular analysis, and the various molecular methods compare well with each other. There has been considerable effort to detect molecular markers close to genes of interest for breeding. Much of the activity in lettuce has been devoted to finding markers closely linked to downy mildew resistance alleles, to the mo-12 allele for lettuce mosaic resistance, and to the cor allele for corky root resistance (Michelmore 1998). These studies have two purposes. One is to characterize the region around disease resistance genes as a preliminary step to cloning and sequence characterization of these genes at the molecular level (Meyers et al. 1998a, 1998b). A second, highly practical purpose is to identify markers in or around a desired gene. This permits indirect selection of a desired gene without growing large field populations or challenging with the pathogen. It also permits elimination of undesirable linked genes associated with the recurrent allele in a backcrossing program by indirect selection of closely linked markers. This practice should accelerate the backcrossing procedure. Much interest has been generated recently in marker-assisted identification of multiple loci controlling the same character in a quantitative manner, so-called quantitative trait loci or QTL. This approach is likely to be most successful in those cases where some or all of the loci have sufficient effect to be detected or are grouped closely on the chromosome such that they behave as major genes. Individual or multiple markers associated with QTL or QTL groups whose effect can be measured with some confidence can be used for indirect selection (Paterson et al. 1991). The proportion of the phenotypic effect that can be detected will depend on the number of genes affecting the trait and magnitude of the effect of each gene or gene cluster that is measurable, i.e.

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not obscured by environmental effects. Jackson (1998) reported the association of taproot characters and other morphological QTL with molecular markers in a cross between an iceberg cultivar, ‘Salinas’, and a wild relative, L. serriola. The proportion of trait effects detected ranged from 37–83% of the total variation for the trait in question. This probably reflects the lack of map saturation and the difficulty of measuring the traits. The breeder using this information would therefore not be able to access 100% of the total variation for this trait; nevertheless, this procedure provides an opportunity to dissect and manipulate these complex traits. Transformation is potentially a powerful tool in lettuce breeding for transferring genes that are not easily or not at all available through traditional breeding techniques. These would include: (a) genes from L. saligna and L. virosa, which cross with lettuce with some difficulty, (b) genes from the other, genetically isolated, Lactuca species, and (c) genes from outside the genus Lactuca. Transformation has been accomplished in two ways in lettuce: mediation by a species of Agrobacterium, usually A. tumefaciens, and electroporation. Transformation of lettuce using A. tumefaciens has been conducted in several laboratories. The first report of routine transformation described the transfer into plant cells of a kanamycin resistance gene by Ti plasmids of A. tumefaciens (Michelmore et al. 1987). They found that the transformation procedure was most effective with the butterhead cultivar ‘Cobham Green’, compared to several iceberg type cultivars. Later, an iceberg lettuce cultivar, ‘South Bay’, was successfully transformed using cotyledon explants (Torres et al. 1993). Curtis et al. (1994) transformed 13 cultivars of iceberg, butterhead, leaf, and romaine lettuces, but the number of shoots produced varied among the cultivars. Transformation to generate useful disease resistance in lettuce has been attempted, but the activity of the transgene has thus far not been conserved. Sequences of the lettuce mosaic virus coat protein gene transformed into ‘Cobham Green’ lettuce conferred some resistance to LMV in the R2 generation but apparently failed to express the resistance in the R3 and R4 generations (Michelmore 1998; Gilbertson 1998). Torres et al.(1999) transformed ‘South Bay’ lettuce with the gene for glyphosate herbicide resistance and reported the development of a seedling assay for resistance. Electroporation is a means of direct transfer of foreign DNA into protoplasts, using electrical discharges in a closed chamber. Chupeau et al. (1989) obtained transgenic plants of lettuce using electroporation at 200 and 300 volts. Kanamycin resistance was the trait transferred. Finally, biotechnology may be useful in the detection of virus presence in seeds; elimination of virus-infected seed lots is an important means

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of protecting against lettuce mosaic virus (LMV) infection in lettuce fields. The standard procedure employs ELISA (Clark and Adams 1977) to detect seed lots with seed-borne virus. Van der Vlugt et al. (1997) have proposed the use of a procedure called immunocapture reverse transcriptase PCR (IC-RT-PCR) for this purpose and have found it to be more sensitive than ELISA. B. Public and Private Breeding Responsibilities As the scope of biotechnology continues to broaden, it may be useful to contemplate its possible effects on the future path of lettuce breeding. These effects will certainly also be felt in the entire world of plant breeding. In a strictly operational fashion, the tools will be tested and applied in plant breeding programs, as have all previously discovered tools. In lettuce, as in other crops, the breeding cycle may be accelerated, particularly with the use of marker-aided selection and backcrossing. Genes of value that are not now available in lettuce may eventually become a part of the lettuce genome. We need more knowledge and perspective on the likelihood that genes from outside the species will function normally, or close to it, in a new genomic environment. With regard to QTL, we know little of the proportion of these loci and of their effect in their original context that can be successfully captured and moved. If less than 100%, what percentage will be sufficient to be useful and justify the effort? This will be partially dependent upon the biological value of the trait under consideration. Potentially, the possible societal ramifications of biotechnological development may be far more important and far-reaching than the technical ones. Biotechnology has excited the economic and legal communities more than any other previous technological development in biology. The reason is obvious: the potential for making large amounts of money is considered to be highly promising. Because of this, companies and public agencies both are taking steps to protect biotechnical advances. No plant technologies developed in earlier years merited such attention. These actions sometimes lead to conflicts over intellectual property rights and ownership. Patenting of asexually propagated plant cultivars has been possible in the United States since 1930. Plant variety protection (PVP) has been available since 1970. In 1985, utility patents were made available for various types of biological phenomena: cultivars, genes, DNA fragments, and various techniques. As the means of protection have proliferated and as the field becomes more competitive, consequences have developed that might well have been expected. These include disputes over

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ownership, objections to the sweeping nature of some patents, and increasing restrictions on the use of patented materials. An abundant literature is developing, discussing the patents involved, the litigation stemming from disputes, and on the relations developing among biotech companies and between biotech companies and non-biotech companies such as traditional seed houses. To narrow the focus considerably, what are likely to be the consequences as they apply to the small world of lettuce breeding? On one level, private companies that choose to protect newly developed cultivars do so for two reasons: to have recourse if a cultivar is appropriated and sold by another company, and to generate income to help cover the costs of research. Public agencies are more interested in the latter goal, which stems from the increasing difficulty in obtaining tax monies for breeding research. Private company activities are driven by the need for income, which is the source of their existence. Therefore, the generation of income as a direct consequence of developing cultivars that can be protected makes eminently good sense. Public agencies, on the other hand, exist primarily to do research that has other goals, which can be grouped together as an overall purpose: to do public good. There is a clear and likely danger that public research programs will be driven by market forces, so that the breeders will be directed, by self or otherwise, to orient their programs in the direction of cultivar development to the partial or complete exclusion of the “public good” goals. Public breeders should devote a major part of their efforts to development of information about their crops, to undertaking long-term breeding programs without the certainty of delivering new and wanted cultivars, and to communicating information and providing materials to other interested scientists. In lettuce breeding, the public agencies have provided continuing, long-term leadership in the development of freely available advanced breeding materials from crosses with wild species and landraces, knowledge of the genetics of disease resistance and other useful traits, and germplasm curatorship, in addition to cultivar development. Public lettuce breeders in the United States have produced several landmark cultivars, making use of wide crosses, which have required difficult manipulations and long-term effort. The pedigrees of several of these are shown in Fig. 4.1. Private companies have often taken these materials and information and converted them into popular and useful cultivars for the multiplicity of niches available for growing lettuce. This has resulted in proliferation from the original cultivars to a wide range of useful and important cultivars (Table 4.1). This balanced relationship between the two types of institutions has worked well. Unfortunately, the place of the public effort and the relationship with

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Table 4.1. A partial listing of cultivars released by private and public agencies that were derived from four original landmark cultivars developed in public breeding programs. Original cultivar

Released by

Derived cultivars

Great Lakes

USDA, Michigan; 1941

Calmar

California USDA; 1960

Salinas

USDA, California; 1975

Vanguard

USDA; 1958

Great Lakes 13, 54, 65, 66, 118, 366, 407, 428, 659, 6238, A36, R-200, J Strain, Shipper Strain, Regular, Premier, Phoenix; Calmar. Montemar, Calmaria, Cal K-60, La Jolla, Salinas. Salinas R-100, 105, 88, 115, 128, 350, 501, 517, Pybas 101, Pybas 102, XL, S; Bix, Blanco, Alpha, Target, El Dorado, Legacy. Vanguard 30, 75, 007X, 9050, BL; Vanmax, Moranguard, Winterhaven, Red Coach 74, Coolguard.

private industry is in some danger of being disrupted. As patenting progresses and as some materials become more intrinsically valuable, it will be difficult for public breeders to obtain materials for crossing except by purchase of a license, which may be quite expensive. It will thus become more and more difficult for the public breeder to operate. Eventually, the public breeders may become only custodians and manipulators of germplasm. The ultimate consequence, as access to worldwide germplasm becomes more restricted, is that germplasm collections themselves will attain great value. Will that mean they will become private property and all public activity will cease to exist? It seems a farfetched idea now, but it may not seem so in coming years. There is a great deal of literature on plant intellectual property rights and its ramifications. The reader may wish to consult Hamilton (1993), de Miranda Santos and Lewontin (1997), and Wright (1998), for some other views. C. Pesticides and Other Chemicals An issue that has been of concern ever since the publication of Silent Spring by Rachel Carson (1962) is the use of pesticides and other chemicals in crop production. There is a continuing pressure to restrict the use of pesticides on lettuce. Regardless of the virtues of either side in

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what has become an ongoing controversy, there is no doubt that breeding as a means of controlling lettuce pests and diseases will become even more important in future years. However, a key need in the development of resistant cultivars is that the resistance should be durable, so that the grower can have confidence that the resistant cultivar can be used for many years. An example, as described earlier, is downy mildew resistance breeding, which has concentrated on development of cultivars with Dm genes that confer complete resistance. Unfortunately, the relationship between the lettuce host and the fungus, B. lactucae, is a dynamic one, and the resistance offered by a rapid succession of cultivars has been unstable. Other approaches, as described, may provide the durability needed. One of the principal objections to the use of chemical fertilizers is that they may find their way into surface and ground water supplies and are of concern because of their toxicity, as potential carcinogens, and as a cause of eutrophication of bodies of water. This is a particular concern with regard to nitrate nitrogen. The opportunity for the lettuce breeder in this area is to develop cultivars with lower fertilizer requirements and wider adaptibility. Crisphead cultivars, for example, should have the ability to grow to adequate size and have a desirable green color along with good head shape and density, despite lower nutrient inputs. Goals towards this end might include increasing total root length to increase foraging ability (Jackson 1998), increasing photosynthetic capacity, and incorporation of genes for increased size and improved color. D. Lettuce in Nutrition and Health Two issues involving lettuce as food for human consumption are likely to be important. One is nutritional value and the contribution of lettuce to the diet. The other is the safety of lettuce for consumption with regard to the possible presence of harmful microorganisms. Lettuce makes only a modest contribution of vitamins and minerals to the diet on a unit weight basis (Table 4.2). However, on the basis of volume consumed, its contribution is greater when compared with many vegetables and fruits that have a greater value per unit weight (Stevens 1974). Stevens compared 35 major vegetables and fruits for actual contribution of 10 important vitamins and minerals, and iceberg lettuce placed 26th on the list. If its nutrient contribution is weighted by the amount consumed, however, iceberg lettuce climbed to 4th place (Table 4.3). Romaine, butterhead, and leaf lettuces have greater content

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Table 4.2. Nutritional values for four major lettuce types. Values are for 100 g of edible product.z Minerals (g)

Vitamins

Lettuce type

Ca

P

Fe

Na

K

A(IU)

C(g)

Water (%)

Fiber (g)

Crisp Butter Cos Leaf

22 35 44 68

26 26 35 25

1.5 1.8 1.3 1.4

7 7 9 9

166 260 277 264

470 1065 1925 1900

7 8 22 18

95.5 95.1 94.9 94.0

0.5 0.5 0.7 0.7

z

Adapted from Rubatzky and Yamaguchi 1997, as compiled from several original sources.

of some of these nutrients, and the overall nutritional contribution of lettuce may have improved in recent years with the increase in consumption of the non-crisphead types. Recently, there has been increasing interest in vegetables as contributors of anti-oxidant, anti-carcinogenic substances to the diet. Various substances can contribute such activity, and the amounts vary among

Table 4.3. Rankings of 15 vegetables for actual nutritional value and weighted value based upon amount consumed. Based on sum of values for 10 vitamins and minerals (Vitamin A, Vitamin C, niacin, riboflavin, thiamin, potassium, phosphorus, calcium, iron, and sodium). Rankings included fruits not shown here.z Vegetable

Actual rank

Weighted rank

1 2 3 4 5 6 7 8 9 10 12 14 15 16 26

21 18 34 23 15 25 36 30 10 7 5 3 8 1 4

Broccoli Spinach Brussels sprouts Lima bean Pea Asparagus Artichoke Cauliflower Sweet potato Carrot Sweet corn Potato Cabbage Tomato Lettuce z

Adapted from Stevens 1974.

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species. They include: ascorbic acid, β-carotene, Vitamin E, anthocyanidins, lycopene, and glutathione. Cao et al. (1996) compared 22 common vegetables with green and black tea for anti-oxidant capacity, measured by their oxygen radical absorbance capacity (ORAC). The scores for both iceberg and leaf lettuce were quite low compared to tea, and to garlic, spinach, beets, red bell pepper, and several brassica vegetables on a per weight basis. However, evaluating the contribution of lettuce on a consumption basis might enhance its value in the same way as for vitamins and minerals. One might consider alternative means for increasing the nutritional and phytoceutical effects of vegetables in general and lettuce in particular. Encouraging increased consumption of all vegetables and fruits to five portions a day as recommended in this country, or to seven a day as actually consumed in many European countries, would be useful. With regard to lettuce, encouraging increased consumption of salads, with special emphasis on the leaf, butterhead, and romaine types would increase the contribution of lettuce. Finally, the breeder might consider a program to increase the level of one or more substances in lettuce cultivars. In relation to the other alternatives, it would be important to consider the costs involved as compared to those of other breeding goals before engaging in nutrient improvement breeding for lettuce. E. Food Safety Concerns Food safety is an issue stemming largely from outbreaks of illnesses from ingestion of the 0157:H7 form of Escherichia coli, Salmonella typhimurium, and other infectious organisms. Most cases of food poisoning from 0157:H7 have been associated with improperly cooked meats and dairy products (Ackers et al. 1998). However, uncooked produce items, including lettuce, have come under scrutiny because of the tremendous growth of these extensively handled products for salad mixtures and the possible use of unsafe water and untreated manure during production. Each of the steps in handling fresh produce provides the opportunity for contamination with a variety of primarily bacterial species. Hazard analysis critical control point (HACCP) programs, including emphasis on personal hygiene, are being developed to prevent bacterial contamination at each of the steps in handling the products (Beuchat 1996). The possible contribution of breeding in dealing with this problem is unknown. It might be possible to develop cultivars that present an inhospitable environment to the organisms, and thus reduce the level of contamination.

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F. Value-added and Other New Products The rise of value-added products in the produce industry has been phenomenal in recent years. For lettuce the most significant divergences from traditional production and marketing were the use of plastic wrap around heads with most outside leaves removed, and the more recent development of bulk harvesting and marketing. The latter includes two groups of products. One is the institutional package containing large quantities of lettuce, which is chopped or shredded. Chopped lettuce is often mixed with other vegetables, especially carrot shreds and red cabbage, and is used for salads in restaurants, hospitals, schools, and other institutions. Shredded lettuce is used by restaurants for hamburgers and other sandwich fillings. The other group includes a variety of prepared salad packs, which are sold in food markets for home use, and contain enough salad for one or two meals. Many combinations of lettuces, other leafy vegetables, protein products, croutons, and dressings are sold this way. This is one of our fastest growing products; approximately onethird of lettuce production is for bulk harvesting. The opportunities for plant breeding improvement are substantial in this area. In the early days of bulk production, the lettuce was gleaned from fields after harvest for cartons. As the demand grew, whole fields were contracted specifically for bulk harvest, and some breeding activities should be directed towards this form of production. Of the traditional breeding goals, disease and insect resistances remain important for bulk lettuce. However, head formation and shape of iceberg lettuce will be less relevant. Both exterior and interior color will continue to be important for all lettuces used in salad mixtures. Intense greens and reds are more desirable than paler shades for outer leaves, and creamy yellow is preferable to white for the interior. Several traits have special importance in the value-added products and require special attention from the breeder. Although tipburn resistance is desirable in any form of lettuce, it is particularly critical in packaged lettuce, since it can be readily seen by the consumer and may be a cause for rejection of the package. Tipburn is associated with rapid growth and delayed transport of calcium to rapidly growing tissues (Collier and Tibbitts 1982). Unfortunately, the genetic basis for tipburn reaction is unknown, and selection for resistance must be done under field conditions that cannot be relied upon to produce the effect when desired (Ryder 1999). Future breeding efforts will depend upon knowing the genetic basis for more efficient selection. Further research on the genetic basis for tipburn, perhaps using QTL analysis and the identification of molecular markers, is necessary.

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Resistance to development of other types of physiologic disorders, such as rib blight and marginal browning in the field and russet spotting and brown stain under storage conditions (Ryder 1999), should also become breeding goals for lettuce for bulk use. Resistance to various forms of bacterial and fungal rotting, caused by Erwinia carotovora or Botrytis cinerea, would also be desirable. An obvious consequence of the bulk process is a tremendous increase in the number of cut surfaces. These can become brown upon exposure to air. Delay of browning can be achieved by washing and by packaging in a modified atmosphere, with reduced oxygen and increased carbon dioxide (Ballantyne et al. 1988). It is possible that genotypes vary in sensitivity to conditions controlling the development and extent of browning. Thus it may be possible to alleviate the effect by genetic improvement. Several other types of specialty lettuces may enter into breeding programs. Baby lettuce leaves for use in mesclun (mixtures of small leaves) are harvested when they are 10 cm or less in length. These leaves should have intense green and/or red color, and novel shapes, such as lobed or frilled margins. Also, young leaves tend to wilt rapidly because of their small size. Breeding improvement is possible for these traits. Two types of lettuce, which are now grown primarily in specific areas in the world, may have future usefulness in other places. Latin lettuce is grown in Europe and South America and a little in the United States, but is scarce elsewhere. It is a romaine-like type, but has shorter leaves and a more succulent texture. A breeding goal might be to increase its size, while retaining the eating quality. Stem lettuce has narrow leaves or broader romaine-like leaves, but is grown primarily for its thick edible stem in Egypt, and other Middle Eastern countries, where it is consumed raw. It is also grown in China, and is used there as a cooked vegetable. Stem lettuce can be improved in at least two ways, reduction of the bolting rate and retention of quality at maturity. In addition to nutrient and phytoceutical constituents, lettuce and its relatives have an array of chemical substances that have actual and potential use in other ways. Strains of primitive L. sativa (often identified as L. serriola), which are fast bolting and have narrow leaves that form only a rudimentary rosette, are grown in Egypt for their large seeds, which are pressed to produce a cooking oil (P. F. Knowles, unpublished plant exploration report; Ramadan 1976). This practice may actually be one of the earliest uses for domesticated or partially domesticated L. sativa in Ancient Egypt. The wild relative L. virosa is used in some countries to produce two sesquiterpene lactones, lactucin and lactucopikrin, from the latex. These materials are used in sedatives and soporifics (Gonzales 1977). Recent work has shown that seed oil obtained from L.

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sativa has pharmacological properties similar to those found in the latex of L. virosa (Said et al. 1996). They found sedative, analgesic, and anticonvulsive effects on mice. Another wild species, L. brevirostris, grows in South America, and produces large leaves that are dried and used in non-nicotine forming cigarettes. Each of these forms may become more useful with domestication through breeding and selection. G. Lettuce as a Research Organism Finally, we may consider the broad contribution of lettuce as a research organism to further knowledge of plant biology. Several species have been informally designated as model research organisms: maize, Arabidopsis, Drosophila, several bacteria, yeast, and the mouse, to name a few. Each of these has biological characteristics, and often historical and economic ones as well, which have generated widespread interest, activity, and generous research funding. Information and concepts derived from the research on these few organisms are often useful in understanding the biology of other species. However, systems for development, survival, reproduction, and other biological processes, despite commonality of origin, have evolved essentially independently in each well-defined species. There are certainly common genes across species and family lines. However, their operational modes may differ, possibly due to position effect, from species to species. This may explain why transgenes may not function in the genome of a new host as well as in the original one. Lettuce is a self-pollinated species, a member of the Asteraceae (Compositae), which is the largest family among the dicotyledons. It has a number of biologically interesting characteristics meriting further study. Single genes for early flowering and dwarfness have been identified (Ryder 1983, 1985, 1988; Waycott et al. 1995). Thus, there are forms that are relatively small and reproduce in 45–55 days. Under growth chamber conditions this cycle can be reduced by at least 10 days. Rapid cycling in restricted space is thus possible for the study of vegetative and reproductive growth and development. These may include the bases for rosette and head formation, the conversion from the vegetative stage to the reproductive stage, the development of the laticifer (latex forming) system, the physiology of flowering, and the formation of the potentially useful chemicals described earlier in this paper. The useful information derived from the study of model organisms must be applied and supplemented in the study of other organisms, which in turn requires support proportional to their biological, historical, and/or economic importance.

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This review discusses some of the history of lettuce breeding, describes some of the areas in which lettuce breeding has flourished, and points out some of the issues that may be important in the years ahead. It is not an attempt to predict the future, but rather to point out where we may be going and some of the promises and pitfalls that may await our arrival.

LITERATURE CITED Ackers, M. L., B. E. Mahon, E. Leahy, B. Goode, T. Damrow, P. S. Hayes, W. F. Bibb, D. H. Rice, T. J. Barrett, L. Hutwagner, P. M. Griffin, and L. Slutsker. 1998. An outbreak of Escherichia coli 0157:H7 infections associated with leaf lettuce consumption. J. Inf. Dis. 177:1588–1593. Anderson, P. A., P. A. Okubara, R. Arroyo-Garcia, B. C. Meyers, and R. W. Michelmore. 1996. Molecular analysis of irradiation-induced and spontaneous deletion mutants at a disease resistance locus in Lactuca sativa. Molec. Gen. Genet. 251:316–325. Babcock, E. B., G. L. Stebbins, Jr., and J. A. Jenkins. 1937. Chromosomes and phylogeny in some genera of the Crepidinae. Cytologia, Fujii Jubilee Volume. p. 188–210. Ballantyne, A., R. Stark, and J. D. Selman. 1988. Modified atmosphere packaging of shredded lettuce. Int. J. Food Sci. and Tech. 23:267–274. Bannerot, H., L. Boulidard, J. Marrou, and M. Duteil. 1969. Étude de l’hérédité de la tolérance au virus de la mosaïque de la laitue chez la variété Gallega de Invierno. Ann. Phytopath. 1:219–226. Beuchat, L. R. 1996. Pathogenic organisms associated with fresh produce. J. Food Prot. 59:204–216. Bohn, G. W., and T. W. Whitaker. 1951. Recently introduced varieties of head lettuce and methods used in their development. U.S. Dept. Agr., Washington, DC. Circ. 881. Bremer, A. H. and Grana, J. 1935. Genetische untersuchungen mit salat. II. Gartenbauwissenshaft 9:231–245. Brown, P. R., and R. W. Michelmore. 1988. The genetics of corky root resistance in lettuce. Phytopathology 78:1145–1150. Cao, G., E. Sofic, and R. L. Prior. 1996. Antioxidant capacity of tea and common vegetables. J. Agr. Food Chem. 44:3426–3431. Carson, R. 1962. Silent Spring. Houghton Mifflin, New York. Chupeau, M. C., C. Bellini, P. Guerche, B. Maisonneuve, G. Vastra, and Y. Chupeau. 1989. Transgenic plants of lettuce (Lactuca sativa) obtained through electroporation of protoplasts. Bio/Technology 7:503–508. Clark, M. F., and A. N. Adams. 1977. Characteristics of the microplate method of enzymelinked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34:475–483. Collier, G. F., and T. W. Tibbitts. 1982. Tipburn of lettuce. Hort. Rev. 4:49–65. Crute, I. R. 1984. The integrated use of genetic and chemical methods for control of lettuce downy mildew (Bremia lactucae Regel). Crop Prot. 3:223–241. Crute, I. R., and A. G. Johnson. 1976. The genetic relationship between races of Bremia lactucae and cultivars of Lactuca sativa. Ann. Appl. Biol. 83:125–137. Crute, I. R., and J. M. Norwood. 1981. The identification and characteristics of field resistance to lettuce downy mildew (Bremia lactucae Regel). Euphytica 30:707–717. Curtis, I. S., J. B. Power, N. W. Blackhall, A. M. M. de Laat, and M. R. Davey. 1994.

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Genotype-independent transformation of lettuce using Agrobacterium tumefaciens. J. Expt. Bot. 45:1441–1449. Davis, R. M., K. V. Subbarao, R. N. Raid, and E. A. Kurtz (eds.). 1997. Compendium of lettuce diseases. APS Press, St. Paul. de Miranda Santos, M., and R. C. Lewontin. 1997. Genetics, plant breeding and patents: conceptual contradictions and practical problems in protecting biological innovations. Plant Gen. Resour. Newsl. 112:1–8. Dinant, S., and H. Lot. 1992. Lettuce mosaic virus: A review. Plant Path. 41:528–542. Doerschug, M. R., and C. O. Miller. 1967. Chemical control of organ formation in Lactuca sativa explants. Am. J. Bot. 54:410–413. Durst, C. E. 1930. Inheritance in lettuce. Illinois Agr. Expt. Sta. Bul. 356. Eenink, A. H., R. Groenwold, and F. L. Dieleman. 1982. Resistance of lettuce (Lactuca) to the leaf aphid Nasonovia ribisnigri. 2. Inheritance of the resistance. Euphytica 31:301–304. Ellis, P. R., D. A. C. Pink, and A. D. Ramsey. 1994. Inheritance of resistance to lettuce root aphid in the lettuce cultivars ‘Avoncrisp’ and ‘Lakeland’. Ann. Appl. Biol. 124:141–151. Engler, D. E. and R. G. Grogan. 1984. Variation in lettuce plants regenerated from protoplasts. J. Hered. 75:426–430. Gilbertson, R. L. 1998. Evaluation of coat protein-mediated resistance for lettuce mosaic virus. Annu. Rep., California Lettuce Research Board. p. 71–74. Gonzales, A. G. 1977. Lactuceae-chemical review. p. 1081–1093. In: V. H. Heywood, J. B. Harborne, and B. L. Turner (eds.), The biology and chemistry of the Compositae. Academic Press, New York. Hamilton, N. D. 1993. Who owns dinner: Evolving legal mechanisms for ownership of plant genetic resources. Univ. Tulsa Law J. 28:589–657. Hulbert, S. H., T. W. Ilott, E. J. Legg, S. E. Lincoln, E. S. Lander, and R. W. Michelmore. 1988. Genetic analysis of the fungus, Bremia lactucae, using restriction fragment length polymorphism. Genetics 120:947–958. Hulbert, S. H., and R. W. Michelmore. 1988. DNA restriction fragment length polymorphism and somatic variation in the lettuce downy mildew fungus, Bremia lactucae. Mol. Plant-Microbe Interact. 1:17–24. Irwin, S. V., R. V. Kesseli, W. Waycott, E. J. Ryder, J. J. Cho, and R. W. Michelmore. 1999. Identification of PCR-based markers flanking the recessive LMV resistance gene mo1 in an intraspecific cross in lettuce. Genome 42:982–986. Jackson, L. E. 1998. Plant-soil relationships in lettuce. Annu. Rep., California Lettuce Research Board, 1997–98. p. 213–227. Jagger, I. C., and T. W. Whitaker. 1940. The inheritance of immunity from mildew (Bremia lactucae) in lettuce. Phytopathology 30:427–433. Jagger, I. C., T. W. Whitaker, J. J. Uselman, and W. M. Owen. 1941. The Imperial strains of lettuce. U.S. Dept. Agr., Circ. 596. Johnson, A. G., I. R. Crute, and P. L. Gordon. 1977. The genetics of race specific resistance in lettuce (Lactuca sativa) to downy mildew (Bremia lactucae). Ann. Appl. Biol. 86:87–103. Kesseli, R. V., and R. W. Michelmore. 1986. Genetic variation and phylogenies detected from isozyme markers in species of Lactuca. J. Hered. 77:324–331. Kesseli, R., O. Ochoa, and R. Michelmore. 1991. Variation in RFLP loci in Lactuca spp. and origin of cultivated lettuce (L. sativa). Genome 34:430–436. Kesseli, R. V., I. Paran, and R. W. Michelmore. 1994. Analysis of a detailed genetic linkage map of Lactuca sativa (lettuce) constructed from RFLP and RAPD markers. Genetics 136:1435–1446.

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Lewis, M. T. 1931. Inheritance of heading characteristics in lettuce varieties. Proc. Am. Soc. Hort. Sci. 27:347–351. Maisonneuve, B., V. Chovelon, and H. Lot. 1991. Inheritance of resistance to beet western yellows virus in Lactuca virosa L. HortScience 26:1543–1545. Meyers, B. C., D. B. Chin, K. A. Shen, S. Sivaramakrishnan, D. O. Lavelle, Z. Zhang, and R. W. Michelmore. 1998a. The major resistance gene cluster in lettuce is highly duplicated and spans several megabases. Plant Cell 10:1817–1832. Meyers, D. B., K. A. Shen, P. Rohani, B. S. Gaut, and R. W. Michelmore. 1998b. Receptorlike genes in the major resistance locus of lettuce are subject to divergent selection. Plant Cell 10:1833–1846. Michelmore, R. W. 1998. Genetic variation in lettuce. Annu. Rep., California Lettuce Research Board, 1997–98. p. 54–59. Michelmore, R. W., and D. S. Ingram. 1980. Heterothallism in Bremia lactucae. Trans. Brit. Mycol. Soc. 75:47–56. Michelmore, R. W., and D. S. Ingram. 1981. Recovery of progeny following sexual reproduction of Bremia lactucae. Trans. Brit. Mycol. Soc. 77:131–137. Michelmore, R. W., E. Marsh, S. Seely, and B. Landry. 1987. Transformation of lettuce mediated by Agrobacterium tumefaciens. Plant Cell Rep. 6:439–442. Norwood, J. M., A. G. Johnson, M. O’Brien, and I. R. Crute. 1985. The inheritance of field resistance to lettuce downy mildew (Bremia lactucae) in the cross ‘Avon Crisp’ × ‘Iceberg’. Z. Pflanzenzucht. 94:259–262. O’Brien, S. J. (ed.). 1990. Genetic maps. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Ochoa, O., B. Delp, and R. W. Michelmore. 1987. Resistance in Lactuca spp. to Microdochium panattoniana (lettuce anthracnose). Euphytica 36:609–614. Paran, I., and R. W. Michelmore. 1993. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor. App. Gen. 85:985–993. Paterson, A. H., S. D. Tanksley, and M. E. Sorrells. 1991. DNA markers in plant improvement. Adv. Agron. 46:39–90. Pink, D. A. C., D. G. A. Walkey, and S. J. McClement. 1991. Genetics of resistance to beet western yellows virus in lettuce. Plant Path. 40:542–545. Ramadan, A. A. S. 1976. Characteristics of prickly lettuce seed oil in relation to methods of extraction. Die Nahrung 20:579–583. Rappaport, L., and S. H. Wittwer. 1956. Flowering in head lettuce as influenced by seed vernalization, temperature, and photoperiod. Proc. Am. Soc. Hort. Sci. 67:429–437. Reinink, K. 1991. Genetics of nitrate content of lettuce, 1: Analysis of generation means. Euphytica 554:83–92. Robinson, R. W., J. D. McCreight, and E. J. Ryder. 1983. The genes of lettuce and closely related species. Plant Breed. Rev. 1:267–293. Rubatzky, V. E., and M. Yamaguchi. 1997. World vegetables. Principles, production, and nutritive values. 2nd ed. Chapman Hall. Ryder, E. J. 1970. Inheritance of resistance to common lettuce mosaic. J. Am. Soc. Hort. Sci. 95:378–379. Ryder, E. J. 1979. ‘Vanguard 75’ lettuce. HortScience 14:284–286. Ryder, E. J. 1983. Inheritance, linkage, and gene interaction studies in lettuce. J. Am. Soc. Hort. Sci. 108:981–985. Ryder, E. J. 1985. Use of early flowering genes to reduce generation time in backcrossing, with specific application to lettuce breeding. J. Am. Soc. Hort. Sci. 110:570–573. Ryder, E. J. 1988. Early flowering time in lettuce as influenced by a second flowering time gene and seasonal variation. J. Am. Soc. Hort. Sci. 113:456–460.

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Ryder, E. J. 1996. Inheritance of a mild reaction to lettuce mosaic. HortScience 31:612. Ryder, E. J. 1999. Lettuce, endive and chicory. CABI Publ., Wallingford, UK. Ryder, E. J., and B. J. Robinson. 1995. Big-vein resistance in lettuce: identifying, selecting, and testing resistant cultivars and breeding lines. J. Am. Soc. Hort. Sci. 120:741–746. Said, S. A., H. A. El Kashef, M. M. El Mazar, and O. Salama. 1996. Phytochemical and pharmacological studies on Lactuca sativa seed oil. Fitoterapia 67:215–219. Sequeira, L. 1970. Resistance to corky root rot in lettuce. Plant Dis. Rptr. 54:754–758. Stevens, M. A. 1974. Varietal influence on nutritional value. p. 87–109. In: P. L. White and N. Selvey (eds.), Nutritional qualities of fresh fruits and vegetables. Futura Publ., Mt. Kisco, NY. Thompson, R. C., and E. J. Ryder. 1961. Descriptions and pedigrees of nine varieties of lettuce. U.S. Dept. Agr., Washington, DC. Tech. Bul. 1244. Thompson, R. C., T. W. Whitaker, and W. F. Kosar. 1941. Interspecific genetic relationships in Lactuca. J. Agr. Res. 63:91–107. Torres, A. C., D. J. Cantliffe, B. Laughner, B. Bieniek, R. Nagata, M. Ashraf, and R. J. Ferl. 1993. Stable transformation of lettuce cultivar South Bay from cotyledon explants. Plant Cell, Tissue, Organ Culture 34:279–285. Torres, A. C., R. T. Nagata, R. J. Ferl, T. A. Bewick, and D. J. Cantliffe. 1999. In vitro assay selection of glyphosate resistance in lettuce. J. Am. Soc. Hort. Sci. 124:86–89. Van der Vlugt, R. A. A., M. Berendsen, and H. Koenraadt. 1997. Immunocapture reverse transcriptase PCR for the detection of lettuce mosaic virus. p. 185–191. In: J. D. Hutchins and J. C. Reeves (eds.), Seed health testing. CAB Int., Wallingford, UK. Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. van de Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res. 23:4407–4414. Waycott, W., and S. B. Fort. 1994. Differentiation of nearly identical germplasm accessions by a combination of molecular and morphologic analyses. Genome 37:577–583. Waycott, W., S. B. Fort, and E. J. Ryder. 1995. Inheritance of dwarfing genes in Lactuca sativa L. J. Hered. 86:39–44. Whitaker, T. W., and D. E. Pryor. 1941. The inheritance of resistance to powdery mildew (Erysiphae cichoracearum) in lettuce. Phytopathology 31:534–540. Williams, J. G. K., A. R. Kubelik, K. J. Livak, A. Rafalsky, and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531–6535. Witsenboer, H., J. Vogel, and R. W. Michelmore. 1997. Identification, gene localization, and satellite diversity of selectively amplified microsatellite polymorphic loci in lettuce and wild relatives (Lactuca spp.) Genome 40:923–936. Wright, B. D. 1998. Public germplasm development at a crossroads: Biotechnology and intellectual property. Calif. Agr. 52(6):8–13. Zohary, D. 1991. The wild genetic resources of cultivated lettuce (Lactuca sativa L.). Euphytica 53:31–35.

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5 Cactus Pear Domestication and Breeding Candelario Mondragon Jacobo Instituto Nacional de Investigaciones Forestales y Agropecuarias, A. P. 25. San Jose Iturbide, Gto. Mexico

I. INTRODUCTION II. ORIGIN AND EARLY DEVELOPMENT A. Domestication B. Breeding Achievements III. GENETIC RESOURCES A. The Cultivated Gene Pool B. Germplasm for Vegetable Production C. The Semi-domesticated Gene Pool D. The Wild Gene Pool IV. BREEDING OBJECTIVES A. Fruit Size and Seed Content B. Fruit Color C. Cladode Spininess and Glochids in Fruits D. Out-of-season Production E. Sugar and Acid Levels F. Flesh Juiciness G. Cold Tolerance H. Disease and Pest Resistance I. Resistance to Handling and Packing J. Vegetable Production V. BREEDING TECHNIQUES A. Propagation B. Floral Biology C. Pollen Collection, Storage, and Testing D. Emasculation and Controlled Pollination E. Seed Extraction and Germination F. Handling of Seedlings G. Reduction of Juvenility

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VI. BREEDING SYSTEMS A. Inbreeding and Outbreeding B. Polyploidy C. Apomixis D. Biotechnology 1. Tissue Culture 2. Transformation VII. SUMMARY AND FUTURE PROSPECTS LITERATURE CITED

I. INTRODUCTION Cactus pear, formerly prickly pear, (Opuntia sp. Mill.) is a dicotyledonous plant of the cactus family (Cactaceae) native to Central Mexico and the Caribbean region. It is extensively used as food for humans and animals. Although wild populations are still exploited, the plant has been and is being domesticated as an important fruit crop in many areas of the world. There are more than 300 species of the genus Opuntia (Scheinvar 1995) distributed across the American continent but only three species, Opuntia albicarpa sp. Novo, O. ficus-indica (L.) Miller, and O. robusta Wendl var. larreyi (Weber), are important for horticultural purposes. Cactus pear is a perennial, characterized by a jointed flattened stem, each piece termed a cladode. The tiny succulent leaves, cylindrical or conical, are ephemeral and present only in young stems. Areoles (the equivalent of a complex bud with glochids [bristles] and spines) are arranged in clusters and distributed in a helicoidal fashion on the cladodes. Fruits also contain areoles and have thick rinds and comparatively large seeds covered by hard, bony, light-colored arils (Weniger 1984). The morphology of the cactus pear plant is described in Fig. 5.1. In Mexico all the platyopuntias (cacti with flattened stems) are known by the popular name of nopales; the tender pads are called nopalitos and the fruits are called tunas. Another edible cactus is Opuntia cochellinifera (Scheinvar 1995) syn. Nopalea cochellinifera Britton & Rose. The young cladodes of this plant are used as a vegetable in Northern Mexico because they are almost devoid of glochids and spines. The mature cladodes are elongated with a thick cuticle. The floral structure is unique, possessing a pink corolla that does not open at anthesis, and an exerted stigma and stamens. Several cacti species besides cactus pear bear edible fruits, and they are being slowly incorporated into commercial cultivation. Pitaya (Stenocereus sp.) is a columnar cactus that thrives in dry tropical areas

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Stigma Stamens Perianth

Ovules Ovary Glochid

Receptacle

A Areoles

Normal seeds

Aborted seeds

B Fig. 5.1.

Cactus pear morphology: (A) Pad and flower; (B) Cross section of fruits.

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with more restrictive growing conditions than those dedicated to cactus pear. Pitahaya (Hylocereus sp.), called dragon fruit in Vietnam and Eden fruit in Israel, is now cultivated in Latin America and some Asian countries, while an outdoors grown columnar cactus (Cereus peruvianus) is marketed as Koubo and shows great market potential in Israel (Mizrahi et al. 1997). Cactus pear is gaining importance as an alternative crop for the tropical semiarid regions of the world. This paper reviews the background, the domestication, and breeding of cactus pear for fruit and vegetable production.

II. ORIGIN AND EARLY DEVELOPMENT A. Domestication Cactus pear was domesticated in the highlands of Central Mexico. Reports of usage of cactus pear fruits and tender pads date back to the ancient groups that inhabited Mesoamerica. Evidence from coprolites, fossilized human feces, indicates that agaves and cacti have been part of the human diet for over 9,000 years (Nobel 1994). Hoffman (1995) reported that gathering activity of fresh as well as dried fruits, and early domestication of wild plants could have taken place before 6000 B.C. A crucial step was the transition from a wild gathered plant to that of the cultivated crop. While some traditionally gathered species [e.g. pitaya Steneocereus spp. and pitahaya Hylocereus triangularis (L.) Britt & Ross] are only now being cultivated, cactus pear has been grown for thousands of years. These cacti, together with maize, beans, and agave, are among the oldest cultivated plants in Mexico. The cactus pear along with the eagle and snake is the symbol of Mexico. Cactus pear plants were brought to Europe by the Spanish conquistadores between the 15th and the beginning of the 16th century. They were attracted by the unusual morphology and the importance that the Aztecs attached to the cactus pear in their economic, social, and religious life (Barbera et al. 1992). The plants spread along the Mediterranean coast as birds ate the fruits and distributed the seeds. They were carried to North Africa from Spain when the Moors were driven from Spain. They were also taken aboard ships to combat scurvy, because the cladodes easily tolerate long journeys (Barbera 1995). Cactus pear moved as far south as Australia. In the 17th century a governor of that country envisioned that the production of natural red dyes from the cochineal insect (Dactilopius coccus Costa) would be a suitable

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industry for the Australian economy. Cacti and insects were brought from Rio de Janeiro and carried to Australia. Unfortunately, the cactus became weedy, invading and overwhelming the native vegetation, but the cochineal insect did not, and the industry was a failure. Cactus pear became one of the worst pests of the country, dominating large areas of grazing land (Baker 1970). The pest was ultimately controlled by the introduction from Mexico of cactus parasites as a method of biological control (Dodd 1940). Cactus pear evolved in Mexico into a formal crop in the current century. Outstanding wild plants were collected and ultimately a few individuals were selected by farmers. Family gardens provided the germplasm base needed for the expansion of the crop. The transition of the rural population to an urban semi-industrial society provided the consumer base for the initial marketing of cactus pear in the 1960s. During the 1970s through the 1980s the Mexican government subsidized the planting of cactus pear orchards in semiarid regions with a history of poor yields of maize and dry beans, the traditional annual crops. The ability of cactus pear to tolerate poor soil conditions was exploited in governmental drought relief programs. These programs provided an important boost to the development of commercial cactus pear orchards. Wild populations of cactus pear are scattered across 2 million ha of Mexico, mostly in the central and northern region. Stands located close to important cities are subject to intensive use for forage. The plants are cut, cleaned of thorns by means of oil and gas burners, and fed to cattle in small suburban dairy operations. Other countries such as Tunisia and Eritrea, having naturalized wild populations of cactus pear, are becoming aware of the importance of their natural stands of cactus pear. Mexico has great potential for fruit production because its topography allows the presence of multiple microclimates suitable for the cultivation of a wide variety of temperate and tropical fruit. Tropical fruits, which thrive in less restrictive weather and soil conditions, occupy 94.3% of the area devoted to fruit crops. Cactus pear is the only fruit crop grown extensively without irrigation. Official statistics report that cactus pear orchards cover 57,000 ha. The gross volume of fresh fruit reaching the market every year has been estimated to be from 210,000 to 330,000 t (SARH 1993) and annual per capita consumption is 3.7 kg, one sixth that of bananas (Moreno and Flores 1996). The average size of the orchard for fruit production is about 20 ha, whereas the size of orchards planted for vegetable production is less than 1 ha. Overall this crop provides income to more than 10,000 small growers across Mexico. Production of tender pads for use as a vegetable is a highly specialized intensive agricultural system that deserves a separate discussion.

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Mexico and Italy are the main producing countries and consumers of cactus fruits. Italy is reported to have close to 2,500 ha of specialized orchards in its San Cono area and it is the sole provider of fresh fruit to the European market (Barbera 1995). Chile, South Africa, and Israel have also started to plant commercial orchards and consume cactus pear in limited volumes. In the United States, California has the largest commercial orchard, with about 300 ha, providing fruits for the chain markets and fruit puree for the restaurant industry. More success has been realized in growing cactus pear as a vegetable and Texas has an aggressive campaign to promote this product. B. Breeding Achievements Widespread interest in cactus pear improvement has occurred only in the 1990s. It is considered among the recent additions to “new” tropical and subtropical crops. Selection of outstanding cultivars by growers is an accomplishment that has led to the present industry. Fruit quality, productivity, drought and frost tolerance were the main selection criteria. Six to eight commercial selections are the basic stock for the Mexican and Italian industry. Hybridization in cactus pears was first claimed by Luther Burbank at the beginning of the century, which led to the development of the so called “spineless” cactus. Burbank saw it as having immense potential for cattle forage in desert areas. Several cultivars were developed and Burbank aggressively marketed five of them (Dreyer 1985). They were said to be the product of extensive crossing and selection among accessions shipped from Mexico, South Africa, and other countries. Today, four of the aforementioned cultivars still remain in the South African collection. Formal breeding was initiated in Mexico in the 1960s. Back then the Universidad Autonoma Agraria Antonio Narro initiated research selecting for cold hardy Opuntias (Martinez 1968; Borrego et al. 1990). In the 1970s the late Dr. F. Barrientos of the Colegio de Postgraduados de Chapingo pioneered the first hybridizations of cactus pear. Using a limited stock of white fleshed selections from the central region of Mexico, he developed several cultivars, the COPENA series, which had only modest success among growers. The most likely reason is that they did not present a clear advantage over the native white pulp cultivars. These efforts in selection and genetic improvement were unsustained. A fresh start at breeding is underway in the United States, South Africa, Israel, Italy, and Mexico, based on the utilization of locally available plant material. A renewed interest, encouraged by the Food and Agriculture

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Organization of the United Nations, has resulted in collection of wild and semi-domesticated accessions, publication of information on crop management practices, and development of new uses of cactus pear (Barbera et al. 1995) D’Arrigo Brothers, a produce company based in California, has been supporting a private breeding program to improve fruit quality of their spineless commercial cultivar ‘Andy Boy’ (a cultivar similar to ‘Rossa’, which is grown and marketed in Italy), which dominates the American “out-of-season” market. They also use some germplasm obtained from commercial orchards in Mexico. Texas A&M University–Kingsville (TAMUK) has been involved since 1982 in collection and introduction of cactus pear to the United States as well as agronomic research and extension. The program aims to develop freeze tolerant cultivars, to alleviate a common problem in this region (Wang et al. 1997). In 1996 the first round of crosses marked the beginning of a long-term breeding program. In 1998 these genetic materials were transferred to the Universidad Nacional Santiago del Estero in Argentina, where the work continued vigorously. TAMUK is also responsible for the popularization of the vegetable cactus pear and cactus pear products in Texas. ‘Spineless 1308’ (an accession originally collected in the humid tropical region of Tamazunchale, Mexico) has been extremely successful among growers and consumers. Another active breeding program is located in Sassari, Italy, involved in improving fruit quality as well as selection of other opuntias with potential ornamental value. This program relies on naturalized accessions collected in the semiarid Mediterranean region of Italy.

III. GENETIC RESOURCES A. The Cultivated Gene Pool Three main groups can be recognized according to the color of peel and pulp: light green or “white,” yellow, and red (which includes an array of colors ranging from light red to deep purple). The most important cultivars in Mexico have been described by Mondragon and Perez (1993), Pimienta and Muñoz (1995), and Mondragon and Perez (1997). The cultivar names reflect some specific peel or flesh features and, in some cases, the response to stress and resistance to handling. The most popular white cultivars are ‘Reyna’, ‘Cristalina’, ‘Esmeralda’, and ‘Burrona’. ‘Reyna’ sets the standard of quality and dominates the national market (Mondragon and Perez 1993). ‘Naranjona’ and ‘Amarilla Montesa’ with

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orange pulp and the red-purple ‘Roja Lisa’ are less important. There are many lesser-known accessions available in regional markets, but they do not affect the market trends due to their low volumes. The cultivars available in the Sicily area of Italy are ‘Gialla’ (yellow), ‘Bianca’ (white), and ‘Rossa’ (red). ‘Gialla’ , the most abundant and productive cultivar, is easy to handle, and is well liked by consumers (Barbera et al. 1992). A “seedless” cultivar is known but its commercial cultivation has never been attempted because of the poor fruit quality. In Chile, ‘Verde’ (green) is the most common cultivar of O. ficusindica (Sudzuki et al. 1993). It is consumed in the central dry areas of the country. Exports to the U.S. market have been reported since 1982; they take advantage of the reversal of seasons. Production in Israel is based mostly on the cultivar known as ‘Ofer’, which has yellow pulp. In the United States, ‘Andy Boy’, a red cultivar, is marketed and available in chain stores from November until April. It is produced only in California, where the availability of irrigation and the presence of mild winters allow for off-season production. In South Africa the cultivars available originated from the introduction of 21 spineless types imported from the Burbank nursery of California in 1914. All known types currently grown were developed from the original material, either as clones or as artificial or natural hybrids; 40% of these selections are of light green color. The cultivars were classified into three groups based on climatic requirements: five types for hot, frost-free areas; one for intermediate climatic areas; and two types for areas with a cold winter. Mexico has three reliable germplasm banks containing from 50 to 300 accessions. South Africa reports 22 outstanding accessions, and Italy and Israel have a few local types that are also represented in other collections. Due to the perennial habit of this plant, maintenance of germplasm banks in situ is costly, complicating the evaluation and utilization of the genetic resources. Basic descriptions of the most important cultivars and outstanding accessions are available from Mexico (Pimienta and Muñoz 1995; Mondragon and Perez 1997; Parish and Felker 1997), Italy, South Africa, Argentina, and Chile (Pimienta and Muñoz 1995). An analysis of morphological traits and the relationships among a set of 11 Mexican cultivars have been published by Valdes et al. (1997a, 1997b). A description of the morphological features of the most important commercial cultivars as well as some outstanding entries in Central Mexico was reported by Mondragon (1999). Chessa et al. (1997) analyzed germplasm variability in 32 Italian accessions using 13 enzymatic systems in four types of tissue; root, cladode,

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petal, and pollen. They were able to separate the entries into several groups based on biotypes, cultivation location, and species, but there was no correlation among fruit colors and isozyme patterns. Pollen had the highest enzymatic activity and the most active enzymes were malate dehydrogenase, phosphoglucoisomerase, and phosphoglucomutase. A similar study carried out by Uzun (1997) with Turkish entries was unable to differentiate among ecotypes and cultivated accessions. These conclusions suggest that the results have limited applicability, indicating low variability among the accessions studied. The difficulty involved in the separation of cultivars is probably because they are closely related. Mondragon (1999) conducted a study with 32 accessions of Mexican entries including commercial cultivars and some outstanding entries by means of the Random Amplified Polymorphic DNA (RAPD) technique. The cultivated and outstanding entries share a coefficient of relatedness above 0.8, which indicates that a small number of parents are involved. B. Germplasm for Vegetable Production Most of the wild species, and to a lesser extent the domesticated ones, possess thorns to protect the vulnerable succulent new shoot, and it is surprising that this organ was utilized for human consumption. Collection and use of tender cladodes from cactus pear in Central Mexico might have started as a response to the scarcity of drinking water and as a resource to substitute for the lack of game for hunting or fruits for collection. The new shoots are developed early in the season, as soon the frosts are over, which is regularly the dry season in these latitudes. They are sustained on moisture stored in the older pads, so in a way they are not dependent on the soil moisture. In the semiarid regions the shoots cover a seasonal gap in which no other source of “greens” is available. Consequently they are a valuable food source for animals as well as humans. Consumption of nopalitos or tender pads is almost exclusive to Mexico. Mexican immigrants in the United States consume limited volumes. Efforts to popularize this produce are underway in Texas and California in the United States, as well as in Argentina and Chile. The main source of germplasm for vegetable purposes is in Mexico. The Universidad Autonoma de Chapingo near Mexico City has assembled an extensive collection of Mexican accessions of cactus pear for vegetable production. ‘Milpa Alta’ (O. ficus-indica), the country’s most important cultivar, is cultivated mostly in the region of the same name located in the suburbs of Mexico City. The indigenous production system is based

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on heavy doses of fresh manure and continuous pruning, ensuring annual yields up to 60 t/ha of fresh pads, a productivity level impossible to obtain with any other crop under the poor soil conditions prevailing in the area. ‘Copena V1’ is the second vegetable cultivar in importance. A third selection , ‘Copena F1’, even though it was selected for fodder production, produces tender pads also suited to consumption as a vegetable. Both cultivars were selected by the late Dr. F. Barrientos in the 1970s. Both have intense green color, thin epidermis, good flavor, and low acidity (Flores 1995). Plantings of these cultivars are widespread across the central highlands of Mexico and are increasing near the border with the United States. ‘Moradilla’, ‘Atlixco’, ‘Polotitlan’ and ‘Redonda’ are examples of locally selected cultivars of O. ficus-indica. The ‘Nopal Blanco’, or white cactus pear, is the cultivar of choice to plant in mixed stands using the empty space in young avocado orchards in the southwestern states of Michoacan and Jalisco. This local selection tolerates humid conditions (up to 1600 mm of annual rainfall). Another extraordinary example of utilization of cactus pear as a vegetable is the locally known ‘Valtierrilla’ in central Guanajuato, which has the largest thorns and densest glochids of all cultivars. The pads ranging in size from 10–15 cm long are useful only if they are picked very tender. They are collected using either gloves or tongs. Texas A&M University–Kingsville has been promoting the cultivar ‘Spineless 1308’ (O. cochellinifera, Syn. Nopalea cochellinifera) in the United States as suitable for field cultivation in the coastal rainfed area of Texas. It is also used for greenhouse cultivation in frost prone areas near San Antonio (Mick 1991). This cultivar was selected by P. Felker from accessions collected in the humid lowlands of the Gulf of Mexico, where it is currently present in wild stands, family gardens, and small commercial plantations. Plants are bushy and usually more prolific than O. ficus-indica and have a delicate taste. In the past five years the presence of peeled and diced tender pads obtained from this cultivar have become a common sight in the convenience stores of Texas. Nerd et al. (1997) successfully cultivated this cultivar in Israel. C. The Semi-domesticated Gene Pool A wide variability of locally known types exist in Mexico in the native areas of cactus pear, some serving two or even three purposes, producing edible fruits, tender pads, and fodder according to the season. Traditionally, cactus pear plantings are found on small family properties in dry regions. Opuntia hedges are concentrated close to farmsteads and also carry out the function of protecting families’ fruit and vegetable

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gardens. These hedges represent the transition from wild plants to modern commercial cultivars. They can be considered as miniature gene banks (Hoffman 1995). The concentration of outstanding accessions in mixed populations in family orchards has accounted for an interesting source of new genotypes derived from natural outcrossings. At present this source of germplasm has been only partially explored. The characterization of new entries in the few germplasm banks existing in Mexico is incomplete and therefore not easily available for exchange among breeders. D. The Wild Gene Pool The last reservoir of interesting germplasm are wild populations. Several efforts have been made to collect representatives from them. However, collecting plant material by relying only on common names often leads to duplicated accessions. The likelihood of replicated clones is high in a plant like cactus pear, which has only subtle differences even to the expert’s eye, and where apomixis is prevalent. Opuntias are also highly plastic plants that react to differences in their environments more quickly and with more drastic growth-form changes than do other cacti. Cladode spininess, shape and size of cladodes and fruits are a few examples (Weniger 1984). Recently Mexico, Israel, and the United States joined collection efforts in the highlands of Northern Mexico, assembling 130 accessions selected primarily for cold hardiness (Felker 1995). Limited exchange has taken place since 1992, when the international cooperation formally began. More flexible exchange of germplasm among countries is restricted due to bureaucratic regulations and the lack of an accessible database.

IV. BREEDING OBJECTIVES A. Fruit Size and Seed Content The flesh of the fruit of cactus pear consists of tightly packed round pieces with the seeds acting as their core. The enlarged portion of the fruit corresponds to the funiculus. Botanically the fruit is recognized as a false berry (Sudzuki 1995). Partial seed set is common in cactus pear. Unbalanced gametes as a result of polyploidy probably lead to the partial seed set observed in O. ficus-indica (Nerd and Mizrahi 1994). Still, every seed has the capacity to form pulp even if undeveloped. Seeds are normally swallowed along with the pulp, a well-known fact in the native places of cactus pear, but a considerable barrier to attract new consumers.

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The seed count ranges from 80 to more than 300 per fruit (Barbera et al. 1991; Pimienta 1990; Mondragon and Perez 1997; Parish and Felker 1997), which means there are 2.8 to 7.5 g of seeds per fruit depending on the fruit size and the cultivar (Mondragon and Perez 1997). Lower seed weight (2.2 to 6.4 g) was reported by Parish and Felker (1997) for Chilean and Mexican clones grown at Kingsville, Texas. The ratio between aborted and normal seeds is higher in the Italian (0.44) than in the Mexican cultivars (0.11) (Barbera et al. 1994; Pimienta and Mauricio 1987). Seed content is positively correlated with the fruit size. The ideal fruit should have a large number of seeds to attain good size, with a high ratio of aborted to normal seeds. According to Mondragon (1999), early selection for large fruits is feasible due to the correlation found between cladode vigor and fruit size. In Mexican entries, the most vigorous accessions had the larger fruits. Plant vigor can be defined during the second or third year of growth, improving the efficiency of breeding programs. Fruit size and the fraction of aborted seeds are traits that should be examined together. Large fruits obtain premium prices on the market and low seed content is regarded as a quality trait because a high percentage of aborted seeds makes the fruit more palatable. Natural parthenocarpy has been mentioned as a solution to this problem. Parthenocarpy was reported in ‘BS1’ , a yellow-fleshed accession native to Israel. This O. ficus-indica clone bears fruits containing only degenerated seeds, but the fruits are similar in size and color to other yellow fruits of O. ficusindica cultivars. A study conducted to elucidate the mechanism involved in the development of this seedless fruit indicated that ‘BS1’ does not require pollination for fruit set and development; however, its overall quality was unacceptable (Weiss et al. 1993; Nerd and Mizrahi 1994). The seed content reported for this cultivar was 2.09 g when cultivated in fall and 0.9 g if harvested in spring, which indicates a significant environmental effect on this trait. The values reported for the fruits harvested in fall were similar to those observed with several fruit cultivars from Chile and Mexico grown in Texas by Parish and Felker (1997). There is a wide variability for fruit size and seedlessness in the available germplasm, but the interactions among environmental and crop management factors that influence these traits have not been elucidated. Differences in fruit size are more evident in Mexican accessions, which in general are larger than the South African or Italian accessions (Inglese 1995). Average fruit size of commercial cultivars in Mexico ranges from 67 to 216 g (Mondragon and Perez 1997). Several attempts have been made to reduce seed size by means of gibberellin application, but results are unsatisfactory (Gil and Espinoza

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1980; Aguilar 1987; Ortiz 1988). Selection of small-seeded genotypes within the available germplasm can provide limited results on a shortterm basis and the evaluation of crosses between the accessions having the desired traits are a long-term goal. To reach a wider market, it is imperative to develop new cultivars with lower seed content and large fruit. The genetic pool available ensures that it is an achievable goal. B. Fruit Color National markets show specific preferences for fruit color. The favored fruits on the international market have red-purple and yellow-orange peel and flesh (Inglese et al. 1993). In Mexico white (green) cultivars are predominant, while in Italy and Northern Europe yellow-orange cultivars are preferred. In the United States, the fruit colors that are available depend on the season and the exporting country. In summer white cultivars from Mexico are available, but the availability of red fruits produced off-season in California is influencing consumer preference. C. Cladode Spininess and Glochids in Fruits Spines and glochids are modified leaves that allow cactus pear to withstand drought. Thus, drought-hardy accessions are typically spiny. There are different degrees of spininess in cactus pear. Spine density and size varies among accessions, but spines are present even in the socalled spineless entries, though in reduced numbers and size. There are no reports of spineless individuals in wild populations, leading to the assumption that this trait was acquired through domestication. Another possibility is that the spineless individuals in the wild are lost due to predation. In regard to crop management, spines represent an inconvenience because they make routine operations such as pruning difficult and are an obstacle at harvest. Commercial cultivars for fruit production in Mexico are all spiny (with the exception of ‘Roja Lisa’). All other producing countries rely on spineless cultivars. Efforts to develop new cultivars should be focused on spineless cultivars. The presence of glochids or spicules in the fruit is a constraint to increased consumption. Glochids can be removed after harvest, but techniques must be improved to accomplish effective removal. Eventually, selection and breeding for glochid-free cultivars should be encouraged (Barbera 1995). Another possibility is the induction of early shedding of glochids. Genes for a low number of areoles and short glochids are present in O. robusta, a species that has the earliest ripening fruit of the season (available in May in the Northern Hemisphere).

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However, it is not very well accepted due to its low sugar content, bland flavor, and low keeping quality. D. Out-of-season Production Because of the market trend for fresh fruits, early cultivars command a premium price. For the Northern Hemisphere any fruit that ripens in May or June can generally be marketed easily. In the Southern Hemisphere, harvest season is less restricted because it is intended for export to the northern markets in winter. Existing germplasm is adapted to ripen from the end of June to September in the Northern Hemisphere, and the most preferred types ripen in July and August. The capacity of some cultivars of cactus pear to bear a second flush of flowers after removal of the first one is the basis for out-of-season production of ‘Gialla’ in the south of Italy (Barbera et al. 1991). Off-season production of cactus pear fruits gives a marketing advantage because of reduced competition with other well-established summer fruits. The same approach was attempted in Mexico with ‘Cristalina’ and ‘Reyna’, which presented a strong tendency toward vegetative growth instead of new fruits (Mondragon et al. 1995; Fernandez 1997). Manipulation through fertilization, irrigation, and use of plastic covers has been reported to modify harvest season in Israel (Barbera and Inglese 1993; Nerd et al. 1989). The breeding of new cultivars with the ability to produce out-of-season fruit would be an advantageous trait. E. Sugar and Acid Levels High soluble solids is a very desirable trait for fresh markets and is an important trait for some value-added products, such as jams, candies, and pulp purees. Commercial cultivars range from 13 to 17° Brix. This trait is highly influenced by environment and crop management. Fruit produced in dry years or dry locations are sweeter than those produced in humid ones. In out-of-season production, late fruits growing during cool, cloudy days have lower soluble solids than those produced during hot and sunny days (Mondragon et al. 1995). Fruits with higher acid content were favored in sensory tests in some markets (Saenz and Costell 1990). F. Flesh Juiciness Fruits with juicy pulp are favored over those with dry pulp. Juicy fruits could also be advantageous for manufacturing value-added products

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such as carbonated beverages and canned juices. All combinations of juiciness and flesh color are found in germplasm. G. Cold Tolerance The most important cactus pear cultivars are generally irreversibly injured at temperatures of –5 to –10°C (Russell and Felker 1987; Nobel and Loik 1993). Certain wild Opuntia species such as O. fragilis (Nutt.) Haworth and O. humifusa (Rafinesque), both native to Canada, can tolerate temperatures below –20°C when properly acclimated (Nobel and Loik 1993). Cold tolerance is an important feature for cactus pear production (fruit as well as forage) in the southern United States, where freezing temperatures occur occasionally (Parish and Felker 1997). Susceptibility to freezing is the primary factor limiting the expansion of prickly pear as fodder and forage in cattle-producing areas of the United States. Russell and Felker (1987) reported that O. ellisiana in Texas endured a –9°C without apparent damage; meanwhile, fruit and fodder accessions from Mexico, Chile, Brazil, and South Africa presented different degrees of frost damage. The South African spineless fodder cultivars were the least affected (13% above ground). Early in this century Uphof (1916) reported that species of cacti having relatively thick integuments (cuticle, epidermis, crystal-bearing layers, and several layers of thick walled cells) were more resistant to low temperature than those with thinner integuments. According to Goldstein and Nobel (1991), reduced water content and accumulation of organic solutes and mucilage may be partially responsible for cold acclimation. A key issue in cold hardiness is the length of the onset period of freezing temperatures. In Opuntia the lack of freeze hardiness is probably not due to the lack of tolerance to cold temperatures per se, but the range of day to night temperatures, which may occur from 28°C down to –12°C in a single day in Texas (Gregory et al. 1993). This does not allow the plant to attain proper acclimation and express cold tolerance. Hybridization of cold-tolerant native species and highly productive, but cold sensitive commercial species, should be a major objective of breeding programs to expand the cultivation of cacti (Gregory et al. 1993; Mizrahi et al. 1997). Borrego et al. (1990) reported that selection for cold hardy genotypes was initiated in the Universidad Antonio Narro in northern Mexico by Dr. Lorenzo Medina in 1963, taking advantage of an unusual –16°C frost event. The best 31 individuals were selected along with outstanding fruit and vegetable regional genotypes that were

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obtained from backyards and that also had survived the frost. Some of these clones were later introduced to southern Texas by P. Felker. Cowan and Felker (1998) found several promising clones in their experimental orchard at Kingsville, Texas, an area with recurrent frost and low temperatures of about –12°C. The clone 1436 obtained from Saltillo, Mexico was found to have high yield and good fruit quality. Two other clones, 1452 and 1458, collected in northern Mexico from areas in the highlands exposed to late frosts and light snow cover, are also promising. These findings indicate the existence of genes for cold tolerance and the possibility of cactus pear cultivation in colder regions. H. Disease and Pest Resistance Germplasm needs to be systematically screened in search of tolerance or resistance to black soft rot incited by Erwinia carotovora (Fucikovsky and Luna 1988), as well as “excessive budding” and “cladode swelling,” a condition of unknown cause but for which a mycoplasm involvement has been suspected (Pimienta 1974; Gutierrez 1992). A number of insect pests attack cactus pear. Insects like wild cochineal (Dactilopius spp.), a relative of the beneficial domesticated cochineal Dactilopius coccus Costa that produces a valuable red dye is increasing its presence in the cultivated orchards of Mexico. Differential infestation has been observed in germplasm, revealing the possibility of genetic tolerance. This insect is resistant to most common pesticides and biological control has been suggested. Other pests difficult to control are those that thrive inside the cladodes, affecting the inner succulent tissues. Tolerance to thrips (Rhopalothrips bicolor) reported in California by Curtis (1977) and Sericothrips opuntiae Hood present in northern Mexico (Borrego and Burgos 1986) and cladode bugs (Chelinidae tabulata) should also be considered when selecting new cultivars. I. Resistance to Handling and Packing Some cultivars, such as ‘Cristalina’ and ‘Burrona’, can be handled better than others. Peel thickness and peel toughness are involved in improved resistance to handling. These two cultivars can also tolerate up to two months in cold storage at 5°C (Corrales et al. 1997). Oval or barrel-shaped fruits are easier to harvest than elongated fruits and therefore they suffer less harvest damage to the stem-end (Cantwell 1991). Higher resistance of the peel to handling, especially on the fruit base, is

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desirable to reduce damage (Wessels 1988). This is extremely important because cactus pear is harvested by hand and some twisting of the fruit results in tears of the peel. J. Vegetable Production The “nopalitos” are tender cladodes or stem pieces that are collected when they are 20–30 days old, depending on the growing conditions; they are consumed fresh or cooked (Rodriguez and Cantwell 1988). The presence of thorns and glochids in nopalitos is one the main constraints for the development of a wider market outside Mexico (Nerd et al. 1997). Handling of peeled whole pads is difficult and shelf life is reduced. Costs associated with hand peeling are high. Attempts at mechanical peeling are underway. A permanent solution to this problem is the development of spineless cultivars. The currently used commercial cultivars are spineless, but the new cladodes develop glochids and spines in the first two to three months. As the pad matures, almost all the glochids and spines are shed, resembling the original cladode. However, at this stage the pad is no longer acceptable for the market. Screening for low number of glochids on the faces of the pads, as well as short, soft glochids is possible because they are expressed in the juvenile stage. The ability to shed the glochids before the pad increases the fiber content and hardens could be a selection criteria when breeding for new vegetable cultivars. Another useful selection criteria is the position of the podarius, the structure that holds the true leaves. Upright shaped podaria facilitate mechanical peeling. Cultivars with large (up to 30 cm long) elliptical and thin (0.5 to 1.0 cm) pads dominate the market for cultivated cactus pear, but the small round pads of O. robusta species obtained from wild stands are available on a small scale in local markets. Currently the most common way of handling and packing nopalitos in Mexico is in large cylindrical bales, 250 to 300 kg each (Flores 1995). This fact, as well as the market preference, explains the preference for the elliptical shape. An effective approach to improve output of a cactus pear breeding program is to routinely include the evaluation of quality and productivity of tender pads along with fruit evaluation. Data on plant productivity and nutrient content can also provide information for prospective vegetable and fodder selections while selecting for fruit quality. Suitability of new accessions to be consumed as a vegetable can be determined in the second year.

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V. BREEDING TECHNIQUES A. Propagation Sexual and asexual propagation is possible in cactus pear and new propagules can be obtained from almost every part of the plant, including flowers and unripe fruits. Commercial orchards utilize vegetative propagation based on pad pieces, whole pads, or short branches. A few specialized nurseries were attempted in Mexico with limited success, and cuttings are currently obtained from productive orchards using pruning residues. Efficient protocols for in vitro propagation are also available (Escobar et al. 1986; Villalobos 1995) but they are not used on a commercial scale due to the ease and low cost of propagation from cladodes. Sexual propagation is only used for breeding purposes.

B. Floral Biology The floral bud is basically a modified stem with the floral parts inserted in the middle. The areoles are conserved and the ability to act as vegetative buds is maintained up to the early stages of fruit formation. A single flower bud arises from each areole. Flower color varies from yellow to reddish, and some light-green cultivars like ‘Reyna’ can have flowers of different hues on the same plant. Pollinated flowers fade and develop pink color. The flowers are hermaphroditic with an inferior ovary. Cacti are among the few plants in which the exterior of an inferior ovary, the receptacle, displays leaves and perfect areoles. This structure later becomes the peel of the fruit. The perianth is composed of several segments and the calyx cannot be clearly differentiated from the corolla (Sudzuki 1995). The pistil has four or more fused carpels, enclosed in a floral cup and a unilocular ovary with parietal placentation (Boke 1964). The pistil is thick and succulent, hollow, with several lobes that become sticky when receptive. The ovary contains numerous ovules. Nerd and Mizrahi (1994) reported an average of 270 ovules for the cultivar ‘Ofer’. Ovaries are located in the outer ovary wall (parietal placentation). The numerous stamens (up to 350) are inserted in the cavity of the receptacle surrounding the style. The filaments are free with the anthers on top. Pollen is shed before and after the flower opens (partial cleistogamy), so self-fertilization is common but not the only mode of pollination. The pollen is dry, spherical, and reticulate. The edible tissue develops from the funiculus and the funicular envelopes of the seeds, and the peel develops from the receptacular tissue. The fruits contain both viable and aborted seeds and the funicular envelopes of both seed types are equally capable of contributing to the pulp tissue (Pimienta 1990).

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Flowering season varies according to climatic conditions, cultivar, and crop management. About 90% of the flower buds are located on the top story of pads, those that are one year old. Older cladodes account for the remaining 10%. Under normal conditions flowering occurs March–April in the Northern Hemisphere and September–October in the Southern Hemisphere (Nerd and Mizrahi 1995). The reproductive potential varies with the species and cultivar. Mature (six to eight years old) well tended plants of ‘Reyna’ carry as many as 600 flowers and up to 95% can develop into mature fruits. Since almost all flowers set fruit, the number of fruit produced per plant is a function of the number of fertile cladodes and the average number of flowers per cladode (Pimienta 1990). Budding flushes are spread over a period of several weeks and plants can bear newly initiated buds, flowers, young cladodes, and ripe fruits at the same time (Nerd et al. 1989; Wessels and Stuart 1990). Some cultivars are able to withstand the loss of the first flush, bearing a new one after 50 to 70 days. This unusual feature is routinely used to obtain outof-season production by manual bud removal (scozzolatura ) in Italy as well as Israel (Barbera et al. 1991; Nerd and Mizrahi 1997). According to Pimienta (1990), floral differentiation occurs in 50 to 60 days (meristem activation until anthesis) mainly on the upper section of the cladode. This is in contrast with other temperate climate fruit trees where floral differentiation occurs during the previous season. All flowers bloom in the daytime. There are two types of flowers; type A flowers open late in the morning, and type B open in the afternoon. All flowers close in the evening. However, both types of flowers reopen for an additional day during the morning hours. The flowers of O. lindheimeri, a wild species, open only for one day. Early in the day the anthers dehisce and expose the yellow pollen (Grant et al. 1979). At the beginning of anthesis the stamens are close to the style and the anthers are in contact with the base of the pistil (Pimienta 1990). The stamens are thigmotropically sensitive and move and bent toward the style when touched (Grant et al. 1979). It has been proposed that this nastic response may promote insect pollination. Pollen tubes grow rapidly, reaching the base of the style within 24 h. The first pollen tubes to reach the micropyle of the ovule are observed three days after flower opening. A high percentage of ovules are viable and most of them are fertilized by the gametes that enter the locule (Rosas and Pimienta 1986). Cactus pear flowers are capable of self-pollination, and bagged flowers are able to set fruit (Nerd and Mizrahi 1994). Selfpollination was confirmed by Wang et al. (1996) with a hybridization trial involving six Opuntia species. Under natural conditions, once the flower opens, pollination by insects (mainly bees) ensures a higher number of

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seeds. The protandry explains the autogamic process (cleistogamy). A different response was reported by Grant et al. (1979) for O. lindheimeri Engelm.; they found that bagged flowers did not produce fruits. Wang et al. (1996) also reported sterility of the clone 1233, a spineless, cold-hardy clone that set no fruit under open pollinated or bagged conditions. However, when crossed with the Texas native O. linheimeri clone, 1233 produced fruit. Sicilian cultivars have been shown to be self-compatible since problems of fruit set are seldom encountered in vegetatively propagated plantations composed of a single cultivar or in single plants grown in backyards (Damigella 1958, cited by Nerd and Mizrahi 1995). A similar situation is observed in Central Mexico in the Pyramids region, a compact area in which around 7,000 ha of ‘Reyna’ are producing without apparent pollination problems. C. Pollen Collection, Storage, and Testing Short-term pollen storage (< 1 week) can be accomplished by collecting buds close to flowering and placing them in a cool and shaded location. Before use, the buds are exposed to full sun for a few hours to promote flowering. Unopened buds can also be used but their pollen yield is lower. Bunch (1997) reported that pollen collected fresh or stored at room temperature or field conditions for up to 6 days will remain viable and effect successful pollination. Pollen remained viable after 9 days of cold storage. Germination tests of pollen can be conducted in vitro using the technique reported by Brewbaker and Kwack (1963). D. Emasculation and Controlled Pollination The emasculation of a cactus pear flower resembles a surgical operation and should be performed carefully (Fig. 5.2). The material needed for emasculation includes rubber gloves, brush, a sharp knife or razor blade, small scissors with a curved tip, rinsing bottle, paper towels, glassine or paper bags, and rubber bands. The following steps are taken when emasculating cactus pear flowers: (1) remove the glochids from the exterior of the buds with the brush to allow easy handling; (2) excise the corolla, using as few strokes as possible, thereby avoiding wounds and mechanical damage to the style; (3) carefully remove the stamens and anthers, cutting close to the base; (4) rinse thoroughly with clean water to get rid of residual pollen and anthers; (5) clean the wounded surface with a paper towel; (6) allow 15–20 min to promote drying of the wounded tissues; (7) cover the flower with a glassine or paper bag and seal it with a rubber band; and (8) label.

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A

B

C

D

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Fig. 5.2. Cactus pear emasculation technique: (A) Emasculation; (B) Cleaning of residual anthers; (C) Pollination; (D) Isolation.

Flowers are able to recover from considerable damage (as long as the stigma remains intact) without significantly harming their reproductive potential. Bunch (1997) reported a modification of the technique whereby only those anthers close to the stigma were removed to reduce damage to the style, and then the cut surface of the receptacle and the remaining anthers were covered with first-aid tape. The stigma and a portion of the style were left protruding through the tape to be pollinated. Wang et al. (1996) reported that the application of dithyocarbamate (Sevin 2%) powder in the receptacular cavity after partial emasculation prevented insect visitation and undesired pollination.

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Although removal of the entire perianth causes extensive damage, the plant is able to withstand the damage due to the abundance of mucilage that flows quickly from the wounds. Mucilages, water-soluble complex polymeric carbohydrates, have been implicated in wound healing (Ting 1994). After emasculation the receptivity of the stigma occurs in 3–4 days under normal conditions. Under warm (>30°C) conditions they are receptive in two days. At this time the tip of the stigma is shiny and sticky with the lobes wide open. Even young flowers can be emasculated without appreciable loss of fertility. However, handling very young buds is difficult because the stamens tend to be less exposed, and the risk of mechanical damage or wounding to the stigma is greater. The most efficient way to pollinate is by means of a detached, fresh, fully open flower devoid of its style and corolla to allow close contact of the stamens with the stigma of the pistillate flower. Stored pollen can be applied onto the stigma of an emasculated flower with a #3 camelhair paintbrush (Bunch 1997). Non-fully opened buds can also be used, taking advantage of the protandric nature of cactus pear pollination. Although emasculation is difficult, about 100 to 250 seeds can be expected from a single fruit depending upon the cross. If the first flowering flush is eliminated, a new round of crosses can be performed in 50–70 days. However, the later the flowering season the lower the number of normal seeds expected. E. Seed Extraction and Germination Seeds can be extracted easily from peeled fruits processed in a blender set at low speed. Seed disinfecting is accomplished by soaking the seeds in commercial bleach (5–6% of sodium hypochlorite) for 10 min and then air dried for 3–4 h. Seeds can germinate after slight scarification. Muratalla et al. (1990) found that storage of seed lots for 9 years reduced the germination percentages to 50% of a fresh batch. Temperature is the most important variable for cactus seed germination. Nobel (1988) reported that for 19 species of cacti the optimal temperature for seed germination ranges from 17 to 34°C with a mean of 25°C. Opuntia lindheimeri and O. phaeacantha required 30°C and 28°C, respectively. This condition can be accomplished by means of a heat mat, with the bottom temperature set around 35°C. Some differences attributed to cultivar and seed condition have been observed (Mondragon 1999). Seeds may have a physical dormancy due to the hard seed coat. Several treatments overcome this barrier. Scarifying seeds in hot water (80–90°C) for 15 min twice and allowing them to cool off to room tem-

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perature and then soaking them in distilled water for 24 h has been enough to promote germination under greenhouse conditions in about 10 to 17 days (Mondragon 1999). Wang et al. (1996) found that seed germination was enhanced by using coarse vermiculite media maintained at 35°C. Seeds soaked in gibberellic acid (35 mg/L) germinated faster. Planting media should be kept moist under greenhouse conditions. F. Handling of Seedlings Seedlings are kept in germination trays until the first cladode grows 10–15 cm. Then they can be transplanted to small pots or black plastic bags. Prior to transplanting, the entire tap root is excised and the seedling allowed to dehydrate for 5–7 days to promote suberization of wounded tissue. Elimination of the tap root promotes formation of lateral roots, increasing the root volume and improving anchorage. Cacti, especially the columnar and spherical forms, are slow growing plants under natural conditions. However, fast growth of seedlings can be obtained. Ornamental succulents, grown in greenhouse on peatperlite media (1:1 by volume), watered, and fertilized frequently, and grown at cool night temperature (10°C) achieved marketable size rapidly (Stefanis and Langhans 1980). Almost all types of cacti, including the spherical and columnar ones, respond to optimal conditions of water and nutrients (Nobel 1988). Sanderson et al. (1986) reported that photoperiod length is important for cactus growth. Chamaecerus silvestri and Opuntia mycrodasys produced more shoots when grown under long photoperiod than under short photoperiod. Atmospheric CO2 concentration has been recognized as an important factor that affects growth in cacti and succulents, especially under greenhouse conditions. The CO2 range in well-controlled greenhouse cultivation varies roughly from 200 to 1200 ppm and may drop below the normal outside concentration. In general recommended levels are 700 to 900 ppm (Nederhoff 1994). Net CO2 uptake and biomass accumulation by O. ficus-indica growing in open top chambers were substantially enhanced when the CO2 level was doubled (Cui et al. 1993; Nobel and Israel 1994). Greenhouse grown seedlings can be transplanted to the field at six months. At this age they bear two to three slender pads and they can be managed as transplants. Plants derived from seeds tend to grow in an upright slender shape, branching only in the upper part. In contrast, plants grown from cuttings tend to have thicker and wider cladodes as well as more pads in the first story. Branching can be promoted in seedlings by pinching in the onecladode stage. This practice encourages thickening of the basal cladode

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as well as branching, thus increasing photosynthetic area and improving anchorage and vigor. Pinching also allows for an early expression of adult cladode shape. G. Reduction of Juvenility Grafting has been reported as a standard practice to maintain endangered or rare forms of cacti such as those that do not possess chlorophyll, and cristate (asymmetrically, bizarre shaped cacti) forms (Haage 1963; Pizzeti 1985; Pilbeam 1987). However, the information available pertains mostly to spherical forms such as Echinocactus used as scions and sharp-angled forms of Myrtyllocactus geometrizans, Hylocereus spp., Cereus, Trichocereus, Pereskia, and Rhipsalis used as rootstocks (Pizzeti 1985). Grafting platyopuntias is a little more complicated. The main concern is the shape and thickness of the stocks and scions. Estrada (1988) tried “micrografting” on six wild Opuntia species and two cultivated ones in vitro. The best combination used stock and scion prepared with squared tip and bottom, respectively, carefully matched and returned to in vitro conditions until the healing stage was complete. The success of the method (90% take rate) was confirmed by anatomical studies performed after 30 days. All combinations of the different species studied were found to be compatible. Grafting pieces of seedlings onto adult plants may be a possibility to reduce juvenility but it has not been documented. Intensive management of the seedlings involve long days (14 h), day temperature around 28°C and cool nights, while irrigation and fertilization enhance growth during the seedling stage in the greenhouse (Mondragon 1999). Once in the field the plants should be irrigated, weeded, and fertilized regularly. Plants maintained under this regime bore fruit in the fourth year in Northern Guanajuato, Mexico. Some genotypic effects on the length of juvenile stage have also been observed.

VI. BREEDING SYSTEMS A. Inbreeding and Outbreeding Self-compatibility has been reported by Wang et al. (1996) in Mexican, Chilean, and South African clones of O. ficus-indica grown in Southern Texas and the native O. lindheimeri. Damigella (1958, cited by Nerd and Mizrahi 1995) reported this behavior for Sicilian cultivars. In Mexico, ‘Reyna’ and other clonal cultivars are grown in solid stands and produce fruits without the need for special pollinating cultivars. This fact is sup-

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ported by the observation of large orchards growing plants of the same cultivar propagated asexually, which regularly set fruit. The long-term effects of selfing are unknown; however, it was observed that one round of self-pollination did not reduce the germination percentage or the seedling vigor in breeding populations under greenhouse conditions (Mondragon 1999). The flowers of cactus pear display the attributes of insect-pollinated flowers with large colorful perianth lobes and rigid stigma that enable the insects to land. The stigma becomes sticky when receptive. They also possess large pollen grains and abundant nectar that accumulates at the base of the corolla (Barbera et al. 1992; Pimienta 1990). Bees of various species, as well as small beetles and thrips appear to be involved in pollination (Grant et al. 1979). The degree of cross pollination has not been evaluated. Hybridization in natural populations of Southern California was elucidated by Walkington (1966, as cited by Gibson and Nobel 1986) based on morphological and chemical studies. The findings indicated that plants of Opuntia occidentalis arose from a cross between two native platyopuntias, O. ficus-indica and O. megacantha. The hybrid was reported as having features of both parents. Both partial and total cross pollination are found in cultivated accessions, thus cultivated types are likely the result of cross pollination. All Mexican cultivars are reported to be the products of hybridization of O. ficus-indica with different wild cactus pear forms (Pimienta et al. 1995). Scheinvar (1995) reported that in wild populations of opuntias, plants located in the periphery of the population show greater variability than those growing in the middle, probably due to a greater exposure to genetic exchange with other species and genotypes. B. Polyploidy The differences in fruit and cladode size found in wild and cultivated populations are likely due to differences in ploidy levels. Cytogenetic studies have revealed the existence of different ploidy levels; 2x, 3x, 4x, 5x, 6x, 8x, 10x, 11x, 12x, 13x, and 19x. Approximately 63% of the species of the subfamily Opuntioidea are reported as polyploid (Sosa 1964; Yuasa 1973, cited by Pimienta and Muñoz 1995; Pinkava et al. 1992). The highest number of chromosomes (2n = 6x = 66 and 2n = 8x = 88) are commonly found in the cultivars. ‘Charola’ and ‘Cardona’ are octoploid, both are highly productive, vigorous plants but fruit size is only 67 and 83 g, respectively, suggesting that ploidy is not always associated with large fruit size (Mondragon and Perez 1997). The octoploid

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cultivars might have originated as autopolyploids by two rounds of chromosome duplication of the (2n = 22) wild ancestor (Pimienta 1990). Muñoz et al. (1995), in a study carried out with two wild tetraploid entries (‘Tapon’ and ‘Tapon de Mayo’ ) and three octoploid cultivars (‘Burrona’, ‘Chapeada’, and ‘Naranjona’), found that ploidy level was positively correlated with cuticle and epidermis thickness and depth of the stomata chamber, but not with the size of the stomata. C. Apomixis Apomixis has been reported in several species of Opuntia, including O. aurantiaca Lindl., O. dillenii Haw., O. glaucophyla Wendl., O. leucantha Link., O. rafinesquii Engelm., O. tortispina Engelm., and O. ficusindica (L.) Mill. derived from nucellar tissue (Tisserat 1979). Within cultivated cactus pear, apomixis is also a widespread phenomena. Mondragon (1999), working with 17 breeding populations of Mexican origin grown under greenhouse conditions, found that all entries produced maternal seedlings. Richards (1986) indicated that somatic embryos originate by adventitious embryony. In this process the sexual embryo and one of the apomictic embryos coexist, sharing the same endosperm. Thus the mature seed may have one sexual embryo and one or more apomictic embryos. In general, cactus pear tends to produce mostly nucellar embryos, probably also associated with their polyploidy (Jacobs and Kistner 1992). In vitro studies using a scanning electron microscope showed that high numbers of somatic embryos originate from nucellar tissue at the micropyllar end of the ovule. Only well developed mature zygotic and somatic embryos germinated in vitro after seven days of culture. Two to three seedlings per seed were obtained and all somatic embryos showed well-developed embryo stages with a close similarity to zygotic embryogenesis (Velez and Rodriguez 1996). In a study conducted under greenhouse conditions with commercial cultivars and breeding populations derived from Mexican entries, the percentage of apomictic seedlings ranged from 2 to 20 (Mondragon 1999). The putative apomictic embryos were visible as early as two days after the zygotic seedlings could be identified. All apomictic seedlings germinated during the cotyledonary stage of the zygotic seedling. The number of maternal seedlings per seed varied from 1 to 3. The somatic origin of seedlings of late emergence was verified by means of the RAPD technique.

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D. Biotechnology 1. Tissue Culture. An efficient micropropagation protocol for cactus pear was developed by Escobar et al. (1986) and tested for four wild and two cultivated opuntia species by Estrada (1988). In vitro culture was intended to produce virus-free plants and to be used as a method for fast propagation of new cultivars. The technique has not been used on a commercial scale due to the ease of propagation through traditional methods. 2. Transformation. The feasibility of transient transformation of shoot apical meristems was demonstrated via the biolistic approach. Campos et al. (1997) were able to express the GUS reporter gene in cells of apical meristems. The percentage of transformed cells reached 37%. The manipulation of genes through molecular biology techniques represent a shortcut to complement the efforts of traditional breeding. The starting point is the isolation of DNA. A protocol suitable for DNA extraction of cactus was developed by Mondragon (1999). Amplification of genomic DNA extracted from eight Opuntia and other cacti species was used to perform RAPD analysis. VII. SUMMARY AND FUTURE PROSPECTS Cactus pear evolved into a formal crop in the last 30 years. During this time the area planted surpassed over 50,000 ha in Mexico alone, reaching the status as the first fruit crop specifically adapted to semiarid, nonirrigated lands. Other countries involved in cactus pear cultivation are Argentina, Chile, Italy, Israel, South Africa, and the United States. Mexico and Italy are the main producers and consumers of cactus fruits. All planting stock is obtained through vegetative propagation. Interest in this plant is gradually increasing as more countries are finding that cactus pear can be used extensively in the fight against desertification and for the development of new products including human food and animal feed, as well as industrial and medicinal applications. Present-day cultivars of cactus pear are the grower selections from plants grown in family gardens. The cultivars that dominate the world market of this produce were selected in Mexico or were obtained from Mexican germplasm. Cactus pear breeding was attempted erratically at the beginning of the century in California and again during the 1960s and 1970s in Mexico. Earlier breeding objectives of these programs were the development of spineless cultivars for forage, cold hardiness and high yielding cultivars of light green fruits, respectively. A fresh start on

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breeding is underway in Mexico, Italy, and the United States. Current interest is in the development of spineless cultivars with low seed content and juicy and yellow, red, or purple fruits. Frost tolerance is necessary for those regions periodically exposed to low temperatures. Breeding programs have to be of long duration since the juvenile period lasts 4 to 6 years. The first generation of hybrids is currently in the second or third year of evaluation in Mexico, California, Argentina, and Italy. The new techniques provided by molecular biology might accelerate breeding of cactus pear. Successful delivery of foreign DNA was recently demonstrated by means of particle bombardment.

LITERATURE CITED Aguilar, B. A. 1987. Efecto de la aplicacion de acido giberelico (GA3) y urea en el fruto del nopal (Opuntia amychlaea Tenore). Tesis de M.C. Colegio de Postgraduados. Chapingo, Mexico. Baker, G. H. 1970. Plants and civilization. Wadsworth Publ. Co., Belmont, CA. Barbera, G. 1995. History, economic and agro-ecologic importance. In: G. Barbera, P. Inglese, and B. E. Pimienta, (eds.), Agroecology cultivation and uses of cactus pear. FAO Plant Production and Protection Paper 132. Rome, Italy. Barbera, G., F. Carimi, and P. Inglese. 1991. The reflowering of prickly pear (Opuntia ficusindica (L). Miller) influence of removal time and cladode load on yield and fruit ripening. J. Am. Soc. Hort. Sci. 77–80. Barbera, G., F. Carimi, and P. Inglese. 1992. Past and present role of the indian-fig prickly pear (Opuntia ficus-indica (L.) Miller, Cactaceae) in the agriculture of Sicily. Econ. Bot. 461:10–20. Barbera, G., and P. Inglese. 1993. La cultura dell fico di India. Frutticoltura Moderna. Edagricole. Bologne, Italy. Barbera, G., P. Inglese, and T. La Mantia. 1994. Influence of seed content on some characteristics of the fruit of cactus pear (Opuntia ficus-indica Mill). Scientia Hort. 58:161–165. Boke, N. 1964. The cactus gynoecium: a new interpretation. Am. J. Bot. 51:598–610. Borrego, E. F., and V. N. Burgos. 1986. El nopal. UAAAN. Saltillo Mexico. Borrego, E. F., S. M. Murillo, A. Flores H., E. Olhagaray R., and M. Villavicencio O. 1990. Potencial de produccion en el norte de Mexico de variedades de nopal (Opuntia spp) tolerantes a frio. In: Proc. First Annual Texas Prickly Pear Council. 49–73. Brewbaker, J. L., and B. H. Kwack. 1963. The essential role of calcium ion in pollen germination and pollen growth. Am. J. Bot. 50:859–865. Bunch, R. 1997. Update on cactus pear breeding program and new products at D’Arrigo Bros. J. Prof. Assoc. Cactus Develop. 2:60–65. Campos, F. A. P., R. M. L. Zarate, L. F. A. Ponte, and J. Landsmann. 1997. Transient transformation of shoot apical meristems of the prickly pear (Opuntia ficus-indica). Memorias del VII Congreso Nacional y V Internacional sobre el Conocimiento y Aprovechamiento del Nopal. Monterrey, Mexico. p. 281–282. Cantwell, M. 1991. Quality and postharvest physiology of “nopalitos” and “tunas.” In: P. Felker (ed.), Proc. 2nd Annual Texas Prickly Pear Conference. McAllen, TX.

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Chessa, I., and G. Nieddu. 1997. Report Int. Workshop on Promotion of Minor Fruit Crops. Cactus pear. May 16–23. Universitá degli Study di Sassari, Italy. Chessa, I., G. Nieddu, and P. Serra. 1997. Isozyme characterization of Opuntia species and varieties from Italian germplasm. Acta Hort. 438:45–53. Corrales, G. J., J. Andrade R., and E. Bernabe C. 1997. Response of six cultivars of tuna fruits to cold storage. J. Prof. Assoc. Cactus Develop. 2:160–168. Cowan, R. and P. Felker. 1998. Fruit quality of new cold-hardy Opuntias from northern and high-elevation sites in Mexico. J. Prof. Assoc. Cactus Develop. 3:8–18. Cui, M., P. M. Miller, and P. S. Nobel. 1993. CO2 exchange and growth of the crassulacean acid metabolism plant Opuntia ficus-indica under elevated CO2 in open top chambers. Plant Physiol. 103:519–524. Curtis, R. J. 1977. Prickly pear farming in the Santa Clara Valley, California. Econ. Bot. 31:175–179. Dodd, A. P. 1940. The biological campaign against prickly pear. Commonwealth Prickly Pear Board. Brisbane, Australia. Dreyer, P. 1985. A gardener touched with genius. The life of Luther Burbank. Univ. California Press, Berkeley. Escobar, A., V. M. Villalobos, and M. Villegas. 1986. Opuntia micropropagation by axillary proliferation. Plant Cell Tissue Organ Culture 7:269–277. Estrada, L. A. A. 1988. Produccion de brotes en injertacion in vitro de seis especies de nopal (Opuntia spp) originarias del altiplano Potosino-Zacatecano. Tesis M.C. Colegio de Postgraduados. Chapingo, Mexico. Felker, P. 1995. A review of cactus pear development in the United States. Memorias del 6o. Congreso Nacional y 4o. Internacional sobre el Conocimiento y Aprovechamiento del Nopal. Jalisco, Mexico. Fernandez, M. R. M. 1997. Atraso de la epoca de cosecha del nopal tunero en el Norte de Guanajuato. Memorias del VII Congreso Nacional y V Internacional sobre el Conocimiento y Aprovechamiento del Nopal. Monterrey, Mexico. Flores, V. A. C. 1995. ‘Nopalitos’ production and processing. In: G. Barbera, P. Inglese, and B. E. Pimienta, (eds.), Agroecology cultivation and uses of cactus pear. FAO Plant Production and Protection Paper 132. Rome, Italy. Fucikovsky, L., and J. Luna. 1988. Pudricion bacteriana de la tuna y su transmision. Memorias del V Congreso Nacional de Fitopatologia. Mexico. Gibson, A., and P. S. Nobel. 1986. The cactus primer. Harvard Univ. Press, Cambridge. Gil, G. S., and A. Espinosa. 1980. Desarrollo de frutos de tuna (Opuntia ficus-indica, Mill.) con aplicacion prefloral de giberellina y auxina. Cien. Inv. Agraria 7:141–147. Goldstein, G., and P. S. Nobel. 1991. Changes in osmotic pressure and mucilage during low temperature acclimation of Opuntia ficus indica. Plant Physiol. 97:954–961. Grant, T. V., and P. D. Haud. 1979. Pollination of the south western opuntias. Plant Syst. Evol. 133:15–28. Grant, T. V., K., Grant, and D. P. Hurd. 1979. Pollination of Opuntia lindheimeri and related species. Plant Syst. Evol. 132:313–320. Gregory, A. R., J. O. Kuti, and P. Felker. 1993. A comparison of Opuntia fruit quality and winter hardiness for use in Texas. J. Arid Environments 24:37–46. Gutierrez, L. H. 1992. Plagas y enfermedades del nopal en Mexico. Universidad Autonoma de Chapingo, Mexico. Haage, W. 1963. Cacti and succulents: A practical handbook. E. P. Dutton and Co., New York. Hellgren, C. E. 1994. Prickly-pear cactus (Opuntia spp) and its use by wildlife. Proc. 5th Annual Texas Prickly Pear Council. August 12, 1994. Kingsville. Hoffman, W. 1995. Ethnobotany. In: G. Barbera, P. Inglese, and B. E. Pimienta (eds.),

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Agroecology cultivation and uses of cactus pear. FAO Plant Production and Protection Paper 132. Rome, Italy. Inglese, P. 1995. Orchard planting and management. In: G. Barbera, P. Inglese, and B. E. Pimienta (eds.), Agroecology cultivation and uses of cactus pear. FAO Plant Production and Protection Paper 132. Rome, Italy. Inglese, P., G. Barbera, and T. La Mantia. 1993. Research strategies and improvement of cactus pear (Opuntia ficus-indica) fruit quality and production. Proc. 4th Annual Texas Prickly Pear Council. August 13–14, 1993. Kingsville. Jacobs, D. H., and M. Kistner. 1992. Microsporogenesis in two Opuntia species with notes on its implication for breeding. II Congreso Int. de Tuna y Cochinilla, Sept. 22–25, Santiago, Chile. Martinez, M. L. 1968. Estudios del nopal rastrero y el nopal frutal. p. 339–344. In: T. W. Box, and P. Rojas-Mendoza (eds.), Proc. Int. Symposium on Increasing Food Production on Arid Lands. Lubbock, TX. Mick, R. J. 1991. Growing variety 1308 for year round nopalito production. p. 32–34. In: P. Felker (ed.), Proc. 2nd Annual Texas Prickly Pear Council. McAllen, TX. Mizrahi, Y., A. Nerd, and P. S. Nobel. 1997. Cacti as crops. Hort. Rev. 18:291–346. Mondragon J., C. 1999. Preliminary genetic studies on cactus pear (Opuntia spp. Cactaceae) germplasm from Central Mexico. Ph.D. Diss. Purdue Univ., West Lafayette, IN. Mondragon J., C., M. R. M. Fernandez, and Ch. J. Estrada. 1995. Ampliacion de la epoca de cosecha de la tuna. Memorias del 6o. Congreso Nacional y 4o. Int. sobre el Conocimiento y Aprovechamiento del Nopal. Jalisco, Mexico. Mondragon J., C., and G. S. Perez. 1993. ‘Reyna’ (Syn. Alfajayucan) is the leading cactus pear cultivar in Central Mexico. Fruit Var. J. 48:134–136. Mondragon J., C., and G. S. Perez. 1997. Native cultivars of cactus pear in Mexico. p. 446–450. In: J. Janick (ed.), Progress in new crops. ASHS Press, Alexandria, VA. Moreno, R. P., and V. C. Flores. 1996. The world cactus pear market. J. Prof. Assoc. Cactus Develop. 1:75–86. Muñoz, U. A., V. A. Garcia, and B. E. Pimienta. 1995. Relacion entre el nivel de ploidia y variables anatomicas y morfologicas en especies silvestres y cultivadas de nopal tunero (Opuntia spp). In: B. E. Pimienta et al. (eds.), 1995. Memorias del 6o. Congreso Nacional y 4o. Int. sobre el Conocimiento y Aprovechamiento del Nopal. Jalisco, Mexico. Muratalla, L. A., P. F. Barrientos, and A. J. Rodriguez. 1990. Germinacion de semilla de nopal (Opuntia amychlaea T. Cv. ‘V5’ y O. ficus-indica Cvs. ‘V1’ y ‘F1’). In: Memorias de la IV Reunion Nacional sobre el Conocimiento y Aprovechamiento del Nopal. Zacatecas, Mexico. Nederhoff, M. E. 1994. Effects of CO2 concentration on photosynthesis, transpiration and production of greenhouse fruit and vegetable crops. Ph.D. Diss. Wageningen, The Netherlands. Nerd, A., M. Dumotier, and Y. Mizrahi. 1997. Properties and postharvest behaviour of the vegetable cactus Nopalea cochellinifera. Postharvest Biol. Tech. 10:135–143. Nerd, A., A. Marady, and V. Mizrahi. 1989. Irrigation, fertilization and polyethylene covers influence bud development in prickly pear. HortScience 24:773–775. Nerd, A., and Y. Mizrahi. 1994. Toward seedless prickly pear. In: P. Felker and J. R. Moss (eds.), Proc. 5th Annual Texas Prickly Pear Council. Kingsville. Nerd, A., and Y. Mizrahi. 1995. Reproductive biology. In: G. Barbera, P. Inglese, and B. E. Pimienta (eds.), Agroecology cultivation and uses of cactus pear. FAO Plant Production and Protection Paper 132. Rome, Italy. Nobel, P. S. 1988. Environmental biology of agaves and cacti. Cambridge Univ. Press, New York.

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Sudzuki, H. F. 1995. Anatomy and morphology. In: G. Barbera, P. Inglese, and B. E. Pimienta (eds.), Agroecology cultivation and uses of cactus pear. FAO Plant production and Protection Paper 132. Rome, Italy. Ting, P. I. 1994. Carbohydrate metabolism in cacti: gums and mucilage. In: P. Felker and R. J. Moss (eds.), Proc. 5th Annual Prickly Pear Council. Kingsville, TX. Tisserat, B., E. B. Esan, and T. Murashige. 1979. Somatic embryogenesis in angiosperms. Hort. Rev. 1:1–78. Uphof, T. C. J. 1916. Cold resistance in spineless cacti. University of Arizona. Agr. Exp. Sta. Bul. 79. Tucson. Uzun, I. 1997. Fruit and cladode isozymes in cactus pear. In: Proc Int. Congress on Cactus Pear and Cochenille. Acta Hort. 438:45–53. Valdez, C. R. D., V. C. Gallegos, and M. F. Blanco. 1997a. Analisis multivariado de once variedades de nopal tunero. Atributos de crecimiento. Memorias del VII Congreso Nacional y V Int. sobre el Conocimiento y Aprovechamiento del Nopal. Monterrey, Mexico. Valdez, C. R. D., V. C. Gallegos, and M. F. Blanco. 1997b. Analisis multivariado de once variedades de nopal tunero: Atributos de fruto. In: Memorias del VII Congreso Nacional y V Int. sobre el Conocimiento y Aprovechamiento del Nopal. Monterrey, Mexico. Velez, G. C., and G. B. Rodriguez. 1996. Microscopic analysis of polyembryony in Opuntia ficus-indica. J. Prof. Assoc. Cactus Develop. 1:39–48. Villalobos, A. V. 1995. Tissue culture application for Opuntia sp micropropagation. In: G. Barbera, P. Inglese, and B. E. Pimienta (eds.), Agroecology cultivation and uses of cactus pear. FAO Plant Production and Protection Paper 132. Rome, Italy. Wang, X., P. Felker, and A. Paterson. 1997. Environmental influences on cactus pear fruit yield, quality, and cold hardiness and development of hybrids with improved cold hardiness. J. Prof. Assoc. Cactus Develop. 2:48–59. Wang, X., P. Felker, A. Paterson, Y. Mizrahi, A. Nerd, and J. C. Mondragon. 1996. Cross hybridization and seed germination in Opuntia species. J. Prof. Assoc. Cactus Develop. 1:49–55. Weiss, J., A. Nerd, and Y. Mizrahi. 1993. Vegetative parthenocarphy in the cactus pear Opuntia ficus-indica (L.) Mill. Ann. Bot. 72:521–526. Weniger, D. 1984. Cacti of Texas and neighboring states: A field guide. Univ. Texas, Austin. Wessels, B. A., and E. Stewart. 1990. Morphogenesis of the reproductive bud and fruit of the prickly pear (Opuntia ficus-indica (L.) Mill.) Cv. Morado. Acta Hort. 275:245–253. Wessels, B. A. 1988. Spineless prickly pear. Perskor. Johannesburg, South Africa. Yeung, E. C., and D. W. Meinke. 1993. Embryogenesis in angiosperms: Development of the suspensor. Plant Cell: 5:1371–1381.

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6 Somatic Hybridization and Applications in Plant Breeding Alexander A. T. Johnson and Richard E. Veilleux Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0327

I. INTRODUCTION A. Types of Somatic Hybrids B. Fusion Methods C. Selection Schemes D. Methods of Detection II. RECENT EFFORTS IN SOMATIC HYBRIDIZATION A. Intraspecific Somatic Hybrids B. Interspecific Somatic Hybrids 1. Rutaceae 2. Solanaceae 3. Brassicaceae 4. Fabaceae 5. Asteraceae 6. Liliaceae 7. Iridaceae 8. Cucurbitaceae 9. Caryophyllaceae 10. Passifloraceae 11. Ebenaceae 12. Laminaceae C. Intergeneric Somatic Hybrids 1. Rutaceae 2. Solanaceae 3. Brassicaceae 4. Poaceae 5. Caryophyllaceae

Plant Breeding Reviews, Volume 20, Edited by Jules Janick ISBN 0-471-38788-6 © 2001 John Wiley & Sons, Inc. 167

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D. Asymmetric Hybrids 1. Solanaceae 2. Brassicaceae 3. Poaceae 4. Convolvulaceae E. Cybrids 1. Solanaceae 2. Brassicaceae 3. Asteraceae 4. Poaceae III. CONCLUSION LITERATURE CITED

I. INTRODUCTION Somatic hybridization of plants by protoplast fusion is a technique that has captivated the imaginations of plant breeders for three decades. It offers the possibility of accessing sexually incompatible germplasm between crop species and distant relatives, merging genomes of sexually dysfunctional cultivars or breeding lines, and substituting one cytoplasm for another with little effect on the nuclear genome. Since Carlson et al. (1972) first reported success with parasexual hybridization of tobacco (Nicotiana tabacum L.), hundreds of reports have been published to extend the procedures to additional plant genera and to evaluate the potential of somatic hybrids in many crops. Somatic hybridization has even been conducted under microgravity as part of a space lab experiment (Hoffmann et al. 1995). Using somatic hybrid as a key word in the on-line database (Web of Science) maintained by the Institute for Scientific Information, we found between 30 and 50 hits of primary literature per year for the last decade, indicating that interest in somatic hybridization as a plant breeding adjuvant has not diminished. Waara and Glimelius (1995) reviewed the literature on somatic hybridization through the early 1990s and somatic hybrids have been reviewed twice previously in Plant Breeding Reviews, once generally (Bravo and Evans 1985) and specifically for Citrus (Grosser and Gmitter 1990). The following review focuses primarily on literature published subsequently to the Waara and Glimelius (1995) review, with particular emphasis on the utility of somatic hybrids in plant breeding programs. A quick mention of the types of somatic hybrids possible and the methods used to obtain and identify them precedes our coverage of primary literature.

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A. Types of Somatic Hybrids If the complete genomes of two different species are combined parasexually, then an amphiploid somatic hybrid results. This is the most common occurrence in somatic hybridization experiments. If one fusion partner is an unadapted species with some desirable trait, then the resulting somatic hybrids can be expected to carry many of the undesirable traits of the unadapted species along with the trait of interest, assuming that this trait of interest will be expressed in the amphiploid product. Therefore plant breeders would indeed be foolishly optimistic to expect table-ready germplasm from such effort; considerable backcrossing and ploidy reduction are required to introgress the desirable trait into germplasm suitable for cultivar release. Indeed, much of the recent effort in somatic hybridization has been directed at development of somatic hybrids and/or verification that the trait of interest has been transmitted. Subsequent utilization of somatic hybrid germplasm in plant breeding has been less frequent. In an effort to limit the genetic contribution of an unadapted “parent” to the product of protoplast fusion, some geneticists have promoted asymmetric somatic hybridization, whereby the genome of the donor species is fractionated by irradiation prior to fusion. The resulting asymmetric hybrids retain the complete genome of the recipient (adapted) species and only fragments of the genome of the donor (unadapted) species. Irradiation is imprecise and damage to the donor genome is random. Therefore transmission of the trait of interest is not guaranteed and the amount of the donor genome transmitted is highly variable, depending upon the irradiation dosage and the tolerance of the recipient genome to chromosome fragments and rearrangements. Cybrids or cytoplasmic hybrids result from protoplast fusion between a cultivated species and enucleated or nucleus-inactivated protoplasts bearing a different plastome. For plant breeding purposes, cybridization offers the possibility of developing lines for hybrid breeding via cytoplasmic male sterility systems in a single step. Such systems are the mainstay of hybrid cultivar production in many crops and availability of suitable plastome variation has been a limitation in breeding. B. Fusion Methods Successful somatic hybridization was originally obtained through the use of PEG-mediated (polyethylene glycol) fusion. More recently, electrofusion techniques have become available. Of 135 primary reports of

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successful somatic hybridization covered in this review, 63 were achieved by chemical fusion and 72 by electrofusion. C. Selection Schemes When protoplasts of two different sources are mixed in the hope of obtaining somatic hybrids, a range of possible products may regenerate, resulting from unfused protoplasts of either “parent” (somaclones produced), homofusions between genetically similar protoplasts of either parent (polyploid somaclones produced), heterofusions between single protoplasts of each parent (amphidiploid somatic hybrids produced), or multiple fusions among several protoplasts (highly polyploid somatic hybrids produced). In addition, aneuploids often arise due to chromosome loss during cell culture or aberrant mitotic cycles within the hybrid nucleus. Without any selection against the undesirable products, researchers are faced with distinguishing the desired amphidiploid somatic hybrids from the undesired variable ploidy somaclones of each parent and highly polyploid hybrids resulting from multiple protoplast fusions. The simplest selection has been to analyze whatever plants may regenerate by morphological or molecular markers to determine their possible genomic constitution. When heterokaryons regenerate more readily than homokaryons, this can be effective. Otherwise, many more sophisticated selection schemes have been developed to encourage regeneration of only the somatic hybrids, but all generally carry some inherent cost. Selective media can be used if sufficient tissue culture research has been done on the prospective fusion partners. If each protoplast source can reliably be predicted to regenerate only in the presence of a distinct unique media component, elimination of both components in the medium used for regeneration following protoplast fusion should result exclusively in somatic hybrids. If one parent is known to be non-regenerable, then this procedure can be simplified by omitting only the medium requirement of the regenerable “parent.” This particular complementation scheme requires the conduct of rigorous plant tissue culture experimentation on both fusion partners prior to their utilization in somatic hybridization schemes in order to predict the outcome with any reliability. In addition, absolute medium requirements in plant tissue culture are difficult to identify. The use of metabolic inhibitors such as iodoacetate (IOA) to prevent regeneration of unfused protoplasts of one fusion partner has greatly enhanced complementation schemes. Because it is common to find that one protoplast source is unregenerable, protoplasts from such a source can be fused with IOA-treated protoplasts of an ordinarily regenerable

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source with the expectation that only fusions will regenerate. Introduction of transgenic antibiotic resistance into one or both fusion partners has also been used to facilitate complementation schemes. This provides an easy selection scheme by which protoplasts lacking antibiotic resistance will not regenerate on medium containing the antibiotic. Antibiotic resistance in an unregenerable protoplast source combined with antibiotic sensitivity in a regenerable protoplast source then provides a suitable selection scheme for exclusive regeneration of somatic hybrids. Ishige (1995) used transgenic resistance to kanamycin in one potato (Solanum tuberosum L.) dihaploid and hygromycin in another to regenerate exclusively somatic hybrids that varied for ploidy. However, the restrictions of this system are the extensive pre-breeding required to introduce transgenic resistance into the protoplast fusion partner(s) and the limitation of the procedure only to transgenic plants. In order to eliminate the necessity of pre-breeding one of the fusion partners, Dörr et al. (1994) developed a selection method to label protoplasts with superparamagnetic beads mediated by biotinylated lectins. Electrofusion of these labeled protoplasts with transgenic kanamycin resistant protoplasts was followed by sorting using a magnetic cell sorter to retain protoplasts labeled with the microbeads. Subsequent culture of selected cells on kanamycin containing medium resulted in a significant enrichment for somatic hybrids among the regenerants. Surface-labeling of prospective fusion partners with biotin and avidin has been used to facilitate heterologous aggregation of fusion partners during electrofusion (van Kesteren et al. 1993). Finally, Waara et al. (1998) used different vital fluorescent stains (fluorescein diacetate and scopoletin) on dihaploid potato lines, followed by laser activated flow cytometric sorting to regenerate 98% somatic hybrids without the need of transgenic resistance: 187 of the 301 hybrids obtained were tetraploid. Hoffmann-Tsay et al. (1994) identified several surface-active chemicals that could be used as adjuvants to treat protoplasts prior to electrofusion to increase the fusion rate. They concentrated on adjuvants that did not need to be removed from the culture medium in order to retain one of the advantages of electrofusion over chemical fusion, i.e., the direct culture of fusion products. Möllers et al. (1994) likewise studied the possibility of increasing the frequency of somatic hybrid formation during electrofusion by selectively inactivating one protoplast source with the mitochondrial inhibitor, nonyl-acridine orange (NAO). NAO was considered to be an alternative to IOA that might have the specific result of predetermining the mitochondrial composition of somatic hybrids. Neither of these two techniques, i.e., chemical adjuvants or NAO, has been used extensively in somatic hybridization subsequent to

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the original reports. Funatsuki et al. (1994) described a modification of chemical fusion whereby the fusion was conducted on a Millicell (Millipore) membrane on a puddle of PEG; this facilitated heterofusions between protoplasts that differed considerably in size. This method has likewise not been extensively adapted. D. Methods of Detection Once putative somatic hybrids have been regenerated, an array of techniques is now available for proof of their hybrid nature. Although intermediate morphology and heteroallelic isozyme patterns are still frequently employed in recent reports, most authors are now using molecular analysis to demonstrate hybridity. Even if specific probes for restriction fragment length polymorphisms (RFLPs) have not been developed for the species under investigation, probes that anneal to universal genes, such as rDNA from other species, will often reveal differences between the two protoplast sources following restriction enzyme digest and Southern blotting. One recent reference employed rDNA probes both to document hybridity and to demonstrate that site-specific methylation had occurred during somatic hybridization between potato and three related species selected for nematode resistance (Harding and Millam 1999). After southern hybridization, other repetitive DNA sequences have been used to detect the presence of both parental genomes in somatic hybrids (Stadler et al. 1995; Zanke et al. 1995, 1997). Molecular markers such as randomly amplified polymorphic DNA (RAPDs; De Filippis et al. 1996; Shi et al. 1998b), simple sequence repeats (SSRs; Johnson et al. 2000), and amplified fragment length polymorphisms (AFLPs; Brewer et al. 1999; Tian and Rose 1999) have been used. Chromosomes of both parents within a somatic hybrid have been visually distinguished using techniques such as FISH (fluorescent in situ hybridization; Yan et al. 1999a). Organellar probes, such as mitochondrialspecific probes from Pisum sativa L. (Moriguchi et al. 1997) or chloroplast-specific probes from Oncidium excavatum (Heath and Earle 1995), have been used successfully to differentiate organellar genomes of fusion partners and reveal the chondriome and plastome of somatic hybrids. There is no phylogenetic limitation to protoplast fusion. Even fusions between plant and animal protoplasts have been conducted (Makonkawkeyoon et al. 1995). However, regeneration of somatic hybrids has only been possible when fusion partners are somewhat related. Somatic hybridization has allowed us to exceed the limits of sexual compatibility and evidence of partial genome transfer has even been presented after

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fusion between a monocot (Hordeum vulgare L.) and a dicot (Daucus carota L.) (Kisaka et al. 1997). However, it has been far more common to obtain only unregenerable callus if the fusion partners are too phylogenetically distant (Wang et al. 1993; Jarl et al. 1995; Vazquez-Tello et al. 1996; Pattanavibool et al. 1998). Because such callus has little potential in plant breeding, we have omitted references where actual somatic hybrid plants were not obtained.

II. RECENT EFFORTS IN SOMATIC HYBRIDIZATION A. Intraspecific Somatic Hybrids Although the greatest efforts in somatic hybridization have been for introgression of alien germplasm into crop plants through interspecific protoplast fusion, there has also been considerable effort at intraspecific somatic hybridization for various purposes (Table 6.1). Intraspecific somatic hybridization has been applied to potato, for genetic reconstruction of tetraploids by fusion of selected dihaploids. Chase in 1963 proposed an analytic breeding scheme for potato and other polyploid crops. The tetraploid genome is first reduced to the dihaploid level by haploidization schemes, then breeding is conducted among dihaploids where segregation ratios and inheritance of desirable quantitative traits is simpler. Finally, the tetraploid condition is restored through sexual polyploidization, colchicine doubling, or some other means. Chase, of course, could not predict protoplast fusion. Wenzel et al. (1979) were quick to realize that the newly developed technique would facilitate analytic breeding in potato, especially because experience with dihaploids had revealed them to be reluctant to flower and frequently sterile, thereby prohibiting sexually combining their genomes. Another purported advantage of this breeding strategy is the possibility of eliminating deleterious alleles harbored in the tetraploid, but revealed at the dihaploid level through partial inbreeding. Rasmussen and Rasmussen (1995) electrofused two potato dihaploids that exhibited resistance to the cyst nematode, Globodera pallida. Although one dihaploid never flowered and the other was male sterile, some of the approximately tetraploid somatic hybrids were both male and female fertile. The somatic hybrids also exhibited a range of resistance to G. pallida, some combining the resistance to separate pathotypes from each dihaploid fusion partner (Rasmussen et al. 1996). Employing a similar strategy, Cooper-Bland et al. (1994) found variable resistance to G. pallida among tetraploid somatic hybrids between selected dihaploid

174 SH SH SH

SH SH SH SH SH SH

Electrofusion Electrofusion Electrofusion

n.s.x Electrofusion Electrofusion Chemical Electrofusion Electrofusion

S. tuberosum dihaploids S. tuberosum dihaploids Daucus carota L. + D. carota Ipomoea batatas Diospyros kaki Colocasia esculenta

RFLP, RAPD RAPD none RAPD RAPD isozymes

RFLP RFLP RAPD

isozymes

Verificationy

y

SH = somatic hybrid. RFLP = restriction fragment length polymorphism; RAPD = randomly amplified polymorphic DNA. x n.s. = not specified in journal.

z

Umbelliferae Convolvulaceae Ebenaceae Araceae

SH

Type of hybridsz

Electrofusion

Solanaceae

Protocol

Dörr et al. 1994 Ishige et al. 1995 Rasmussen and Rasmussen 1995; Rasmussen et al. 1996, 1998 Frei et al. 1998 Waara et al. 1998 Koyama et al. 1995 Wang et al. 1997 Tamura et al. 1995 Murakami et al. 1998

Cooper-Bland et al. 1994, 1996

Reference

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Solanum tuberosum L. dihaploids S. tuberosum dihaploids S. tuberosum dihaploids S. tuberosum dihaploids

Fusion partners

Intraspecific somatic hybrids of crop plants (1994–1998).

Family

Table 6.1.

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clones. Considerable variation has been found among intraspecific somatic hybrids regenerated from a single fusion; however, there appears to be little cytoplasmic influence on these phenotypes (Frei et al. 1998). Using five potato dihaploids selected for either foliar or tuber resistance to late blight [Phytophthora infestans (Mont.) de Bary] in pairwise fusions, Rasmussen et al. (1998) restored fertility and combined foliar and tuber resistance in some euploid somatic hybrids. Cooper-Bland et al. (1996) also evaluated late blight resistance of somatic hybrids between potato dihaploids and compared field performance of the somatic hybrids with diploid and tetraploid sexual hybrids between the parents. Tetraploid hybrids were obtained by colchicine doubling the dihaploids prior to crossing. All tetraploids expressed greater yield than the diploids, regardless their derivation through sexual or somatic hybridization. Both sexual and somatic hybrids showed intermediate resistance to late blight; however, there was less variation for resistance among somatic compared to sexual hybrids. In other crop genera, intraspecific somatic hybridization has been used to overcome various breeding barriers. Koyama et al. (1995) used protoplast fusion to regenerate plants from a carrot (Daucus carota L.) cell line, selected for utilization of Al-phosphate, that had been maintained in vitro for over 10 years and had lost regenerative capacity. Protoplast fusion of this cell line with IOA-inactivated wild-type carrot resulted in regenerable somatic hybrid calluses that were tolerant of Alphosphate. Tamura et al. (1995) fused protoplasts of two hexaploid (2n = 6x = 90) cultivars of Japanese persimmon (Diospyros kaki L.) that produced only female flowers to obtain dodecaploid (2n = 12x = 180) somatic hybrids. Sexually incompatible cultivars of hexaploid sweet potato [Ipomoea batatas (L.) Lam.] were also combined into dodecaploid (2n = 12x = 180) somatic hybrids estimated to have limited pollen fertility (Wang et al. 1997). Protoplasts of two cultivars of taro (Colocasia esculenta Schott) have been electrofused to develop autotetraploid somatic hybrids (Murakami et al. 1998). The utility of these highly polyploid intraspecific somatic hybrids has yet to be demonstrated. It may well depend on the efficacy of chromosome reduction procedures such as anther culture or pseudogamy in these species to generate useful variation for breeding programs. B. Interspecific Somatic Hybrids The majority of somatic hybridization has been conducted to obtain interspecific somatic hybrids (Table 6.2). Occasionally sexual hybrids are possible, but generally the objective is to transcend breeding barriers.

176 SH SH SH SH SH

SH SH

SH

SH, CY SH, CY CY

SH, CY SH SH, CY

Electrofusion Chemical n.s.x n.s. n.s.

Chemical Chemical

Electrofusion

Electrofusion Electrofusion Chemical

Electrofusion n.s. Electrofusion

[Citrus reticulata Blanco × C. paradisi Macf.] + C. jambhiri Lush C. aurantium L. + C. limonia Osb. [C. jambhiri × C. sinensis L. Osb.] + C. reticulata 11 different Citrus somatic hybrids 15 different Citrus somatic hybrids C. unshiu Marc. cv. Hayashi + C. sinensis C. paradisi Macf. + C. sinensis [C. reticulata × C. sinensis] + C. sinensis C. junos Sieb. ex Tanka + C. sinensis C. jambhiri + C. sinensis C. sinensis + C. ichangensis C. sinensis + C. reticulata C. sinensis + [C. paradisi × C. reticulata] C. sinensis + [C. reticulata × C. sinensis] C. sinensis + [C. reticulata × C. paradisi] C. unshiu Marc. + haploid C. clementina hort. ex Tanka C. sinensis + haploid C. clementina C. sudachi Hort. + C. aurantifolia Swingle C. sudachi + C. limon Burn. C. aurantifolia + C. limon C. microcarpa Bunge + C. aurantium L. C. reticulata + C. sinensis C. sinensis + C. limon Six different Citrus somatic hybrids C. sinensis + C. limon [C. reticulata × C. paradisi] + C. jambhiri

Rutaceae

Type of hybridsz

Protocol

Fusion partners

Interspecific somatic hybrids of crop plants (1993–1999).

Family

Table 6.2.

RFLP n.s. RFLP

RFLP RFLP, isozymes

RFLP

RAPD

RAPD, isozymes

n.s.

Moriguchi et al. 1996 Fatta Del Bosco 1998 Moriguchi et al. 1997

Saito et al. 1994 Grosser et al. 1996

Saito et al. 1993

Kobayashi et al. 1997

Moura Fo et al. 1996

Deng et al. 1995

Grosser et al. 1998b Grosser et al. 1998a Kobayashi et al. 1995

Grosser et al. 1994

Guo et al. 1998

Reference

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VNTR-PCR RAPD n.s.

isozymes

RAPD, isozymes

Verificationy

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Solanaceae

S. tuberosum + S. brevidens S. tuberosum + S. pinnatisectum Dun. S. tuberosum + S. pinnatisectum S. tuberosum + S. pinnatisectum S. tuberosum + [S. pinnatisectum × S. brevidens] S. tuberosum + S. nigrum L. S. tuberosum + S. chacoense S. tuberosum + S. bulbocastanum Dun. S. tuberosum + S. bulbocastanum S. tuberosum + S. bulbocastanum S. etuberosum Lindl. + [S. tuberosum × S. berthaultii Hawkes] S. tuberosum + S. papita Rydb. S. tuberosum + S. commersonii Dun. S. tuberosum + S. commersonii S. tuberosum + S. commersonii S. tuberosum + S. phureja Juz. & Buk. S. tuberosum + S. megistacrolobum Bitt. S. tuberosum + S. sanctae-rosae Hawkes S. tuberosum + S. sparsipilum (Bitt.) Juz. & Buk. S. khasianum + S. aculentissimum S. melongena L. + S. sanitwongsei Lycopersicon esculentum Mill. + S. tuberosum L. esculentum + L. hirsutum Humb. and Bonpl.

S. tuberosum L. + S. brevidens Phil. S. tuberosum + S. brevidens S. tuberosum + S. brevidens SH SH SH SH

SH SH SH SH SH SH SH SH SH SH SH SH

SH SH SH SH

Electrofusion Electrofusion n.s. n.s. Chemical n.s. Electrofusion n.s. n.s. Electrofusion Electrofusion Electrofusion

Electrofusion Electrofusion n.s. Chemical

SH SH SH

Electrofusion n.s. Chemical, Electrofusion Chemical Electrofusion Electrofusion Electrofusion

GISH isozymes

isozymes RAPD

RFLP, RAPD n.s. n.s. n.s. RAPD RFLP

(continued)

Wolters et al. 1994 Jourdan et al. 1993

Stattmann et al. 1994 Asao et al. 1994

Kaendler et al. 1996 Nyman and Waara 1997 Laferrière et al. 1999 Cardi 1998 Craig et al. 1994 Harding and Millam 1999

Horsman et al. 1997, 1999 Cheng et al. 1995 Mojtahedi et al. 1995 Brown et al. 1996 Helgeson et al. 1998 Novy and Helgeson 1994

Polgár et al. 1996 Ward et al. 1994 Menke et al. 1996 Thieme et al. 1997

Valkonen et al. 1994b Rokka et al. 1994, 1995 Jacobsen et al. 1993

9/26/2000 1:19 PM

n.s. isozymes n.s. n.s. RFLP, RAPD RFLP

RFLP RFLP, isozymes RFLP RAPD

RAPD RAPD isozymes

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177

178

B. oleracea + B. rapa B. oleracea + B. rapa B. oleracea var. italica + B. rapa B. napus L. + B. oleracea [B. tournefortii Gouan × B. oleracea] + B. nigra (L.) Koch B. tournefortii + B. napus

L. esculentum + L. peruvianum (L.) Mill. L. esculentum + L. peruvianum Nicotiana plumbaginigolia Viv. + N. sylvestris Speg. & Comes N. tabacum L. + N. sanderae Hort. N. tabacum + N. benthamiana Domin. N. tabacum + N. glutinosa L. N. tabacum + N. megalosiphon Van Heurck & Mull. Arg N. tabacum + N. sylvestris N. tabacum + N. debneyi Domin. N. tabacum + N. rustica L. N. tabacum + N. rustica N. tabacum + N. plumbaginifolia Hyoscyamus muticus L. + H. albus H. muticus + H. albus Petunia hybrida + P. variabilis Brassica oleracea L. + B. rapa L. B. oleracea + B. rapa

Solanaceae (cont.)

SH

Chemical

SH SH SH SH SH SH SH SH SH

Chemical Chemical Electrofusion Chemical Chemical n.s. Electrofusion n.s. Chemical SH SH SH SH SH

SH SH SH SH

Chemical Electrofusion Chemical Chemical

Chemical Chemical Chemical Chemical Chemical

SH SH SH

Type of hybridsz

n.s. n.s. Chemical

Protocol

RFLP, isozymes

RFLP, RAPD RFLP, isozymes RFLP, isozymes RFLP, isozymes RFLP, RAPD

RFLP, isozymes RFLP, isozymes RAPD isozymes isozymes n.s. RFLP, isozymes n.s. isozymes

isozymes RFLP RFLP, isozymes RFLP, isozymes

n.s. n.s. RFLP

Verificationy

Liu et al. 1995a

Sproule et al. 1991 Donaldson et al. 1993 De Filippis et al. 1996 Desprez et al. 1995 Rahman et al. 1994 Zehra et al. 1998 Taguchi et al. 1993 Celis and Jourdan 1993 Ozminkowski and Jourdan 1994a,b Hansen and Earle 1994 Heath and Earle 1995, 1997 Heath and Earle 1996 Landgren et al. 1994 Mukhopadhyay et al. 1994

Dragoeva et al. 1997b Hagimori et al. 1993 Donaldson et al. 1994 Donaldson et al. 1995

Brüggemann et al. 1995 Sakata and Monma 1993 Hung et al. 1993

Reference

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Brassicaceae

Fusion partners

(continued)

Family

Table 6.2.

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SH SH SH

n.s. Chemical Chemical n.s. Chemical Electrofusion Electrofusion Electrofusion

Electrofusion

Senecio fuchsii Gmel. + S. jacobaea L. A. ampeloprasum + A. cepa Asparagus officinalis L. + A. macowanii

Iris ensata Thumb. + I. germanica L. Cucumis melo L. + C. myriocarpus Naud.

C. melo + C. anguria L. var. longipes (Hook. fil.) Dianthus chinensis L. + D. caryophyllus L. D. chinensis + D. barbatus

Liliaceae

Iridaceae Cucurbitaceae

Caryophyllaceae

Asteraceae

Chemical

SH SH SH

Electrofusion n.s. Chemical

SH

SH

SH SH

isozymes, RFLP RFLP, GISH isozymes, RAPD, RFLP RAPD RFLP, PCR amplification of rDNA PCR amplification of rDNA RFLP, RAPD, isozymes

isozymes

RAPD

RAPD

RFLP, RAPD RFLP RAPD

RFLP, isozymes RFLP

isozymes

RAPD RAPD

(continued)

Nakano and Mii 1993a,b

Dabauza et al. 1998

Shimizu et al. 1999 Bordas et al. 1998

Krasnyanski and Mencze 1995 Wang and Binding 1993 Buiteveld et al. 1998a,b Kunitake et al. 1996

Henn et al. 1998

Maisonneuve et al. 1995

Crea et al. 1997 Pupilli et al. 1995 Chupeau et al. 1994

Nenz et al. 1996 Cluster et al. 1996

Yamagishi et al. 1994

Hansen and Earle 1995 Ryschka et al. 1996

9/26/2000 1:19 PM

SH SH SH

SH SH SH

n.s. Electrofusion Electrofusion

Fabaceae

SH SH

n.s. Chemical

B. napus + B. oleracea B. oleracea + B. carinata Braun B. oleracea + Sinapis alba L. B. oleracea + B. campestris L. Medicago sativa L. + M. arborea L. M. sativa + M. arborea L. M. sativa + M. coerulea M. sativa + M. falcata L. M. sativa + M. falcata M. sativa + M. coerulea Lactuca sativa L. + L. tatarica (L.) C.A. Meyer L. sativa + L. perennis L. sativa L. + L. tatarica L. sativa + L. perennis H. annuus + H. giganteus H. annuus + H. maximiliani H. annuus + H. giganteus

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Passiflora edulis f. flavicarpa Degener + P. incarnata P. edulis var. flavicarpa + P. alata Ait. P. edulis var. flavicarpa + P. amethystina Mikan P. edulis var. flavicarpa + P. cincinnata Mast. P. edulis var. flavicarpa + P. giberti N.E. Brown P. edulis var. flavicarpa + P. coccinea Aubl. P. edulis var. flavicarpa + P. amethystina Diospyros glandulosa + D. kaki cv. Jiro Mentha piperita L. + M. gentilis L. M. piperita + M. spicata L.

Passifloraceae SH

SH SH SH SH

Chemical

Chemical Electrofusion Electrofusion Chemical

n.s. RAPD, PCR-RFLP RAPD RAPD, RFLP

isozymes

RAPD, isozymes

Verificationy

Barbosa and Vieira 1997 Tamura et al. 1998 Sato et al. 1996 Krasnyanski et al. 1998

Dornelas et al. 1995

Otoni et al. 1995

Reference

y

SH = somatic hybrid; CY = cybrid. RAPD = randomly amplified polymorphic DNA; VNTR = variable number of tandem repeats-PCR; PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism; GISH = genomic in situ hybridization. x n.s. = not specified in journal.

z

SH

Type of hybridsz

Electrofusion

Protocol

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Ebenaceae Laminaceae

Fusion partners

(continued)

Family

Table 6.2.

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1. Rutaceae. The cultivated Citrus fruits consist of eight principal horticultural groups that are mostly interfertile (Cameron and Soost 1976). Much research on interspecific hybridization in Citrus has been carried out with the intention of creating new rootstocks for use in commercial cultivation of various Citrus species, or for the production of tetraploid clones that could be crossed to diploid species for the production of seedless, thin-rinded, easy to peel, triploid cultivars. The parameters for protoplast fusion of Citrus species have been well-described (Grosser and Gmitter 1990; Guo et al. 1998), and reports of somatic hybrid production are numerous. Grosser et al. (1994, 1998b) have produced many different allotetraploid somatic hybrids by fusion of various Citrus species. These somatic hybrids are intended to serve as widely adapted rootstocks displaying both the desirable horticultural characteristics and disease resistances of the fusion partners. Using allotetraploids as the pollen donor, Grosser et al. (1998a), Kobayashi et al. (1995), Deng et al. (1996) and Mourao Fo et al. (1996) produced triploid Citrus cultivars through pollination of diploid Citrus species. Allotetraploid somatic hybrids may be cytologically unstable, however, thus affecting their fertility. On further analysis of presumed allotetraploid somatic hybrids between rough lemon (Citrus jambhiri Lush.) and Ohta ponkan (C. reticulata Blanco) using differential chromosome staining, Miranda et al. (1997a) found many aneusomatic somatic cells in shoots. Kobayashi et al. (1995) observed pollen germinability rates ranging from 9.9–27% among four different Citrus interspecific somatic hybrids. One of the somatic hybrids (Murcot tangor + Frost Washington navel orange) set fruit on the diploid ‘Clementine’ mandarin and produced eleven vigorously growing triploids. Notably, the sterile navel orange (C. sinensis Osb. var. brasiliensis Tanka cv. Frost Washington) was used as a fusion partner in the production of the four partially fertile somatic hybrids described by Kobayashi et al. (1995). Thus somatic hybridization may serve as a “bridge” to allow the genome of sterile cultivars such as the navel orange to be used in Citrus breeding programs. Side-stepping sexual crosses between allotetraploids and diploids to produce triploid Citrus clones, Kobayashi et al. (1997) fused protoplasts from a haploid clementine (C. clementine hort. ex Tanka) to protoplasts from three different diploid Citrus species to produce triploid somatic hybrids. Most research in Citrus interspecific somatic hybridization has focused on fusing nucellar-derived protoplasts to mesophyll protoplasts (Saito et al. 1993; Saito et al. 1994; Grosser et al. 1996a; Moriguchi et al. 1996, 1997). Citrus allotetraploids and cybrids were often obtained. The allotetraploids contained complete genomes from both fusion partners,

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while the cybrids contained one complete genome from the mesophyll parent and the mitochondrial genome of the nucellar fusion parent, leading to speculation that mitochondria of nucellar-derived protoplasts influence embryogenesis of somatic hybrids in Citrus. Despite the wide use of protoplast fusion technology in Citrus and frequent production of somatic hybrids, few field evaluations have been published at this time due to the long juvenility and many years required for meaningful yield data to be obtained. Deng et al. (1995) evaluated two interspecific Citrus somatic hybrids for agronomic performance and found low fertility, with 95% of the flowers pistil abortive and only 6% pollen germinability. In addition, cold-hardiness did not appear to be transferred from cold-tolerant Citrus species to the somatic hybrids. Fatta Del Bosco et al. (1998) found that a somatic hybrid between ‘Valencia’ sweet orange (C. sinensis L. Osbeck) and ‘Femminello’ lemon (C. limon L. Burn f.) had larger fruit than the fusion parents and larger leaves with fewer oil glands per unit area. 2. Solanaceae. Even when tuber-bearing species have similar chromosome numbers, a difference in the endosperm balance number (EBN) effectively prohibits sexual crosses. Inter-EBN hybrids have been the focus of somatic hybridization in order to access previously inaccessible germplasm in potato breeding (Ortiz and Erlenfeldt 1992). Several somatic hybrids have been created through protoplast fusion between potato (dihaploid S. tuberosum) and the wild diploid species, S. brevidens Phil. The objective of most such fusions has been the transfer of viral, bacterial, and fungal resistance from S. brevidens to the S. tuberosum gene pool. Valkonen et al. (1994b) produced tetraploid and hexaploid somatic hybrids demonstrating extreme resistance to PLRV and PVYo and moderate resistance to PVX. Rokka et al. (1994) produced similar hybrids and found that only hexaploid hybrids were fertile. Progeny generated through crosses to S. tuberosum cv. Matilda demonstrated PVYN resistance equivalent to that of the S. brevidens parent. Jacobsen et al. (1993) found that tetraploid somatic hybrids set more seed; however, hexaploid hybrids had higher seed germination. Rokka et al. (1995) determined that hexaploid somatic hybrids responded to anther culture and produced triploid regenerants that retained high resistance to PLRV. Polgár et al. (1996) generated S. tuberosum + S. brevidens somatic hybrids that were female fertile and backcrossed them to four different S. tuberosum genotypes. Eleven of 28 progeny were highly resistant to bacterial soft rot (Erwinia carotovora ssp. carotovora). Three somatic hybrids regenerated from the same callus differed in their resistance to soft rot; the least resistant of the three was found to have lost specific

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RAPD markers associated with portions of S. brevidens chromosomes (Polgár et al. 1999). Bains et al. (1999) reported that the stem and tuber soft rot resistance obtained by somatic hybridization between S. tuberosum cv. Katahdin and S. brevidens was transferred to sexual progeny of the somatic hybrid. Solanum pinnatisectum Dun. has been viewed as a potential source of late blight resistance in potato but it is incompatible with S. tuberosum. Ward et al. (1994) generated four mixoploid somatic hybrids, whereas Menke et al. (1996) produced 155 somatic hybrids, of which 75% were tetraploid, through protoplast fusion of dihaploid S. tuberosum and S. pinnatisectum. Thieme et al. (1997) produced tetraploid, hexaploid, and mixoploid somatic hybrids through a similar fusion procedure. Some hybrids were more resistant to late blight than S. tuberosum; however, none expressed resistance equivalent to that of the wild parent. The somatic hybrids were sterile. Fusion between dihaploid S. tuberosum and a triploid S. pinnatisectum × S. bulbocastanum Dun. sexual hybrid resulted in pentaploid somatic hybrids demonstrating intermediate late blight resistance; these hybrids set seed when backcrossed as the female parent to S. tuberosum (Thieme et al. 1997). Resistance to late blight has also been sought by somatic hybridization of potato with the black nightshade, S. nigrum (Horsman et al. 1997, 1999). Of 761 somatic hybrids obtained by fusions between four species within the S. nigrum complex and either dihaploid or tetraploid potato, only 60 were sufficiently vigorous to attain flowering in the greenhouse (Horsman et al. 1997). After backcrossing an octoploid somatic hybrid comprised of 6x S. nigrum + 2x S. tuberosum with pollen of tetraploid S. tuberosum, Horsman et al. (1999) obtained a single flowering plant by ovule culture (4,362 pollinations for 505 cultured ovules for two plants, only one of which flowered). Heroic efforts were again needed to proceed from this single BC1 plant to obtain BC2 progeny (5,474 pollinations for 3,065 cultured ovules for nine plants, all of which were less vigorous than the BC1 plant). Although the six of eight BC2 plants that were tested exhibited resistance to late blight similar to the S. nigrum parent, they were as infertile as the original somatic hybrid. Potato has been fused to various other wild Solanum species. Cheng et al. (1995) transferred leptines from S. chacoense to S. tuberosum + S. chacoense somatic hybrids. In a feeding experiment, Colorado potato beetle (Leptinotarsa decemlineata Tsay) consumed three-fold greater leaf mass of potato than of the somatic hybrids or S. chacoense. Mojtahedi et al. (1995) produced fertile somatic hybrids between potato and S. bulbocastanum in an attempt to transfer resistance to Meloidogyne chitwoodi race 1 to the S. tuberosum gene pool. Brown et al. (1996) analyzed

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second backcross progeny, derived from a S. tuberosum (tetraploid) + S. bulbocastanum somatic hybrid, that segregated for resistance to M. chitwoodi to map the resistance to one end of potato chromosome 11. Novy and Helgeson (1994) transferred PVY resistance to fertile tetraploid somatic hybrids through fusion of a S. tuberosum × S. berthaultii Hawkes sexual hybrid to S. etuberosum Lindl, which were then backcrossed to the S. tuberosum cvs. Atlantic and Katahdin. The majority of somatic hybrids and some progeny had significantly higher PVY resistance than the S. tuberosum cultivars. Kaendler et al. (1996) fused dihaploid S. tuberosum to S. papita Rydb. and generated fertile tetraploid somatic hybrids that were backcrossed to S. tuberosum cvs. Datura and Desiree. Nyman and Waara (1997) fused dihaploid S. tuberosum to S. commersonii Dun. in an attempt to increase cold tolerance of potato. The tetraploid hybrids appeared fertile and some had a lower lethal temperature than S. tuberosum. Laferrière et al. (1999) also produced somatic hybrids between S. tuberosum and S. commersonii in order to transfer resistance to bacterial wilt (Ralstonia solanacearum) from S. commersonii to cultivated potato. Five of six of the near tetraploid somatic hybrids had bacterial wilt resistance levels equal to that of the S. commersonii parent. Cardi (1998) examined 56 S. tuberosum + S. commersonii somatic hybrids by multivariate analysis and found that their morphology was closer to that of S. tuberosum than S. commersonii, suggesting that the somatic hybrids could be useful in a potato breeding program. The somatic hybrids were fertile and could be backcrossed to cultivated potato. After acclimation, several of them showed freezing tolerance comparable to that of S. commersonii (Chen et al. 1999). Resistance to blackleg and tuber soft rot (Erwinia carotovora subsp. atroseptica and E. carotovora subsp. carotovora) was demonstrated in tetraploid and hexaploid somatic hybrids between dihaploid potato and S. commersonii (Carputo et al. 1997). The fertility of these and similar somatic hybrids studied by Carputo et al. (1998) demonstrated the usefulness of somatic hybridization to access genes in germplasm that differs in EBN number from potato. The fate of alien germplasm in potato somatic hybrids and their progeny has been followed by observing species-specific molecular markers. Craig et al. (1994) used RAPDs to demonstrate that specific S. phureja chromosomes were absent in S. tuberosum + S. phureja somatic hybrids. Similarly, Masuelli et al. (1995) found that several S. bulbocastanumspecific SCAR (sequence characterized amplified regions) markers were lost on backcrossing a hexaploid somatic hybrid to tetraploid potato due to abnormal meiosis. By comparison, most S. brevidens-specific RFLP markers were retained after backcrossing a hexaploid somatic hybrid

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with potato to tetraploid potato (Williams et al. 1993). Analysis of rDNA loci of progeny of hexaploid S. tuberosum + S. brevidens somatic hybrids using fluorescence in situ hybridization (FISH) revealed structural changes in rDNA-carrying chromosomes (McGrath and Helgeson 1998). The authors suggested that asynchrony of the cell cycle of somatic hybrids allowed the opportunity for recombination leading to an isochromosome of S. brevidens. Tomato (Lycopersicon esculentum Mill.) has been fused to various wild species for the transfer of desirable traits to the tomato gene pool (Wolters et al. 1994a). Jourdan et al. (1993) produced tetraploid somatic hybrids contained L. esculentum and L. hirsutum Humb. and Bonpl. to increase the cytoplasmic diversity of cultivated tomato. All somatic hybrids contained L. hirsutum cpDNA and a mix of mtDNA of both fusion parents. Brüggemann et al. (1995) fused L. esculentum to L. peruvianum (L.) Mill. and found that the somatic hybrids were equal to or superior to L. peruvianum in terms of chilling tolerance. Sakata and Monma (1993) produced similar tetraploid somatic hybrids and found that most progeny obtained through backcrosses to cultivated tomato were aneuploid. Sybenga et al. (1994) conducted a cytogenetic analysis of pachytene pairing of a similar tetraploid somatic hybrid and found little differentiation in pairing affinity between the genomes, despite considerable molecular differentiation. Cultivated tobacco has been fused to a variety of wild species to incorporate disease resistances and for the creation of novel plasmons. All recent interspecific fusions have utilized complementation of transgenic resistance (such as kanamycin and methotrexate) to regenerate somatic hybrids. Dragoeva et al. (1997b) used N. tabacum as a “universal hybridizer,” a protoplast donor possessing both a dominant and recessive trait, to create somatic hybrids between N. tabacum and N. sanderae. N. tabacum was kanamycin resistant and nitrate reductase deficient, so regeneration on kanamycin-containing medium enabled only hybrid calluses and plants to grow. N. sanderae is resistant to tomato spotted wilt virus (TSWV) through a hypersensitive response, and all somatic hybrids were similarly resistant to TSWV upon inoculation with the virus. Hagimori et al. (1993) produced a universal hybridizer by treating kanamycin-resistant N. benthamiana protoplasts with IOA to prevent division. Culture of heterokaryons from fusion of N. benthamiana and N. tabacum protoplasts on kanamycin-containing medium enabled only somatic hybrid calluses and plants to develop. Aphids (Myzus persicae Sulzer) feeding on N. benthamiana generally die within 3 days in feeding assays; those feeding on somatic hybrid leaves died within 4–7 days.

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Donaldson et al. (1994) created 70 allohexaploid somatic hybrids through fusion of methotrexate resistant N. tabacum and kanamycin resistant N. glutinosa. Of 41 hybrids examined, most had either the N. glutinosa or N. tabacum cpDNA, whereas all but two hybrids had rearranged mtDNA, indicating recombination between the two parental mitochondrial genomes. Donaldson et al. (1995) used methotrexate and kanamycin resistance to regenerate somatic hybrids between N. tabacum and N. megalosiphon. Most hybrids had N. megalosiphon cpDNA and rearranged mtDNA. Sproule et al. (1991) also used methotrexate and kanamycin resistance to produce N. tabacum + N. debneyi somatic hybrids. All but one somatic hybrid had the N. debneyi cpDNA and all had the N. debneyi mtDNA. N. debneyi cytoplasm causes male-sterility in cybrids with N. tabacum; however, these somatic hybrids were fertile and backcrossed to N. tabacum. The authors speculated that an interaction between N. debneyi nuclear and mtDNA may be necessary for fertility. A somatic hybrid between N. tabacum cv. Delgold and N. debneyi Domin. has been used to develop promising advanced germplasm with resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Bai et al. 1996). Donaldson et al. (1993) used the same transgenic complementation strategy to regenerate N. tabacum + N. rustica somatic hybrids. Nearly all hybrids had the N. rustica cpDNA and rearranged mtDNA. Desprez et al. (1995) fused binucleate kanamycinresistant pollen protoplasts of N. tabacum to N. plumbaginifolia mesophyll protoplasts (gametosomatic hybridization). Seventeen somatic hybrids containing the N. plumbaginifolia cpDNA were obtained and one was successfully backcrossed to N. tabacum. Chloroplast segregation was found to be 1:1 after somatic hybridization between haploids of N. plumbaginifolia and N. sylvestris regardless of the ploidy or genomic constitution of the hybrids (Hung et al. 1993). A few interspecific somatic hybridization efforts in Solanum have involved crops other than potato, tomato, and tobacco. Asao et al. (1994) fused eggplant (S. melongena L.) to S. sanitwongsei to transfer Psedomonas solanacearum resistance to cultivated eggplant. The tetraploid somatic hybrids were self-fertile and produced offspring (S1). The S1 progeny served as better rootstocks for eggplant than S. sanitwongsei. Stattmann et al. (1994) produced tetraploid somatic hybrids between S. khasianum and S. aculeatissimum as part of an effort to increase solasodine levels in solanaceous crops. To overcome sexual incompatibility between Hyoscyamus muticus and H. albus, Rahman et al. (1994) produced amphidiploid somatic hybrids. The hybrids were fertile and set seed when selfed or backcrossed to either parent. For similar objectives, Taguchi et al. (1993) combined the genomes of Petunia hybrida,

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one of the most popular ornamental flowers, with its wild relative P. variabilis through protoplast fusion. The resulting amphidiploid hybrids produced seed when selfed or backcrossed to the P. variablis parent. 3. Brassicaceae. The cultivated brassicaceous crops include the mustards [Brassica juncea L. Coss (brown mustard), B. nigra Koch (black mustard), B. carinata Braun (Ethiopian mustard), and Sinapis alba L. (white mustard)], the cole crops [B. oleracea L. var. gemmifera (Brussels sprout), var. acephala (kale), var. capitata (cabbage), var. italica (broccoli), var. gongyloides (kohlrabi), and var. botrytis (cauliflower)], the swedes and rapes (B. napus L.), turnip (B. rapa L.) and Chinese cabbages (B. campestris L.), radish (Raphanus sativus L.), and horseradish (Armoracia rusticana Gaertn., Mey., & Scherb.), among others. In addition, there is a wealth of related, uncultivated species. Rapeseed (B. napus), a naturally occurring amphidiploid believed to have resulted from hybridization between B. oleracea and B. rapa L., is one of the most important oilseed crops in temperate climates. Several recent interspecific fusions in Brassica have focused on the “resynthesis” of B. napus through protoplast fusion of B. oleracea and B. rapa to increase genetic diversity of this important crop and to alter oil content. Direct comparisons between resynthesized sexual and somatic B. napus hybrids have been hindered by regeneration of highly polyploid somatic hybrids (Heath and Earle 1996). Celis and Jourdan (1993) produced partially male fertile B. napus through fusion of CMS B. oleracea and fertile B. rapa protoplasts. Ozminkowski and Jourdan (1994a,b) reconstructed B. napus allotetraploid hybrids both by sexual crosses and by somatic fusion; they found that somatic hybrids could be obtained faster because the chromosome doubling stage could be omitted. The majority of somatic hybrids (46 of 58) had the B. rapa cpDNA, ten had both chloroplast types (heteroplastidic), and two hybrids had B. oleracea cpDNA. Most somatic hybrids had the B. rapa mitochondrial DNA. Hansen and Earle (1994) developed self-fertile B. oleracea + B. rapa somatic hybrids all of which had the cpDNA of B. rapa. Heath and Earle (1997) attempted production of B. napus by fusing B. oleracea and B. rapa accessions low in linolenic acid. Some of the fertile somatic hybrids had linolenic acid levels lower than the midparent value for the two fusion partners. As with the previously described studies showing preferential transmission of B. rapa organelles, all hybrids possessed the B. rapa cpDNA. Somatic hybrid organellar segregation has been studied in other Brassica interspecific fusions. Landgren et al. (1994) fused hypocotyl and mesophyll protoplasts derived from B. napus and B. oleracea to determine

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if cell type influenced mitochondrial segregation in somatic hybrids. Regardless of cell type, most of the resulting somatic hybrids possessed the mtDNA of the B. napus parent. Of somatic hybrids resulting from protoplast fusion of a B. tournefortii Gouan × B. oleracea sexual hybrid with B. nigra, Mukhopadhyay et al. (1994) found that the hybrids possessed cpDNA of one or the other fusion parent, while the mtDNA showed novel patterns not present in either parent. Liu et al. (1995a) reported that 61% of B. napus + B. tournefortii somatic hybrids had B. tournefortii cpDNA (the remaining had B. napus cpDNA). Some novel mtDNA patterns were observed in these hybrids. Interspecific protoplast fusion has been used in Brassica to transfer disease resistance and other desirable traits from wild to cultivated species. Liu et al. (1995a) transferred blackleg [Phoma lingam (Tode ex. Fr.) Desm.] resistance from B. tournefortii to B. napus through protoplast fusion; 75% of the resulting somatic hybrids were resistant when inoculated with blackleg pycnospores. Hansen and Earle (1995) fused B. napus and B. oleracea protoplasts in order to transfer resistance to the bacterium Xanthomonas campestris pv. campestris to B. oleracea. Resistant somatic hybrids resulted that were subsequently used to develop highly resistant BC1 and BC2 progeny upon backcrosses to B. oleracea. Ryschka et al. (1996) produced somatic hybrids between B. oleracea and B. carinata A. Braun that demonstrated resistance to Alternaria brassicae Berk. and P. lingam but weak resistance to A. brassicicola. In an attempt to develop new Brassica vegetables adapted to tropical areas of the world, Yamagishi et al. (1994) produced somatic hybrids between a heat-tolerant cabbage (B. oleracea) and a chinese cabbage cultivar (B. campestris var. pekinensis L.). The amphidiploid somatic hybrids displayed greater fertility (both pollen and seed set) than corresponding sexual hybrids. 4. Fabaceae. Medicago arborea L. has traits of interest, such as drought tolerance and resistance to bacterial wilt and anthracnose, that may be introgressed into cultivated alfalfa, M. sativa L.) through somatic hybridization. Nenz et al. (1996) regenerated somatic hybrids 1 year after fusion between M. sativa mesophyll protoplasts and callus-derived M. arborea protoplasts. Cluster et al. (1996) fused M. sativa to three different species (M. coerulea, M. arborea, and M. falcata) for an analysis of the genetic makeup of Medicago interspecific somatic hybrids using rDNA markers in RFLP analysis. Hybrids from all three combinations showed loss of some markers, even when ploidy was the total of the parents, indicating small deletions or rearrangements of rDNA in the somatic hybrids. Crea et al. (1997) found evidence of chromosomal

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translocation in hyperaneuploid somatic hybrids obtained through protoplast fusion between tetraploid M. sativa and diploid M. falcata. Pupilli et al. (1995) created hexaploid somatic hybrids by fusing M. sativa mesophyll protoplasts to M. coerulea callus protoplasts. RFLP analysis showed that many of the M. coerulea markers were absent in the somatic hybrids. The hexaploid somatic hybrids were fertile and segregation of M. coerulea RFLP markers (that were present in the somatic hybrids) to progeny was normal. 5. Asteraceae. Chupeau et al. (1994) developed a universal hybridizer of lettuce (Lactuca sativa L.) first by selecting single gene recessive albino mutants after ethyl-methane sulphonate treatment of seeds of cv. Girelle. Then, heterozygous albinos were crossed with cv. Ardente that had been transformed for kanamycin resistance. After the hybrids had been selfed, albino kanamycin resistant seedlings could be grown in vitro to be used as universal hybridizers. Protoplasts from the universal hybridizer were subsequently fused with those of three Lactuca species and five distantly related genera within Asteraceae. Only fusions with L. tatarica and L. perennis resulted in somatic hybrids. Limited fertility of the L. tatarica fusions permitted backcrossing with lettuce. Maisonneuve et al. (1995) reported that the hybrids carried downy mildew resistance transferred from L. tatarica. In other Asteraceae, protoplast fusion between sunflower (Helianthus annuus L.), Helianthus giganteus L., and H. perennis has resulted in somatic hybrids of limited fertility (Krasnyanski and Menczel 1995). Henn et al. (1998) produced H. annuus + H. giganteus and H. annuus + H. maximiliani somatic hybrids for the eventual goal of transferring Sclerotinia resistance to cultivated sunflower. Somatic hybrids and cybrids were obtained from protoplast fusion between Senecio fuchsii Gmel. and S. jacobaea L. (Wang and Binding 1993). 6. Liliaceae. Within liliaceous crop plants, somatic hybrids have been obtained for both Asparagus and Allium. Kunitake et al. (1996) fused protoplasts of Asparagus officinalis L. with those of A. macowanii, a wild species with resistance to asparagus decline, caused by several fungal pathogens. Many somatic hybrid plants were obtained with 2n = 50, in contrast to the expected 2n = 40 from adding the genomes of both parents; however, the hybrids could not be grown to maturity in the greenhouse. Buiteveld et al. (1998b) produced somatic hybrids between leek, Allium ampeloprasum L., and onion, A. cepa L. These sexually incompatible species resulted in aneuploid somatic hybrids (2n = 41–45) rather than the additive number of the two parents (48). As opposed to

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the Asparagus somatic hybrids, however, their growth ex vitro was more normal and their morphology intermediate between the parents. Buiteveld et al. (1998a) found novel cytoplasm with mtDNA rearrangements and new combinations of organelles in these somatic hybrids that may be useful in breeding programs. 7. Iridaceae. Shimizu et al. (1999) reported the regeneration of both symmetric and asymmetric somatic hybrids between the sexually incompatible Iris ensata Thunb. and I. germanica L. to improve scent and increase flower color variation of I. ensata. 8. Cucurbitaceae. Bordas et al. (1998) produced somatic hybrids between Cucumis melo L. and C. myriocarpus Naud. Several C. myriocarpus RFLP markers were absent in the somatic hybrids, indicating selective genome elimination, and the stunted hybrids could not be established as mature plants in the greenhouse. Similarly, Dabauza et al. (1998) produced two somatic hybrids between C. melo and C. anjuria L. var. longipes (Hook. Fil.) that failed to form roots and could not be established as mature plants. 9. Caryophyllaceae. Protoplast fusion may be used to produce new ornamental cultivars of Dianthus. Nakano and Mii (1993a) fused D. chinensis L. and D. caryophyllus L. protoplasts to produce vigorous allotetraploid somatic hybrids that were established and flowered in the greenhouse. Dianthus chinensis and D. barbatus protoplasts were also fused but this combination resulted in a less vigorous aneuploid somatic hybrid (Nakano and Mii 1993b). 10. Passifloraceae. Cultivated passionfruit (Passiflora edulis f. flavicarpa Degener) is considered the most economically important species in the Passiflora genus. Otoni et al. (1995) produced allotetraploid somatic hybrids between P. edulis f. flavicarpa and P. incarnata in order to transfer cold tolerance and other desirable traits from P. incarnata to cultivated passionfruit. The hybrids produced seed when backcrossed to either parent. Dornelas et al. (1995) obtained allotetraploid somatic hybrids through fusion of P. edulis var. flavicarpa and five different Passiflora species. Barbosa and Vieira (1997) studied the meiotic behavior of P. edulis f. flavicarpa + P. amethystina Mikan somatic hybrids and found them to have high pollen viability and a large number of quadrivalents in microsporocytes, indicating recombination between chromosomes of the parental species.

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11. Ebenaceae. Tamura et al. (1998) produced allooctoploid somatic hybrids between the diploid Diospyros glandulosa and the hexaploid D. kaki cv. Jiro. The interspecific hybrids possessed the chloroplast genome of D. glandulosa. 12. Laminaceae. Mints are economically important for the production of essential oils that are used worldwide for food flavorings. Unfortunately, most mints are sterile, making interspecific crosses between them impossible. Protoplast fusion has been explored as a means of circumventing mint sterility and producing new cultivars. Sato et al. (1996) used protoplast fusion between the sterile, triploid mints Mentha piperita L. cv. Blackmint and M. gentilis L. cv. Variegata to produce six allohexaploid somatic hybrids. One of the hybrids expressed all three of the essential oils present in the parents: menthone, menthal, and linalool. Krasnyanski et al. (1998) fused the triploid M. spicata L. cv. Native Spearmint to the tetraploid M. piperita L. cv. Black Mitcham to transfer resistance to the fungus Verticillium dahliae from Black Mitcham to Native spearmint. Verticillium resistance was not expressed in the somatic hybrids and the authors concluded that somatic hybridization was not suitable for transferring this trait between mint species. C. Intergeneric Somatic Hybrids Intergeneric somatic hybrids generally bridge a much wider taxonomic gap between fusion partners than interspecific somatic hybrids. In many cases, regeneration of hybrid callus has been impossible or only weak, sterile plants have resulted. However, in other cases, viable hybrids with evidence of at least partial genome transfer have resulted (Table 6.3). 1. Rutaceae. Citrus is one of 28 genera in the tribe Citreae, of the orange subfamily Aurantioideae. The true Citrus fruits comprise six genera including Citrus, its two closest relatives, Fortunella (kumquat) and Poncirus, a deciduous, cold resistant species often used as a rootstock, and three more distant relatives, Microcitrus, Eremocitrus, and Clymenia (Cameron and Soost 1976). The cultivated Citrus are not only interfertile among themselves but also with Fortunella and Poncirus. Therefore viable somatic hybrids between representatives of these genera would not be surprising and indeed have been realized between C. sinensis (L.) (sweet orange) and both P. trifoliata (Kaneko et al. 1995) and F. crassifolia Swingle (Shi et al. 1998a). Miranda et al. (1997b) also

192

Fusion partners

Citrus reticulata Blanco + Microcitrus australis (Planch.) Swingle C. reticulata + Glycosmis pentaphylla (Retz.) Correa C. reticulata + Swinglea glutinosa (Blanco) Merr. [C. reticulata × C. paridisi Macf.] + Severinia buxifolia (Poir.) Tenore [C. reticulata × C. paridisi] + Atalantia monophylla DC.) [C. reticulata × C. paridisi] + Severinia buxifolia (Poir.) Tenore [C. reticulata × C. paridisi] + Murraya paniculata (L.) Jack C. reticulata + Citropsis gabunensis (Engl.) Swing. & M. Kell C. paradisi + Poncirus trifoliata (L.) Raf. C. sinensis (L.) Osbeck + Fortunella crassifolia Swing. F. crassifolia + C. reticulata C. sinensis + Microcitrus papuana C. sinensis + Severinia busifolia (Poir.) Tenore C. sinensis + S. disticha (Blanco) Swing. C. reticulata + S. disticha C. sinensis + Citropsis gilletiana Swingle & M. Kell. C. sinensis + Atalantia ceylanica (Arn.) Oliv.

Rutaceae

SH SH

SH

SH SH SH

Electrofusion

Electrofusion

Electrofusion Electrofusion Chemical

Type of hybridsz

Electrofusion

Protocol

Intergeneric somatic hybrids of crop plants (1993–1999).

Family

Table 6.3.

isozymes, RAPD

isozymes

RAPD

Grosser et al. 1996

Ling and Iwamasa 1994

Guo and Deng 1998

Motomura et al. 1995

Motomura et al. 1997

Motomura et al. 1996

Reference

9/26/2000 1:19 PM

RFLP

None

RFLP

Verificationy

3499 P-06 Page 192

L. peruvianum + N. plumbaginifolia S. tuberosum + L. pennellii Corr. [L. esculentum × L. peruvianum (L.) Mill. var. humifusum] + S. lycopersicoides Atropa belladonna L. + Hyoscyamus muticus L.

SH SH SH SH SH SH SH SH

Chemical Chemical Electrofusion Electrofusion Electrofusion Chemical Electrofusion Electrofusion

SH

SH

Electrofusion

Chemical

SH

Chemical

Electrofusion Electrofusion

SH

Electrofusion

ASH SH SH

SH SH

Electrofusion Chemical

isozymes RAPD, RFLP, morphology isozymes, morphology

isozymes RFLP RAPD, isozymes isozymes, RAPD Dot blot using repetitive DNA probe

isozymes n.s.x GISH

RFLP

isozymes

RAPD

isozymes isozymes, RAPD

(continued)

Ahuja et al. 1993

Sherraf et al. 1994 Matsumoto et al. 1997

Wolters et al. 1993

Schoenmakers et al. 1993 Wolters et al. 1995 Kobayashi et al. 1996 Hossain et al. 1994

Grosser et al. 1994 Deng et al. 1995 Wolters et al. 1994b

Miranda et al. 1997b

Louzada et al. 1993

Guo and Deng 1999

Kaneko et al. 1995 Shi et al. 1998a

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Solanaceae

C. sinensis + Feronia limonia Swing. Nova tangelo [C. reticulata × (C. paradisi × C. reticulata)] + Severinia disticha Nova tangelo + Citrus ichangensis Swing. C. sinensis + Poncirus trifoliata C. sinensis + Fortunella crassifolia Swingle C. sinensis + Clausena lansium (Lour.) Skeels C. sinensis + Atalantia ceylanica (Arn.) Oliv. Poncirus trifoliata + Fortunella hindsii (Champ.) Swing. C. aurantium L. + P. trifoliata C. sinensis + F. crassifolia Lycopersicon esculentum Mill. + Solanum tuberosum L. L. esculentum + S. tuberosum L. esculentum + S. tuberosum L. esculentum + S. ochranthum L. esculentum + S. lycopersicoides L. esculentum + Nicotiana tabacum L.

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193

194 SH SH SH SH, ASH SH

Chemical Chemical Chemical Chemical Chemical

Chemical Chemical Electrofusion Electrofusion Electrofusion Chemical Electrofusion Electrofusion

SH SH SH SH SH SH SH SH ASH SH ASH SH, ASH SH, ASH

SH

Chemical

Electrofusion n.s.x Chemical Chemical

SH

Chemical

Type of hybridsz

RFLP n.s. isozymes RAPD RFLP, isozymes RFLP, isozymes RFLP AFLP RFLP RFLP RFLP RAPD, GISH RFLP

RAPD, isozymes isozymes RFLP

RAPD RAPD

RFLP

GISH

Verificationy

Sigareva & Earle 1999b Hansen & Earle 1997 Kirti et al. 1995b Mohapatra et al. 1998 Gaikwad et al. 1996 Baldev et al. 1998 O’Neill et al. 1996 Yan et al. 1996b Chevre et al. 1994 Forsberg et al. 1994 Siemens & Sacristan 1995 Brewer et al. 1999 Kisaka et al. 1997 Kisaka et al. 1998 Nakajo et al. 1994 Jelodar et al. 1999 Nakano et al. 1996

Hansen 1998 Sigareva and Earle 1999a

Navrátilová et al. 1997

Skarzhinskaya et al. 1998

Reference

y

SH = somatic hybrid; ASH = asymmetric somatic hybrid. RFLP = restriction fragment length polymorphism; RAPD = randomly amplified polymorphic DNA; GISH = genomic in situ hybridization; AFLP = amplified fragment length polymorphism. x n.s. = not specified in journal.

z

Caryophyllaceae

Poaceae

Brassica napus L. ssp. oleifera + Lesquerella fendleri Gray (Wats.) B. oleracea var. botrytis + Armoracia rusticana B. oleracea + Camelina sativa (L.) Crantz B. oleracea + Camelina sativa B. oleracea ssp. italica + C. sativa B. oleracea + Capsella bursa-pastoris L. B. oleracea + Sinapis alba (L.) B. juncea + Diplotaxis catholica

Brassicaceae

Protocol

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B. juncea + S. alba B. junceae + Trachystoma ballii B. napus + Moricandia arvensis B. oleracea + M. nitens B. napus + Sinapis alba B. napus + A. thaliana B. napus + A. thaliana B. napus + Thlaspi caerulescens Hordeum vulgare L. + Daucus carota L. Oryza sativa L. + Hordeum vulgare L. O. sativa + Lotus corniculatus L. O. sativa + Porteresia coarctata Dianthus barbatus + Gypsophila paniculata

Fusion partners

(continued)

Family

Table 6.3.

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fused P. trifoliata with F. hindsii, resulting in 5x and 6x somatic hybrid plants that could be established in the greenhouse by grafting onto citrange (C. sinensis × P. trifoliata). Success with somatic hybridization in Citrus has extended beyond the limits of sexual compatibility. Somatic hybrids of sweet orange have been obtained with the more distantly related Atalantia ceylanica (Arn.) Oliv. (Louzada et al. 1993) and Clausena lansium (Lour.) Skeels (Guo and Deng 1999). The former species represents a source of cold hardiness, whereas the latter species is a cultivated fruit in China. Pummelo (C. reticulata Blanco) or tangelo (C. reticulata × C. paridisi Macf.) have also been fused with various sexually incompatible related genera including Microcitrus australis (Planch.) Swingle, a source of drought resistance (Motomura et al. 1996), Atalantia monophylla DC (Motomura et al. 1995), a source of dwarfing, Severinia buxifolia (Poir.) Tenore (Motomura et al. 1995), a source of resistance to Phytophthora citrophthora, Murraya paniculata (L.) Jack (Guo and Deng 1998), a source of resistance to Citrus huanglongbin (Liberobacter), and Citropis gabunensis (Engl) Swing. & M. Kell (Ling and Iwamasa 1994). This last study was undertaken to clarify the taxonomic relationship between the members of the two genera. Motomura et al. (1997) attempted fusion of cultivated Citrus with various members of four different tribes of Aurantioideae to test the limits of intergeneric somatic hybridization. Somatic hybrids resulted from combinations between tangelo and Severinia buxifolia (tribe Citrinae) or between pummelo and both Swinglea glutinosa (tribe Balsamocitrinae) and Glycosmis pentaphylla (Retz.) Correa (tribe Clauseneae). Grosser et al. (1996b) developed 18 new intergeneric allotetraploid somatic hybrids of citrus, ten among sexually incompatible genera (Table 6.2). Two of these [Citrus sinensis + Microcitrus papuana and C. sinensis + Feronia limonia (L.) Swing.] were reported for the first time in this study. All but one of these hybrids [C. sinensis + Severinia disticha (Blanco) Swing.] were viable and entered into field trials to evaluate their rootstock potential. Although these studies established limits to somatic hybridization in Citrus, the limits are not very restrictive and many more somatic hybrids between cultivated Citrus and its many relatives can be expected in years to come. 2. Solanaceae. The abundance of cultivated plants within the Solanaceae has led to numerous attempts to develop intergeneric somatic hybrids both between different cultivated species and between cultivated and wild species. There has been little success in the development of sexual intergeneric hybrids in this family. Access to pathogen or pest resistance from diverse germplasm sources has been one of the

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A. JOHNSON AND R. VEILLEUX

major objectives underlying these efforts. One of the original wonders of somatic hybrid technology was the tomato-potato somatic hybrid first described by Melchers et al. (1978). However, the sterility of this hybrid proved to be a dead end for breeding purposes. After electrofusion of monoploid potato (2n = 1x = 12) in combination with diploid tomato, Schoenmakers et al. (1993) selected 25 triploid (2n = 3x = 36) somatic hybrids from a total of 450 that included allotetraploids, allohexaploids, and aneuploids. They hoped that triploids would be more fertile than tetraploid somatic hybrids, as reported for interspecific sexual hybrids within the Solanaceae. However, no progeny were obtained from the triploids with either potato or tomato pollinators. Wolters et al. (1994b) used genomic in situ hybridization (GISH) to determine that a recombinant chromosome occurred in an aneuploid (2n = 46) somatic hybrid obtained from electrofusion of a transformed tomato and a potato dihaploid. In a study of organellar composition of monoploid potato + diploid tomato somatic hybrids, Wolters et al. (1995) reported that mtDNA type segregated independently of cpDNA type. Their hybrids were either triploid or tetraploid, differing by one or two potato nuclear genomes, a factor that did not influence the organellar DNA segregation. Backcrosses of a hexaploid potato + tomato somatic hybrid to tetraploid (2n = 4x = 48) potato led to the development of a disomic addition line with an aberrant homologue of tomato chromosome 10 (Garriga-Calderé et al. 1999). The aberrant chromosome was transmitted at a reduced frequency and was suggested to be useful for physical mapping of tomato chromosome 10. In an attempt to introduce resistance genes into tomato, Kobayashi et al. (1996) fused tomato to Solanum ochranthum, a woody-vined tomato relative. The resulting tetraploid and hexaploid somatic hybrids had some stainable pollen (up to 33%) but no backcrosses to tomato were successful. Sherraf et al. (1994) tried to transfer salt tolerance of Lycopersicon pennellii Corr. to dihaploid potato by electrofusion. Although the hybrids were highly male sterile, they exhibited some of the salt tolerance of the wild tomato. Hossain et al. (1994) attempted to transfer chilling resistance of Solanum lycopersicoides to tomato using somatic hybridization. The fertile somatic hybrids were intermediate in chilling resistance to the two parents. Similar somatic hybrids were obtained between [tomato × L. peruvianum] + S. lycopersicoides (Matsumoto et al. 1997). The euploid (4x and 6x) somatic hybrids were fertile. Transfer of tropane alkaloids between Atropa belladonna L. and Hyoscyamus muticus L. was the objective of Ahuja et al. (1993). They obtained five viable hybrids that set seed on backcrossing with H. muticus. Wolters et al. (1993) undertook a massive effort to develop symmetric and asym-

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metric hybrids between tomato (both L. esculentum and L. peruvianum) and tobacco (both N. tabacum and N. plumbaginifolia). From 56 different electrofusion experiments, a single highly aneuploid somatic hybrid regenerated with sufficient vigor to be established in the greenhouse. It had 96 L. peruvianum and 4–6 N. plumbaginifolia chromosomes, flowered only briefly, and was completely sterile. The authors concluded that intertribal protoplast fusions have limited potential for plant breeding. 3. Brassicaceae. Intergeneric sexual hybridization among Brassicaceae is generally difficult, requiring embryo or ovule culture, if possible at all (Dickson and Wallace 1986). Hence, various intergeneric somatic fusions have been attempted to try to introgress traits of interest from wild species to the cultivated Brassicas. The development of highly regenerable “rapid-cycling” Brassica oleracea, i.e., plants that complete an entire life cycle in just 5 weeks, has facilitated intergeneric fusions, expanding germplasm combinations well beyond sexual compatibilities. Camelina sativa (L.) Crantz has been one fusion partner, valued for its resistance to Alternaria brassicae and A. brassicicola, pathogens causing black leaf spot disease, as well as its drought and cold tolerance. The sexual cross has not been possible. Hansen (1998) obtained approximately euploid somatic hybrids between these species; however, the plants died before reaching maturity. Similar fusions obtained by Sigareva and Earle (1999a) led to more robust but sterile somatic hybrids that varied for resistance to A. brassicicola. Resistance was correlated with the presence of camalexin, a phytoalexin produced by C. sativa. Female fertile somatic hybrids were produced between B. juncea and Diplotaxis catholica, a weedy source of resistance to Alternaria blight disease of oilseed Brassicas (Kirti et al. 1995b). A subsequent analysis of these hybrids revealed intergenomic mitochondrial recombination that could possibly lead to novel plasmons useful in male sterility systems (Mohapatra et al. 1998). A similar situation was reported for somatic hybrids between B. juncea and Sinapis alba, i.e., female fertile hybrids with evidence of mitochondrial recombination that could be backcrossed to B. juncea (Gaikwad et al. 1996). The somatic hybrids and most of the backcrosses exhibited field resistance to Alternaria brassicae, presumably due to the S. alba genome. Baldev et al. (1998) analyzed a CMS line of B. juncea, derived by backcrossing a B. juncea + Trachystoma ballii somatic hybrid to B. juncea, and found that the CMS B. juncea exhibited a recombinant chloroplast composed of fragments specific to both T. ballii and B. juncea. Such chloroplast recombination after somatic hybridization has been much less common than mitochondrial recombination.

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A. JOHNSON AND R. VEILLEUX

Similar rapid cycling B. oleracea have been fused to Sinapis alba, again with the hope of transferring resistance to Alternaria brassicae to susceptible Brassica germplasm (Hansen and Earle 1997). The hybrids ranged from susceptible to highly resistant but all were completely sterile. Sigareva and Earle (1999b) obtained somatic hybrids between rapid cycling B. oleracea and the common weed, shepherd’s-purse (Capsella bursa-pastoris L. Medic.), valued for resistance to Alternaria brassicae, flea beetle (Phyllotreta cruciferae Goeze and P. striolata Fabricius) and cold tolerance. These sterile hybrids survived for only 5–6 weeks in soil and died shortly after flowering. Of two hybrids that could be tested, one exhibited resistance to A. brassicicola. In an attempt to introgress resistance to clubroot (Plasmodiophora brassicae Woron) into cole crops, Navrátilová et al. (1997) fused the resistant Armoracia rusticana with selections of cabbage, cauliflower, and kohlrabi. Only the cauliflower fusion succeeded, resulting in mostly asymmetric hybrids with only small fractions of the Armoracia genome, despite the fact that no intentional effort had been made to induce asymmetric hybridization. Siemens and Sacristan (1995) attempted to transfer clubroot resistance by somatic hybridization between B. napus and Arabidopsis thaliana (L.) Heynh. in order to study the resistance in the genetic background of A. thaliana. Only two symmetric somatic hybrids were produced but neither could be maintained beyond flowering in the greenhouse. They were too weak for disease challenges; however, some asymmetric hybrids were obtained that were more vigorous. More promising somatic hybrids between A. thaliana and B. napus were obtained by Forsberg et al. (1994), several of which set seed in the greenhouse. O’Neill et al. (1996) were able to transmit the intermediate C3-C4 pathway of Moricandia arvensis (L.) DC into B. napus through somatic hybridization; of 13 hybrids obtained with a wide range of chromosome numbers, three showed evidence of the C3-C4 intermediate pathway. To incorporate the C3-C4 genes from Moricandia nitens into the Brassicas, Yan et al. (1999b) fused protoplasts of M. nitens to those of B. oleracea. Six of eight somatic hybrids showed a CO2 gas exchange character that was intermediate between M. nitens and B. oleracea, indicating that the C3-C4 genes were partially expressed in the hybrids. B. napus has also been hybridized with Lesquerella fendleri Gray (Wats.) for the purpose of altering the fatty acid composition of rapeseed oil (Skarzhinskaya et al. 1998); although symmetric hybrids were sterile, asymmetric hybrids exhibited increased fertility with decreasing dosages of the L. fendleri genome. Intergenomic chromosome recombinations were evident through genomic in situ hybridization (GISH).

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Somatic and sexual hybrids obtained by ovary culture were compared between B. napus and Sinapis alba, a source of resistance to Alternaria brassicae, clubroot, and drought (Chevre et al. 1994). A detailed study of meiosis in these hybrids revealed that more chromosome rearrangements had occurred in somatic hybrids compared to sexual hybrids between the same parents. Thlaspi caerulescens is a small plant species noted for its ability to survive on soil contaminated with zinc and cadmium and thus is valuable for phytoextraction of toxic metals from soil. To produce plants with more biomass than T. caerulescens yet still capable of phytoextraction, Brewer et al. (1999) fused T. caerulescens to B. napus. The resulting somatic hybrids survived on a high zinc medium that was lethal to B. napus. Although the limits to sexual hybridization in the Brassicaceae have been transcended through protoplast fusion, most of the amphidiploid hybrids obtained have been disappointing due to sterilities or severe genomic incompatibilities leading to premature death of plants. Asymmetric hybrids between such taxonomically distinct parents appear to be more promising because fractions of alien genomes seem to be tolerated more easily by the crop Brassicas. 4. Poaceae. Barley (Hordeum vulgare L.) tolerates cold and salinity, whereas rice (Oryza sativa L.) is more sensitive to these environmental stresses. Kisaka et al. (1998) obtained a single intergeneric somatic hybrid after more than 20 fusion attempts between leaf protoplasts of barley and suspension culture protoplasts of rice. The somatic hybrid with 14 barley and six rice chromosomes was sterile. Attempting an extremely wide cross, Nakajo et al. (1994) fused protoplasts from rice callus with those from callus of birdsfoot trefoil (Lotus corniculatus L.). There was evidence of mtDNA alterations in the hybrid callus; however, the 12 regenerated plants were morphologically similar to birdsfoot trefoil. Electrofusion between barley and carrot (Daucus carota L.) protoplasts resulted in an aneuploid somatic hybrid that closely resembled carrot (Kisaka et al. 1997). Analysis of the somatic hybrid organellar genome showed evidence of both chloroplast and mitochondrial recombination. Jelodar et al. (1999) fused rice protoplasts with those of Porteresia coarctata, a salinetolerant wild species, to produce an allohexaploid somatic hybrid carrying complete genomes of both parents. The saline tolerance of the somatic hybrid, which has not flowered, has not been tested. 5. Caryophyllaceae. Nakano et al. (1996) obtained asymmetric hybrids after intergeneric protoplast fusion between Dianthus barbatus and

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A. JOHNSON AND R. VEILLEUX

Gypsophila paniculata. The two hybrid plants had approximately 55 chromosomes instead of the expected 64 (30 + 34) and were extremely dwarfed with precocious flowering. D. Asymmetric Hybrids Asymmetric somatic hybrids have been created by fusion of protoplasts with unequal genomic contributions of each parent (Table 6.4). They have been sought in attempts to introduce only part of the genome of a wild (donor) species into a cultivated (recipient) species to limit the amount of alien germplasm to smaller genomic sectors that may control a trait of interest. Compared to somatic hybrids, smaller proportions of an alien genome may be tolerated better in asymmetric somatic hybrids, resulting in more vigorous plants than could be obtained by merger of two complete genomes. Asymmetric hybrids can be induced by irradiation (x-rays, γ-rays, or UV light) of the donor genome prior to fusion in order to fragment chromosomes. Often a metabolic inhibitor such as IOA is used on the recipient protoplasts to prevent regeneration of unfused protoplasts. Otherwise, nonregenerable recipient protoplasts can be used or donor plants can be transformed with a selectable marker. 1. Solanaceae. The use of γ- or x-irradiation to produce asymmetric hybrids has often resulted in transmission of an undesirably high proportion of the donor genome to asymmetric hybrids. In a study of N. plumbaginifolia + N. tabacum asymmetric hybrids, Trick et al. (1994) found that chromosome elimination of the donor increased with gammaradiation dose and the amount of time in culture; however, the hybrids retained on average 30% of the donor DNA. Liu et al. (1995b) applied a sublethal dose of γ-irradiation to Lycopersicon esculentum × L. pennellii hybrid protoplasts before fusion to eggplant (Solanum melongena) protoplasts. All but one of the resulting hybrids possessed more chromosomes than the sum of the two parents, indicating that γ-irradiation was ineffective in producing asymmetric hybrids. Rasmussen et al. (1997) fused protoplasts of S. tuberosum to x-irradiated protoplasts of S. spegazzinii or S. microdontum × S. vernei. RAPD analysis revealed loss of the donor genome ranging from 33–95%; however, no correlation between irradiation dose and the degree of genome elimination could be established. Kisaka and Kameya (1994) reported somatic hybrids that closely resembled carrot from protoplast fusion between carrot and xray irradiated tobacco. The somatic hybrids segregated for chloroplast type and were found to have carrot mitochondria—there was no evidence of organellar recombination between the parents.

Fusion partners

S. tuberosum + S. brevidens S. tuberosum + S. brevidens S. tuberosum + S. spegazzinii S. tuberosum + S. microdontum × S. vernei S. tuberosum + S. bulbocastanum S. tuberosum + S. circaeifolium L. peruvianum + S. tuberosum microprotoplasts Nicotiana tabacum + S. tuberosum microprotoplasts L. peruvianum + S. tuberosum microprotoplasts L. peruvianum + S. tuberosum microprotoplasts S. melongena + L. esculentum × L. pennellii S. melongena + [L. esculentum × L. pennellii] N. tabacum + L. esculentum Daucus carota + N. tabacum N. tabacum + N. plumbaginifolia N. plumbaginifolia + N. tabacum N. tabacum + N. plumbaginifolia N. tabacum + N. sanderae

Solanaceae

Type of hybridsz ASH ASH ASH

ASH

MA MA

MA ASH, SH ASH ASH ASH ASH ASH ASH

Protocol n.s.x Electrofusion Electrofusion

Electrofusion

Chemical Chemical

Chemical Chemical Chemical Chemical Electrofusion Electrofusion n.s. Chemical

isozymes

RFLP RFLP RFLP GISH

RFLP, isozymes

RFLP, RAPD

GISH, RFLP

n.s. GISH, morphology

201

(continued)

Dragoeva et al. 1997a

Vlahova et al. 1997 Kisaka and Kameya 1994 Trick et al. 1994 Parokonny et al. 1994

Samoylov et al. 1996, Samoylov and Sink 1996 Liu et al. 1995b

Ramulu et al. 1996b

Ramulu et al. 1995 Ramulu et al. 1996a

Oberwalder et al. 1997, 1998

Xu and Pehu 1994 Valkonen et al. 1994a Rasmussen et al. 1997

Reference

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RFLP

RFLP n.s. RAPD

Verificationy

Asymmetric hybrids of crop plants reported 1994–1998 (fractionated parent, if present, is in bold).

Family

Table 6.4.

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202

B. napus + A. thaliana B. napus + B. nigra

B. napus + Lesquerella fendleri Festuca arundinacea Schreb. + Lolium multiflorum Lam. F. arundinaceae + L. multiflorum Triticum aestivum L. + Leymus chinensis T. aestivum + Haynaldia villosa Schur. Triticum aestivum + Bromus inermis Leyss O. sativa + Zizania latifolia (Griseb) Turez. Ex Stapf O. sativa + Daucus carota L. Ipomoea batatas L. Lam + I. trifida Don. Medicago truncatula + M. scutellata

Brassicacae

Poaceae

ASH ASH ASH ASH ASH

Chemical Chemical Electrofusion Electrofusion Chemical, Electrofusion Chemical ASH

ASH ASH

Electrofusion Chemical

AFLP

RFLP, isozymes isozymes

RFLP

RAPD, isozymes

isozymes

RFLP isozymes

RFLP RFLP

RFLP isozymes

Verificationy

Tian and Rose 1999

Kisaka et al. 1994 Belarmino et al. 1996

Liu et al. 1999

Xiang et al. 1999

Zhou et al. 1996

Spangenberg et al. 1994 Xia and Chen 1996

Forsberg et al. 1998a,b Gerdemann-Knörck 1994, 1995 Skarzhinskaya et al. 1996 Spangenberg et al. 1995

Reference

y

SH = somatic hybrid; ASH = asymmetric somatic hybrid; MA = monosomic additions. RFLP = restriction fragment length polymorphism; RAPD = randomly amplified polymorphic DNA; GISH = genomic in situ hybridization; AFLP = amplified fragment length polymorphism. x n.s. = not specified in journal.

z

Fabaceae

SH, ASH SH, ASH

ASH ASH, SH

Type of hybridsz

Chemical Electrofusion

n.s. n.s.

Protocol

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Convolvulaceae

Fusion partners

Asymmetric hybrids of crop plants reported 1994–1998 (fractionated parent, if present, is in bold) (continued).

Family

Table 6.4.

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To increase the effectiveness of asymmetric hybridization efforts, researchers have utilized tools other than γ-irradiation to fragment the donor genome. Dragoeva et al. (1997a) used UV-irradiation of Nicotiana sanderae protoplasts prior to fusion with N. tabacum protoplasts to produce asymmetric hybrids. The asymmetric hybrids had 52–63 chromosomes (66 is the additive number of the parents). Twelve of the 54 hybrids were resistant to tomato spotted wilt virus (TSWV), a trait from N. sanderae. Up to 55% of progeny, resulting from self-pollination or backcrossing of the hybrids to N. tabacum, were also resistant to TSWV. In solanaceous asymmetric hybrids, the recipient genome has often doubled, tempting researchers to speculate that polyploidization may “buffer” detrimental effects caused by the addition of a fragmented donor genome. Oberwalder et al. (1998) fused either dihaploid or tetraploid S. tuberosum to irradiated S. bulbocastanum or S. circaeifolium; they found that the tetraploid S. tuberosum recipients carried less DNA of the donor species than the dihaploid recipient. Thus tetraploids, due to their apparently greater buffering capacity, may be better suited than dihaploids for asymmetric hybridization in potato. Samoylov and Sink (1996) carried out asymmetric hybridization between irradiated L. esculentum × L. pennellii hybrid protoplasts and S. melongena protoplasts. The resulting hybrids had a polyploid genome of eggplant (4C) and a DNA equivalent of 6.29 average-sized tomato chromosomes. Their organellar composition was mostly that of eggplant (Samoylov et al. 1996). Vlahova et al. (1997) fused UV-irradiated L. esculentum protoplasts to N. plumbaginifolia protoplasts and found that most of the hybrids possessed a hexaploid N. plumbaginifolia genome with the addition of 2–4 tomato chromosomes. Several studies have focused on the identification of asymmetric hybrids. Oberwalder et al. (1997) used three different techniques (RFLP with an oligonucleotide repeat probe, RFLP with a single-copy probe, and flow cytometry) to identify asymmetric hybrids between dihaploid S. tuberosum and x-irradiated S. bulbocastanum or S. circaeifolium. The oligonucleotide probe was ineffective for the identification of asymmetric hybrids. The single-copy probe identified many asymmetric hybrids, as did flow cytometry. Flow cytometry had the additional advantage of identifying chimeric plants. Valkonen et al. (1994a) also used flow cytometry to identify asymmetric hybrids between dihaploid potato and irradiated S. brevidens and noted that flow cytometry could readily identify chimeric plants that were otherwise labeled as euploid hybrids by chromosome counts. Xu and Pehu (1994) used organelle specific probes to identify recombinant mitochondria in similar asymmetric hybrids between S. tuberosum and S. brevidens.

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Parokonny et al. (1994) used GISH (genomic in situ hybridization) technology to monitor the behavior of chromosomes in N. plumbaginifolia and N. sylvestris asymmetric hybrids. Results indicated that intergenomic translocations occurred in the asymmetric plants and these recombinant chromosomes were transmitted through both male and female gametes to progeny. Because of the complexity of asymmetric somatic hybrids regarding segregation of traits encoded on multiple chromosomes, Ramulu et al. (1995, 1996a,b) attempted to transfer individual chromosomes from microprotoplasts of a potato cell line to diploid L. peruvianum through protoplast fusion. Microprotoplasts with only one or a few chromosomes were obtained by high-speed centrifugation of protoplasts on a Percoll gradient. Fertile monosomic addition lines of tomato were thus developed with single potato chromosomes (determined by GISH) from these sexually incompatible species. Limited pollen fertility in the monosomic addition lines indicated that single alien chromosomes could be tolerated better than larger genomic fragments of asymmetric hybrids or complete genomes in symmetric hybrids. Monosomic and disomic addition lines of tobacco with one or two potato chromosomes have been developed using the same techniques (Ramulu et al. 1996a). These monosomic addition lines were female fertile. 2. Brassicaceae. Fusion between Brassica napus and Arabidopsis thaliana has served as a model for asymmetric hybridization. Forsberg et al. (1998b) compared the efficiency of x-rays, UV light, or a restriction endonuclease (PvuII) to fractionate the donor genome for asymmetric hybridization of canola and Arabidopsis. Both x-rays and UV light induced asymmetry in a dose-dependent fashion whereas the restriction enzyme was ineffective. Seed set after self-pollination of asymmetric hybrids increased with increasing irradiation of the donor genome (Forsberg et al. 1998a,b). With more practical breeding goals in mind, Gerdemann-Knörck et al. (1994, 1995) produced asymmetric hybrids between Brassica napus (recipient) and B. nigra (donor) in order to transfer blackleg (Leptosphaeria maculans, Phoma lingam) and/or clubroot (Plasmodiophora brassicae) resistance to canola. They used x-irradiation of a transgenic donor strain with hygromycin resistance and a selective medium to regenerate fusion products exclusively. Of 30 hybrids obtained, exhibiting a range of chromosome numbers (2n = 46–85 compared to an additive number of 72–74), four expressed resistance to both fungal pathogens. Fertility was low for these hybrids after either selfing or backcrossing with B. napus. Skarzhinskaya et al. (1996) used

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both symmetric and asymmetric intergeneric fusion between B. napus and Lesquerella fendleri (Gray) Wats to transfer oil quality, or drought or low temperature tolerance of the latter weedy species to canola. Five symmetric and 24 asymmetric hybrids could be established in the greenhouse. The asymmetric hybrids were somewhat more fertile than symmetric hybrids upon self-pollination. 3. Poaceae. Asymmetric somatic hybrids have been attempted in several members of the Poaceae. The first intergeneric flowering somatic hybrids in Poaceae were obtained by Spangenberg et al. (1995) between Festuca arundinacea Schreb. and Lolium multiflorum Lam. although sexual progeny had been previously generated between these species. These asymmetric hybrids exhibited a range of chromosome numbers from 18 to 90 [56 would have been expected for amphiploid somatic hybrids (42 + 14)]. Asymmetric Festulolium hybrids were shown to retain from 5% to most of the donor genome with primarily recipient-type cytoplasm; some pollen stainability with fluorescein diacetate was noted (Spangenberg et al. 1994). Xia and Chen (1996) fused nonregenerable protoplasts derived from wheat (Triticum aestivum L.) suspension cultures with protoplasts isolated from irradiated callus of Leymus chinensis (Trin.), a forage grass with resistance to cold, drought, salinity, and various pathogens. Of 20 hybrid regenerants, three survived in soil and two overwintered outside the greenhouse. Their chromosome number was variable and included numerous fragments, indicating asymmetric hybridization. Asymmetric aneuploid hybrids with fragmented chromosomes were obtained by fusion of wheat protoplasts with those isolated from irradiated callus of Haynaldia villosa, a related genus with disease resistance, high seed protein, and strong tillering (Zhou et al. 1996). Similarly, Xiang et al. (1999) fused wheat and UV-treated Bromus inermis Leyss to transfer desirable traits such as cold, drought, and disease tolerance to the wheat gene pool. The three resulting asymmetric hybrids had numerous fragments of the B. inermis genome. Asymmetric protoplast fusion between rice (Oryza sativa L., recipient) and γirradiated Zizania latifolia (Griseb) Turcz. ex. Stapf (donor) resulted in hybrids with the same chromosome number as rice (Liu et al. 1999); however, the authors claimed that intergenomic recombination had occurred because there was evidence of donor DNA in the hybrids by Southern analysis. Kisaka et al. (1994) attempted extremely wide hybridization between IOA-treated rice and x-irradiated carrot protoplasts. They reported asymmetric somatic hybrids that were carrot-like in appearance but with isozyme and molecular markers characteristic of

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both parents. Organellar DNA was that of carrot, except that a novel mtDNA band pattern was present. 4. Convolvulaceae. A single study of asymmetric hybridization between Ipomoea batatas L. Lam. (recipient) and two wild sweetpotato donor species, I. trifida Don. and I. lacunosa L., was reported by Belarmino et al. (1996). Both electrofusion and chemical fusion were attempted after x-irradiation of the donor genome but only electrofusion resulted in nonflowering hybrids, exclusively with I. trifida. E. Cybrids Cybridization, a technique that combines the plastome and/or chondriome of one plant species with the nuclear genome of another, has been used to transfer economically important traits such as CMS (cytoplasmic male-sterility) between species and to create de novo CMS systems due to nuclear-mitochondrial incompatibilities between unrelated species (Table 6.5). Often, the cytoplasmic donor is treated with xirradiation to eliminate nuclear DNA, while the cytoplasm of the recipient is treated with IOA to inhibit cell division of unfused protoplasts. Subsequent fusion of differentially treated protoplasts results in the production of cybrids. Cybridization can replace the cytoplasm of a cultivar in one step, saving considerable time over lengthy backcrossing programs used to introduce alien cytoplasm into crops. 1. Solanaceae. Matibiri and Mantell (1994) used cybridization to transfer cytoplasmic male-sterility from Nicotiana suaveolens Leh. to tobacco. Dual selection for spectinomycin resistance (encoded transgenically by the N. suaveolens cytoplasm) and kanamycin resistance (encoded transgenically by the N. tabacum nucleus) enabled only cybrids to regenerate. Several CMS lines of tobacco were regenerated from this experiment and were ready for field testing 12 months after fusion. Zubko et al. (1996) produced a novel CMS source in tobacco—”green flowers” lacking corollas and stamens—by using Hyoscyamus niger and Scopolia carniolica as cytoplasmic donors in protoplast fusion to N. tabacum. Backcrossing confirmed that this novel CMS trait was maternally inherited. Atanassov et al. (1998) also produced a novel CMS source—flowers with secondary petaloids and stigmoids in place of stamens—by fusing gamma-irradiated N. alata protoplasts to cholorophyll-deficient N. tabacum protoplasts. Regenerated green plants were cybrids with maternally inherited CMS demonstrated through backcrossing.

Nicotiana tabacum L. + N. suaveolens Leh. N. tabacum + Hyoscyamus niger N. tabacum + Scopolia carniolica Nicotiana tabacum + N. alata S. tuberosum L. + S. bulbocastanum S. tuberosum + S. pinnatisectum S. nigrum + S. tuberosum

Brassica oleracea spp. capitata + B. oleracea L. ssp. italica B. oleracea + B. rapa Brassica oleracea spp. capitata + R. sativus L. cv. Shougin B. napus var. Brutor + CMS B. napus CMS B. juncea + B. juncea

Cichorium intybus L. cv. Magdebourg + Helianthus annuus cv. Mirasol

Oryza sativa L. line RCPL1-2C + O. sativa line V20A

Solanaceae

Brassicaceae

Asteraceae

Poaceae

CY

y

CY = cybrid; SH = somatic hybrid. RFLP = restriction fragment length polymorphism; PCR = polymerase chain reaction. x n.s. = not specified in journal.

Electrofusion

CY

CY SH, CY

n.s. Chemical n.s.

CY CY

CY

n.s.

Chemical n.s.

CY CY

n.s.x n.s.

CY

CY

Chemical

Chemical

CY

Type of hybridsz

Electrofusion

Protocol

RFLP

n.s.

n.s. RFLP

PCR amplification of mtDNA fragment Morphology, RFLP RFLP

n.s.

RFLP isozymes

RFLP, isozymes

transgenic resistance

Verificationy

Bhattacharjee et al. 1999

Dubreucq et al. 1999

Grelon et al. 1994 Kirti et al. 1995

Cardi and Earle 1997 Kanno et al. 1997

Sigareva and Earle 1997

Hassanein et al. 1998

Atanassov et al. 1998 Sidorov et al. 1994

Zubko et al. 1996

Matibiri and Mantell 1994

Reference

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z

Fusion partners

Cybrids of crop plants reported 1994–1996 (irradiated parent, if present, is in bold).

Family

Table 6.5.

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Sidorov et al. (1994) created potato cybrids using Solanum bulbocastanum or S. pinnatisectum as cytoplasmic donors and S. tuberosum as the recipient. To select for cybrids without the use of genetic markers, they treated the irradiated donor protoplasts with N-nitroso-N-methyl urea to induce plastome mutations for streptomycin resistance. The protoplasts were then regenerated on streptomycin sulfate and only tobacco cybrids with a mutant plastome from the wild species could regenerate. The effect of substituting black nightshade cytoplasm with that of potato on various physiological traits was studied in S. nigrum cybrids (Hassanein et al. 1998). Shoot weight (fresh and dry) was lower and potassium content was higher for the cybrid compared to the original S. nigrum. 2. Brassicaceae. Sigareva and Earle (1997) used cybridization to transfer the cytoplasm of a cold-tolerant, CMS broccoli line (B. oleracea L. ssp. italica) to cabbage (B. oleracea ssp. capitata). The regenerated cabbage cybrids possessed a mitochondrial-specific PCR marker from broccoli that correlated with male sterility. The authors found that irradiation was more effective than UV light or cytoplasts (enucleated protoplasts) for the production of cybrids. Similarly, Cardi and Earle (1997) transferred the male-sterile ‘Anand’ cytoplasm of B. rapa to B. oleracea using cybridization. Male sterility was expressed in many cybrids and was maternally inherited. Grelon et al. (1994) found that expression of a 19 kDa protein from the mitochondrial orf138 locus was associated with CMS in a Brassica napus cybrid. Finally, Kirti et al. (1995a) used protoplast fusion to improve a male-sterile B. juncea cultivar with ogu cytoplasm from Raphanus sativus developed by backcrossing. The CMS B. juncea displayed leaf chlorosis, likely due to incompatibility between Raphanus chloroplasts and the Brassica nuclear genome. Protoplasts of the male-sterile line and a wild type B. juncea cultivar were fused to regenerate green tetraploid hybrids by chloroplast substitution. After three rounds of backcrossing the somatic hybrids to B. juncea, vigorous, green, diploid progeny resulted that retained the male sterility present in the original but chlorotic B. juncea cultivar. 3. Asteraceae. Cytoplasmic male sterile chicory was produced through protoplast fusion between fertile industrial chicory (Cichorium intybus L.) and CMS lines of sunflower (Helianthus annuus L.) (Dubreucq et al. 1999). Male-sterility present in the resulting chicory cybrids was believed to be caused by the expression of one to several sunflower mitochondrial genes in the context of a chicory nuclear background; however, the genes responsible for CMS were not identified.

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4. Poaceae. Bhattacharjee et al. (1999) introduced the WA cytoplasm from a CMS rice line (V20A) into an advanced rice breeding line (RLPL12C) using asymmetric hybridization. The CMS continued to be effective when combined with a new nuclear background.

III. CONCLUSION As plant breeding enters the 21st century, somatic hybridization remains an area of active research. Protoplast isolation, fusion, and regeneration protocols have been developed for many commercially important plant species; however, the release of plant cultivars derived from somatic hybrids remains rare. The tobacco cultivar Delfield, developed by Radhey Pandeya at Agriculture and Agri-Food Canada through protoplast fusion of Nicotiana tabacum and N. rustica, is one of the first examples of a commercially successful biotechnology-derived crop. Delfield yielded a 1991 crop in Ontario valued at more than $85 million. A similar somatic hybrid can be found in the parentage of another recently released and promising tobacco cultivar, AC Cheng (Brandle et al. 1996). Despite this initial success with somatic hybridization, most releases of biotechnology-derived crops since 1991 have resulted from genetic transformation. Examples abound of fusion-derived material with strong potential for cultivar development in the near future. Heath and Earle (1995) produced rapeseed somatic hybrids high in erucic acid (22:1) through protoplast fusion of Brassica oleracea and B. rapa. The high erucic acid content was transmitted stably through several generations of selfed progeny and the progeny exhibited additional desirable agronomic features, such as resistance to shattering and lodging. Helgeson et al. (1998) developed high-yielding backcross progeny populations of potato from a S. tuberosum + S. bulbocastanum somatic hybrid that showed strong resistance to late blight (Phytophthora infestans). Field evaluation of backcross progenies in Toluca, Mexico, where virtually all races of the late blight pathogen exist, demonstrated that some resistant progeny had less than 10% infection while S. tuberosum cultivars were killed. The late blight resistance (which has been stable for over 4 years) in these somatic hybrid progeny appears to be more general than the race-specific resistance that has been incorporated into many potato cultivars from S. demissum. Many sources of CMS that could be used to produce new hybrid cultivars have been found in the Brassicaceae and other plant families as a result of somatic hybridization (Kanno et al. 1997; Prakash et al. 1998).

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After protoplast fusion between Trachystoma ballii and Brassica juncea and repeated backcrossing of somatic hybrids to the B. juncea cultivar, Kirti et al. (1997) obtained both CMS and restorer lines that were genetically equivalent to the original cultivar. This was the first report of a restorer line in B. juncea. Citrus somatic hybrids have the potential to be released directly as cultivars (without backcrossing) by serving as versatile rootstocks expressing desirable traits from many different species (Grosser 1993). Trials of rootstocks and seedless triploids, both derived from somatic hybridization, have been underway for several years in Florida; some appear promising but will require additional evaluation (J. W. Grosser, personal communication). Somatic hybrids have been developed for production of valuable secondary metabolites, such as tropane alkaloids collected from roots of the Hyoscyamus muticus + H. albus somatic hybrid described by Zehra et al. (1998). Most somatic hybrids must be backcrossed to the adapted parent in order to contribute to cultivar development. Of critical importance, therefore, is the elucidation of somatic hybrid chromosomal behavior during sexual crosses. A recent study by Bohman et al. (1999) utilized the dense molecular map of Arabidopsis thaliana to study the transmission of A. thaliana chromosomes to progeny during two backcrosses of Brassica napus + A. thaliana somatic hybrids to B. napus. Two RFLP markers were chosen to mark each of the ten A. thaliana chromosomes. The study revealed a rapid loss of chromosomes through the backcrosses. The original somatic hybrids had the expected chromosome number of 48 (38 from B. napus and ten from A. thaliana). After one backcross, an average of 42.8 chromosomes remained and, after a second backcross, an average of 39.4 remained. Two cycles of backcrossing had nearly returned the average chromosome number to that of the recurrent parent (B. napus) in this study. Despite the rapid reduction in chromosome number, molecular markers deriving from A. thaliana remained in the backcross progeny. This indicates that intergenomic recombination, translocation, or even substitution of B. napus chromosomes with A. thaliana chromosomes had occurred. Due to rapid elimination of chromosomes through backcrossing, it is important that homoeologous pairing occurs in somatic hybrids so that opportunities for intergenomic recombination arise. Such pairing is likely to happen in interspecific and even intergeneric somatic hybrids, as evidenced by the previously discussed paper and others that have analyzed backcross progeny of intergeneric somatic hybrids (Begum et al. 1995). However, combinations of more phylogenetically distant plant species (intertribal somatic hybrids) may not be useful for transferring

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nuclear traits due to a lack of homoeologous pairing. Such fusions may be more useful for creating novel nuclear-cytoplasmic combinations often important for the development of CMS systems. Transgenic plant production has certainly overwhelmed somatic hybridization as a technique heavily utilized by plant breeders and geneticists over the past decade. This is understandable considering that transgenic modification theoretically affects only a single trait of interest without introducing innumerable extraneous genes of uncertain agronomic influence. The opportunity of genetic revision of existing cultivars is afforded by genetic engineering. Conversely, somatic hybridization involves introgressing traits of interest into crop plants without any prior knowledge of their genetic control. Resulting somatic hybrids are generally not expected to be ready for cultivar release without additional breeding effort to acquire the trait of interest in a more acceptable genetic background resembling current cultivars. However, an advantage of somatic hybridization compared to genetic engineering concerns the controversy surrounding genetically modified organisms, a stigma that does not apply to somatic hybrids. Current restrictions that hinder the release of transgenic crops are avoided using somatic hybrid plant material. In addition, the transfer of polygenic traits between species is often only possible using protoplast fusion (Millam et al. 1995). An underlying assumption of plant breeding is that current cultivars do not represent the pinnacle of plant breeding potential but that new combinations of genes other than those acquired via single gene insertion (genetic engineering) will result in cultivars with unprecedented improvements. A recent report by Bernacchi et al. (1998) identified alleles contributing to agronomic characters of tomato from wild species that did not express the particular agronomic traits (e.g., fruit color, yield) and thus highlights the importance of utilizing unadapted germplasm for cultivar improvement. Somatic hybrids represent valuable and unique genetic material because two (often sexually incompatible) complete or partial genomes are combined in one organism. Desirable interactions between the genomes may be retained in subsequent generations and exploited for development of more productive cultivars. The impact of somatic hybridization can be expected to be much quieter than that of genetic engineering because traditional breeding must intervene between the biotechnological event (protoplast fusion) and the release of a product for market. Valuable resistances to various pathogens or environmental stresses have been identified in direct somatic hybrids and many have been entered into current plant breeding programs. In integrated programs where biotechnology and applied

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plant breeding research are conducted by teams of researchers, the value of germplasm developed by protoplast fusion has the greatest potential of being realized. Plant breeding throughout the 20th century has been successful because of the incorporation of resistance from exotic germplasm into modern cultivars. Somatic hybridization broadens the base of accessible germplasm and offers additional opportunities for introgression of desirable traits into cultivars. Even though measurable success of somatic hybridation in terms of cultivar release may still be limited 28 years after the first somatic hybrid was reported, the potential for their use in plant breeding remains great.

LITERATURE CITED Ahuja, P. S., L. U. Rahman, S. C. Bhargava, and S. Banerjee. 1993. Regeneration of intergeneric somatic hybrid plants between Atropa belladonna L. and Hyoscyamus muticus L. Plant Sci. 92:91–98. Asao, H., S. Arai, T. Sato, and M. Hirai. 1994. Characteristics of a somatic hybrid between Solanum melongena L. and Solanum sanitwongsei Craib. Breed. Sci. 44:301–305. Atanassov, I. I., S. A. Atanassova, A. I. Dragoeva, and A. I. Atanassov. 1998. A new CMS source in Nicotiana developed via somatic cybridization between N. tabacum and N. alata. Theor. Appl. Genet. 97:982–985. Bai, D., R. Reeleder, and J. E. Brandle. 1996. Production and characterization of tobacco addition lines carrying Nicotiana debneyi chromosomes with a gene for resistance to black root rot. Crop Sci. 36:852–857. Bains, P. S., V. S. Bisht, D. R. Lynch, L. M. Kawchuk, and J. P. Helgeson. 1999. Identification of stem soft rot (Erwinia carotovora subspecies atroseptica) resistance in potato. Am. J. Potato Res. 76:137–141. Baldev, A., K. Gaikwad, P. B. Kirti, T. Mohapatra, S. Prakash, and V. L. Chopra. 1998. Recombination between chloroplast genomes of Trachystoma ballii and Brassica juncea following protoplast fusion. Mol. Gen. Genet. 260:357–361. Barbosa, L. V., and M. L. C. Vieira. 1997. Meiotic behavior of passion fruit somatic hybrids, Passiflora edulis f. flavicarpa Degener + P. amethystina Mikan. Euphytica 98:121–127. Begum, F., S. Paul, N. Bag, S. R. Sikdar, and S. K. Sen. 1995. Somatic hybrids between Brassica juncea (L.) Czern. and Diplotaxis harra (Forsk.) Boiss and the generation of backcross progenies. Theor. Appl. Genet. 91:1167–1172. Belarmino, M. M., T. Abe, and T. Sasahara. 1996. Asymmetric protoplast fusion between sweet potato and its relatives, and plant regeneration. Plant Cell Tissue Organ Cult. 46:195–202. Bernacchi, D., T. Beck-Bunn, Y. Eshed, J. Lopez, V. Petiard, J. Uhlig, D. Zamir, and S. Tanksley. 1998. Advanced backcross QTL analysis in tomato. I. Identification of QTLs for traits of agronomic importance from Lycopersicon hirsutum. Theor. Appl. Genet. 97:381–397. Bhattacharjee, B., A. P. Sane, and H. S. Gupta. 1999. Transfer of wild abortive cytoplasmic male sterility through protoplast fusion in rice. Mol. Breed. 5:319–327.

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Bohman, S., J. Forsberg, K. Glimelius, and C. Dixelius. 1999. Inheritance of Arabidopsis DNA in offspring from Brassica napus and A. thaliana somatic hybrids. Theor. Appl. Genet. 98:99–106. Bordas, M., L. Gonzalez-Candelas, M. Dabauza, D. Ramon, and V. Moreno. 1998. Somatic hybridization between an albino Cucumis melo L. mutant and Cucumis myriocarpus Naud. Plant Sci. 132:179–190. Brandle, J. E., J. C. D. Ankersmit, and W. D. Rogers. 1996. AC Cheng flue-cured tobacco. Can. J. Plant Sci. 77:155–156. Bravo, J. E., and D. A. Evans. 1985. Protoplast fusion for crop improvement. Plant Breed. Rev. 3:193–218. Brewer, E. P., J. A. Saunders, J. S. Angle, R. L. Chaney, and M. S. McIntosh. 1999. Somatic hybridization between the zinc accumulator Thlaspi caerulescens and Brassica napus. Theor. Appl. Genet. 99:761–771. Brown, C. R., C. P. Yang, H. Mojtahedi, G. S. Santo, and R. Masuelli. 1996. RFLP analysis of resistance to Columbia root-knot nematode derived from Solanum bulbocastanum in a BC2 population. Theor. Appl. Genet. 92:572–576. Brüggemann, W., A. Wenner, and Y. Sakata. 1995. Long-term chilling of young tomato plants under low light. VII. Increasing chilling tolerance of photosynthesis in Lycopersicon esculentum by somatic hybridization with L. peruvianum. Plant Sci. 108:23–30. Buiteveld, J., W. Kassies, R. Geels, M. M. van Lookeren Campagne, E. Jacobsen, and J. Creemers-Molenaar. 1998a. Biased chloroplast and mitochondrial transmission in somatic hybrids of Allium ampeloprasum L. and Allium cepa L. Plant Sci. 131: 219–228. Buiteveld, J., Y. Suo, M. M. van Lookeren Campagne, and J. Creemers-Molenaar. 1998b. Production and characterization of somatic hybrid plants between leek (Allium ampeloprasum L.) and onion (Allium cepa L.). Theor. Appl. Genet. 96:765–775. Cameron, J. W., and R. K. Soost. 1976. Citrus. p. 261–264. In: N. W. Simmonds (ed.), Evolution of crop plants. Longman, New York. Cardi, T. 1998. Multivariate analysis of variation among Solanum commersonii (+) S. tuberosum somatic hybrids with different ploidy levels. Euphytica 99:35–41. Cardi, T., and E. D. Earle. 1997. Production of new CMS Brassica oleracea by transfer of ‘Anand’ cytoplasm from B. rapa through protoplast fusion. Theor. Appl. Genet. 94:204–212. Carlson, P. S., H. H. Smith, and R. D. Dearing. 1972. Parasexual interspecific plant hybridization. Proc. Nat. Acad. Sci. (USA) 69:2292–2294. Carputo, D., T. Cardi, M. Speggiorin, A. Zoina, and L. Frusciante. 1997. Resistance to blackleg and tuber soft rot in sexual and somatic interspecific hybrids with different genetic background. Am. Potato J. 74:161–172. Carputo, D., P. Garreffa, M. Mazzei, L. Monti, and T. Cardi. 1998. Fertility of somatic hybrids Solanum commersonii (2x, 1EBN) (+) S. tuberosum haploid (2x, 2EBN) in intra- and inter-EBN crosses. Genome 41:776–781. Celis, C., and P. Jourdan. 1993. Characterization of a partial male fertility derived from Ogura-cms in progeny of a resynthesized Brassica napus. Sex. Plant Reprod. 6:266–274. Chase, S. C. 1963. Analytic breeding in Solanum tuberosum L.: a scheme utilizing parthenotes and other diploid stocks. Can. J. Genet. Cytol. 5:359–363. Chen, Y. K. H., J. P. Palta, J. B. Bamberg, H. Kim, G. T. Haberlach, and J. P. Helgeson. 1999. Expression of nonacclimated freezing tolerance and cold acclimation capacity in somatic hybrids between hardy wild Solanum species and cultivated potatoes. Euphytica 107:1–8.

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Subject Index Volume 20 A

L

Allium cepa, see Onion

Lactuca sativa, see Lettuce Lettuce breeding, 105–133

B Biography, N. W. Simmonds, 1–13 Breeding: cactus, 135–166 lettuce, 105–133 onion, 67–103 somatic hybridization, 167–225

M Maize, origins, 15–66 O Onion, breeding history, 67–103 Opuntia, see Cactus

C Cactus: breeding, 135–166 domestication, 135–166 Cytogenetics, maize-tripsacum hybrids, 15–88 Cybrids, 206–209 Cytoplasm, cybrids, 206–209

P Protoplast fusion, 167–225 S Somatic hybridization, 167–225 T

E

Tripsacum, maize ancestry, 15–66

Evolution, maize, 15–66 V F Fruit breeding, cactus, 135–166

Vegetable breeding: lettuce, 105–133 onion, 67–103

G Germplasm, cactus, 141–145

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Cumulative Subject Index (Volumes 1–20)

A Adaptation: blueberry, rabbiteye, 5:351–352 durum wheat, 5:29–31 genetics, 3:21–167 testing, 12:271–297 Alfalfa: honeycomb breeding, 18:230–232 inbreeding, 13:209–233 in vitro culture, 2:229–234 somaclonal variation, 4:123–152 unreduced gametes, 3:277 Allard, Robert W. (biography), 12:1–17 Allium cepa, see Onion Almond: breeding self-compatible, 8:313–338 transformation, 16:103 Alstroemaria, mutation breeding, 6:75 Amaranth: breeding, 19:227–285 genetic resources, 19:227–285 Aneuploidy: alfalfa, 10:175–176 alfalfa tissue culture, 4:128–130 petunia, 1:19–21 wheat, 10:5–9 Anther culture: cereals, 15:141–186 maize, 11:199–224 Anthocyanin pigmentation, maize aleurone, 8:91–137 Antifungal proteins, 14:39–88 Antimetabolite resistance, cell selection, 4:139–141, 159–160

Apple: genetics, 9:333–366 rootstocks, 1:294–394 Apple transformation, 16:101–102 Apomixis: breeding, 18:13–86 genetics, 18:13–86 reproductive barriers, 11:92–96 rice, 17:114–116 Apricot transformation, 16:102 Arachis, in vitro culture, 2:218–224 Artichoke breeding, 12:253–269 Avena sativa, see Oat Azalea, mutation breeding, 6:75–76 B Bacillus thuringensis, 12:19–45 Bacterial diseases: apple rootstocks, 1:362–365 cell selection, 4:163–164 cowpea, 15:238–239 potato, 19:113–122 raspberry, 6:281–282 soybean, 1:209–212 sweet potato, 4:333–336 transformation fruit crops, 16:110 Banana: Breeding, 2:135–155 transformation, 16:105–106 Barley: anther culture, 15:141–186 breeding methods, 5:95–138 doubled haploid breeding, 15:141–186 gametoclonal variation, 5:368–370

Plant Breeding Reviews, Volume 20, Edited by Jules Janick ISBN 0-471-38788-6 © 2001 John Wiley & Sons, Inc.

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230 Barley (cont.) haploids in breeding, 3:219–252 photoperiodic response, 3:74, 89–92, 99 vernalization, 3:109 Bean (Phaseolus): breeding, 1:59–102 breeding mixtures, 4:245–272 breeding (tropics), 10:199–269 heat tolerance, 10:149 in vitro culture, 2:234–237 photoperiodic response, 3:71–73, 86–92, 16:102–109 protein, 1:59–102 Biochemical markers, 9:37–61 Biography: Allard, Robert W., 12:1–17 Bringhurst, Royce S., 9:1–8 Burton, Glenn W., 3:1–19 Downey, Richard K., 18:1–12 Draper, Arlen D., 13:1–10 Duvick, Donald N., 14:1–11 Gabelman, Warren H., 6:1–9 Hallauer, Arnel R., 15:1–17 Harlan, Jack R., 8:1–17 Jones, Henry A., 1:1–10 Laughnan, John R. 19:1–14 Munger, Henry M., 4:1–8 Ryder, Edward J., 16:1–14 Sears, Ernest Robert, 10:1–2 Simmonds, Norman W., 20:1–13 Sprague, George F., 2:1–11 Vogel, Orville A., 5:1–10 Weinberger, John H., 11:1–10 Yuan, Longping, 17:1–13 Birdsfoot trefoil, tissue culture, 2:228–229 Blackberry, 8:249–312 mutation breeding, 6:79 Black walnut, 1:236–266 Blueberry: breeding, 13:1–10 rabbiteye, 5:307–357 Brachiaria, apomixis, 18:36–39, 49–51 Bramble transformation, 16:105 Brassica, see Cole crops Brassicaceae: incompatibility, 15:23–27 molecular mapping, 14:19–23 Brassica: napus, see Canola, Rutabaga rapa, see Canola

CUMULATIVE SUBJECT INDEX Breeding: alfalfa via tissue culture, 4:123–152 almond, 8:313–338 amaranth, 19:227–285 apple, 9:333–366 apple rootstocks, 1:294–394 apomixis, 18:13–86 banana, 2:135–155 barley, 3:219–252; 5:95–138 bean, 1:59–102; 4:245–272 biochemical markers, 9:37–61 blackberry, 8:249–312 black walnut, 1:236–266 blueberry, rabbiteye, 5:307–357 cactus, 20:135–166 carbon isotope discrimination, 12:81–113 carrot, 19:157–190 cassava, 2:73–134 cell selection, 4:153–173 chestnut, 4:347–397 chimeras, 15:43–84 chrysanthemum, 14:321–361 citrus, 8:339–374 coffee, 2:157–193 coleus, 3:343–360 competitive ability, 14:89–138 cowpea, 15:215–274 cucumber, 6:323–359 diallel analysis, 9:9–36 doubled haploids, 15:141–186 durum wheat, 5:11–40 exotic maize, 14:165–187 fescue, 3:313–342 forest tree, 8:139–188 gene action 15:315–374 genotype x environment interaction, 16:135–178 grapefruit, 13:345–363 grasses, 11:251–274 guayule, 6:93–165 heat tolerance, 10:124–168 herbicide-resistant crops, 11:155–198 heterosis, 12:227–251 homeotic floral mutants, 9:63–99 honeycomb, 13:87–139; 18:177–249 hybrid, 17:225–257 hybrid wheat, 2:303–319; 3:169–191 induced mutations, 2:13–72 insect and mite resistance in cucurbits, 10:199–269 isozymes, 6:11–54

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CUMULATIVE SUBJECT INDEX lettuce, 16:1–14; 20:105–133 maize, 1:103–138, 139–161; 4:81–122; 9:181–216; 11:199–224; 14:139–163, 165–187, 189–236 molecular markers, 9:37–61 mosaics, 15:43–84 mushroom, 8:189–215 negatively associated traits, 13:141–177 oat, 6:167–207 oil palm, 4:175–201 onion, 20:67–103 pasture legumes, 5:237–305 pearl millet, 1:162–182 perennial rye, 13:265–292 persimmon, 19:191–225 plantain, 2:150–151; 14:267–320 potato, 3:274–277; 9:217–332; 16:15–86, 19:59–155 proteins in maize, 9:181–216 quality protein maize (QPM), 9:181–216 raspberry, 6:245–321 recurrent restricted phenotypic selection, 9:101–113 recurrent selection in maize, 9:115–179; 14:139–163 rice, 17:15–156 rose, 17:159–189 rutabaga, 8:217–248 sesame, 16:179–228 somatic hybridization, 20:167–225 soybean, 1:183–235; 3:289–311; 4:203–243 soybean nodulation, 11:275–318 soybean recurrent selection, 15:275–313 spelt, 15:187–213 statistics, 17:296–300 strawberry, 2:195–214 sugar cane, 16:272–273 supersweet sweet corn, 14:189–236 sweet cherry, 9:367–388 sweet corn, 1:139–161; 14:189–236 sweet potato, 4:313–345 tomato, 4:273–311 triticale, 5:41–93; 8:43–90 Vigna, 8:19–42 virus resistance, 12:47–79 wheat, 2:303–319; 3:169–191; 5:11–40; 11:225–234; 13:293–343 wheat for rust resistance, 13:293–343

231 white clover, 17:191–223 wild rice, 14:237–265 Bringhurst, Royce S. (biography), 9:1–8 Broadbean, in vitro culture, 2:244–245 Burton, Glenn W. (biography), 3:1–19 C Cactus: breeding, 135–166 domestication, 135–166 Cajanus, in vitro culture, 2:224 Canola, R. K. Downey, designer, 18:1–12 Carbohydrates, 1:144–148 Carbon isotope discrimination, 12:81–113 Carnation, mutation breeding, 6:73–74 Carrot breeding, 19: 157–190 Cassava, 2:73–134 Castanea, see Chestnut Cell selection, 4:139–145, 153–173 Cereal breeding, see Grain breeding Cherry, see Sweet cherry transformation, 16:102 Chestnut breeding, 4:347–397 Chickpea, in vitro culture, 2:224–225 Chimeras and mosaics, 15:43–84 Chinese cabbage, heat tolerance, 10:152 Chromosome, petunia, 1:13–21, 31–33 Chrysanthemum: breeding, 14:321–361 mutation breeding, 6:74 Cicer, see Chickpea Citrus, protoplast fusion, 8:339–374 Clonal repositories, see National Clonal Germplasm Repository Clover: in vitro culture, 2:240–244 molecular genetics, 17:191–223 Coffea arabica, see Coffee Coffee, 2:157–193 Cold hardiness: breeding nectarines and peaches, 10:271–308 wheat adaptation, 12:124–135 Cole crops: Chinese cabbage, heat tolerance, 10:152 gametoclonal variation, 5:371–372 rutabaga, 8:217–248 Coleus, 3:343–360 Competition, 13:158–165

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232 Competitive ability breeding, 14:89–138 Controlling elements, see Transposable elements Corn, see Maize; Sweet corn Cotton, heat tolerance 10:151 Cowpea: breeding, 15:215–274 heat tolerance, 10:147–149 in vitro culture, 2:245–246 photoperiodic response, 3:99 Cybrids. 3:205–210; 20: 206–209 Cryopreservation, 7:125–126,148–151,167 buds, 7:168–169 genetic stability, 7:125–126 meristems, 7:168–169 pollen, 7:171–172 seed, 7:148–151,168 Cucumber, breeding, 6:323–359 Cucumis sativa, see Cucumber Cucurbitaceae, insect and mite resistance, 10:309–360 Cytogenetics: alfalfa, 10:171–184 blueberry, 5:325–326 cassava, 2:94 citrus, 8:366–370 coleus, 3:347–348 durum wheat, 5:12–14 fescue, 3:316–319 Glycine, 16:288–317 guayule, 6:99–103 maize mobile elements, 4:81–122 maize-tripsacum hybrids, 20:15–66 oat, 6:173–174 pearl millet, 1:167 perennial rye, 13:265–292 petunia, 1:13–21, 31–32 rose, 17:169–171 rye, 13:265–292 Saccharum complex, 16:273–275 sesame, 16:185–189 triticale, 5:41–93; 8:54 wheat, 5:12–14; 10:5–15; 11:225–234 Cytoplasm: cybrids, 3:205–210; 20:206–209 molecular biology of male sterility, 10:23–51 organelles, 2:283–302; 6:361–393 pearl millet, 1:166 petunia, 1:43–45 wheat, 2:308–319

CUMULATIVE SUBJECT INDEX D Dahlia, mutation breeding, 6:75 Daucus, see Carrot Diallel cross, 9:9–36 Diospyros, see Persimmon Disease and pest resistance: antifungal proteins, 14:39–88 apple rootstocks, 1:358–373 banana, 2:143–147 blackberry, 8:291–295 black walnut, 1:251 blueberry, rabbiteye, 5:348–350 cassava, 2:105–114 cell selection, 4:143–145, 163–165 citrus, 8:347–349 coffee, 2:176–181 coleus, 3:353 cowpea, 15:237–247 durum wheat, 5:23–28 fescue, 3:334–336 herbicide-resistance, 11:155–198 host-parasite genetics, 5:393–433 induced mutants, 2:25–30 lettuce, 1:286–287 potato, 9:264–285, 19:69–155 raspberry, 6:245–321 rutabaga, 8:236–240 soybean, 1:183–235 spelt, 15:195–198 strawberry, 2:195–214 virus resistance, 12:47–79 wheat rust, 13:293–343 DNA methylation, 18:87–176 Doubled haploid breeding, 15:141–186 Downey, Richard K. (biography), 18:1–12 Draper, Arlen D. (biography), 13:1–10 Drought resistance: durum wheat, 5:30–31 soybean breeding, 4:203–243 wheat adaptation, 12:135–146 Durum wheat, 5:11–40 Duvick, Donald N. (biography), 14:1–11

E Elaeis, see Oil palm Embryo culture: in crop improvement, 5:181–236 oil palm, 4:186–187 pasture legume hybrids, 5:249–275

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CUMULATIVE SUBJECT INDEX Endosperm: maize, 1:139–161 sweet corn, 1:139–161 Endothia parasitica, 4:355–357 Evolution: coffee, 2:157–193 grapefruit, 13:345–363 maize, 20:15–66 sesame, 16:189 Exploration, 7:9–11, 26–28, 67–94 F Fabaceae, molecular mapping, 14:24–25 Fescue, 3:313–342 Festuca, see Fescue Floral biology: almond, 8:314–320 blackberry, 8:267–269 black walnut, 1:238–244 cassava, 2:78–82 chestnut, 4:352–353 coffee, 2:163–164 coleus, 3:348–349 fescue, 3:315–316 guayule, 6:103–105 homeotic mutants, 9:63–99 induced mutants, 2:46–50 pearl millet, 1:165–166 pistil in reproduction, 4:9–79 pollen in reproduction, 4:9–79 reproductive barriers, 11:11–154 rutabaga, 8:222–226 sesame, 16:184–185 sweet potato, 4:323–325 Forage breeding: alfalfa inbreeding, 13:209–233 fescue, 3:313–342 perennials, 11:251–274 white clover, 17:191–223 Forest crop breeding: black walnut, 1:236–266 chestnut, 4:347–397 ideotype concept, 12:177–187 molecular markers, 19:31–68 quantitative genetics, 8:139–188 Fruit, nut, and beverage crop breeding: almond, 8:313–338 apple, 9:333–366 apple rootstocks, 1:294–394 banana, 2:135–155 blackberry, 8:249–312

233 blueberry, 13:1–10 blueberry, rabbiteye, 5:307–357 cactus, 20:135–166 cherry, 9:367–388 citrus, 8:339–374 coffee, 2:157–193 ideotype concept, 12:175–177 genetic transformation, 16:87–134 grapefruit, 13:345–363 mutation breeding, 6:78–79 nectarine (cold hardy), 10:271–308 peach (cold hardy), 10:271–308 persimmon, 19:191–225 plantain, 2:135–155 raspberry, 6:245–321 strawberry, 2:195–214 sweet cherry, 9:367–388 Fungal diseases: apple rootstocks, 1:365–368 banana and plantain, 2:143–145, 147 cassava, 2:110–114 cell selection, 4:163–165 chestnut, 4:355–397 coffee, 2:176–179 cowpea, 15:237–238 durum wheat, 5:23–27 host-parasite genetics, 5:393–433 lettuce, 1:286–287 potato, 19:69–155 raspberry, 6:245–281 soybean, 1:188–209 spelt, 15:196–198 strawberry, 2:195–214 sweet potato, 4:333–336 transformation, fruit crops, 16:111–112 wheat rust, 13:293–343 G Gabelman, Warren H. (biography), 6:1–9 Gametes: almond, self compatibility, 7:322–330 blackberry, 7:249–312 competition, 11:42–46 forest trees, 7:139–188 maize aleurone, 7:91–137 maize anthocynanin, 7:91–137 mushroom, 7:189–216 polyploid, 3:253–288 rutabaga, 7:217–248 transposable elements, 7:91–137 unreduced, 3:253–288

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234 Gametoclonal variation, 5:359–391 barley, 5:368–370 brassica, 5:371–372 potato, 5:376–377 rice, 5:362–364 rye, 5:370–371 tobacco, 5:372–376 wheat, 5:364–368 Garlic, mutation breeding, 6:81 Genes: action, 15:315–374 apple, 9:337–356 Bacillus thuringensis, 12:19–45 incompatibility, 15:19–42 incompatibility in sweet cherry, 9:367–388 induced mutants, 2:13–71 lettuce, 1:267–293 maize endosperm, 1:142–144 maize protein, 1:110–120, 148–149 petunia, 1:21–30 quality protein in maize, 9:183–184 rye perenniality, 13:261–288 soybean, 1:183–235 soybean nodulation, 11:275–318 sweet corn, 1:142–144 wheat rust resistance, 13:293–343 Genetic engineering: bean, 1:89–91 DNA methylation, 18:87–176 fruit crops, 16:87–134 host-parasite genetics, 5:415–428 maize mobile elements, 4:81–122 transformation by particle bombardment, 13:231–260 virus resistance, 12:47–79 Genetic load and lethal equivalents, 10:93–127 Genetics: adaptation, 3:21–167 almond, self compatibility, 8:322–330 amaranth, 19:243–248 Amaranthus, see Amaranth apple, 9:333–366 apomixis, 18:13–86 Bacillus thuringensis, 12:19–45 bean seed protein, 1:59–102 blackberry, 8:249–312 black walnut, 1:247–251 blueberry, 13:1–10 blueberry, rabbiteye, 5:323–325 carrot, 19:164–171 chestnut blight, 4:357–389

CUMULATIVE SUBJECT INDEX chimeras, 15:43–84 chrysanthemums, 14:321 clover, white, 17:191–223 coffee, 2:165–170 coleus, 3:3–53 cowpea, 15:215–274 DNA methylation, 18:87–176 durum wheat, 5:11–40 forest trees, 8:139–188 fruit crop transformation, 16:87–134 gene action, 15:315–374 herbicide resistance, 11:155–198 host-parasite, 5:393–433 incompatibility, 15:19–42 incompatibility in sweet cherry, 9:367–388 induced mutants, 2:51–54 insect and mite resistance in Cucurbitaceae, 10:309–360 isozymes, 6:11–54 lettuce, 1:267–293 maize aleurone, 8:91–137 maize anther culture, 11:199–224 maize anthocynanin, 8:91–137 maize endosperm, 1:142–144 maize male sterility, 10:23–51 maize mobile elements, 4:81–122 maize mutation, 5:139–180 maize seed protein, 1:110–120, 148–149 male sterility, maize, 10:23–51 mapping, 14:13–37 maturity, 3:21–167 markers to manage germplasm, 13:11–86 metabolism and heterosis, 10:53–59 molecular mapping, 14:13–37 mosaics, 15:43–84 mushroom, 8:189–216 oat, 6:168–174 organelle transfer, 6:361–393 overdominance, 17:225–257 pearl millet, 1:166, 172–180 perennial rye, 13:261–288 petunia, 1:1–58 photoperiod, 3:21–167 plantain, 14:264–320 potato disease resistance, 19:69–165 potato ploidy manipulation, 3:274–277; 16:15–86 quality protein in maize, 9:183–184 quantitative trait loci, 15:85–139 reproductive barriers, 11:11–154 rice, hybrid, 17:15–156

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CUMULATIVE SUBJECT INDEX rose, 17:171–172 rutabaga, 8:217–248 sesame, 16:189–195 soybean, 1:183–235 soybean nodulation, 11:275–318 spelt, 15:187–213 supersweet sweet corn, 14:189–236 sweet corn, 1:139–161; 14:189–236 sweet potato, 4:327–330 temperature, 3:21–167 tomato fruit quality, 4:273–311 transposable elements, 8:91–137 triticale, 5:41–93 virus resistance, 12:47–79 wheat gene manipulation, 11:225–234 wheat male sterility, 2:307–308 wheat molecular biology, 11:235–250 wheat rust, 13:293–343 white clover, 17:191–223 yield, 3:21–167 Genome: Glycine, 16:289–317 Poaceae, 16:276–281 Genotype x environment, interaction, 16:135–178 Germplasm, see also National Clonal Germplasm Repositories; National Plant Germplasm System acquisition and collection, 7:160–161 apple rootstocks, 1:296–299 banana, 2:140–141 blackberry, 8:265–267 black walnut, 1:244–247 cactus, 20:141–145 cassava, 2:83–94, 117–119 chestnut, 4:351–352 coffee, 2:165–172 distribution, 7:161–164 enhancement, 7:98–202 evaluation, 7:183–198 exploration and introduction, 7:9–18,64–94 genetic markers, 13:11–86 guayule, 6:112–125 isozyme, 6:18–21 maintenance and storage, 7:95–110, 111–128,129–158,159–182; 13:11–86 maize, 14:165–187 management, 13:11–86 oat, 6:174–176 pearl millet, 1:167–170 plantain, 14:267–320

235 potato, 9:219–223 preservation by tissue culture, 2:265–282 rutabaga, 8:226–227 sesame, 16:201–204 spelt, 15:204–205 sweet potato, 4:320–323 triticale, 8:55–61 wheat, 2:307–313 Gesneriaceae, mutation breeding, 6:73 Gladiolus, mutation breeding, 6:77 Glycine, genomes, 16:289–317 Glycine max, see Soybean Grain breeding: amaranth, 19:227–285 barley, 3:219–252, 5:95–138 doubled haploid breeding, 15:141–186 ideotype concept, 12:173–175 maize, 1:103–138, 139–161; 5:139–180; 9:115–179, 181–216; 11:199–224; 14:165–187 oat, 6:167–207 pearl millet, 1:162–182 rice, 17:15–156 spelt, 15:187–213 transformation, 13:231–260 triticale, 5:41–93; 8:43–90 wheat, 2:303–319; 5:11–40; 11:225–234, 235–250; 13:293–343 wild rice, 14:237–265 Grape, transformation, 16:103–104 Grapefruit: breeding, 13:345–363 evolution, 13:345–363 Grass breeding: breeding, 11:251–274 mutation breeding, 6:82 recurrent selection, 9:101–113 transformation, 13:231–260 Growth habit, induced mutants, 2:14–25 Guayule, 6:93–165 H Hallauer, Arnel R. (biography), 15:1–17 Haploidy, see also Unreduced and polyploid gametes apple, 1:376 barley, 3:219–252 cereals, 15:141–186 maize, 11:199–224 petunia, 1:16–18, 44–45 potato, 3:274–277; 16:15–86

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236 Harlan, Jack R. (biography), 8:1–17 Heat tolerance breeding, 10:129–168 Herbicide resistance: breeding needs, 11:155–198 cell selection, 4:160–161 decision trees, 18:251–303 risk assessment, 18:251–303 transforming fruit crops, 16:114 Heterosis: gene action, 15:315–374 overdominance, 17:225–257 plant breeding, 12:227–251 plant metabolism, 10:53–90 rice, 17:24–33 Hordeum, see Barley Honeycomb: breeding, 18:177–249 selection, 13:87–139, 18:177–249 Host-parasite genetics, 5:393–433 Hyacinth, mutation breeding, 6:76–77 Hybrid and hybridization, see also Heterosis barley, 5:127–129 blueberry, 5:329–341 chemical, 3:169–191 interspecific, 5:237–305 overdominance, 17:225–257 rice, 17:15–156 wheat, 2:303–319 I Ideotype concept, 12:163–193 In vitro culture: alfalfa, 2:229–234; 4:123–152 barley, 3:225–226 bean, 2:234–237 birdsfoot trefoil, 2:228–229 blackberry, 8:274–275 broadbean, 2:244–245 cassava, 2:121–122 cell selection, 4:153–173 chickpea, 2:224–225 citrus, 8:339–374 clover, 2:240–244 coffee, 2:185–187 cowpea, 2:245–246 embryo culture, 5:181–236, 249–275 germplasm preservation, 7:125,162–167 introduction, quarantines, 3:411–414 legumes, 2:215–264

CUMULATIVE SUBJECT INDEX mungbean, 2:245–246 oil palm, 4:175–201 pea, 2:236–237 peanut, 2:218–224 petunia, 1:44–48 pigeon pea, 2:224 pollen, 4:59–61 potato, 9:286–288 sesame, 16:218 soybean, 2:225–228 Stylosanthes, 2:238–240 wheat, 12:115–162 wingbean, 2:237–238 zein, 1:110–111 Inbreeding depression, 11:84–92 alfalfa, 13:209–233 cross pollinated crops, 13:209–233 Incompatibility: almond, 8:313–338 molecular biology, 15:19–42 pollen, 4:39–48 reproductive barrier, 11:47–70 sweet cherry, 9:367–388 Incongruity, 11:71–83 Industrial crop breeding, guayule, 6:93–165 Insect and mite resistance: apple rootstock, 1:370–372 black walnut, 1:251 cassava, 2:107–110 clover, white, 17:209–210 coffee, 2:179–180 cowpea, 15:240–244 Cucurbitaceae, 10:309–360 durum wheat, 5:28 maize, 6:209–243 raspberry, 6:282–300 rutabaga, 8:240–241 sweet potato, 4:336–337 transformation fruit crops, 16:113 white clover, 17:209–210 Interspecific hybridization: blackberry, 8:284–289 blueberry, 5:333–341 citrus, 8:266–270 pasture legume, 5:237–305 rose, 17:176–177 rutabaga, 8:228–229 Vigna, 8:24–30 Intersubspecific hybridization, rice, 17:88–98 Introduction, 3:361–434; 7:9–11, 21–25

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CUMULATIVE SUBJECT INDEX Ipomoea, see Sweet potato Isozymes, in plant breeding, 6:11–54 J Jones, Henry A. (biography), 1:1–10 Juglans nigra, see Black walnut K Karyogram, petunia, 1:13 Kiwifruit transformation, 16:104 L Lactuca sativa, see Lettuce Laughnan, Jack R. (bibliography), 19:1–14 Legume breeding, see also Oilseed, Soybean cowpea, 15:215–274 pasture legumes, 5:237–305 Vigna, 8:19–42 Legume tissue culture, 2:215–264 Lethal equivalents and genetic load, 10:93–127 Lettuce: genes, 1:267–293 breeding, 16:1–14; 20:105–133 Linkage: bean, 1:76–77 isozymes, 6:37–38 lettuce, 1:288–290 maps, molecular markers, 9:37–61 petunia, 1:31–34 Lotus: hybrids, 5:284–285 in vitro culture, 2:228–229 Lycopersicon, see Tomato M Maize: anther culture, 11:199–224; 15:141–186 anthocyanin, 8:91–137 apomixis, 18:56–64 breeding, 1:103–138, 139–161 carbohydrates, 1:144–148 doubled haploid breeding, 15:141–186 exotic germplasm utilization, 14:165–187

237 honeycomb breeding, 18:226–227 hybrid breeding, 17:249–251 insect resistance, 6:209–243 male sterility, 10:23–51 mobile elements, 4:81–122 mutations, 5:139–180 origins, 20:15–66 overdominance, 17:225–257 protein, 1:103–138 quality protein, 9:181–216 recurrent selection, 9:115–179; 14:139–163 supersweet sweet corn, 14:189–236 transformation, 13:235–264 transposable elements, 8:91–137 unreduced gametes, 3:277 Male sterility: chemical induction, 3:169–191 coleus, 3:352–353 lettuce, 1:284–285 molecular biology, 10:23–51 pearl millet, 1:166 petunia, 1:43–44 rice, 17:33–72 sesame, 16:191–192 wheat, 2:303–319 Malus spp, see Apple Malus ×domestica, see Apple Malvaceae, molecular mapping, 14:25–27 Mango transformation, 16:107 Manihot esculenta, see Cassava Medicago, see also Alfalfa in vitro culture, 2:229–234 Meiosis, petunia, 1:14–16 Metabolism and heterosis, 10:53–90 Microprojectile bombardment, transformation, 13:231–260 Mitochondria genetics, 6:377–380 Mixed plantings, bean breeding, 4:245–272 Mobile elements, see also Transposable elements: maize, 4:81–122; 5:146–147 Molecular biology: apomixis, 18:65–73 comparative mapping, 14:13–37 cytoplasmic male sterility, 10:23–51 DNA methylation, 18:87–176 herbicide-resistant crops, 11:155–198 incompatibility, 15:19–42 molecular mapping, 14:13–37; 19:31–68

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238 Molecular biology (cont.) molecular markers, 9:37–61, 10:184–190; 12:195–226; 13:11–86; 14:13–37 quantitative trait loci, 15:85–139 somaclonal variation, 16:229–268 soybean nodulation, 11:275–318 transposable (mobile) elements, 4:81–122; 8:91–137 virus resistance, 12:47–79 wheat improvement, 11:235–250 Molecular markers, 9:37–61 alfalfa, 10:184–190 apomixis, 18:40–42 clover, white, 17:212–215 forest crops, 19:31–68 fruit crops, 12:195–226 mapping, 14:13–37 plant genetic resource mangement, 13:11–86 rice, 17:113–114 rose, 17:179 somaclonal variation, 16:238–243 white clover, 17:212–215 Monosomy, petunia, 1:19 Mosaics and chimeras, 15:43–84 Mungbean, 8:32–35 in vitro culture, 2:245–246 photoperiodic response, 3:74, 89–92 Munger, Henry M. (biography), 4:1–8 Musa, see Banana, Plantain Mushroom, breeding and genetics, 8:189–215 Mutants and mutation: alfalfa tissue culture, 4:130–139 apple rootstocks, 1:374–375 banana, 2:148–149 barley, 5:124–126 blackberry, 8:283–284 cassava, 2:120–121 cell selection, 4:154–157 chimeras, 15:43–84 coleus, 3:355 cytoplasmic, 2:293–295 gametoclonal variation, 5:359–391 homeotic floral, 9:63–99 induced, 2:13–72 maize, 1:139–161, 4:81–122; 5:139–180 mobile elements, see Transposable elements mosaics, 15:43–84 petunia, 1:34–40

CUMULATIVE SUBJECT INDEX sesame, 16:213–217 somaclonal variation, 4:123–152; 5:147–149 sweet corn, 1:139–161 sweet potato, 4:371 transposable elements, 4:181–122; 8:91–137 tree fruits, 6:78–79 vegetatively-propagated crops, 6:55–91 zein synthesis, 1:111–118 Mycoplasma diseases, raspberry, 6:253–254 N National Clonal Germplasm Repository (NCGR), 7:40–43 cryopreservation, 7:125–126 genetic considerations, 7:126–127 germplasm maintenance and storage, 7:111–128 identification and label verification, 7:122–123 in vitro culture and storage, 7:125 operations guidelines, 7:113–125 preservation techniques, 7:120–121 virus indexing and plant health, 7:123–125 National Plant Germplasm System (NPGS), see also Germplasm history, 7:5–18 information systems, 7:57–65 operations, 7:19–56 National Seed Storage Laboratory (NSSL), 7:13–14, 37–38, 152–153 Nectarines, cold hardiness breeding, 10:271–308 Nematode resistance: apple rootstocks, 1:368 banana and plantain, 2:145–146 coffee, 2:180–181 cowpea, 15:245–247 soybean, 1:217–221 sweet potato, 4:336 transformation fruit crops, 16:112–113 Nicotiana, see Tobacco Nodulation, soybean, 11:275–318 O Oat, breeding, 6:167–207 Oil palm:

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CUMULATIVE SUBJECT INDEX breeding, 4:175–201 in vitro culture, 4:175–201 Oilseed breeding: canola, 18:1–20 oil palm, 4:175–201 sesame, 16:179–228 soybean, 1:183–235; 3:289–311; 4:203–245; 11:275–318; 15:275–313 Onion, breeding history, 20:57–103 Opuntia, see Cactus Organelle transfer, 2:283–302; 3:205–210; 6:361–393 Ornamentals breeding: chrysanthemum, 14:321–361 coleus, 3:343–360 petunia, 1:1–58 rose, 17:159–189 Ornithopus, hybrids, 5:285–287 Orzya, see Rice Overdominance, 17:225–257 Ovule culture, 5:181–236 P Panicum maximum, apomixis, 18:34–36, 47–49 Papaya transformation, 16:105–106 Parthenium argentatum, see Guayule Paspalum, apomixis, 18:51–52 Paspalum notatum, see Pensacola bahiagrass Passionfruit transformation, 16:105 Pasture legumes, interspecific hybridization, 5:237–305 Pea: flowering, 3:81–86, 89–92 in vitro culture, 2:236–237 Peach: cold hardiness breeding, 10:271–308 transformation, 16:102 Peanut, in vitro culture, 2:218–224 Pear transformation, 16:102 Pearl millet: apomixis, 18:55–56 breeding, 1:162–182 Pecan transformation, 16:103 Pennisetum americanum, see Pearl millet Pensacola bahiagrass, 9:101–113 apomixis, 18:51–52 selection, 9:101–113 Pepino transformation, 16:107

239 Peppermint, mutation breeding, 6:81–82 Perennial grasses, breeding, 11:251–274 Perennial rye breeding, 13:261–288 Persimmon breeding, 19:191–225 Petunia spp., genetics, 1:1–58 Phaseolin, 1:59–102 Phaseolus vulgaris, see Bean Phytophthora fragariae, 2:195–214 Pigeon pea, in vitro culture, 2:224 Pistil, reproductive function, 4:9–79 Pisum, see Pea Plant introduction, 3:361–434; 7:9–11, 21–25 Plant exploration, 7:9–11, 26–28, 67–94 Plantain breeding, 2:135–155; 14:267–320 Plastid genetics, 6:364–376, see also Organelle Plum transformation, 16:103–140 Poaceae: molecular mapping, 14:23–24 Saccharum complex, 16:269–288 Pollen: reproductive function, 4:9–79 storage, 13:179–207 Polyploidy, see also Haploidy alfalfa, 10:171–184 alfalfa tissue culture, 4:125–128 apple rootstocks, 1:375–376 banana, 2:147–148 barley, 5:126–127 blueberry, 13:1–10 gametes, 3:253–288 isozymes, 6:33–34 petunia, 1:18–19 potato, 16:15–86 reproductive barriers, 11:98–105 sweet potato, 4:371 triticale, 5:11–40 Population genetics, see Quantitative Genetics Potato: breeding, 9:217–332, 19:69–165 disease resistance breeding, 19:69–165 gametoclonal variation, 5:376–377 heat tolerance, 10:152 honeycomb breeding, 18:227–230 mutation breeding, 6:79–80 photoperiodic response, 3:75–76, 89–92 ploidy manipulation, 16:15–86 unreduced gametes, 3:274–277

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240 Protein: antifungal, 14:39–88 bean, 1:59–102 induced mutants, 2:38–46 maize, 1:103–138, 148–149; 9:181–216 Protoplast fusion, 3:193–218; 20: 167–225 citrus, 8:339–374 mushroom, 8:206–208 Prunus: amygdalus, see Almond avium, see Sweet cherry Pseudograin breeding, amaranth, 19:227–285 Psophocarpus, in vitro culture, 2:237–238 Q Quantitative genetics: forest trees, 8:139–188 genotype x environment interaction, 16:135–178 overdominance, 17:225–257 statistics, 17:296–300 trait loci (QTL), 15:85–139; 19:31–68 Quantitative trait loci (QTL), 15:85–138; 19:31–68 Quarantines, 3:361–434; 7:12,35 R Rabbiteye blueberry, 5:307–357 Raspberry, breeding, 6:245–321 Recurrent restricted phenotypic selection, 9:101–113 Recurrent selection, 9:101–113, 115–179; 14:139–163 soybean, 15:275–313 Red stele disease, 2:195–214 Regional trial testing, 12:271–297 Reproduction: barriers and circumvention, 11:11–154 pollen and pistil, 4:9–79 Rhododendron, mutation breeding, 6:75–76 Rice, see also Wild rice anther culture, 15:141–186 apomixis, 18:65 doubled haploid breeding, 15:141–186 gametoclonal variation, 5:362–364 heat tolerance, 10:151–152

CUMULATIVE SUBJECT INDEX honeycomb breeding, 18:224–226 hybrid breeding, 17:1–15, 15–156 photoperiodic response, 3:74, 89–92 Rosa, see Rose Rose breeding, 17:159–189 Rubus, see Blackberry, Raspberry Rust, wheat, 13:293–343 Rutabaga, 8:217–248 Ryder, Edward J. (biography), 16:1–14 Rye: gametoclonal variation, 5:370–371 perennial breeding, 13:261–288 triticale, 5:41–93 S Saccharum complex, 16:269–288 Salt resistance: cell selection, 4:141–143 durum wheat, 5:31 Sears, Ernest R. (biography), 10:1–22 Secale, see Rye Seed: apple rootstocks, 1:373–374 banks, 7:13–14, 37–40, 152–153 bean, 1:59–102 lettuce, 1:285–286 maintenance and storage, 7:95–110, 129–158, 159–182 maize, 1:103–138, 139–161, 4:81–86 pearl millet, 1:162–182 protein, 1:59–138, 148–149 rice production, 17:98–111, 118–119 soybean, 1:183–235, 3:289–311 synthetic, 7:173–174 variegation, 4:81–86 wheat (hybrid), 2:313–317 Selection, see also Breeding cell, 4:139–145, 153–173 honeycomb design, 13:87–139; 18:177–249 marker assisted, forest tree, 19:31–68 recurrent restricted phenotypic, 9:101–113 recurrent selection in maize, 9:115–179; 14:139–163 Sesame breeding, 16:179–228 Sesamum indicum, see Sesame Solanaceae: incompatibility, 15:27–34 molecular mapping, 14:27–28 Solanum tuberosum, see Potato

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CUMULATIVE SUBJECT INDEX Somaclonal variation, see also Gametoclonal variation alfalfa, 4:123–152 isozymes, 6:30–31 maize, 5:147–149 molecular analysis, 16:229–268 mutation breeding, 6:68–70 rose, 17:178–179 transformation interaction, 16:229–268 utilization, 16:229–268 Somatic embryogenesis, 5:205–212; 7:173–174 oil palm, 4:189–190 Somatic genetics, see also Gametoclonal variation; Somaclonal variation alfalfa, 4:123–152 legumes, 2:246–248 maize, 5:147–149 organelle transfer, 2:283–302 pearl millet, 1:166 petunia, 1:43–46 protoplast fusion, 3:193–218 wheat, 2:303–319 Somatic hybridization, see also Protoplast fusion, 20:167–225 Sorghum: photoperiodic response, 3:69–71, 97–99 transformation, 13:235–264 Southern pea, see Cowpea Soybean: cytogenetics, 16:289–317 disease resistance, 1:183–235 drought resistance, 4:203–243 in vitro culture, 2:225–228 nodulation, 11:275–318 photoperiodic response, 3:73–74 recurrent selection, 15:275–313 semidwarf breeding, 3:289–311 Spelt, agronomy, genetics, breeding, 15:187–213 Sprague, George F. (biography), 2:1–11 Sterility, see also Male sterility, 11:30–41 Starch, maize, 1:114–118 Statistics, history, 17:259–316 Strawberry: red stele resistance breeding, 2:195–214 transformation, 16:104 Stress resistance: cell selection, 4:141–143,161–163 transformation fruit crops, 16:115

241 Stylosanthes, in vitro culture, 2:238–240 Sugarcane: and Saccharum complex, 16:269–288 mutation breeding, 6:82–84 Sweet cherry, pollen-incompatibility and selffertility, 9:367–388 transformation, 16:102 Sweet corn, see also Maize endosperm, 1:139–161 supersweet (shrunken2), 14:189–236 Sweet potato breeding, 4:313–345; 6:80–81 T Tamarillo transformation, 16:107 Taxonomy: amaranth, 19:233–237 apple, 1:296–299 banana, 2:136–138 blackberry, 8:249–253 cassava, 2:83–89 chestnut, 4:351–352 chrysanthemum, 14:321–361 clover, white, 17:193–211 coffee, 2:161–163 coleus, 3:345–347 fescue, 3:314 Glycine, 16:289–317 guayule, 6:112–115 oat, 6:171–173 pearl millet, 1:163–164 petunia, 1:13 plantain, 2:136; 14:271–272 rose, 17:162–169 rutabaga, 8:221–222 Saccharum complex, 16:270–272 sweet potato, 4:320–323 triticale, 8:49–54 Vigna, 8:19–42 White clover, 17:193–211 wild rice, 14:240–241 Testing: adaptation, 12:271–297 honeycomb design, 13:87–139 Tissue culture, see In vitro culture Tobacco, gametoclonal variation, 5:372–376 Tomato: breeding for quality, 4:273–311 heat tolerance, 10:150–151

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242 Toxin resistance, cell selection, 4:163–165 Transformation: alfalfa, 10:190–192 cereals, 13:231–260 fruit crops, 16:87–134 mushroom, 8:206 rice, 17:179–180 somaclonal variation, 16:229–268 white clover, 17:193–211 Transpiration efficiency, 12:81–113 Transposable elements, 4:81–122; 5:146–147; 8:91–137 Tree crops, ideotype concept, 12:163–193 Tree fruits, see Fruit, nut and beverage crop breeding Trifolium, see Clover, White Clover Trifolium hybrids, 5:275–284 in vitro culture, 2:240–244 Tripsacum: apomixis, 18:51 maize ancestry, 20:15–66 Trisomy, petunia, 1:19–20 Triticale, 5:41–93; 8:43–90 Triticum: Aestivum, see Wheat Turgidum, see Durum wheat Triticosecale, see Triticale Tulip, mutation breeding, 6:76 U United States National Plant Germplasm System, see National Plant Germplasm System Unreduced and polyploid gametes, 3:253–288; 16:15–86 Urd bean, 8:32–35 V Vaccinium, see Blueberry Vegetable breeding: artichoke, 12:253–269 bean, 1:59–102; 4:245–272 bean (tropics), 10:199–269 carrot 19: 157–190 cassava, 2:73–134 cucumber, 6:323–359 cucurbit insect and mite resistance, 10:309–360

CUMULATIVE SUBJECT INDEX lettuce, 1:267–293; 16:1–14; 20:105:133 mushroom, 8:189–215 onion, 20:67–103 potato, 9:217–232; 16:15–86l; 19:69–165 rutabaga, 8:217–248 tomato, 4:273–311 sweet corn, 1:139–161; 14:189–236 sweet potato, 4:313–345 Vicia, in vitro culture, 2:244–245 Vigna, see Cowpea, Mungbean in vitro culture, 2:245–246; 8:19–42 Virus diseases: apple rootstocks, 1:358–359 clover, white, 17:201–209 coleus, 3:353 cowpea, 15:239–240 indexing, 3:386–408, 410–411, 423–425 in vitro elimination, 2:265–282 lettuce, 1:286 potato, 19:122–134 raspberry, 6:247–254 resistance, 12:47–79 soybean, 1:212–217 sweet potato, 4:336 transformation fruit crops, 16:108–110 white clover, 17:201–209 Vogel, Orville A. (biography), 5:1–10 W Walnut (black), 1:236–266 Walnut transformation, 16:103 Weinberger, John A. (biography), 11:1–10 Wheat: anther culture, 15:141–186 apomixis, 18:64–65 chemical hybridization, 3:169–191 cold hardiness adaptation, 12:124–135 cytogenetics, 10:5–15 doubled haploid breeding, 15:141–186 drought tolerance, 12:135–146 durum, 5:11–40 gametoclonal variation, 5:364–368 gene manipulation, 11:225–234 heat tolerance, 10:152 hybrid, 2:303–319; 3:185–186 in vitro adaptation, 12:115–162

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CUMULATIVE SUBJECT INDEX molecular biology, 11:235–250 photoperiodic response, 3:74 rust interaction, 13:293–343 triticale, 5:41–93 vernalization, 3:109 White clover, molecular genetics, 17:191–223 Wild rice, breeding, 14:237–265 Winged bean, in vitro culture, 2:237–238

243 Y Yuan, Longping (biography), 17:1–13.

Z Zea mays, see Maize, Sweet corn Zein, 1:103–138 Zizania palustris, see Wild rice

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Cumulative Contributor Index (Volumes 1–20) Abdalla, O. S., 8:43 Acquaah, G., 9:63 Aldwinckle, H. S., 1:294 Anderson, N. O., 10:93; 11:11 Aronson, A. I., 12:19 Ascher, P. D., 10:93 Ashri, A., 16:179 Baltensperger, D. D., 19:227 Basnizki, J., 12:253 Beck, D. L., 17:191 Beineke, W. F., 1:236 Bingham, E. T., 4:123; 13:209 Binns, M. R., 12:271 Bird, R. McK., 5:139 Bjarnason, M., 9:181 Bliss, F. A., 1:59; 6:1 Boase, M. R., 14:321 Borlaug, N. E., 5:1 Boyer, C. D., 1:139 Bravo, J. E., 3:193 Brenner, D. M., 19:227 Bressan , R. A., 13:235; 14:39 Bretting, P. K., 13:11 Broertjes, C., 6:55 Brown, J. W. S., 1:59 Brown, S. K., 9:333,367 Burnham, C. R., 4:347 Burton, G. W., 1:162; 9:101 Byrne, D., 2:73 Campbell, K. G., 15:187 Cantrell, R. G., 5:11 Carvalho, A., 2:157 Casas, A. M., 13:235 Choo, T. M., 3:219

Christenson, G. M., 7:67 Christie, B. R., 9:9 Clark, R. L., 7:95 Clarke, A. E., 15:19 Clegg, M. T., 12:1 Condon, A. G., 12:81 Cooper, R. L., 3:289 Cornu, A., 1:11 Costa, W. M., 2:157 Cregan, P., 12:195 Crouch, J. H., 14:267 Crow, J. F., 17:225 Cummins, J. N., 1:294 Dana, S., 8:19 De Jong, H., 9:217 Deroles, S. C., 14:321 Dhillon, B. S., 14:139 Dickmann, D. I., 12:163 Dodds, P. N., 15:19 Draper, A. D., 2:195 Dumas, C., 4:9 Duncan, D. R., 4:153 Echt, C. S., 10:169 Ehlers, J. D., 15:215 England, F., 20:1 Eubanks, M. W., 20:15 Evans, D. A., 3:193; 5:359 Everett, L. A., 14:237 Ewart, L. C., 9:63 Farquhar, G. D., 12:81 Fasoula, D. A., 14:89; 15:315; 18:177 Fasoula, V. A., 13:87; 14:89; 15:315; 18:177

Plant Breeding Reviews, Volume 20, Edited by Jules Janick ISBN 0-471-38788-6 © 2001 John Wiley & Sons, Inc.

245

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246 Fasoulas, A. C., 13:87 Fazuoli, L. C., 2:157 Fear, C. D., 11:1 Ferris, R. S. B., 14:267 Flore, J. A., 12:163 Forsberg, R. A., 6:167 Forster, R. L. S., 17:191 French, D. W., 4:347 Galiba, G., 12:115 Galletta, G. J., 2:195 Gmitter, F. G., Jr., 8:339; 13:345 Gold, M. A., 12:163 Goldman, I. L. 19:15; 20:67 Gradziel, T. M., 15:43 Gressel, J., 11:155; 18:251 Gresshof, P. M., 11:275 Grombacher, A. W., 14:237 Grosser, J. W., 8:339 Grumet, R., 12:47 Gudin, S., 17:159 Guimarães, C. T., 16:269 Gustafson, J. P., 5:41; 11:225 Guthrie, W. D., 6:209 Hall, A. E., 10:129; 12:81; 15:215 Hall, H. K., 8:249 Hallauer, A. R., 9:115; 14:1,165 Hamblin, J., 4:245 Hancock, J. F., 13:1 Hancock, J. R., 9:1 Hanna, W. W., 13:179 Harlan, J. R., 3:1 Hasegawa, P. M. 13:235; 14:39 Havey, M. J., 20:67 Hillel, J., 12:195 Hunt, L. A., 16:135 Hutchinson, J. R., 5:181 Hymowitz, T., 8:1; 16:289 Janick, J., 1:xi Jansky, S., 19:77 Jayaram, Ch., 8:91 Johnson, A. A. T., 16:229; 20:167 Jones, A., 4:313 Jones, J. S., 13:209 Ju, G. C., 10:53 Kang, H., 8:139 Kann, R. P., 4:175 Karmakar, P. G., 8:19 Kartha, K. K., 2:215,265

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CUMULATIVE CONTRIBUTOR INDEX Kasha, K. J., 3:219 Keep, E., 6:245 Kleinhofs, A., 2:13 Knox, R. B., 4:9 Kollipara, K. P., 16:289 Kononowicz, A. K., 13:235 Konzak, C. F., 2:13 Krikorian, A. D., 4:175 Krishnamani, M. R. S., 4:203 Kronstad, W. E., 5:1 Kulakow, P. A., 19:227 Lamkey, K. R., 15:1 Lavi, U., 12:195 Layne, R. E. C., 10:271 Lebowitz, R. J., 3:343 Lehmann, J. W., 19:227 Levings, III, C. S., 10:23 Lewers, K. R., 15:275 Li, J., 17:1,15 Liedl, B. E., 11:11 Lin, C. S., 12:271 Lovell, G. R., 7:5 Lukaszewski, A. J., 5:41 Lyrene, P. M., 5:307 McCoy, T. J., 4:123; 10:169 McCreight, J. D., 1:267; 16:1 McDaniel, R. G., 2:283 McKeand, S. E., 19:41 McRae, D. H., 3:169 Maheswaran, G., 5:181 Maizonnier, D., 1:11 Marcotrigiano, M., 15:43 Martin, F. W., 4:313 Medina-Filho, H. P., 2:157 Miller, R., 14:321 Mondragon Jacobo, C. 20:135 Morrison, R. A., 5:359 Mowder, J. D., 7:57 Mroginski, L. A., 2:215 Murphy, A. M., 9:217 Mutschler, M. A., 4:1 Myers, O., Jr., 4:203 Myers, R. L., 19:227. Namkoong, G., 8:139 Neuffer, M. G., 5:139 Newbigin, E., 15:19 O’Malley, 19:41 Ortiz, R., 14:267; 16:15

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CUMULATIVE CONTRIBUTOR INDEX Palmer, R. G., 15:275 Pandy, S., 14:139 Parliman, B. J., 3:361 Paterson, A. H., 14:13 Pedersen, J. F., 11:251 Perdue, R. E., Jr., 7:67 Peterson, P. A., 4:81; 8:91 Polidorus, A. N., 18:87 Porter, R. A., 14:237 Proudfoot, K. G., 8:217 Rackow, G., 18:1 Raina, S. K., 15:141 Ramage, R. T., 5:95 Ramming, D. W., 11:1 Ray, D. T., 6:93 Redei, G. P., 10:1 Reimann-Phillipp, R., 13:265 Reinbergs, E., 3:219 Rhodes, D., 10:53 Richards, R. A., 12:81 Roath, W. W., 7:183 Robinson, R. W., 1:267; 10:309 Ron Parra, J., 14:165 Roos, E. E., 7:129 Rotteveel, T., 18:251 Rowe, P., 2:135 Russell, W. A., 2:1 Rutter, P. A., 4:347 Ryder, E. J., 1:267; 20:105 Samaras, Y., 10:53 Sansavini, S., 16:87 Saunders, J. W., 9:63 Savidan, Y., 18:13 Sawhney, R. N., 13:293 Schaap, T., 12:195 Schroeck, G., 20:67 Scott, D. H., 2:195 Seabrook, J. E. A., 9:217 Sears, E. R., 11:225 Shands, Hazel L. 6:167 Shands, Henry L. 7:1,5 Shannon, J. C., 1:139 Shattuck, V. I., 8:217; 9:9 Shaun, R., 14:267 Sidhu, G. S., 5:393 Simmonds, N. W., 17:259 Simon, P. W., 19:157 Singh, B. B., 15:215 Singh, R. J., 16:289 Singh, S. P., 10:199

247 Singh, Z., 16:87 Slabbert, M. M., 19:227 Sleper, D. A., 3:313 Sleugh, B. B., 19:227 Smith, S. E., 6:361 Socias i Company, R., 8:313 Sobral, B. W. S., 16:269 Sondahl, M. R., 2:157 Spoor, W., 20: 1 Steffensen, D. M., 19:1 Stevens, M. A., 4:273 Stoner, A. K., 7:57 Stuber, C. W., 9:37; 12:227 Sugiura, A., 19:191 Tai, G. C. C., 9:217 Talbert, L. E., 11:235 Tarn, T. R., 9:217 Tehrani, G., 9:367 Thompson, A. E., 6:93 Towill, L. E., 7:159, 13:179 Tracy, W. F., 14:189 Tsaftaris, A. S., 18:87 Tsai, C. Y., 1:103 Ullrich, S. E., 2:13 Van Harten, A. M., 6:55 Varughese, G., 8:43 Vasal, S. K., 9:181; 14:139 Veilleux, R., 3:253; 16:229; 20:167 Villareal, R. L., 8:43 Vogel, K. P., 11:251 Vuylsteke, D., 14:267 Wallace, D. H., 3:21; 13:141 Wan, Y., 11:199 Weeden, N. F., 6:11 Wehner, T. C., 6:323 Westwood, M. N., 7:111 Whitaker, T. W., 1:1 White, D. W. R., 17:191 White, G. A., 3:361; 7:5 Widholm, J. M., 4:153, 11:199 Widmer, R. E., 10:93 Widrlechner, M. P., 13:11 Wilcox, J. R., 1:183 Williams, E. G., 4:9; 5:181, 237 Williams, M. E., 10:23 Wilson, J. A., 2:303 Woodfield, D. R., 17:191 Wright, G. C., 12:81

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CUMULATIVE CONTRIBUTOR INDEX Yang, W. -J., 10:53 Yonemori, K., 19:191 Yopp, J. H., 4:203 Yun, D. -J., 14:39 Zeng, Z. -B., 19:41 Zimmerman, M. J. O., 4:245 Zohary, D., 12:253