Plant Breeding Reviews (Volume 25)

PLANT BREEDING REVIEWS Volume 25

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 25 M. Gilbert I. L. Goldman C. H. Michler

PLANT BREEDING REVIEWS Volume 25

edited by

Jules Janick Purdue University

John Wiley & Sons, Inc.

This book is printed on acid-free paper. Copyright © 2005 by John Wiley & Sons. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey 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 Section 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, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, e-mail: [email protected]. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data: ISBN: 0471666939 ISSN: 0730-2207 Printed in the United States of America 10

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Contents

List of Contributors 1. Dedication: Stanley J. Peloquin Potato Geneticist and Cytogeneticist

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Rodomiro Ortiz, Luigi Frusciante, and Domenico Carputo

2. Politics of Plant Breeding

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Cary Fowler and Richard L. Lower I. II. III. IV.

Introduction Germplasm, Plant Breeding, and the Fight for Rights The Debate Over Biotechnology Plant Breeders’ Choices Literature Cited

3. Doubled Haploids in Genetics and Plant Breeding

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Brian P. Forster and William T. B. Thomas I. II. III. IV. V.

Introduction Doubled Haploid Technology Doubled Haploid Populations in Genetics Doubled Haploids in Breeding Prospects Literature Cited

4. Biochemistry and Genetics of Flower Color

57 58 63 72 79 80

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R. J. Griesbach I. II. III. IV.

Introduction Flavonoid Chemistry Anthocyanin Biosynthesis Mendelian Inheritance

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CONTENTS

V. Transgene Technology Literature Cited

5. The Influence of Mitochondrial Genetics on Crop Breeding Strategies

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Sally A. Mackenzie I. II. III. IV.

Introduction Structure of the Mitochondrial Genome in Plants Cytoplasmic Male Sterility Occurrence and Developmental Implications of Nuclear-Cytoplasmic Incompatibility V. Some Implications of Cytoplasmic Genetics for the Plant Breeder Literature Cited

6. Genetic and Cytoplasmic-Nuclear Male Sterility in Sorghum

115 117 119 125 127 131

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Belum V. S. Reddy, S. Ramesh, and Rodomiro Ortiz I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Genetic Male Sterility (GMS) Cytoplasmic-Nuclear Male Sterility (CMS) Molecular Characterization of Cytoplasms DNA Polymorphism and Mapping Restorer Genes Factors Influencing CMS Systems Use Diversification of CMS Systems Heterosis and Hybrid Development Conclusion Literature Cited

7. Improving Drought Tolerance in Maize

140 140 146 150 151 152 162 166 167 167

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T. Barker, H. Campos, M. Cooper, D. Dolan, G. Edmeades, J. Habben, J. Schussler, D. Wright, and C. Zinselmeier I. II. III. IV.

Introduction Physiology of the Response of Maize Under Drought Experimental Methods Applied Breeding Methods

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CONTENTS

V. Molecular Breeding VI. Conclusions Literature Cited

8. The Origins of Fruits, Fruit Growing, and Fruit Breeding

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Jules Janick I. Introduction II. The Horticultural Arts III. Origin, Domestication, and Early Culture of Fruit Crops IV. Genetic Changes and Cultural Factors in Domestication Literature Cited

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Subject Index

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Cumulative Subject Index

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Cumulative Contributor Index

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List of Contributors Barker, T., Pioneer Hi-Bred International, Inc., 7300 NW 62nd Ave., Johnston, IA 50131, [email protected] Campos, H., Pioneer Hi-Bred International, Inc., 7300 NW 62nd Ave., Johnston, IA 50131 Carputo, Domenico, Departmento of Soil, Plant and Environmental Sciences, University of Naples “Federico II”, Via Università, 100, 80055 Portici, Italy Cooper, M., Pioneer Hi-Bred International, Inc., 7300 NW 62nd Ave., Johnston, IA 50131 Dolan, D., Pioneer Hi-Bred International, Inc., 7300 NW 62nd Ave., Johnston, IA 50131 Edmeades, G., Pioneer Hi-Bred International, Inc., 7300 NW 62nd Ave., Johnston, IA 50131 Forster, Brian P., Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK, [email protected] Fowler, Cary, Center for International Environment & Development Studies (NORAGRIC), Agricultural University of Norway, P.O. Box 5003, 1432 Aas, Norway, [email protected] Frusciante, Luigi, Departmento of Soil, Plant and Environmental Sciences, University of Naples “Federico II”, Via Università, 100, 80055 Portici, Italy Griesbach, R. J., Floral and Nursery Plants Research Unit, U.S. National Arboretum, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705-2350, [email protected] Habben, J., Pioneer Hi-Bred International, Inc., 7300 NW 62nd Ave., Johnston, IA 50131 Janick, Jules, Purdue University, Department of Horticulture and Landscape Architecture, 625 Agriculture Mall Drive, West Lafayette, Indiana 479072010, [email protected] Lower, Richard L., Dept. of Horticulture, 1575 Linden Drive, University of Wisconsin, Madison, WI 53706 [email protected] Mackenzie, Sally A., Plant Science Initiative, University of Nebraska, Lincoln, Nebraska 68588-0660, [email protected] Ortiz, Rodomiro, International Institute of Tropical Agriculture (IITA), Eastern and Southern Africa Regional Center (ESARC), Plot 7, Bandali Rise, Bugolobi, Kampala, Uganda, and International Institute of Tropical Agriculture (IITA), Nigeria; IITA c/o Lambourn Ltd. (UK), 26 Dingwall Road, Croydon, CR9 3EE, UK, [email protected] Ramesh, S., Global Theme–Crop Improvement, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India.

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Reddy, Belum V. S., Global Theme–Crop Improvement, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India Schussler, J., Pioneer Hi-Bred International, Inc., 7300 NW 62nd Ave., Johnston, IA 50131 Thomas, William T. B., Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK Wright, D., Pioneer Hi-Bred International, Inc., 7300 NW 62nd Ave., Johnston, IA 50131 Zinselmeier, C., Pioneer Hi-Bred International, Inc., 7300 NW 62nd Ave., Johnston, IA 50131

Stanley J. Peloquin

1 Dedication: Stanley J. Peloquin Potato Geneticist and Cytogeneticist Rodomiro Ortiz* International Institute of Tropical Agriculture (IITA), Nigeria; IITA c/o Lambourn Ltd. (UK), 26 Dingwall Road, Croydon, CR9 3EE, UK Luigi Frusciante and Domenico Carputo Departmento of Soil, Plant and Environmental Sciences, University of Naples “Federico II”, Via Università, 100, 80055 Portici, Italy

Dr. Stanley J. Peloquin is known to plant breeders for his decisive contributions to genetic enhancement of potato (Solanum tuberosum L.) using haploids, 2n gametes, and wild Solanum species; for his pioneering work on potato cultivation through true seed; and as mentor of a new generation of plant breeders worldwide. The genetic enhancement of potato, the fourth most important food crop worldwide, benefited significantly from Peloquin’s work on ploidy manipulations led by the genetic knowledge he and his co-workers, mostly former graduate students, created and systematically transformed into applied breeding methods. His scientific papers, book chapters, classes, seminars, and talks on potato as a model plant for genetic breeding and evolutionary research are a source of inspiration to all researchers in crop improvement.

*The authors thank Dr. Phil Simon (Univ. of Wisconsin, Madison) for assistance with this manuscript. Plant Breeding Reviews, Volume 25. Edited by Jules Janick. ISBN 0471666939 © 2005 John Wiley & Sons, Inc. 1

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BIOGRAPHICAL SKETCH Stan was born in Barron, Wisconsin, in 1921. He went to grade school in three small towns in Wisconsin and to a small high school at Ondossagon near Lake Superior in northern Wisconsin, where he was taught to get out of northern Wisconsin to make a living. There were 20 in his graduating class. After graduating with a degree in chemistry in 1942 from River Falls State College (now the University of Wisconsin—River Falls), he joined the U.S. Navy and served on a destroyer in the South Pacific for 3-1/2 years during the Second World War. Upon his release from the service, he enrolled at Marquette University, Milwaukee, Wisconsin, and obtained a MSc degree in biology in 1948. He then enrolled in the University of Wisconsin and studied genetics under the guidance of Dr. R. A. Brink and Dr. D. C. Cooper and was awarded a PhD degree in genetics in 1952. His thesis was entitled “Abnormal Embryo and Endosperm Development in Zea mays Following the Use of Pollen that has been Exposed to Mustard Gas.” From 1951 to 1956, he taught biology at Marquette University. From 1957, when he joined the faculty at the University of Wisconsin, Madison, until his retirement in 1994, he taught genetics to undergraduates in the Biocore Biology Program, and two graduate level courses: cytogenetics, and, with Dr. Ted Brigham, Chromosome Manipulations in Plants. He married Helga Sorensen and reared three sons: Philip, John, and James. Some time after her passing, he married Virgie Eastburn Fry, who is the mother of two children: David and Diane. Stan and Virgie are the proud grandparents of Brandon and Brienne (from Philip), and Christopher, Julia, and Melissa (from Diane), and two great grandchildren, Gavin and David. In 1984, Dr. Peloquin was elected to the U.S. National Academy of Sciences and was awarded a life membership by the Potato Association of America. In 1983, Dr. Peloquin was named the Campbell Bascom Professor by the University of Wisconsin and received Laurea Honoris Causa from the Università degli Studi di Napoli “Federico II” (Italy) in 2002. These recognitions he has received for his innovative research and scientific leadership are an acknowledgment of his superb skills as a potato geneticist and breeder. Peloquin always points out that potato should be seen not only as an important food (fresh or processed) but also as the raw material for the starch-processing industry, as feed because its vines can be fed to animals, and as a potential resource for medicine because of the compounds in its true seed, and as an ornamental.

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RESEARCH Peloquin’s achievements are the result of fundamental, ingenious scientific insight, using potato genetics as the focal point for his research. Stan integrates his efforts with a broad range of networking activities, creating a world-famous “school” composed largely of his graduate students and research fellows. This intense collaboration has had a great impact in generating basic knowledge and for achieving practical methods for genetic enhancement. Peloquin’s early efforts were in broadening the basic knowledge about cytogenetics and genetics of potato. Stan’s collaboration with Robert W. Hougas at the beginning of his career was an important first step, which led to more than 40 years of seminal potato research. Peloquin and Hougas, sometimes with early students and other collaborators, published 19 papers together that established a foundation of potato reproductive biology, genetics, and the genetics of reproductive biology that supported the broad and deep fundamental and applied developments that were to follow. Because of these early successes, Stan Peloquin set up germplasm enhancement methods relying on scaling up and scaling down chromosome sets via ploidy manipulations. Such ploidy manipulations are easily achieved in potato to transfer genes from wild Solanum species to the primary crop gene pool—particularly alleles for improving horticultural traits. His students in the classrooms of the University of Wisconsin still remember one of his favorite comments when teaching cytogenetics: “The best plant with which to manipulate individual chromosome numbers is wheat. The best plant with which to manipulate sets of chromosomes is potato.” As Stan pointed out in many of his reviews: “The ability to obtain plants with the gamete chromosome number (haploids) and gametes with the plant chromosome number (2n gametes) is the basis for the ease of manipulating whole sets of chromosomes in potato.” Haploid and 2n Gamete Cytology and Cytogenetics Scaling down the ploidy of the tetraploid potato (2n = 4x = 48 chromosomes) to the diploid level is achieved routinely by producing potato haploids (2n = 2x = 24 chromosomes). Maternal haploids can be easily obtained through parthenogenesis after interspecific hybridization of tetraploid cultivars with pollen of S. phureja Juz. et Buk. Haploid frequency is affected by both the maternal genotype and the pollen source,

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and both seed parent and pollen source influence the success of haploid production. Peloquin and co-workers indicated that the endosperm associated with a haploid embryo was always hexaploid, which clearly demonstrated the union of the two chromosome sets from S. phureja with the polar nuclei, and lack of fertilization of the egg. Hence, the pollen source influences haploid frequency via its effect on the endosperm. Paternal haploids are also obtained via anther culture, but maternal haploids offer more advantages for potato breeding because paternal haploid production requires gene(s) for androgenic competence, which are not always available in all tetraploid potato cultivars. Gametes with the sporophytic chromosome number are referred to as 2n gametes. Some authors called them “numerically unreduced gametes,” but Peloquin (and thereafter the students under his mentorship) avoided this term because normal gametes in any species have the haploid (n) number; i.e., 2n gametes would be 2x in diploids, 4x in tetraploids, and so on. Another reason he cautioned against the use of the term is that while chromosome number is not reduced in the formation of 2n gametes, the so-called “reduction division” typically does occur. The failure of students to appreciate this fact leaves them with an incomplete impression of the larger process. Premeiotic, meiotic, and postmeiotic abnormalities during gamete formation are correlated with the production of 2n gametes and there are at least six distinct possible modes of 2n gamete formation: premeiotic doubling, first division restitution (FDR), chromosome replication during meiotic interphase, second division restitution (SDR), postmeiotic doubling, and apospory. FDR and SDR mechanisms are respectively the most common modes of 2n pollen and 2n eggs formation in potato. In potato, FDR 2n gametes transmit on average 80% of the heterozygosity of the diploid progenitor to the tetraploid hybrid offspring. In contrast, SDR 2n gametes transfer on average less than 40% of the diploid heterozygosity to tetraploid hybrids. Parallel orientation of the spindles in the second meiotic division is the most frequent mechanism of 2n pollen formation in most tuber-bearing Solanum spp. This meiotic abnormality is under the genetic control of the recessive gene ps (parallel spindles), which appears to be ubiquitous among Solanum species, but 2n pollen frequency could be affected by variable expressivity and incomplete penetrance. Omission of the second division after a normal first division appears to be the most common mode of SDR 2n egg formation in potato haploids, and haploidspecies hybrids, which is controlled by a recessive meiotic mutant (os) in diploid potato. Genetic background and environment may affect the

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expressivity of this gene that also shows incomplete penetrance, and the frequency of modifier genes may enhance 2n egg expressivity. Ploidy Manipulations for Genetic Enhancement The findings of mechanisms that underlie parthenogenesis for producing maternal haploid plants and the formation of gametes with the parental chromosome number (or 2n gametes), established Peloquin’s international prestige and credentials. Ploidy manipulations with haploids, 2n gametes, and wild species still remains today as one of the most impressive and exciting crop germplasm enhancement methods ensuing from cytogenetics research. In Stan’s words “the potato is unsurpassed in the facility with which sets of chromosomes can be manipulated. This allows a germplasm enhancement strategy that involves species, haploids, 2n gametes and endosperm balance number (EBN). The species are the source of genetic diversity, haploids provide a method for ‘capturing’ the diversity, and 2n gametes and EBN are involved in an effective and efficient method of transmitting diversity to cultivars.” There are two main methods for ploidy manipulations in potato: unilateral sexual polyploidization (4x-n gametes × 2x-2n gametes or vice versa) and bilateral sexual polyploidization (ensuing from crosses between 2x-2n gametes producing parents). For these breeding schemes, the diploid progenitors are developed by crossing potato haploids with tuber-bearing diploid species (2n = 2x = 24 chromosomes). Maternal haploids, which are easily extracted through parthenogenesis from most tetraploid cultivars, are crossed with diploid species for breeding at the diploid level. The locally adapted haploid-species hybrids are selected because they possess 2n gametes, acceptable tuber characteristics, and sometimes additional desired attributes, e.g., disease or pest resistance. Most of the hybrids ensuing from sexual polyploidization matings in potato are tetraploids. Triploid hybrids from tetraploid-diploid or diploid-diploid crosses are very rare due to a strong triploid block in potato. The best diploid parents for tetraploid-diploid crosses are those producing FDR 2n gametes because hybrid vigor associated with high yields may be maximized by multi-allelism per locus in potato. Not surprisingly, tetraploid hybrids derived from diploid progenitors producing FDR 2n pollen often outyield their half-sib tetraploid hybrids from intermating tetraploid progenitors. However, maximum heterozygosity does not appear to be universal and depends on the genetic background of the crossing material, because of the importance of adaptation for the optimum expression of hybrid vigor in potato.

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With this knowledge, Stan and his “school”—particularly in Italy, Poland, and the Centro Internacional de la Papa (CIP, Lima, Peru)—were able to develop new potato genotypes that combine high and stable yield, plus disease or pest resistance, which also allow the widening of potato growing in areas of the world that were previously unsuitable for this crop. The potato genotypes ensuing from their work were amenable to both fresh table markets and the chipping industry. Some of these materials became parents of cultivars now grown across the world. One potato cultivar (‘Snowden’) ensuing from conventional breeding work of Peloquin and colleagues at the University of Wisconsin is a leading chipping cultivar in North America. Endosperm Balance Number (EBN) and Hybridization Barriers The endosperm is a distinct trait among the Angiosperms, which results from double fertilization; one male gamete unites with the egg to form the zygote and the other male gamete with the central cell to form the endosperm, thereby making this tissue necessary for normal seed development. As Peloquin pointed out many times, “One of my colleagues defines endosperm as the tissue which along with potato feeds the world.” Endosperm research by him and co-workers at the University of Wisconsin led to the Endosperm Balance Number (EBN) hypothesis to explain endosperm development in interploidy crosses, both intraspecific and interspecific. Under this theory, normal endosperm development only occurs when a balance of 2 EBN from the female parent matches with 1 EBN from the male parent in the resulting endosperm. Any deviations from this 2 maternal:1 paternal EBN ratio leads to faulty endosperm and lack of normal seed. For example, in 4x × 2x crosses and 2x × 4x crosses, endosperm development is regularly abnormal since the female:male ratios are 4:1 and 1:1, respectively. However, if 2n gametes function in the diploid, the ratio is 2:1 and endosperm development is normal. The fact that Mexican tetraploid species are easy to cross with most diploid species from South America—yielding triploid hybrids— suggested that both species sets possess 2 EBN, while they are unable to cross with Andean tetraploid species that are 4 EBN. This endosperm dosage system is typical of species possessing a “triploid block.” However, triploids from crosses between tetraploids and diploids arise occasionally from misfertilization, mitotic abnormalities in the gametophyte, and/or mitotic misdivisions in the endosperm. The above results led to the hypothesis—tested thereafter with success by many researchers worldwide, that EBN and 2n gametes are more

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important than actual chromosome number for predicting the success of crosses and the ploidy of the resulting progeny. The EBN of a species, which initially was thought to be controlled by a few genes rather than by the whole genome, determines, therefore, the effective ploidy, and natural gene flow may occur among species with distinct chromosome numbers but the same EBN. The EBN can be assigned for most Solanum species on the basis of the crossing behavior of a species with a standard species of known EBN. Further research by some of his former co-workers demonstrated that three genes control EBN in potato, and gave convincing evidence for the participation of the EBN incompatibility system and 2n gametes in the origin and evolution of polyploids in tuber-bearing Solanum species. In this regard, the EBN determined gene pools among potato and wild Solanum species. The primary gene pool consists of old and modern tetraploid cultivars, tetraploid Andean landraces, and tetraploid breeding populations (i.e., 4 EBN polysomic polyploid tetraploid species). Diploid cultivars or breeding populations and diploid tuber-bearing Solanum species (2 EBN) producing 2n gametes and hexaploid (4 EBN) species also belong to this primary gene pool. The secondary gene pool includes disomic tetraploid (2 EBN) and diploid (1 EBN) tuber-bearing Solanum species, which may cross with the crop primary gene pool after isolation barriers (mainly due to EBN) are overcome. Wild diploid nontuber bearing Solanum species (1 EBN) of the series Etuberosa are in the tertiary potato gene pool, which could only cross with the primary crop gene pool through bridge species and embryo rescue. Other researchers expanded the EBN theory to many plant taxa, as a unifying concept to predict endosperm function in intraspecific-interploidy or interspecific crosses. True Potato Seed (TPS) Potato production from true (sexual) seed was not new because this propagation system was used in the Andes by the Incas. Andean farmers still grow potato from true seed for disease elimination, stock rejuvenation, and creation of new cultivars. However, another major global impact of Peloquin’s research was the development of breeding methods for using true seed rather than tubers for growing potato—particularly in warm tropical environments, where potato growers are affected by the high cost of seed-tubers, and lack of clean planting materials because of high pest pressure. TPS lowers production costs, reduces the incidence of pests such as viruses that are not transmitted by true seed, and allows true seed to be the source of planting material even if parental

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plants are diseased. TPS technology enables low-income small landholders in the developing world to grow potato, thereby expanding the geographic range of this crop worldwide, especially in locations where transport and cold storage of seed-tubers are not feasible. The production of potatoes from true seed has increased dramatically in areas of India, Bangladesh, and China where they are grown between two crops of rice. Potato cultivars in modern high-input agricultural systems are homogeneous tetraploid genotypes. These cultivars, which are generally produced by cross-pollination, show a great uniformity due to vegetative propagation by tubers. In this agricultural system, tubers are harvested from a potato plant that grew from a single-sprouted tuber. Hence, TPS cultivars must combine plant characteristics for true seed production, and for tuber production from true seed propagules. Conventional potato breeding for vegetative propagation relies on selecting desired allelic combinations for further clonal multiplication, while breeding for potato production from true seed should be based on phenotypically uniform hybrid offspring with a high frequency of favorable heterogeneous allelic combinations. The genetic improvement for TPS production must, therefore, consider a breeding strategy for a sexually propagated crop that is grown for the harvest of its vegetative part, i.e., the tuber. TPS hybrids from crosses between tetraploid parents are the most popular for potato production from true seed. Although heterogeneous gametes may result from meiosis of heterozygous progenitors, some tetraploid hybrid offspring from such crosses show very uniform plant and tuber phenotypes. Tetraploid clones are selected according to their reproductive and agronomic characteristics. Earliness, desired tuber characteristics such as color, shape, number and size, profuse flowering, fertility, and high berry and seed set are the most important attributes of the selected tetraploid parents. Specific crosses for commercial production of TPS hybrids are recommended after testing specific combining ability between locally selected parents. Peloquin and his co-workers suggested other methods for producing tetraploid TPS hybrids through unilateral sexual or bilateral sexual polyploidization. A high frequency of 2n gametes will be very important for commercial TPS hybrids derived from sexual polyploidization after pollinating by hand. Furthermore, recurrent selection effectively increases 2n gametes frequency in diploid potatoes. Nonetheless, production costs of TPS can be lowered by eliminating pollen collection and hand pollination. Hence, since the 1980s Peloquin and his co-workers investigated new breeding systems for alternative TPS production. The cheapest material for potato production from true seed will be derived

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from open pollination. Open-pollinated TPS costs are significantly lower than those of TPS hybrids because the labor skills and other additional required investments are smaller for its production. However, TPS hybrids always outyield open-pollinated and selfed offspring. The heterozygosity level in the tetraploid sporophyte affects the phenotypic expression of the diploid gametophytic generation in potato; e.g., open pollinated berry and fruit sets are correlated with pollen stainability. It appears that natural pollinators of potato such as bumblebees prefer male fertile plants, especially when pollen viability is at its highest. This finding may explain why tuber yield does not always decrease after successive open-pollinated generations derived from heterogeneous true seed offspring. A synthetic cultivar propagated by open pollination may be, therefore, achievable for potato production from TPS. Open-pollinated offspring could result from selfing but the percentage of hybrid offspring may be increased in open-pollinated derived seedlings because of the variable rate of outcrossing in cultivated tetraploid potato. Selection of vigorous plants in artificial mixtures of hybrid and self-pollinated TPS generations proved, therefore, to be successful in the identification of hybrids in respective TPS generations. The identification of TPS hybrids may be improved by adding a marker gene in the selection scheme, e.g., interplanting a diploid first division restitution 2n pollen-producing male progenitor with a genetic marker (yellow tuber flesh color) among tetraploid female progenitors permitted the selection of TPS hybrids among derived open-pollinated offspring from true seed harvested from the female progenitors. Such TPS hybrids (ensuing from open pollination) had better seedling vigor, more flowers, higher pollen stainability, larger open-pollinated berry set, and greater tuber yield than their open-pollinated half-sibs. Also, the elimination of weak plants during population thinning in the TPS nursery and subsequent interplant competition in densely sown beds for production of tuberlets (the first generation tubers from seedlings) reduce dramatically the frequency of inbred genotypes. There are some disadvantages for potato production from true seeds using transplanted seedlings, e.g., a reliable water supply is required for the successful establishment of seedling in the production field, and labor costs should be added for transplanting. Likewise, potato tuber yield from tuberlets is always higher than that recorded in TPS seedlings, but specific gravity does not change in both generations. Many small tubers are harvested from TPS seedlings, whereas few normal-size tubers are often obtained from tuber propagules. In addition, TPS seedlings require 2 to 3 extra weeks for tuber maturation as compared to tuber propagules. Hence, tuberlets rather than TPS seedlings

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appear to be the most promising propagules for potato production from true seed. Furthermore, tuberlets sold as “seed tubers” are suitable for developing a seed tuber system. Off-season production of tuberlets in well-controlled nursery beds will be another advantage of this seed system, because it provides propagules for the next planting season or in environments where direct sowing of TPS does not seem feasible due to short water supply, high labor cost for thinning and manual weeding, or short growing season. Lastly, selection in the seedling generation may improve the performance of tuberlets from open-pollinated and hybrid offspring. Selection for tuber color, shape, and skin in the TPS seedling nursery may lead to agronomically uniform tuberlets. Peloquin and co-workers reported higher tuber yields in selected tetraploid hybrid offspring from bilateral sexual polyploidization than in those of commercial cultivars. However, both tetraploid and diploid hybrids ensue from diploid-diploid crosses in potato, because polyploid frequency depends on the percentage of 2n gametes in both parents. The formation of first division restitution 2n eggs through desynapsis offers an option for only tetraploid progeny ensuing from bilateral sexual polyploidization. Through their work on TPS breeding, a new method of producing inexpensive tetraploid hybrid true seed offspring through bilateral sexual polyploidization and natural insect pollination was suggested. It consists of using unrelated, locally adapted diploid haploidspecies hybrids as parents—according to their combining ability, with profuse flowering, attractiveness to bumblebees (natural pollinators), and other desired attributes. The diploid male parent has high male fertility and very high frequency of (or almost only) first division restitution 2n pollen, and a heterozygous monogenic dominant marker tightly linked to the centromere. The female parent combines male and female fertility, self-incompatibility, very high frequency (or almost only) 2n egg production, and lacks a dominant marker (recessive genotype). Both diploid haploid-species parents are grown following an interplanting field design to allow natural pollination and gene flow between them. TPS are harvested only from female parents and are grown in a nursery to eliminate those showing poor vigor and lacking dominant phenotype of the male progenitor. Hence, the tuberlet harvest will include mostly (if not only) tetraploid hybrids for potato production in the field. With this TPS scheme, emasculation, pollen collection, and hand pollination are eliminated, thereby reducing by 50% the costs of producing hybrid tetraploid seed. It would be desirable to select diploid parents that are able to set 10,000 hybrid seeds per plant with this method for producing TPS.

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THE MENTOR Stan Peloquin was among the most prolific trainers of graduate students in the history of the University of Wisconsin—Madison. A total of 93 graduate students (51 PhD, 42 MSc) and 27 visiting scientists from 34 countries were guided by Stan. Many of them returned to their country of origin or pursued a professional career in international agriculture. Through their work, they help to enhance food production in the developing world. Many of his students are now heads of advanced research organizations or lead international agricultural research institutes. Stan has the ability to infuse a “love” for research in science to young professionals. He is fiercely loyal and devoted to the students’ best interest. His enthusiasm and broad interest stimulate not only his students but also colleagues and peers. His former students and visiting research fellows know Stan as someone who enjoys the company of others, has strong education principles, and has a great desire to convey his knowledge. He derives intense satisfaction from the success of each and every student or visiting scientist who comes to his laboratory at the University of Wisconsin. His students and visiting fellows can never forget that their professional education and careers were developed, molded, and launched under his strong and vibrant personality. Many feel that much of their success was related not only to what they learned from him about science but also about life. We all learned one principle: “hard work always pays off.” Professor Stanley J. Peloquin has devoted a lifetime to merging basic cytogenetic and genetic research to achieve direct applicability in crop improvement. His plant breeding philosophy of “putting genes into a usable form” is a prime example of farsightedness in science. His efforts are an inspiration to us all and we proudly dedicate this volume of Plant Breeding Reviews to him and his achievements.

SELECTED REFERENCES Arndt, G. C., and S. J. Peloquin. 1990. The identification and evaluation of hybrid plants among open pollinated true seed families. Am. Potato J. 67:293–304. Arndt, G. C., J. L. Rueda, H. M. Kidane-Mariam, and S. J. Peloquin. 1990. Pollen fertility in relation to open pollinated true seed production in potatoes. Am. Potato J. 67:499–505. Ascher, P. D., and S. J. Peloquin. 1966. Effect of floral aging on the growth of compatible and incompatible pollen tubes in Lolium longiflorum (Thumb.). Am. J. Bot. 53:99–102. Ascher, P. D., and S. J. Peloquin. 1966. Influence of temperature on incompatible and compatible pollen tube growth in Lolium longiflorum. Can. J. Genet. Cytol. 8:661–664.

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Ascher, P. D., and S. J. Peloquin. 1968. Pollen tube growth and incompatibility following intra- and inter-specific pollinations in Lolium longiflorum. Am. J. Bot. 55:1230–1234. Ascher, P. D., and S. J. Peloquin. 1970. Temperature and the self-incompatibility reaction in Lolium longiflorum Thumb. J. Am. Soc. Hort. Sci. 95:586–588. Buso, J. A., F. A. S. Aragao, F. J. B. Reifschneider, L. S. Boiteux, and S. J. Peloquin. 2002. Assessment under short-day conditions of genetic materials derived from three potato breeding strategies 4x–4x (intra-Tuberosum), 4x–2x (FDR 2n-pollen) and 4x–4x (diplandrous tetraploid). Euphytica 126:437–446. Buso, J. A., F. J. B. Reifschneider, L. S. Boiteux, and S. J. Peloquin. 1999. Effects of 2n pollen formation by first-meiotic division restitution with and without crossover on eight quantitative traits in 4x–2x potato progenies. Theor. Appl. Genet. 98:1311–1319. Buso, J. A., L. S. Boiteux, and S. J. Peloquin. 1999. Comparison of haploid TuberosumSolanum chacoense versus Solanum phureja-haploid Tuberosum hybrids as staminate parents of 4x–2x progenies evaluated under distinct crop management systems. Euphytica 109:191–199. Buso, J. A., L. S. Boiteux, and S. J. Peloquin. 1999. Evaluation of 4x–2x progenies from crosses between potato cultivars and haploid tuberosum—Solanum chacoense hybrids under long day conditions. Ann. Appl. Biol. 135:35–42. Buso, J. A., L. S. Boiteux, and S. J. Peloquin. 1999. Multitrait selection system using populations with a small number of interploid (4x–2x) hybrid seedlings in potato: Degree of high-parent heterosis for yield and frequency of clones combining quantitative agronomic traits. Theor. Appl. Genet. 99:81–91. Buso, J. A., L. S. Boiteux, and S. J. Peloquin. 2000. Heterotic effects for yield and tuber solids and type of gene action for five traits in 4x potato families derived from interploid (4x–2x) crosses. Plant Breed. 119:111–117. Buso, J. A., L. S. Boiteux, G. C. C. Tai, and S. J. Peloquin. 1999. Chromosome regions between centromere and proximal crossovers are the physical sites of major effect loci for yield in potato: genetic analysis employing meiotic mutants. Proc. Natl. Acad. Sci. (USA) 96:1773–1778. Buso, J. A., L. S. Boiteux, G. C. C. Tai, and S. J. Peloquin. 2000. Direct estimation of the effects of meiotic recombination on potato traits via analysis of 4x–2x progenies from synaptic mutants with 2n-pollen formation by FDR without crossing over. Theor. Appl. Genet. 101:139–145. Camadro, E. L., and S. J. Peloquin. 1980. The occurrence and frequency of 2n pollen in three diploid Solanums from northwest Argentina. Theor. Appl. Genet. 56:11–15. Camadro, E. L., and S. J. Peloquin. 1981. Cross-incompatibility between two sympatric polyploid Solanum species. Theor. Appl. Genet. 60:65–70. Camadro, E. L., and S. J. Peloquin. 1982. Selfing rates in two wild polyploid Solanums. Am. Potato J. 59:197–203. Carputo, D., A. Barone, T. Cardi, A. Sebastiano, L. Frusciante, and S. J. Peloquin. 1997. Endosperm balance number manipulation for direct in vivo germplasm introgression to potato from a sexually isolated relative (Solanum commersonii Dun.). Proc. Natl. Acad. Sci. (USA) 94:12013–12017. Carputo, D., L. Frusciante, and S. J. Peloquin. 2003. The role of 2n gametes and endosperm balance number in the origin and evolution of polyploids in the tuber-bearing Solanums. Genetics 163:287–294. Carputo, D., T. Cardi, L. Frusciante, and S. J. Peloquin. 1995. Male fertility and cytology of triploid hybrids between tetraploid Solanum commersonii (2n=4x=48, 2 EBN) and Phureja-Tuberosum haploid hybrids (2n=2x=24, 2 EBN). Euphytica 83:123–129. Chujoy, J. E., and S. J. Peloquin. 1986. Barriers to interspecific hybridization between Solanum chacoense Bitt. and S. commersonii Dun. Am. Potato J. 63:416–417.

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Cipar, M. S., S. J. Peloquin, and R. W. Hougas. 1964. Inheritance of incompatibility in hybrids between Solanum tuberosum haploids and diploid species. Euphytica 13:163–172. Cipar, M. S., S. J. Peloquin, and R. W. Hougas. 1964. Variability in the expression of selfincompatibility in hybrids tuber-bearing diploid Solanum species. Am. Potato J. 41:163–172. Cipar, M. S., S. J. Peloquin, and R. W. Hougas. 1967. Haploidy and the identification of self-incompatibility alleles in cultivated potato groups. Can. J. Genet. Cytol. 9:511–518. Concilio, L., and S. J. Peloquin. 1987. Evaluation of yield and other agronomic characteristics of true potato seed families and advanced clones from different breeding schemes. p. 262–263. In: G. J. Jellis and D. E. Richardson (eds.), The production of new potato varieties: Technological advances. Cambridge Univ. Press, Cambridge. Concilio, L., and S. J. Peloquin. 1987. Tuber yield of true potato seed families from different breeding schemes. Am. Potato J. 64:81–85. Concilio, L., and S. J. Peloquin. 1991. Evaluation of the 4x × 2x breeding scheme in a potato breeding program adapted to local conditions. J. Genet. Breed. 45:13–18. Darmo, E., and S. J. Peloquin. 1990. Performance and stability of nine 4x clones from 4x–2x crosses and four commercial cultivars. Potato Res. 33:352–360. Darmo, E., and S. J. Peloquin. 1991. Use of 2x Tuberosum haploid-wild species hybrids to improve yield and quality in 4x cultivated potatoes. Euphytica 53:1–9. Davies, C. S., M. J. Ottman, and S. J. Peloquin. 2002. Can germplasm resources be used to increase the ascorbic acid of stored potatoes? Am. J. Potato Res. 79:295–299. de La Puente Ciudad, F., and S. J. Peloquin. 1976. Haploids of group Andigena. Am. Potato J. 51:278. den Nijs, T. P. M., and S. J. Peloquin. 1977. 2n gametes in potato species and their function in sexual polyploidization. Euphytica 26:585–600. den Nijs, T. P. M., and S. J. Peloquin. 1977. Polyploid evolution via 2n gametes. Am. Potato J. 54:377–386. den Nijs, T. P. M., E. F. Leue, and S. J. Peloquin. 1980. Topiary: a mutant character in Solanum infundibuliforme. J. Hered. 71:57–60. den Nijs, T. P. M., E. F. Leue, M. Iwanaga, and S. J. Peloquin. 1978. Detection of the mode of 2n egg formation by pollen stainability distributions. Am. Potato J. 55:387–389. Desborough, S., and S. J. Peloquin. 1966. Disc electrophoresis of tuber proteins from Solanum species and interspecific hybrids. Phytochemistry 5:727–733. Desborough, S., and S. J. Peloquin. 1967. Esterase isozymes from Solanum tubers. Phytochemistry 6:989–994. Desborough, S., and S. J. Peloquin. 1968. Disc-electrophoresis of proteins and enzymes from styles, pollen and pollen tubes of self-incompatible cultivars of Lolium longiflorum. Theor. Appl. Genet. 38:327–331. Desborough, S., and S. J. Peloquin. 1968. Potato variety identification by use of electrophotetic patterns of tuber proteins and enzymes. Am. Potato J. 45:220–229. Desborough, S., and S. J. Peloquin. 1969. Acid gel electrophoresis of tuber proteins from Solanum species. Phytochemistry 8:425–429. Desborough, S., and S. J. Peloquin. 1969. Tuber proteins from haploids, selfs and cultivars of Group Tuberosum separated by acid gel electrophoresis. Theor. Appl. Genet. 39:43–47. Frusciante, L., A. Barone, C. Conicella, and S. J. Peloquin. 1989. Use of different breeding schemes and selection of parental lines for TPS production. p. 178–181. In: K. M. Louwes, H. A. J. M. Toussaint, and L. W. L. Dellaert (eds.), Parental line breeding and selection in potato breeding. Pudoc, Wageningen, Netherlands. Groza, H. I., B. D. Brown, D. Kichefski, S. J. Peloquin, and J. Jiang. 2002. Red Companion: A new red potato variety for fresh market. Am. J. Potato Res. 79:133–137.

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Hanneman, R. E. Jr., and S. J. Peloquin. 1981. Genetic-cytoplasmic male sterility in progeny of 4x–2x crosses in cultivated potatoes. Theor. Appl. Genet. 59:53–56. Hanneman, R. E., and S. J. Peloquin. 1967. Crossability of 24-chromosome potato hybrids with 48-chromosome cultivars. Eur. Potato J. 10:62–73. Hanneman, R. E., and S. J. Peloquin. 1968. Ploidy levels of progeny from diploid-tetraploid crosses in the potato. Am. Potato J. 45:255–261. Hermundstad, S. A., and S. J. Peloquin. 1985. Germplasm enhancement with potato haploids. J. Hered. 76:463–467. Hermundstad, S. A., and S. J. Peloquin. 1986. Tuber yield and tuber traits of haploid-wild species F1 hybrids. Potato Res. 29:289–297. Hermundstad, S. A., and S. J. Peloquin. 1987. Breeding at the 2x level and sexual polyploidization. p. 197–210. In G. J. Jellis and D. E. Richardson (eds.), The production of new potato varieties: Technological advances. Cambridge Univ. Press, Cambridge. Hopper, J. E., and S. J. Peloquin. 1968. X-ray inactivation of the stylar component of the self-incompatibility reaction in Lolium longiflorum. Can. J. Genet. Cytol. 10:941–944. Hopper, J. E., and S. J. Peloquin. 1976. Analysis of stylar self-incompatibility competence by use of heat induced inactivation. Theor. Appl. Genet. 47:291–297. Hopper, J. E., P. D. Ascher, and S. J. Peloquin. 1967. Inactivation of self-incompatibility following temperature pretreatments of styles in Lolium longiflorum. Euphytica 16:215–220. Hougas, R. W., and S. J. Peloquin. 1957. A haploid plant of the potato variety Katahdin. Nature 180:1209–1210. Hougas, R. W., and S. J. Peloquin. 1958. The potential of potato haploids in breeding and genetic research. Am. Potato J. 35:701–707. Hougas, R. W., and S. J. Peloquin. 1960. Crossability of Solanum tuberosum haploids with diploid Solanum species. Eur. Potato J. 3:325–330. Hougas, R. W., and S. J. Peloquin. 1962. Exploitation of Solanum germplasm. p. 21–24. In: D. S. Correll (ed.), The potato and its wild relatives. Texas Research Foundation, Renner, Texas. Hougas, R. W., S. J. Peloquin, and A. C. Gabert. 1964. The effect of the seed-parents and pollinator on haploid frequency in Solanum tuberosum. Crop Sci. 4:593–595. Hougas, R. W., S. J. Peloquin, and R. W. Ross. 1958. Haploids of the common potato. J. Hered. 49:103–106. Iwanaga, M., and S. J. Peloquin. 1979. Synaptic mutant affecting only megasporogenesis in potatoes. J. Hered. 70:385–389. Iwanaga, M., and S. J. Peloquin. 1982. Origin and evolution of cultivated tetraploid potatoes via 2n gametes. Theor. Appl. Genet. 61:161–169. Iwanaga, M., R. Ortiz, M. S. Cipar, and S. J. Peloquin. 1991. A restorer gene for geneticcytoplasmic male sterility in cultivated potatoes. Am. Potato J. 68:19–28. Jansky, S. H., G. L. Yerk, and S. J. Peloquin. 1990. The use of potato haploids to put 2x wild species germplasm in a usable form. Plant Breed. 104:290–294. Johnston, S. A., T. P. N. den Nijs, S. J. Peloquin, and R. E. Hanneman Jr. 1980. The significance of genetic balance to endosperm development in interspecific crosses. Theor. Appl. Genet. 57:5–9. Kichefski, D. F., A. A. Quinn, and S. J. Peloquin. 1976. The effectiveness of selection during early clonal generations in varietal breeding. Am. Potato J. 53:370–371. Kidane-Mariam, H. M., and S. J. Peloquin. 1974. The effect of direction of hybridization (4x × 2x vs. 2x × 4x) on yield on cultivated potatoes. Am. Potato J. 51:330–336. Kidane-Mariam, H. M., and S. J. Peloquin. 1975. Method of diplandroid formation and yield of progeny from reciprocal (4x–2x) crosses. J. Am. Soc. Hort. Sci. 100:602–604.

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Kidane-Mariam, H. M., and S. J. Peloquin. 1975. Potato clonal variation with respect to the relation between fruit set and tuber yield. E. African Agr. For. J. 40:256–260. Kidane-Mariam, H. M., G. C. Arndt, A. C. Macaso-Khwaja, and S. J. Peloquin. 1985. Comparisons between 4x × 2x hybrid and open-pollinated true potato seed families. Potato Res. 28:35–42. Kidane-Mariam, H. M., G. C. Arndt, A. C. Macaso-Khwaja, and S. J. Peloquin. 1985. Hybrid vs. open-pollinated TPS families. p. 25–37. In: Innovative methods for propagating potatoes. Centro Internacional de la Papa, Lima, Perú. Kotch, G. P., and S. J. Peloquin. 1987. A new source of haploid germplasm for genetic breeding research. Am. Potato J. 64:137–141. Kotch, G. P., R. Ortiz, and S. J. Peloquin. 1992. Genetic analysis by use of potato haploid populations. Genome 35:103–108. Leue, E. F., and S. J. Peloquin. 1980. Selection for 2n gametes and tuberization in Solanum chacoense. Am. Potato. J. 57:189–195. Leue, E. F., and S. J. Peloquin. 1982. The use of topiary gene in adapting Solanum germplasm for potato improvement. Euphytica 31:65–72. Lynch, D. R., D. Kichefski, S. J. Peloquin, C. S. Schaupmeyer, L. la Croix, D. Waterer, W. T. Andrew, J. Holley, N. Crowe, B. McConnell, G. A. Nelson, and B. Rex. 1991. Niska: A maincrop chipping potato cultivar with high specific gravity and good quality after storage. Am. J. Potato Res. 68:143–149. Lynch, D. R., S. J. Peloquin, L. M. Kawchuk, C. A. Schaupmeyer, J. Holley, D. K. Fujimoto, D. Driedger, D. Waterer, J. Wahab, and M. S. Goettel. 2001. AC Glacier Chip: a highyielding chip cultivar for long-term storage. Am. J. Potato Res. 78:327–332. Macaso-Khwaja, A. C., and S. J. Peloquin. 1983. Tuber yield of families from openpollinated and hybrids true potato seed. Am. Potato J. 60:645–651. Masson, M. F., and S. J. Peloquin. 1987. Heterosis for tuber yields and total solids contents in 4x × 2x FDR-CO crosses. p. 213–227. In: G. J. Jellis and D. E. Richardson (eds.), The production of new potato varieties: Technological advances. Cambridge Univ. Press, Cambridge. Mendiburu, A. O., and S. J. Peloquin. 1976. Sexual polyploidization and depolyploidization: Some terminology and definitions. Theor. Appl. Genet. 48:137–143. Mendiburu, A. O., and S. J. Peloquin. 1977. Bilateral sexual polyploidization in potatoes. Euphytica 26:573–583. Mendiburu, A. O., and S. J. Peloquin. 1977. The significance of 2n gametes in potato breeding. Theor. Appl. Genet. 49:53–61. Mendiburu, A. O., and S. J. Peloquin. 1979. Gene centromere mapping by 4x–2x matings in potato. Theor. Appl. Genet. 54:177–180. Mendiburu, A. O., S. J. Peloquin, and D. W. S. Mok. 1974. Potato breeding with haploids and 2n gametes. In: K. J. Kasha (ed.), First Intl. Symp. Haploids in Higher Plants. Univ. of Guelph, Ontario, Canada. p. 249–258. Mok, D. W. S., and S. J. Peloquin. 1975. Breeding value of 2n pollen (diplandroid) in tetraploid × diploid crosses in potato. Theor. Appl. Genet. 46:307–314. Mok, D. W. S., and S. J. Peloquin. 1975. The inheritance of three mechanisms of diplandroid (2n pollen) formation in diploid potatoes. Heredity 35:295–302. Mok, D. W. S., and S. J. Peloquin. 1975. Three mechanisms of 2n pollen formation in diploid potatoes. Can. J. Genet. Cytol. 17:217–225. Mok, D. W. S., H. K. Lee, and S. J. Peloquin. 1974. Identification of potato chromosomes with giemsa. Am. Potato J. 51:337–341. Mok, D. W. S., S. J. Peloquin, and A. O. Mendiburu. 1976. Genetic evidence for mode of 2n pollen formation and S-locus mapping in potatoes. Potato Res. 19:157–164.

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Mok, D. W. S., S. J. Peloquin, and T. R. Tarn. 1975. Cytology of potato triploids producing 2n pollen. Am. Potato J. 52:171–174. Mok, I. C., and S. J. Peloquin. 1982. Sexual polyploidization and protein diversity in potato. Am. Potato J. 59:480. Mortenson, L. R., S. J. Peloquin, and R. W. Hougas. 1964. Germination of Solanum tuberosum pollen on artificial media. Am. Potato J. 41:322–328. Okwuagwu, C. O., and S. J. Peloquin. 1981. A method of transferring the intact parental genotype to the offspring via meiotic mutants. Am. Potato J. 58:512. Ortiz, R., and S. J. Peloquin. 1991. A new method of producing 4x hybrid true potato seed. Euphytica 57:103–107. Ortiz, R., and S. J. Peloquin. 1991. Breeding for 2n egg production in haploid × species 2x potato hybrids. Am. Potato J. 68:691–703. Ortiz, R., and S. J. Peloquin. 1992. Associations between genetic markers with quantitative traits in potato. J. Genet. Breed. 46:395–400. Ortiz, R., and S. J. Peloquin. 1992. Recurrent selection for improvement of 2n gamete production in 2x potatoes. J. Genet. Breed. 46:383–390. Ortiz, R., and S. J. Peloquin. 1993. Mapping the flower pigmentation locus in potato. J. Genet. Breed. 47:171–173. Ortiz, R., and S. J. Peloquin. 1993. Population improvement in the development of 2x parents in potato using exotic germplasm. J. Genet. Breed. 47:81–88. Ortiz, R., and S. J. Peloquin. 1994. Effect of sporophytic heterozygosity on the male gametophyte of the tetraploid potato (Solanum tuberosum). Ann. Bot. 73:61–64. Ortiz, R., and S. J. Peloquin. 1994. Manipulaciones de ploidía en el mejoramiento genético de la papa. Turrialba 43:196–209. Ortiz, R., and S. J. Peloquin. 1994. Use of 24-chromosome potatoes (diploids and dihaploids) for genetical analysis. p. 133–154. In: J. E. Bradshaw and G. R. Mackay (eds.), Potato genetics. CAB International, Wallingford, Oxon, UK. Ortiz, R., D. S. Douches, G. P. Kotch, and S. J. Peloquin. 1993. Use of haploids and isozyme markers for genetic analysis in the polysomic polyploid potato. J. Genet. Breed. 47:283–288. Ortiz, R., M. Iwanaga, and S. J. Peloquin. 1993. Male sterility and 2n pollen in 4x progenies derived from 4x × 2x and 4x × 4x crosses in potato. Potato Res. 36:227–236. Ortiz, R., M. Iwanaga, and S. J. Peloquin. 1994. Breeding potatoes for developing countries using wild tuber bearing Solanum spp. and ploidy manipulations. J. Genet. Breed. 48:89–98. Ortiz, R., M. Iwanaga, and S. J. Peloquin. 1997. Evaluation of FDR diploid and tetraploid parents in potato under two contrasting day length environments. Plant Breed. 116:353–358. Ortiz, R., R. Freyre, S. J. Peloquin, and M. Iwanaga. 1991. Adaptation to day length and yield stability of families from 4x × 2x crosses in potato. Euphytica 56:187–198. Ortiz, R., S. J. Peloquin, R. Freyre, and M. Iwanaga. 1991. Efficiency of potato breeding using FDR 2n gametes for multi trait selection and progeny testing. Theor. Appl. Genet. 82:602–608. Parfitt, D. E., and S. J. Peloquin. 1977. Variation of vine and tuber yield as a function of harvest date and cultivar. Am. Potato J. 54:410–417. Parfitt, D. E., and S. J. Peloquin. 1981. The genetic basis for tuber greening in 24chromosome potatoes. Am. Potato J. 58:299–304. Parfitt, D. E., and S. J. Peloquin. 1982. Yield trials and economic analysis to select cultivars for silage production. Am. Potato J. 59:395–401. Parfitt, D. E., S. J. Peloquin, and N. A. Jorgensen. 1982. The nutritional value of pressed potato vine silage. Am. Potato J. 59:415–423. Peloquin, J. G., S. J. Peloquin, and D. W. S. Mok. 1975. Vertical slab polyacrylamide gel electrophoresis of tuber proteins. Am. Potato J. 52:280–281.

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Peloquin, S. J. 1979. Breeding methods for achieving phenotypic uniformity. In: Planning Conf. on the Production of Potatoes from True Seed. Centro Internacional de la Papa, Lima, Perú. p. 151–155. Peloquin, S. J. 1981. Chromosomal and cytoplasmic manipulations. p. 1–17. In: K. J. Frey (ed.), Plant breeding II. Iowa State Univ., Ames, IA. Peloquin, S. J. 1982. Meiotic mutants in potato breeding. In: G. P. Redei (ed.), Stadler Genetic Symp. St. Louis, Missouri. p. 99–109. Peloquin, S. J. 1982. New approaches to breeding for the potato of the year 2000. In: W. L. Hooker (ed.), Proc. Intl. Potato Center 10th Annual Congress. Centro Internacional de la Papa, Lima, Perú. Peloquin, S. J. 1983. Genetic engineering with meiotic mutants. p. 311–316. In: D. Mulcahy and E. Ottaviano (eds.), Pollen biology and use in plant breeding. Elsevier Pub. Co, Amsterdam. Peloquin, S. J. 1984. Utilization of exotic germplasm in potato breeding: Germplasm transfer with haploids and 2n gametes. p. 147–158. In: W. L. Brown (ed.), Conservation and utilization of exotic germplasm to improve varieties. Pioneer Hi-Bred Intl. Inc., Des Moines, IA. Peloquin, S. J. 1986. Chromosome engineering with meiotic mutants. p. 47–52. In: D. Mulcahy and E. Ottaviano (eds.), Biotechnology and ecology of pollen. Springer Verlag, New York. Peloquin, S. J., A. C. Gabert, and R. Ortiz. 1996. Nature of pollinator effect in potato (Solanum tuberosum L.) haploid production. Ann. Bot. 77:539–542. Peloquin, S. J., and R. Ortiz. 1992. Techniques for introgressing unadapted germplasm to breeding populations. p. 485–508. In: H. T. Stalker and J. P. Murphy (eds.), Plant breeding in the 1990s. CAB International, Wallingford, Oxon, UK. Peloquin, S. J., and R. W. Hougas. 1958. Fertility in two Solanum tuberosum haploids. Science 128:1340–1341. Peloquin, S. J., and R. W. Hougas. 1959. Decapitation and genetic markers as related to haploidy in Solanum tuberosum. Eur. Potato J. 2:176–183. Peloquin, S. J., and R. W. Hougas. 1960. Genetic variation among haploids of the common potato. Am. Potato J. 37:289–297. Peloquin, S. J., C. O. Okwuagwu, E. F. Leue, S. A. Hermundstad, D. M. Stelly, S. H. Schroeder, and J. E. Chujoy. 1984. Use of meiotic mutants in breeding. p. 65–74. In: Present and Future Strategies for Potato Breeding and Improvement. Centro Internacional de la Papa, Lima, Perú. Peloquin, S. J., G. C. Arndt, and H. M. Kidane-Mariam. 1985. Utilization of ploidy manipulations for true potato seeds. In: Innovative Methods for Propagating Potatoes. Centro Internacional de la Papa, Lima, Perú. p. 17–24. Peloquin, S. J., G. L. Yerk, and J. E. Werner. 1989. Ploidy manipulations in potato. p. 167–178. In: K. W. Adolph (ed.), Chromosomes: Eukaryotic, prokaryotic and viral. CRC Press, Boca Raton, FL. Peloquin, S. J., G. L. Yerk, J. E. Werner, and E. Darmo. 1989. Potato breeding with haploids and 2n gametes. Genome 32:1000–1004. Peloquin, S. J., J. E. Chujoy, and J. E. Werner. 1992. 4x hybrid progeny from 2x–2x crosses in potato. p. 23–29. In: A. Mariani and S. Tavoletti (eds.), Gametes with somatic chromosome number in the evolution and breeding of polyploid polysomic species: Achievements and perspectives. Consiglio Nazionale delle Ricerche, Perugia, Italy. Peloquin, S. J., J. E. Werner, and G. L. Yerk. 1990. The use of potato haploids in genetics and breeding. p. 79–92. In: P. K. Gupta and T. Tsuchiya (eds.), Chromosome engineering in plants. Elsevier Pub. Co., Amsterdam.

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Peloquin, S. J., L. S. Boiteux, and D. Carputo. 1999. Meiotic mutants in potato: Valuable variants. Genetics 153:1493–1499. Peloquin, S. J., R. W. Hougas, and A. C. Gabert. 1966. Haploidy as a new approach to the cytogenetics and breeding of Solanum tuberosum. p. 21–29. In: R. Riley and K. R. Lewis (eds.), Chromosome manipulations and plant genetics. Oliver & Boyd, Edinburgh. Peloquin, S. J., S. H. Jansky, and G. L. Yerk. 1989. Potato cytogenetics and germplasm utilization. Am. Potato J. 66:629–638. Peloquin, S. J., T. P. M. den Nijs, and A. O. Mendiburu. 1978. Sexual polyploidization versus somatic doubling. Proc. 7th EAPR Triennal Conf. p. 201–212. Pérez-Ugalde, O., R. W. Hougas, and S. J. Peloquin. 1964. Fertility of S. phureja-haploids and S. tuberosum F2 hybrids. Am. Potato J. 41:256–262. Quinn, A. A., and S. J. Peloquin. 1973. Use of experimental tetraploids in potato breeding. Am. Potato J. 50:415–420. Quinn, A. A., D. W. S. Mok, and S. J. Peloquin. 1974. Distribution and significance of diplandroids among Solanums. Am. Potato J. 51:16–21. Ross, R. W., S. J. Peloquin, and R. W. Hougas. 1964. Fertility of hybrids from Solanum phureja and haploid S. tuberosum matings. Eur. Potato J. 7:81–89. Schonnard, G., and S. J. Peloquin. 1991. Performance of true potato seed families. I. Effect of level of inbreeding. Potato Res. 34:397–407. Schonnard, G., and S. J. Peloquin. 1991. Performance of true potato seed families. II. Comparison of transplants versus seedlings. Potato Res. 34:409–418. Schroeder, S. H., and S. J. Peloquin. 1983. Seed set in 4x–2x crosses as related to 2n pollen frequency. Am. Potato J. 60:527–536. Serquén, F. C., and S. Peloquin. 1996. Variation for agronomic and processing traits in Solanum tuberosum haploids × wild species hybrids. Euphytica 89:185–191. Simon, P. W., and S. J. Peloquin. 1976. Pollen vigor as a function of mode of 2n gamete formation in potatoes. J. Hered. 67:204–208. Simon, P. W., and S. J. Peloquin. 1977. The incidence of paternal species on the origin of callus in anther culture of Solanum hybrids. Theor. Appl. Genet. 50:53–56. Simon, P. W., and S. J. Peloquin. 1980. Inheritance of electrophoretic variants of tuber proteins in Solanum tuberosum haploids. Biochem. Genet. 18:1055–1061. Souter, B. W., J. C. Daws, and S. J. Peloquin. 1980. 2n pollen formation via parallel spindles in the potato cultivar Sebago. Am. Potato J. 57:449–455. Stelly, D. M., and S. J. Peloquin. 1986. Diploid female gametophytes formation in 24chromosome potatoes. Genetic evidence for the prevalence of second meiotic division restitution mode. Can. J. Genet. Cytol. 28:101–108. Stelly, D. M., and S. J. Peloquin. 1986. Formation of 2n megagametophytes in triploid tuber-bearing Solanums. Am. J. Bot. 73:1351–1363. Stelly, D. M., and S. J. Peloquin. 1986. Screening for 2n female gametophytes, female fertility and 2x × 4x crossability in potatoes (Solanum spp.). Am. Potato J. 62:519–529. Stelly. D. M., S. J. Peloquin, R. G. Palmer, and C. F. Crane. 1984. Mayer’s hemalum-methyl salicytate: A stain-clearing technique for observation within whole ovules. Stain Technol. 59:155–161. Thill, C. A., and S. J. Peloquin. 1995. A breeding method for accelerated development of cold chipping clones in potato. Euphytica 84:73–80. Thill, C. A., and S. J. Peloquin. 1995. Inheritance of potato chip color at the 24 chromosome level. Am. Potato J. 71:629–646. Wangenheim, K. H. von, S. J. Peloquin, and R. W. Hougas. 1960. Embryological investigations in the formation of haploids in the potato (Solanum tuberosum). Z. Vererbungslhre 91:391–399.

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Watanabe, K. N., M. Orrillo, S. Vega, M. Iwanaga, R. Ortiz, R. Freyre, G. L. Yerk, S. J. Peloquin, and K. Ishike. 1995. Selection of diploid potato clones from diploid (haploid × species) F1 hybrid families for short day conditions. Breed. Sci. 45:341–347. Watanabe, K., and S. J. Peloquin. 1989. Occurrence of 2n pollen and ps gene frequencies in cultivated groups and their related wild species in tuber-bearing Solanums. Theor. Appl. Genet. 78:329–336. Watanabe, K., and S. J. Peloquin. 1991. The occurrence and frequency of 2n pollen in 2x, 4x, and 6x wild, tuber-bearing Solanum species from Mexico and Central and South America. Theor. Appl. Genet. 82:621–626. Watanabe, K., and S. J. Peloquin. 1992. Cytological basis of 2n pollen formation in a wide range of 2x, 4x, and 6x taxa from tuber-bearing Solanum species. Genome 36:8–13. Watanabe, K., S. J. Peloquin, and M. Endo. 1991. Genetic significance of mode of polyploidization: somatic doubling or 2n gametes. Genome 34:28–34. Werner, J. E., and S. J. Peloquin. 1987. Frequency and mechanisms of 2n egg formation in haploid Tuberosum-wild species F1 hybrids. Am. Potato J. 64:641–654. Werner, J. E., and S. J. Peloquin. 1990. Inheritance of two mechanisms of 2n egg formation in 2x potatoes. J. Hered. 81:371–374. Werner, J. E., and S. J. Peloquin. 1991. Occurrence and mechanisms of 2n egg formation in 2x potato. Genome 34:862–870. Werner, J. E., and S. J. Peloquin. 1991. Potato haploid performance in 2x × 4x crosses. Am. Potato J. 68:801–811. Werner, J. E., and S. J. Peloquin. 1991. Significance of allelic diversity and 2n gametes for approaching maximum heterozygosity in 4x potatoes. Euphytica 58:21–29. Werner, J. E., and S. J. Peloquin. 1991. Yield and tuber characteristics of 4x progeny from 2x × 2x crosses. Potato Res. 34:352–357. Yeh, B. P., S. J. Peloquin, and R. W. Hougas. 1964. Meiosis in Solanum tuberosum haploids and haploid-haploid F1 hybrids. Can. J. Genet. Cytol. 6:393–402. Yeh, B. P., S. J. Peloquin, and R. W. Hougas. 1965. Pachytene chromosomes of the potato (Solanum tuberosum Group Andigena). Am. J. Bot. 52:1014–1020. Yerk, G. L., and S. J. Peloquin. 1988. 2n pollen in eleven 2x, 2 EBN wild species and their haploid-wild species hybrids. Potato Res. 31:581–589. Yerk, G. L., and S. J. Peloquin. 1989. Comparison of 2n and non-2n pollen producing haploid × wild species hybrids in potato. J. Hered. 80:450–453. Yerk, G. L., and S. J. Peloquin. 1989. Evaluation of tuber traits of 10, 2x (2EBN) wild species through haploid × wild species hybrids. Am. Potato J. 66:731–739. Yerk, G. L., and S. J. Peloquin. 1990. Performance of haploid × wild species 2x hybrids (involving five newly evaluated species) in 4x × 2x families. Am. Potato J. 67:405–417. Yerk, G. L., and S. J. Peloquin. 1990. Selection of potato haploid parents for use in crosses with 2x (2 Endosperm Balance Number) wild species. Crop Sci. 30:943–946.

2 Politics of Plant Breeding Cary Fowler Center for International Environment & Development Studies (NORAGRIC), Agricultural University of Norway, P.O. Box 5003, 1432 Aas, Norway Richard L. Lower Dept. of Horticulture, 1575 Linden Drive, University of Wisconsin, Madison, WI 53706

I. INTRODUCTION II. GERMPLASM, PLANT BREEDING, AND THE FIGHT FOR RIGHTS A. Historical B. “Rights” in the International Arena C. Expanded Claims of Rights D. The FAO Treaty on Plant Genetic Resources and the Global Plan of Action 1. The Treaty 2. The Global Plan of Action E. The International Treaty on Plant Genetic Resources for Food and Agriculture 1. The Treaty 2. Expected Impact of the Treaty III. THE DEBATE OVER BIOTECHNOLOGY A. Hurdles to Construct, Hurdles to Jump B. Taking a Step Backward IV. PLANT BREEDERS’ CHOICES A. Public vs. Private B. GMOs or Non-GMOs C. Intellectual Property Protection and Rights D. Looking Ahead LITERATURE CITED

Plant Breeding Reviews, Volume 25. Edited by Jules Janick. ISBN 0471666939 © 2005 John Wiley & Sons, Inc. 21

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I. INTRODUCTION Over the past century, plant breeding has contributed to and benefited from the growth of the biological sciences that support both the science and art of plant improvement. Major building blocks include contributions from genetics, statistics, and supporting biological curricula. In the past 30 years, the rapid increase in molecular sciences, principally genomics, paired with advanced technologies in information processing and communication have fostered the development and usage of plant breeding tools. The uses of molecular marker techniques are an example of these contributions to plant breeding. The development of germplasm collections and the establishment of gene banks throughout the world have improved the conservation and increased the availability of genetic resources. These events coupled with the development of teams of scientists in related sciences, such as plant pathology, entomology, plant physiology, and biochemistry, have resulted in major advances in plant breeding. Free exchanges of germplasm and information have been major factors in the success and growth of plant breeding. In spite of the timely advances in plant breeding and its supporting/cooperating sciences, the luxury of and access to plant collections, the myriad of molecular tools and techniques, and in spite of the tremendous and increasing need the world has for the products of plant breeding, the practicing plant breeder is likely to encounter complex and often frustrating obstacles and dilemmas in his or her professional career and research. More and more frequently these difficulties are not technical in nature. At their heart, they are political. Politics and plant breeding have an ancient, if unappreciated and uncomfortable, relationship. Liberally interpreted, the genetic vestiges of this history—those associated with the spread of agriculture, and the first plant collecting expeditions 4500 years ago—are still with us today in a world in which virtually every country is highly dependent on crops that originated outside its own region (Plucknett et al. 1987; Palacios 1998). Positing such a lengthy history depends on how one defines “plant breeding” and “politics.” No such definitional problems exist today, however, if we assert that politics is a part of plant breeding, and viceversa. The entire profession of plant breeding and virtually each of its component activities is currently a matter of political concern and/or controversy. Consider: • The acquisition and transfer of plant genetic resources—the “raw material” for plant breeding and the first step in the breeding

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•

•

•

•

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process—is the subject of two international laws and several international contractual agreements. Moreover, the subject continues to evoke strong emotions and is still under active dispute. The priorities and goals of plant breeders are under constant scrutiny. Not infrequently, the “choices” made by breeders are painted in stark and evocative terms. Are plant breeders producing hybrids or open-pollinated cultivars? Genetically modified organisms (GMOs) or non-genetically modified organisms (non-GMOs)? Cultivars tailored for small, resource-poor farmers and marginal ecological niches, or varieties aimed more at commercial agriculture and high-input systems? Cultivars with or without intellectual property rights? Cultivars released by the public or by the private sector or jointly? To many, each of the questions has a single “correct” answer. A constant undercurrent in deliberations over funding and prioritysetting concerns the division of labor between public and private plant breeding efforts. Recent surveys show declining numbers of public plant breeders and increasing numbers in the private sector (Frey 1996). As public efforts depend on public funding, politics is always present. The political process determines how much funding there will be, to which crops funding will go, and how far “upstream” or “downstream” the research should be, i.e., how far from a commercial product or a consumer. Indeed, the public views may be changing as public funding is declining and products of breeding programs come to be viewed as potential sources of financial support for research programs. The methodology and tools used by the breeders are also under question. Are “participatory” methods employed to produce cultivars with the farmers? Are certain biotechnologies used or eschewed? How is field testing done and where is it done? Guidelines vary greatly from country to country, and even within countries, and apply particularly when biotechnological tools are used and when genetically modified organisms are to be marketed. Marketing of seed is highly regulated and controlled by laws that have not always been welcomed or accepted in the seed trade. Quarantine laws, straightforward and laudable on the surface, are frequently the subject of manipulation for political and trade purposes. Questions of nomenclature—for example, producing and selling something called Basmati rice in the U.S.—can also be controversial (Tripp 1997). Marketing seed via grower contracts or agreements on the usage of the resulting crop can be very restrictive and are common with GMOs.

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• Even questions of “rights” are now routinely raised in regard to the practice of plant breeding. For instance, what rights do suppliers of germplasm (or their descendants) have to “benefit-sharing” associated with the final product, a cultivar produced in a breeding program? How is the “Right to Food,” an initiative being promoted by a number of countries in international forums, related to the rights (or obligations) of plant breeders? Note that the “right to food” has been proclaimed in a number of texts, including the Universal Declaration of Human Rights (1948), the International Covenant on Economic, Social and Cultural Rights (1976), and the Rome Declaration on World Food Security issued during the World Food Summit (1996), and is a subject of current discussion and debate in various intergovernmental forums. In addition, 22 countries have enshrined this right in their constitutions. • Ethical concerns are being raised in a political fashion particularly as it regards the use of biotechnologies and the notion of what is “natural,” or not. While many consumers may think of “natural” as being both safe and “better,” many scientists would be uncomfortable in defining such terms with any scientific precision; and many would observe that numerous “naturals” are toxic and some can even be deadly. • We live in changing times. During the past three decades numerous changes in mechanisms for intellectual property rights have taken place. Over the same time frame, dramatic advances in biotechnologies and in related molecular sciences and information technologies have greatly altered and affected the way plant breeding is conducted. Handling these new technologies, when generated in the public sector, is causing considerable stress within public institutions. • Finally, the structure of plant breeding is undergoing a major transformation. As a result of declining political influence, public sector activities are in a state of decline, worldwide. Budgets are down. Fewer students are showing up at universities to study plant breeding, and certain kinds of crops and breeding activities are being abandoned, or “rationalized” as it is more commonly explained. Moreover, the glitz and glamour of some of the biotechnologies continue to attract many who likely would have been plant breeders in earlier decades. In this paper, we examine some, but not all of the political issues facing plant breeding, as there are far too many. We have selected several that we believe are most interesting to the actual practice of plant breed-

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ing in the future. We have chosen not to address in a detailed manner at least one subject that meets these criteria—the issue of the role, status, and funding of public sector plant breeding, though we have touched upon such subjects as they relate to the United States. This is a critically important subject and one that has been developed by numerous authors. We believe it is worthy of discussion. But, unlike the other topics we have chosen to address, this one is predominantly country-specific. The reality is different in the U.S. and Norway and different still in other developed and developing countries. Suffice it to say that the public sector has a virtual monopoly in training new plant breeders, in taking on certain kinds of fundamental basic research, and in working with crops, regions, farming systems, markets, and types of farmers (e.g., the resource-poor) that are not and cannot be priorities for the private sector. This is a broad and vitally important “mandate.” At the international level, it is addressed by international agricultural research centers (the Consultative Group on International Agricultural Research System (CGIAR), Asian Vegetable Research and Development Center (AVRDC), and the Tropical Agriculture Research and Higher Learning Center (CATIE)) and at the national level by national agricultural research systems, including universities. In recent years, all of these have suffered dramatic funding cuts that concern us greatly. However, for pragmatic reasons, we have chosen to concentrate in this paper on issues that can be dealt with more generically, those that are more international in scope.

II. GERMPLASM, PLANT BREEDING, AND THE FIGHT FOR RIGHTS A. Historical By the time the first Europeans had set foot in the New World, maize, for example, had already migrated to the high mountain valleys of the Andes as well as to jungle areas of the Amazon, and was being grown under irrigation in the dry coastal lands of western South America (Plucknett et al. 1987). The spread of many food crops was “natural.” We know of no disputes involved. People and plants had been on the move for thousands of years. While maize spread throughout the world, reaching West Africa in the second half of the 16th century, and while grains whose Center of Diversity was the Near East found their way into Europe, Asia, and the Americas (Columbus introduced wheat along with chickpea, melons, onions, grape vines, and sugar cane on his second voyage), the acquisition,

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transfer, and spread of other species was anything but routine. Some of the earliest plant collecting expeditions dating back 3500 years were mounted by armies, which is precisely what it would take to collect materials in some countries today! Moreover, during the colonial era, a number of species—principally of industrial, plantation, or medicinal crops—were acquired by questionable means. Accusations of theft are not uncommon in the histories of the time (Wolf and Wolf 1936). Colonial powers were interested in establishing production under their own control for certain high-value crops. In some cases they openly desired monopoly (e.g., for indigo) and attempted to establish it. As early as 1556, for instance, Spain’s Council of the Indies, convened in Madrid, passed a law making it illegal for foreigners to explore for plants in Spain’s New World possessions (Haughton 1979). Those who were subjugated and from whom the planting materials were acquired frequently did not give their consent (Brockway 1979; Crosby 1986). Our purpose is not to delve into ancient history; it is to make a simple point in introducing the subject of contemporary conflicts over plant genetic resources, namely that questions of ownership and rights have been an important feature of agriculture for centuries. Disputes have been the norm, not the exception, historically. The colonial era was characterized by the desire to obtain particular species. Mendelian laws were unknown and there were few efforts to acquire and limited ability to use intra-specific diversity, one exception being France’s interest in broadening the variability of certain economic species (Plucknett et al. 1987). The development of sizeable urban markets in Europe and North America in the 1800s provided the impetus for the rise of commercial agriculture. Varietal differentiation became commercially important. In the U.S., the government distributed millions of packets of seeds of countless cultivars or “landraces” acquired abroad and encouraged experimentation by farmers. The establishment of county agricultural fairs in the mid-1800s gave added encouragement to both experimentation and to the “fixing” of ideal traits. Farm journals and the ever-popular Farmer’s Almanac were replete with advice on how farmers could improve their crops. And a significant portion of the seed used was produced on the farm (USDA 1918; Elder 1921). Today, interestingly, much of the same information is available on hundreds of Internet sites. Cultivars were valued for different traits including their keeping and shipping qualities. By the turn of the century (19th to 20th), for example, there were 60,000 refrigerated railway cars operating in North America, and the Illinois Central was boasting of its 25-car Thunderbolt train, the first “all-strawberry train in the nation” designed to link the farms of southern Illinois with Chicago (Stover 1975; Taylor 1901). Despite the

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absence of large, formal “scientific” (Mendel-informed) plant breeding programs, crop cultivars—such as those tailored for shipping—were well established, and certain ones of them were known and appreciated in the marketplace. By 1925 one publication lists 1362 cultivars of strawberry, for example, but an early 20th century U.S. government survey documents the astounding existence of more than 7000 distinct, named cultivars of apples in the U.S. in use in the 1800s (Ragan 1926). In this atmosphere, it should come as little surprise that certain interests—in this case, the nursery trade—began to claim rights and lobby for legal recognition of their ownership of varieties. The first such proposal was made by the Committee on Registration of the Peninsula Horticultural Society in the U.S. in 1891, though prior to this there had been other efforts to achieve control through regulations standardizing nomenclature. Such proposals were made and rejected a number of times before the U.S. Congress finally passed the Plant Patent Act of 1930 (Fowler 2000). This legislation covered asexually-reproducing species excluding, in practice, some tuber crops used for food, such as potatoes. It was only a matter of time before pressure mounted to pass similar legislation for sexually-reproducing (i.e., seed) crops. In 1961, representatives of six European countries gathered to create the Union for the Protection of New Varieties of Plants (UPOV). The UPOV “process” created a consensus among commercial breeding interests about how new varieties should be legally protected through the establishment of intellectual property rights. The UPOV solution took protection out of the realm of patent law by creating its own sui generis system. Plant Breeders Rights—“Plant Variety Protection” in the U.S.—was born, based on the criteria of the new cultivar being distinct, uniform, and stable. It was well recognized that farmers themselves, even in developed countries, were producing new varieties of value. Speaking of the 19th century, Anderson and Brown (1952a,b) recognized the contributions of farmers and observed that “there is detailed evidence of the purposeful blending of diverse cultivars . . . the controlled breeding of new varieties by farmers themselves was more frequent than anyone would believe.” They went on to note the “highly elaborate methods of selection.” Nevertheless, such cultivars fell short of the new legal requirements for recognition. B. “Rights” in the International Arena While this is not the place for a detailed exploration of the immediate roots of contemporary conflicts in the 1980s and 1990s over plant genetic resources (PGR), it seems evident that the content, flavor, and tone of the controversies that arose over intellectual property rights in the U.S. and

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Europe and over PGR more generally at the Food and Agriculture Organization (FAO), were inspired by earlier political movements (e.g., civil rights and environmental), as well as by the politics of the Cold War and the political discourses over development and “under-development” (Fowler 1994). It was also rooted in a certain sense of history, as recounted above. Several small but energetic non-government organizations (NGOs) began to agitate first for increased attention to and funding for PGR conservation programs. Principally, these were the International Coalition for Development Action, the National Sharecroppers Fund/Rural Advancement Fund (later RAFI), and Genetic Resources Action International (GRAIN). These groups were also concerned about two other matters: (1) the perceived imbalance created by the recognition of the rights of plant breeders (mainly in developed countries) without a corresponding recognition of the contributions and rights of (mainly developing) countries and farmers who had “created,” conserved, and ultimately donated the myriad landraces used in such breeding programs, and (2) the perceived diminution of the public domain—of what was then described as the “common heritage of mankind”—by the privatization of the PGR through intellectual property rights. These concerns led to proposals (introduced by Mexico) for a binding legal agreement on plant genetic resources at FAO, one that would fix the status of PGR as a resource in the public domain, and would recognize the existence of “Farmers’ Rights” in juxtaposition to Plant Breeders Rights. Years of acrimonious debate followed at FAO, divided strictly along developed/developing (or north/south) country lines. The early FAO battles produced two outcomes, controversial at the time, but less so now in retrospect. First, an intergovernmental Commission on Plant Genetic Resources was created. Second, governments adopted a non-binding International Undertaking on Plant Genetic Resources. The Commission, which met for the first time in 1985, was to serve as a specialized forum for the discussion and resolution of PGRrelated problems. The Undertaking, adopted in 1983, was to serve as a statement of how the international community viewed PGR—it viewed PGR as a “common heritage.” A subsequent resolution (Resolution 5/89) by the FAO Conference defined Farmers’ Rights as “rights arising from the past, present and future contributions of farmers in conserving, improving, and making available plant genetic resources, particularly those in the centres of origin/diversity.” Resolution 5/89 constituted an important milestone. The concept of Farmers’ Rights had now found a place in intergovernmentally approved text. But, it was a Pyrrhic victory for developing countries and NGOs. A UN document observing where rights arise from is not the same as defin-

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ing what those rights actually are. Lacking a definition, Farmers’ Rights was a concept and only a concept. There was nothing legally enforceable. It was an empty vessel into which everyone was free to provide content. Initially, however, this was not of great concern to its proponents, as Farmers’ Rights had not been envisaged as a system of legal “rights” but as a concept or political slogan designed to pressure governments into increased support for PGR conservation “in support” or “recognition” of those undefined rights (Fowler 1998). Common heritage came with common responsibilities, responsibilities that were not being met. Resolution 3/91, adopted in 1991, made the link explicit by stating that “Farmers’ Rights will be implemented through international funding on plant genetic resources, which will support plant genetic conservation and utilization programmes, particularly, but not exclusively, in the developing countries.” C. Expanded Claims of Rights Claims of “rights” are typically asserted and contested at the national level. In many countries, “rights” are enshrined in constitutions. Politically speaking, additions/alterations to the list are not considered trivial. Nevertheless, in the relatively restricted field of genetic resources, the contest over rights has often taken place in international forums, in part because those advocating such rights lack “critical mass” politically to take up the fight country-by-country. As the mid-1990s approached, there was no obvious way to continue the Farmers’ Rights debate at FAO. An accommodation, of sorts, had been made in the available negotiating document—the International Undertaking and associated resolutions. FAO was moving on to other, rather more pragmatic and scientific concerns, such as the development of a Global Plan of Action for Plant Genetic Resources. While not devoid of politics, the Plan was not the best or most logical vehicle for a renewed fight over Farmers Rights’ or any other kind of rights. Moreover, countries were busy asserting sovereignty over their own PGR and their interest in devolving rights and power to farmers was not high. NGOs, the leading advocates of Farmers’ Rights in the first place, were still passionate about the subject, but Farmers’ Rights competed with other political campaigns concerning intellectual property rights, biopiracy, the CGIAR, and the World Trade Organization. In this context—and lacking any legal/political forum on which to focus, the concept of Farmers’ Rights began to evolve. One NGO publication from 1995 (GRAIN 1995) lists and seems to support an astonishing array of rights: Farmers’ Rights, Expanded Farmers’ Rights, Community Rights, Community Intellectual Rights, Communal Rights, Peoples’

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Rights, Indigenous Peoples’ Rights, Indigenous Community Rights, Local and Indigenous Community Rights, Traditional Resource Rights, Collective Rights, Neighboring Rights, Development Rights, Human Rights, Religious Rights, Heritage Rights, Territorial Rights, Territorial and Tenurial Rights, and, “bundles” of rights! Rather than become more focused and precise—more defined—Farmers’ Rights became more diffuse and fuzzy. Smith (1997) cataloged 16 different claims being associated with the term “Farmers’ Rights” alone, ranging from moral principles of recognition and rights to compensation, subsidies and land, to provision of funding for maintenance of current lifestyles, participatory breeding, and genetic resource conservation. In addition, support for such rights and interests now led some proponents to become highly critical of genebanks, plant breeding and breeders and formal scientific research systems, and to advocate instead for a combination of in-situ and on-farm initiatives coupled with associated “rights.” While the in-situ approach has much to commend itself, we note that its promotion by certain groups has frequently been framed in political terms as being in contrast to and in some ways in opposition to ex situ approaches. It is this political context—and its implications for plant breeding and plant breeders—that we draw attention to here. Otherwise, we do not see it as being within the scope of this particular article to address the merits of the case for or against any “rights.” A more interesting shift was and still is taking place. However imprecisely defined the above “rights” might be when found in various documents, many are taking on the flavor of being a form of intellectual or property rights. Far from emphasizing the “common heritage” history and quality of the PGR, many groups seem now to treat the resource as a commodity, one to be owned, sold, or bartered by its owners. That such a position has evolved in reaction to more dominant intellectual property rights systems seems obvious. Yet, it is also ironic, given the history of the debate and indeed of the actors themselves as opponents of intellectual property rights. GRAIN has gone so far as to describe as “biopiracy” an agreement whereby PGR collections held by the CGIAR Centers are placed essentially in the public domain for the international community under the auspices of FAO (GRAIN 2002). In the early 1990s, they had been among the groups pressuring in favor of this agreement. It is difficult to say whether the dispute could be resolved by national promulgation of rights or intellectual property laws applicable to landraces/farmers’ varieties, or whether the real problem could only be addressed by more fundamental shifts in political and economic power. Without resolution, however, advocacy groups are unlikely to devote time or resources to supporting ex situ conservation efforts or plant breeding efforts (unless the latter are strictly on their terms, i.e., partic-

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ipatory with farmers, and in accordance with the “rights” noted previously). Thus, PGR conservation and public sector plant breeding has lost a potentially powerful advocate. And, it may in many cases have picked up an energetic opponent, as the CGIAR has experienced in recent years. At the national level, the effects of restrictive access laws and political pressures have combined to limit access to PGR, especially from insitu sources. This situation may persist as long as the question of rights is unresolved, unless the new International Treaty on Plant Genetic Resources takes hold and is seen to provide an equitable solution to the issues of access and benefit sharing.

D. The FAO Treaty on Plant Genetic Resources and the Global Plan of Action 1. The Treaty. Neither the FAO Undertaking (a non-binding agreement) nor the resolutions on Farmers’ Rights (which failed to include an operational definition of the term) served to dispel the underlying tensions. Heightened awareness of the importance of “biodiversity” coupled with historic sensitivities associated with its appropriation and use (noted above) led to negotiations culminating in the Convention on Biological Diversity (CBD) in 1992. The CBD turned the world of the FAO Undertaking upside down, by reasserting claims of national sovereignty over genetic resources. In the space of a decade, developing countries shifted from advocating “common heritage,” to demanding “national sovereignty.” NGOs faced a dilemma: abandon what they perceived to be the politically progressive notion of common ownership, or abandon their alliance with developing countries. Most chose the former, quietly avoiding any public recognition of the obvious conflict and subsequent political flip-flop. Many countries proceeded to try to “commoditize” PGR, exactly what NGOs had complained about when the private sector claimed ownership over varieties through intellectual property rights. The CBD was based, according to John Barton, Professor of Law at Stanford University, on a fundamental misconception at least in regards to PGR of agricultural species—the belief that there was a commercial market for the resources (Agres 2003). While access to PGR was to be granted by “prior informed consent” and on the basis of “mutually agreed terms,” no one was buying. Negotiators, however, had recognized that the CBD could not appropriately apply to existing collections of PGR and they specifically invited FAO to initiate negotiations on this subject. FAO got on with the task. In the meantime, access/transfers of genetic resources slowed to a trickle. A study of the Andean region found that several countries had rejected all applications for access and

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that for the region as a whole “no case can be identified where benefit sharing has actually taken place.” (Correa 2003) FAO’s strategy was two-pronged. It would provide a forum for the development of two related agreements: a binding legal agreement on access and benefit-sharing, and a Global Plan of Action for the conservation and utilization of PGR. It was assumed by many that the “benefitsharing” arrangement would provide the means for implementing the Global Plan of Action, implementation of which would generate benefits. Both documents would be formally adopted at a large intergovernmental conference in Leipzig in 1996. This, however, was not to be. 2. The Global Plan of Action. FAO established a Secretariat to draft a major report on the state of the world’s plant genetic resources for food and agriculture as well as the Global Plan of Action itself, and to prepare for the Leipzig Conference. The State of the World’s Plant Genetic Resources, the most comprehensive assessment undertaken to date, was prepared largely on the basis of information in 154 country reports submitted by the countries themselves, as well as additional information provided by scientists, in particular from the International Plant Genetic Resources Institute (IPGRI) and other centers of the Consultative Group on International Agricultural Research. NGO concerns by this time had come to focus almost exclusively on political matters (e.g., Farmers’ Rights) and though formally invited and facilitated to do so, they provided few inputs to the State of the World report or to the prescriptive measures found in the Global Plan of Action. The State of the World’s Plant Genetic Resources functioned as a “problem statement,” identifying gaps in conservation, use and, to a much lesser degree, in the distribution of benefits from this use. It clearly indicated that additional efforts (and funding) were needed, but it just as clearly revealed that better coordination and priority-setting could, even with current levels of funding, solve many problems and bring substantial benefits. The Global Plan of Action is organized into four major categories: • • • •

In Situ Conservation and Development Ex Situ Conservation Utilization of Plant Genetic Resources Institutions and Capacity Building

Within these general categories are 20 concrete “activities.” For each activity, the Global Plan of Action provides: • An assessment of the situation, drawing on the State of the World report

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Long-term objectives Intermediate objectives Policy/strategy Capacity requirements/recommendations Research/technology recommendations And, coordination/administrative recommendations

Negotiations on a binding legal agreement—what would eventually become the International Treaty—bogged down and no draft was ready by the time the Leipzig Conference opened in June, 1996. Instead, the Conference concentrated on the Global Plan of Action, which it adopted, and 150 countries formally agreed on what needed to be done to conserve and better utilize plant genetic resources. For the thoughtful observer, however, one nagging question remained: By the time the Treaty was adopted would countries still support the Global Plan, and would they see its implementation as providing the type and size of benefits they required in order to resolve the access/benefit-sharing conundrum? Would the Treaty “package” feature the Plan “front and center,” or would it simply give a nod to it and proceed to try to lock in financial benefits as did the Convention on Biological Diversity? A partial answer to these questions was quick in coming. The Secretariat’s estimate of the costs of implementing the Global Plan of Action was prevented from being put on the table. A number of countries were opposed to considering it. It seems that they saw the value of the Plan not in terms of the benefits that implementation would bring, but in terms of how big the budget would be for implementation. And it was too small. Thus they continued to feel that they were giving more away in PGR than they were receiving in dollars for the Plan’s implementation. E. The International Treaty on Plant Genetic Resources for Food and Agriculture 1. The Treaty. The acrimonious 7-year negotiation of the International Treaty came down to one simple question: how much access for how much benefits? It was understood that countries would provide access to genetic resources. But, what kind of access would this involve, how many crops would this arrangement cover, and what would the terms of access be? Would specific acts of access be linked to concrete benefits that would accrue to the provider? In this negotiation, developing countries were at something of a disadvantage, and not simply because of any differences of “power” (decisions are taken by consensus at FAO). They were still operating under the notion that their PGR were a highly coveted commodity with a substantial

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monetary value. They were also smarting from the belief that they were being exploited—that modern cultivars protected by intellectual property rights were being marketed to them at high prices when, in fact, these cultivars were based on genetic materials donated to developed countries’ breeders by developing countries and developing country farmers. In retrospect, it is possible to question whether such analyses (irrespective of their merits) and the emotions they generated served the negotiators well in the negotiations. Was, for example, the Treaty a good vehicle for redressing the types of grievances harbored by developing country delegates? Did it lead them to develop the best and most practical remedies? The jury is still out, at least on the latter question. Developing countries also saw themselves as donors, not as recipients, of germplasm. Consequently, they started with a hard bargaining position. FAO studies, however, revealed that countries—including developing countries—are highly interdependent in terms of PGR. And, the State of the World report indicated that much of the diversity had already “escaped” to genebanks outside of the control of individual developing countries. The implications were reasonably clear: one of the major benefits of the Treaty would be to guarantee access. Access itself was a benefit for everyone. Developed countries started with an equally hard bargaining position. In essence they were returning to the view of “common heritage” in the International Undertaking, which they had fought strongly against a decade earlier. They wanted the new Treaty to guarantee access to all crops. Europe countered Africa’s proposal of an agreement on 6 crops with a proposal for nearly 300 crops, including a few that most delegates (including many Europeans!) had never heard of, and some, such as kudzu, that others would just as soon forget. The sheer breadth of the European proposal, however, served to reinforce developing country suspicions that “their” genetic resources were valuable and sought after by commercial interests. In the end, a measure of common sense prevailed on all sides, and the International Treaty was adopted in 2001, the first treaty of the new millennium (FAO 2001). The Treaty is a watershed in international relations regarding plant genetic resources. It is not altogether inaccurate to say that it is the first international agreement addressing what could be described as a 3000-year running dispute. At the heart of the International Treaty is the creation of a Multilateral System of access and benefit-sharing. This System encompasses 35 crops and a number of temperate forage species. The 35 include most major food crops, but exclude soybean, tomato, groundnut, and tropical forages, as well as most industrial crops (rubber and oil palm, for example). A tremendous asset associated with the Treaty is the genetic

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resources, mostly of the world’s major food crops, that are held at the centers of the Consultative Group on International Agricultural Research (CGIAR). Historically, these have been considered as an international heritage and have been freely available to everyone, most recently under the terms of a formal agreement between FAO and the centers in which it is agreed that the centers are holding the materials “in trust” for the benefit of the international community. Several other gene banks outside of the CGIAR system also share the CGIAR philosophy regarding the nature of germplasm being held and its availability. These are usually, but not always, national collections that may be shared widely, but that are nevertheless subject to political whims. For the agreed crops, the Treaty provides for “facilitated” access to PGR that is under the management and control of a Contracting Party (a country that ratifies the Treaty). Access is consistent with respect for applicable intellectual property rights, and is at the discretion of the developer during the period of development. The benefit-sharing provision of the Treaty is activated when someone acquires material from the Multilateral System (for example, from a genebank of a country that has ratified the Treaty), incorporates that genetic material into a product that is a PGR (e.g., a cultivar or line as opposed to a breakfast cereal), commercializes it, and then protects it (through intellectual property rights, for instance) in a way that restricts subsequent access and use of the product. In practice this is assumed by many to mean that if someone takes out a utility patent on the product, benefit-sharing will be required, whereas if one takes out Plant Breeders Rights, benefit-sharing will not be required, as this form of IPR explicitly allows the protected cultivar to be used for further research and breeding. The Treaty calls for a “fair and equitable” share of benefits “in line with commercial practice” to be paid into a fund which in turn will be used to support genetic resources related work, primarily in developing countries and countries with economies in transition. The all-important details of what this means precisely have been left to the first meeting of the Treaty’s Governing Body. The Governing Body, at its first meeting, is tasked with agreeing on a Material Transfer Agreement (MTA) to be used when materials within the Treaty’s Multilateral System are transferred. The Governing Body, composed of the countries that have ratified the Treaty, will convene within a year of the date when the Treaty comes into force, which will take place 90 days after the 40th country has ratified it. We expect the Treaty to come into force sometime in 2004. 2. Expected Impact of the Treaty. How the Treaty will affect the “politics of plant breeding” remains to be seen; however, we would argue that the effects will ultimately be determined by three factors:

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1. The number of countries that ratify the Treaty and their composition. Canada, Ethiopia, and India have already ratified and EU countries are expected to join shortly. But, will the U.S., Japan, and China ratify? The absence of major players will weaken the Treaty. Not only do the U.S. and Japan have major germplasm collections, they are also among the few countries that have intellectual property rights systems that might trigger the Treaty’s benefit-sharing mechanism. 2. The nature of the benefit-sharing agreement yet-to-be-made. Unless this agreement provides for realistic and concrete benefits, some countries (those who do not see the value of access) may choose not to ratify. Moreover, lack of benefits will undermine the Treaty and eventually lead countries to look for other ways in which to gain benefits from their agro-biodiversity. This could lead to the reemergence of conflict. 3. The scope of the Treaty’s Multilateral System. As noted above, 35 crops are covered, including rice, wheat, maize, potato, and the Brassica family. It is a great start, but for the majority of these crops the genetic resources are widely dispersed and rather freely available already, though it can be quite difficult to access materials, even of these crops, from in-situ conditions. For many, access from ex situ sources has never been problematic, especially for crops held by Centers of the CGIAR. Crops not included within the Multilateral System will be accessed for the most part under terms of the Convention on Biological Diversity, that is, on the basis of prior informed consent and mutually agreed terms. In the past, this has meant very limited access at best. Thus, breeders working with soybean, tomato, onion, tobacco, sugar cane, and so on will find plant collecting to be difficult and access from ex situ sources to be far from assured. Assembling genetic resource collections from scratch in order to initiate a breeding program—for example, on an underutilized or “minor” crop—will be almost impossible. Thus, unless the scope of the Treaty is expanded, the Treaty will serve to circumscribe future breeding efforts to crops whose collections fall under the Treaty—collections that are held by Contracting Parties to the Treaty of crops that are within the Multilateral System. The question then is how much is the Treaty’s scope likely to broaden in the future to encompass more crops. Our reluctant conclusion is not much, at least within the next decade. The Treaty requires complete consensus for changes to the list of crops it covers. One objection is enough to keep a crop out of the Multilateral System. It will take at least a decade before all countries realize what seems obvious to us: that there is less to gain from trying to make carrot genetic resources a marketable commodity, for exam-

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ple, than there is in promoting improvement of carrots through the exchange of materials. Despite shortcomings, the Treaty should help defuse and de-politicize tensions over plant genetic resources. Making access and exchange of plant genetic resources more routine will certainly contribute positively to plant improvement efforts. The resolution of long-running disputes and the establishment of international law and norms for access and benefit-sharing also clears the way for an additional and much needed initiative, specifically the creation of the Global Crop Diversity Trust, an endowment fund to support the long-term conservation of PGR, most notably of collections maintained in accordance with the rules of the Treaty. Prior to the adoption of the Treaty, it was hard to argue (or raise money) for an international fund to support “international” collections, when countries were asserting national sovereignty over these materials. Those that assert sovereignty must be prepared to pay the costs of maintenance themselves. Under the Treaty, however, certain crop collections are, in effect, placed in the public domain and it makes sense— as original proponents of the International Undertaking argued—that there is a common and public responsibility vis-à-vis these plant genetic resources. The Trust, created by FAO and the CGIAR, and linked to and made politically feasible by the Treaty, could provide security of funding that no PGR genebank collection has ever had heretofore (Plucknett et al. 1987). To succeed, however, the Trust will need to establish strict criteria that allow it to maintain a focus on supporting the scientific conservation of diversity, not the more politically-enticing conservation of extraneous genebanks. In addition, and in light of “9/11,” the international community cannot ignore the dangers posed to PGR collections as a result of terrorist actions, catastrophes, and so on. This aspect of politics, never a particular concern to plant breeders before, cannot now be ignored. The time may have come to establish a “fail-safe” world PGR collection. In this regard, the offer made by Norway some years ago to host a facility in a secure underground mineshaft in the permafrost of Svalbard near the North Pole could be revisited. The costs would be low; the insurance benefits enormous.

III. THE DEBATE OVER BIOTECHNOLOGY A. Hurdles to Construct, Hurdles to Jump Prior to the 1980s and the development and employment of the new biotechnologies, major innovations in the practice of plant breeding were assimilated with little evident public concern. The notion of

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“improvement” as it applied to crop varieties and animal breeds was already well established before Mendel’s laws of heredity were rediscovered in 1900. Darwin had recognized it explicitly in the first chapter of Origin of Species. To be sure, neither Darwin’s theories nor Mendel’s work were immediately accepted in the scientific community. While most quickly agreed that Mendel’s findings explained inheritance in peas, some thought it might apply only to peas or to a limited number of plants. No one, however, questioned the “application” of Mendel in plant breeding, or opposed the breeding strategy advocated by Beal in the 1870s (Fitzgerald 1990). No one, it seems, thought that greater control and precision in the “improvement” (breeding) process was somehow violating the “natural order” or that the products of Mendelian-informed plant breeding were automatically unnatural or unsafe because of the way in which they were produced. More to the point, the same can be said of George Schull’s crossing of inbred lines to produce hybrids at Cold Spring Harbor, Long Island, and Donald Jones’ subsequent work double-crossing inbreds using four inbred lines (Kloppenburg 1988). The age of hybrids in plant breeding was ushered in with silent consent from the consuming public and approval from the farmers. But times were different then. World trade was less developed and regulated and corporate, consumer, and environmental interests in this area were immature at best. The new biotechnologies were introduced into a totally different political and economic context. Political critiques of the goals, techniques, and impacts of plant breeding were well established by the 1980s (Griffin 1972; Palmer 1972a,b; Cleaver 1972; Hightower 1973; George 1977; Lappé et al. 1977; Mooney 1979). Yet, compared to the current discourse on biotechnology, there was something quite different about these early critiques. The issues being raised principally concerned hunger, the economic plight of small farmers, and the rising power of the private sector. The contemporary critique is not so much about the lack of food as it is about the safety of the food being offered, and the potential harm that its production might cause to the environment. In some quarters the debate is also about how GM foods are produced, about the technology and not just its product. An early White Paper published by the U.S. National Academy of Sciences concluded that safety assessments of introducing recombinant DNA-engineered organisms into the environment “should be based on the nature of the organism and the environment into which the organism is introduced, and not on the method by which it was produced.” (NAS 1987) Nevertheless, it is evident that GM products are subjected to a different kind of scrutiny precisely because of the methods used.

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Technologies are developed for a purpose, even if all consequences of deployment can never be fully discerned by the scientists involved. As Burns and Ueberhorst (1988) note: “Technologies are instruments of social action with consequences. The introduction of new technologies and the development of technological systems entail more than setting up and using new machines and other physical artifacts. It entails social re-organizing and the making of new rules.”—or as Winner (1987) puts it, “in a fundamental sense . . . determining things is what technology is all about.” But “determining” and “rule-making” are rarely trivial processes when major technologies are involved and when significant social, economic, and legal changes are unfolding. Perhaps this is as it should be. The implication, however, is that those who introduce technologies can never manage how they are perceived, accepted, or regulated, or be in complete control of the full range of consequences the technologies ultimately have. Unlike the earlier “Green Revolution,” biotechnology’s “Gene Revolution” has been taking place mostly in the private sector. The technologies are being developed primarily by the private sector, and many are proprietary, outside the public domain. A sense of the scale and speed of these developments can be gathered from a simple comparison. During the period 1981–1985, a total of 257 patents were issued in the U.S. containing the terms “rice,” “wheat,” or “corn” plus “gene.” During the period 1996–August 2001, 11,475 were issued. In addition, backlogs of patent applications are growing and the time required for processing them has increased to over two years (Rader 2000). The asymmetry of claims reflects the asymmetry of funding. In 1998, the CGIAR spent $25 million on biotechnology research, much to the consternation of biotech critics. But, Monsanto spent $1.26 billion that year (Pardey and Beintema 2001). The mobilization of capital to fund such research combined with the use of the utility patent system in the U.S. not just to protect products, but to lay claim to basic technologies, has raised concerns over the creation of an “anti-commons,” and among other things, over whether the public sector will be able to participate in a meaningful way or will be technologically confined (Heller and Eisenberg 1998; Falcon and Fowler 2002). A variety of “solutions” have been offered ranging from ”no patents on life” to the creation of biotechnology “patent pools.” (Resnik 2003) Castagnera et al. (2002) question whether the public sector is prepared to deal with the challenges of managing intellectual property rights. A survey of public plant breeders in the U.S. revealed that close to half had experienced difficulties in obtaining genetic stocks from private companies, 45% indicated that this had interfered with their research, and almost a quarter indicated that it

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had interfered with the training of graduate students (Price 1999). Little wonder then that in a more recent survey of U.S. Land Grant College scientists, the majority see shrinking budgets as an obstacle to their seizing the opportunities provided by biotechnology and view the private sector as the greatest beneficiary of the new technologies and the public sector as being least likely to benefit (Ag Biotechnology Implementation Task Force 2003). B. Taking a Step Backward In the space of a few short years, the amount of land devoted to genetically modified crops has increased dramatically, to approximately 145 million acres worldwide, including 45% of the soybean, 11% of the corn, 20% of the cotton, and 11% of the rapeseed (USDA/USTR 2003). While a great deal of resources have been devoted to the development of these varieties, with obvious implications for the plant breeders of the crops, the trend toward GM crops has not been without controversy. Strong consumer opposition has emerged, particularly in Europe. After approving 18 biotech products, and with 13 applications in the pipeline, the European Union initiated an informal moratorium in June of 1999. This prompted sharp reactions, and eventually a challenge by the U.S. in the World Trade Organization over unfair trade practices. While some are convinced that no evidence exists indicating that such crops pose any danger to human health or the environment, others are not so sure and cite the “precautionary principle” to support a “go slow” approach. Meanwhile, the rhetoric has reached the boiling point. GM opponents have labeled the crops “poison,” described them evocatively as “Frankenfoods” and as “worse than nuclear weapons or radioactive wastes” (Conko and Prakash 2002). GM proponents have retorted that opponents are “Luddites” who are guilty of “murder” for seeking to stop GM food relief to the hungry in Zambia. The verbal food fight is perpetuated and fueled by political leaders, commercial interests, and well-funded advocacy groups on both sides of the divide. While direct causal links are difficult to prove, it seems almost selfevident that the controversy has harmed the reputation of plant breeders and breeding and contributed to reductions and distortions in funding for public sector work. Goodman (2002) notes the worrying downward trend in training of new plant breeding Ph.D.s, which raises the question of whether this too might be linked. It is difficult to discern a silver lining. With polls showing that a large percentage of the American population does not “believe” in Darwin, it is hard to argue that the public debate in the U.S., or in Europe, is based on scientific literacy. As the mother of a

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wheat breeder friend of ours remarked recently: “Son, I’ve made a decision: in the future I’m not going to eat any food with genes in it.” The debate, obviously, is not just about science. Some people do not like Picasso no matter how much art experts say that his is great art. Similarly, a sizeable portion of the public sees little compelling reason to welcome genetically modified crops. To them, such foods do not serve their interests or solve any problems they believe they have. The antipathy is so pronounced that there is speculation that “GM concerns could be spilling over to adversely affect demand for non-GM products” such as “food products from animal industries that use GM foodstuffs, and honey . . .” (Foster et al. 2003). While some advocacy groups seem to think that there is something inherently unnatural and bad about the technology itself, the general public’s view seems to be more nuanced. Biotechnology applications in medicine, for instance, do not uniformly or automatically generate instant opposition. In the field of medicine, many applications are viewed as addressing real problems and providing tangible benefits. Moreover, the “risks” are borne by those who have the problem and need a solution. Society at large is not an unwitting guinea pig. Thus, the movement in Europe and elsewhere to require labeling strikes a cord. In a recent commentary, Taylor (2003) points out that “people won’t accept biotechnology under pressure” and argues that the U.S., for example, “should not be on the wrong side of the choice issue.” But, to make an informed choice, the public must become more informed. Therein lies the difficulty as well as the power and danger of the images conjured up by both sides in the debate. Before they give their support, the public must be convinced that plant breeding using the new biotechnologies brings benefits, not just to the bottom line of corporations or even to farmers, but to consumers through better, cheaper, and more nutritious food products, or through positive impacts on the environment. Until that day comes (and because of the intense publicity surrounding these issues), plant breeders and breeding will be inextricably linked with the public’s wary view of biotechnology. The effect will not just be directed to the “image” of the profession, but through funding mechanisms and regulatory measures, it will also have an impact on the scientist’s “freedom to operate,” including the freedom to choose which technology is most appropriate for solving a particular problem. Increased support to public sector research programs would be one step toward generating real and visible benefits, according to Taylor. At the same time, safety issues must be addressed. Assurances from some scientists that there is “no problem” are not sufficient in a world in which sophisticated technologies have frequently been discovered by the public to come with unintended and unforeseen consequences.

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Strengthening the capacity of scientific institutions to assess products properly—in particular in developing countries—is a prerequisite for uncoerced support in some countries. USAID has provided a $15 million grant for supporting biosafety work in developing countries through a program led by the International Service for National Agricultural Research, ISNAR (ISNAR 2003). In the U.S., Congressman Dennis Kucinich (Ohio) has recently introduced a series of broad-ranging bills that address a potential framework for genetically modified plants, animals, and bacteria. They can be found at http//Thomas.loc.gov.

IV. PLANT BREEDERS’ CHOICES A. Public vs. Private Perhaps one of the first choices a plant breeder makes is to choose the type of research and employment that he or she is interested in, public or private. Frey (1996) surveyed plant breeding interests in the U.S. and found that there were 2241 scientific years devoted to plant breeding in 1994. Of these, 1499 were in private companies and, more specifically, 545 were involved in field corn breeding (with 94% of those involved in field corn breeding being in the private sector). The remainders were in public venues, such as universities and their State Agricultural Experiment Stations (SAESs) and the United States Department of Agriculture–Agriculture Research Service (USDA–ARS). Heisey et al. (2001) in Public Sector Plant Breeding in a Privatizing World flatly state: “Public sector agricultural research in general, and public plant breeding research in particular, is in trouble in both industrialized and developing nations.” They call attention to and emphasize the dramatic growth in the private sector and increase in support of private plant breeding. Further, they discuss the factors that have brought about these changes and how they affect the plant breeding world. They state that public sector plant breeding in industrialized nations is “entering into increasingly turbulent times” and a cloudy future. Declining budgets are a major problem and responsible for the bleak future. Under-investment in agricultural research is ubiquitous in both developed and developing countries and can only be explained as a consequence of short-sightedness and lack of political power of those who support such research. Significant social returns might be possible if appropriate areas of research, such as those that involve components of clear public good, are chosen for future action areas. Pinstrup-Andersen and Cohen (2000) observe that private and social rates of return on investment in agricultural research

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still exceed 20% a year, compared with long-term real interest rates of 3–5% for government borrowing. The demarcation line between public and private plant breeding is probably clearer now than in recent history. The choice is likely to revolve around several options and some of them are discussed in the following synopsis. As the roles of public and private plant breeders have shifted or evolved it is clear that private industry goals are driven and motivated by profit. Private programs have a stronger funding or support base and a more clearly defined research goal than that of public breeding programs. Private interests are directed or formulated to capture market share and make money for the investors. This posture drives companies and their plant breeders to crops that have substantial acreage or seed usages. Frey (1998) estimates that 80% of private plant breeding effort is directed toward cultivar development. The target for the private breeder is usually a cultivar that outperforms the market standard or offers some other advantage such as improved or new pest resistance or nutritional quality. (IPRs also enter into this situation and they will be discussed in another section of this paper.) Support for public breeding, meanwhile, is declining in terms of real money. This trend appears to have begun in the 1990s in the U.S. Heisey et al. (2001) and Frey (1997) reported that funding for public plant breeding research and development had not kept pace with the increasing costs of conducting breeding programs. Because of reduced availability in public support for plant breeding, these scientists have sought out both traditional and non-traditional sources of support. This usually involves grant writing, which takes considerable time and effort away from plant breeding. Public plant breeders have been ascribed numerous roles by various authors (Coffman 1998; Eberhart et al. 1998; Frey 2000; Heisey et al. 2001), but most agree that the prime role for public plant breeders is the education and training of the world’s future plant breeders. All authors suggest that public plant breeders may play important roles in developing and advancing new technologies and in the breeding of minor crops. Further, Frey discussed the need for a formal plan to promote the breeding of minor crops and made suggestions for facilitating or increasing the role for plant breeders, both public and private, in meeting the needs for food, health, fiber and other industrial uses, medicines, and environmental remediation or enhancement. The public sector plays the major role in the collection, conservation, and distribution of genetic resources. Private sector activities in long-term conservation of plant genetic resources for food and agriculture are modest (FAO 1998). Even though there are numerous new technologies available, the science and

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art of plant breeding, whether public or private, will be critical to the development of the world’s crop needs for food, fiber, and medicine. Although most observers expect the public plant breeder to continue to have a major role in the future, we have great concern about how to increase the funding necessary to complete the task of educating and training new plant breeders and thus secure the foundation of both public and private breeding. Tracy (2004) argues that chances for increased public support are strengthened if the public is educated about the positive contributions that plant breeding plays in enhancing food security and sustainability. Further, he reviews the positive aspects of the independence of the public breeder and the roles that the public breeder can play in educating new breeders in the development of cultivars because of the long-term nature/continuity of public programs. B. GMOs or Non-GMOs “Choices” for plant breeders and their clientele are subject to new and changing rules, laws, and even viewpoints. A case in point is the following example of how one industry ventured into the biotechnology arena and then backed away due to consumer response to GMOs. The pickling cucumber processing industry in the U.S. had long sought plant resistance to nematodes and pickle worms and to soil-borne pathogens that caused considerable damage to fruit. The industry was also hampered by weed populations as it changed from hand to machine harvesting. Teams of plant breeders and other scientists screened genetic resources from all over the world, but could not find resistance to either the major nematode species or the pickle worm. After little success in solving these seemingly intractable problems via traditional breeding methods, the industry (in the early 1990s) turned to the possibilities of genetic engineering. They made contacts with the players in the biotechnology businesses to gain access to the Bt gene and gene(s) for herbicide resistance, as well as for genetic constructs that might be of assistance to plant breeders in resolving some of the problems noted above. Concomitantly, they helped support public research to develop a successful transformation system for cucumber. For a number of reasons, the biotech companies demurred and showed little interest in this processing industry’s problems. Undaunted, the processing industry continued to support biotech research, mostly on the identification and use of molecular markers for marker-aided selection to enhance the success of plant breeding, in addition to the more routine conventional plant breeding. In the late 1990s, as consumer backlash to genetically modified organisms became more pronounced and more frequent, the cucumber processing industry withdrew its financial support of research dealing

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with GMOs. Although the industry continues to be plagued by these same pest problems, it continues to seek solutions to them through pre-GMO forms of pest control. Plant breeders that work hand in hand with the processing industry have shown great flexibility and durability in their efforts to find pest resistance even as their choices have become more limited. The interactions of “conventional” plant breeders and biotech’s genetic engineers will continue to be critical and important to the success of both public and private breeding efforts to develop improved cultivars. The positive synergistic potential of conventional and biotechnological approaches is frequently overlooked as the focus has shifted to the prophesied “miracles” of biotech and the currently more tangible transfer of single genes to make a new cultivar immediately better than the donor recipient. Without the interaction of the plant breeder (as well as researchers from many other disciplines), the possibilities of failure of such a new biotech innovation are heightened, as highlighted in the review of the Flavr Savr tomato (Charles 2001). The roles of biotechnology in agriculture are an enticing and interesting subject. Biotech interacts with plant breeding and nearly all other aspects of growing, handling, and marketing a crop. The biology and breeding of a genetically modified crop is sometimes quite simple when compared to the actions necessary to comply with the safety regulations and legal implications of a genetically modified crop. Fortunately, the literature covering these subjects is common and too voluminous to begin listing citations. Thus, our treatment of the subject is at best cursory. C. Intellectual Property Protection and Rights Fretz and MacKenzie (2002) discussed the roles of both the public scientist and the administrator in regard to intellectual property in agricultural research and development. Under the U.S. system, it is the responsibility of a public institution (and its plant breeders) to look for ways and methods to deploy new discoveries for economic, social, health, and environmental benefits. Traditionally, public institutions have openly shared research findings (public goods). Indeed, this behavior was expected by government and by the tax-payer. However, over the past two or three decades the funding at public institutions for research programs has changed dramatically. Research budgets have faced new and numerous fiscal constraints and, in addition, the cost of doing research has increased. Thus, a public institution is now forced to look into ways of protecting intellectual property and to have as one of its goals the generation of returns/revenues to support its research agenda. The public institution has the moral responsibility to transfer its research products to the public. This can be accomplished in different

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ways. The product may be dealt with as a public good and made available to all or it may be protected as intellectual property. If the latter is chosen, the form of protection may be by claiming the intellectual property only to assure that the general public has access to it (so-called “defensive” patenting), or, by protecting the invention with the intent of commercialization. The plant breeder is faced with the dilemma of determining how best to market new developments whether they are in the form of germplasm or technological innovations. Now, at nearly every institution of higher learning in the U.S., there is a technology transfer office that assists with the movement of new products into the market chain. Financial returns resulting from plant breeding inventions are usually distributed according to institutional policy and the formulas for distribution vary greatly across institutions. However, it is a concern to administrative management that the plant breeders are rewarded for their innovations and inventions, but there is also a concern that the reward does not become the principal driving force behind the research program. Knudsen et al. (2000) discuss the intellectual property protection options for plant breeding achievements in the U.S. Similar options (breeders’ rights) are available in many other industrialized nations as well. Details of the various options are also discussed. Most plant breeders, but especially those in the public sector and those that are members of small businesses or are self-employed, will have the opportunity to exercise the protection option that best fits their needs. Jondle (2003) recently addressed members of an American Society for Horticultural Science (ASHS) workshop on the patentability of naturally occurring genes. He listed protection options as follows: • • • •

Plant patents Utility patents Plant Variety Protection Certificates (“Breeders Rights”) UPOV Contract law (varies from state to state) Written contracts Material Transfer Agreements (MTAs) Restrictive use wording on bags, tags, and signed invoices Trade secrets (covered by state laws) • Trade names and trademarks Jondle emphasized the differences between utility patents (UPs) and the Plant Variety Protection Certificates (PVPCs) and explained that UPs have a broader scope than PVPCs and a very narrow research exemption that does not allow for commercialization or farmers exemption. The UP is the strongest form of intellectual property protection for cultivars,

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inbreds, and hybrids. He argues that patenting cultivars is likely to stimulate plant breeding and research and that patents provide a legal form for germplasm exchange via licensing. The use of restricted wording on bags, tags, and invoices, along with similar wording in written contracts and MTAs, allows for strong protection of genetic material. Enforcement, however, is another subject. We believe that it is fair (accurate) to say that these restrictions are frequently overlooked by both growers and breeders and suspect that violations of the wording may also occur rather frequently. The field of intellectual property rights is controversial, however, and divergent viewpoints are easy to find. Jaffé (2000) notes the uncertainty that exists concerning the level of the economic impact of utility patenting. And, the U.K.’s Council of the Royal Society, for example, has recently issued a report (2003) that called attention to the “tension” that IPRs can create between the private profit and public good, and noted their potential to “hinder the free exchange of ideas and information on which science thrives.” The debate, which began in earnest with the adoption of the Paris Convention for the Protection of Industrial Property in 1883, continues! Despite on-going disputes over the efficacy and effects of IPRs, it is obvious that plant breeders have a wide-ranging set of choices for protecting their inventions and developments. Each choice has advantages and disadvantages and it behooves plant breeders, and in some cases, their technology transfer offices, to determine which choice best fits each particular invention. There is not a “one size fits all” philosophy for inventions. Lesser and Mutschler (2002) reviewed the types of IPRs available to plant breeders in the U.S. They analyzed the entire PVP data set from 1970 to 2001 and the use of utility patents on some species from 1985 to 2001. Soybeans (19%), maize (12%), bread wheat (7.7%), cotton (6.1%), and lettuce (5.4%) account for half of the applications for PVPs. They also note that patents on maize and soybeans increased rapidly in the late 1980s (maize) and the early 1990s (maize and soybeans) and discuss the reasons for these increases (changes in types of protection available, results of legal cases, and transgenic crops). Types of protection are also influenced by reproductive biology, mechanisms of seed production, and economics of the crop. Prior to the use of PVP and UPs, plant breeders, at times both public and private, shared information and germplasm freely or with as little as a verbal agreement and a handshake. This level of cooperation was also prevalent in regional and national trials at which breeders agreed to the use of their material (trial entries) in other breeding programs. Once entered into some trials, the germplasm was freely available to other contributors. Although courtesy announcements of the use of

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breeding material from another program were respected, they were not required. We do not know of any similar arrangements in the 21st century. The level of protection(s) now afforded to plant breeding products and the requirement at some levels of both public and private programs to use PVPCs or UPs or some type of contract minimizes the opportunity for the type of germplasm and information exchange that was available 20–30 years ago. No doubt this lost opportunity is not only one of reduced germplasm sharing and fewer improved products, but also the loss of the synergism that results from bringing more brain power to the development of a common goal—the superior genetic performer. Factor into this type of behavior the need for programs to generate their own support, and the transfer of genetic material between competing programs has slowed to at best a trickle of what it was 30 years ago. D. Looking Ahead In reviewing the literature for this effort, we were pleased at the agreement found in previous publications about the role(s) and goals of the public plant breeder and how he or she should garner support to accomplish these targets (Coffman 1998; Eberhart et al. 1998; Fretz and MacKenzie 2002; Frey 1997, 1998, 2000; Heisey et al. 2001). There is no quarrel with the view that one of the primary goals and roles of the public plant breeder is the training and education of future plant breeders. But there are fewer and fewer opportunities to acquire basic training and as Heisey et al. point out, “to the extent that plant breeding skills are not firm-specific, firms will not invest optimally in training . . .” One of the difficulties here is the downsizing of many public breeding programs because of the escalating costs associated with those programs and the lack of political commitment toward maintaining them amidst competing demands on limited public resources going to agriculture. Field and laboratory programs are both time-consuming and costly. The hands-on training that is critical to the student is lacking in many programs now, and the new plant breeder, although perhaps better educated in the sciences and with a better tool box than that of his or her predecessor, may lack the training necessary to improve and grow the crop of interest through breeding. Duvick (1998) stated that . . . a well-rounded plant breeder today must know many more kinds of science and technology than were required as recently as 10 years ago, not to mention 20 or 30 years earlier. In actuality, no one person can master all of the science and technology that is needed for efficient plant breeding for the late 1990s. At best, a well-rounded plant breeder will know

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some of the specialties, and know how to converse and collaborate with specialists in other parts of plant breeding.

The 21st century public plant breeder will be trained in how to develop and utilize new technologies that will enhance the progress of the breeding program and increase the chances of success. Likewise, he or she will likely be involved in germplasm collection, development, preservation and enhancement of the gene pool, breeding methodologies, and variety development. Although the majority of new cultivars for most major and some minor crops will result from efforts in the private sector, the public breeder will have opportunities to develop some of the major and many of the minor crop cultivars if so inclined. Referring to crops as “major” and “minor” is confusing, as the reference could refer to size of production area, nutrition contribution, value of a specific crop, or other descriptors. The private sector is interested in the economics of the crop and its future. On this basis, some so-called major crops today may be called minor in the future and vice-versa. We suspect that there is a shift in emphasis in the private sector toward the high-value market crops and away from other crops that are not as profitable. This has implications for progress in agriculture with the other crops and for the public sector. We also sense that this reality is not clearly understood or acted upon by policy makers, funding organizations, or indeed even by people in the public sector. If such a shift does occur in the private sector, a number of important crops, both major and minor, will by default fall solely into the hands of the public sector, which, unfortunately, is not adequately staffed or funded to meet the challenge. In addition, the public breeding ground will also give chances for seeking answers to seemingly intractable problems, both short-term and long-term, such as technological glitches in the lab and field. As they have in the past, public breeders can and will be expected to continue to make major contributions to genetic resources, including increased pest resistances, improvements in nutritional content and beneficial health components, greater adaptability of cultivars, and environmental preservation and stability. At a recent plant breeding workshop, Ryder (2003) expressed concerns felt by many plant breeders. He challenged the intellectual property guidelines that have been thrust upon the plant breeding community by the legal profession, the courts, and business interests. This suggests another role for plant breeders that starts with understanding those guidelines and their constraints on both public and private plant breeders. Protection of intellectual property to assure returns on investment, although a goal of private industry, is not always in the best interest of

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the public. The opportunity to enhance and further science, and the knowledge that undergirds it, is a continual goal for all plant breeders, as well as a justifiable professional and personal reward and a tangible public good that supports future plant breeding. Frey (1997, 1998, 2000) offers several initiatives to keep private and public sectors interactive and cooperative as they solidify and make positive the future for plant breeding. For example, the need for cooperative ventures between sectors to insure that the world does not forget about the roles of minor crops in the supply of food for healthy diets, nutrition, and antioxidant and health-promoting foods. Many of the International Agricultural Research Centers (IARCs) have long advocated for the need to insert minor crops into the monoculture cropping systems that exist in many developing countries of the world. The minor crops offer much in the way of food and diet diversity, crop rotation, soil, and environmental quality and maintenance. (See: http://www.avrdc.org for information about the importance of vegetable crops, including for instance the role of leafy vegetables in the diet.) Yet, relatively little investment is made in these crops. The potential of many minor crops has not been tapped at all, as quite a few have probably never been the subject of a formal, scientific plant breeding program. Even crops of major economic and nutritional importance (e.g., yams, bananas/plantains, minor millets) receive surprisingly little investment and thus may be worked with by no more than a half-dozen plant breeders around the world, if that. Public-private sector interactions ebb and flow with opportunities. However, some universities and their SAESs, i.e., Cornell and Texas A&M University, have fostered solid relationships between their public plant breeders and the private sector and these plant breeders will continue to enhance contributions to the health of the populace and the health and quality of the environment. Perhaps these models deserve consideration by other institutions as they promote increased cooperative efforts between sectors and generate support for public efforts. The trend line for the number of practicing public plant breeders is going down, while the numbers of private breeders are increasing. Since the late 1970s these trends have been accompanied by an increase in mergers between seed companies and buyouts of one by another. In this same timeframe the vertical integration of biotech companies with seed companies that utilize and market biotech intellectual property has been common. We believe that this decrease in total number of seed companies has led to decreased coverage of many crop species by plant breeders, most of which are among the so-called minor crops. Numerous vegetable crops were bred by no fewer than 20 seed companies in the

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1970s and today the number for many crops is no more than three. Contributions of these crops to health and nutrition, to increased sustainability, to local niche markets, and to special economic considerations may result in a re-focusing of breeding efforts and lead to increased improvement efforts by plant breeders in the future. The public breeder will be called upon to respond to the needs of growers of peripheral or “orphan” crops, some of which are actually very important when judged by virtually any set of criteria. New genetic technologies might create opportunities for substantial advancement in the improvement of such crops currently experiencing “under-investment” in research and development (Naylor et al. 2004). In short, new opportunities for the public sector will surely emerge, sparked by the presence of new tools and new demands created by changes in production and environmental systems. But, will the public sector be in a position to respond? We are deeply troubled by several related phenomena and their effects on both public and private plant breeding. First, the lack of support for public sector plant breeding and the need for researchers to generate funding via IPRs. Second, the belief held by many that the public sector should not compete with the private sector. Finally, the growing necessity that the public sector must behave in a quasi-commercial manner in order to secure funding for its own survival. Increasingly, the public sector is expected to generate support, but usually only on crops on which there is little or no money to be made. These situations threaten the existence of public breeders and their historic mission. The decline in the number of plant breeders in the public sector is accompanied by a changing education and training pattern at the university level. As noted previously, the plant breeding graduates of this millennium will likely not have the same experiences of growing and knowing their crops of interest that were standard for their major professors trained only a few decades earlier. New high-profile courses at the molecular level have replaced specialized courses in plant growth, structure, and supporting courses in associated areas such as soils, pathology, entomology, and food science. Tracy (2004) points out that today’s new plant breeders are “weaker, in agricultural sciences, quantitative thinking, whole plant biology, and selection theory.” This situation reinforces the critical role—and the challenge—of the public institution in the education and training of new plant breeders. Long-term breeding programs, both field and laboratory, are critical to the training of students. However, the costs of these programs continue to grow and many of them are downsized annually to meet budget restraints. University administrations have not been convinced of the deleterious effects of changes to and constraints on plant breeding programs. Funding allocations remain marginal

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when compared to the size of the job. Despite current trends in funding, plant breeders will continue to be the link between the new technologies and the acceptance of a final product for agricultural growers, processors, and consumers. Plant breeders need not fear about their future livelihood as a profession, but they do need to tell their story to their administrators and other supporters in a more convincing manner. We are hopeful that the pendulum swing will reverse its direction and the number of public plant breeders will rebound. We trust that the public demand for agricultural sustainability, food security, and safety will eventually create political pressures that will support public sector efforts and lead to a larger and vibrant cadre of public plant breeders to serve the needs of the greater system. The plant breeder, involved as he or she is in one of the world’s most important endeavors, is never far removed from the political context of the work. At no point since the rediscovery of Mendel’s laws has plant breeding had so many tools and opportunities at its disposal. Yet, never have so many constraints been placed on the art, science, and practice of this ancient and noble profession. Whether, how, and to what end plant breeders will be able to continue to serve society productively will be answered in large part by the accommodation that is being and will be made between plant breeding and politics.

LITERATURE CITED Ag Biotechnology Implementation Task Force. 2003. Survey http://www.cals.ncsu.edu/ agcomm/biotech/ Agres, J. 2003. Biodiversity treaty called disastrous. The Scientist. Sept. 10. www.biomedcentral.com/news/20030910/03/ Anderson, E., and W. Brown. 1952a. The history of common maize varieties of the United States corn belt. Agr. Hist. 26:2–8. Anderson, E., and W. Brown. 1952b. Origin of corn belt maize and its genetic significance. In: J. Gowan (ed.), Heterosis. Iowa State College, Ames. p. 124–148. Brockway, L. 1979. Science and colonial expansion: The role of the British Royal Botanic Gardens. Academic Press, New York. Burns, T., and R. Ueberhorst. 1988. Creative democracy: Systematic conflict resolution and policymaking in a world of high science and technology. Praeger, New York. Castagnera, J., C. Fine, and A. Belfiore. 2002. Protecting intellectual capital in the new century: Are universities prepared? Duke Law Technol. Rev. (June). Charles, D. 2001. Lords of the Harvest. Pereus Publishing, Cambridge, MA 02142. p. 126–148. Cleaver, H. 1972. Contradiction of the green revolution. American Economic Review, Vol. 62, Number 2. p. 177–186. Coffman, W. R. 1998. Future of plant breeding in public institutions. Corn and Sorghum Res. Conf., Am. Seed Trade Assoc., Chicago.

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Committee on Registration of the Peninsula Horticultural Society. 1891. Registration of new fruits. The American Garden, Vol. 12. No 6. p. 338–339. Conko, G., and C. Prakash. 2002. The attack on plant biotechnology. p. 179–218. In: R. Bailey (ed.), Global warming and other eco-myths. Prima Publishing/Random House, New York. Correa, C. 2003. The access regime and the implementation of the FAO treaty on plant genetic resources for food and agriculture in the Andean group countries. Unpubl. manuscript for a study sponsored by SAREC (Sweden) and executed by the International Potato Center. Council of the Royal Society. 2003. Keeping science open: The effects of intellectual property policy on the conduct of science. The Royal Society, London. Crosby, A. 1986. Ecological imperialism: The biological expansion of Europe, 900–1900. Cambridge Univ. Press, Cambridge, UK. Duvick, D. N. 1998. Future sources of plant breeders for industry. Production and Res. Conf., Texas Seed Trade Assoc., College Station. Eberhart, S. A., H. L. Shands, W. Collins, and R. L. Lower. 1998. Intellectual property rights III. Global genetic resources: Access and property rights. CSSA Misc. Pub., Madison, WI. Elder, G. 1921. Seed marketing hints for the farmer. Farmers’ Bul. 1232. USDA. Government Printing Office, Washington, DC. Falcon, W., and C. Fowler. 2002. Carving up the commons: Emergence of a new international regime for germplasm development and transfer. Food Policy 27:197–222. Fitzgerald, D. 1990. The business of breeding: Hybrid corn in Illinois, 1890–1940. Cornell Univ. Press, Ithaca, NY. Food and Agriculture Organization of the United Nations. 1998. The state of the world’s plant genetic resources for food and agriculture. FAO, Rome. Food and Agriculture Organization of the United Nations. 2001. International treaty on plant genetic resources for food and agriculture. FAO, Rome. Foster, M., P. Berry, and J. Hogan. 2003. Market access issues for GM products: Implications for Australia. Australian Bureau of Agricultural and Resource Economics Report for the Dept. Agr. Fisheries and Forestry, Canberra, Australia. Fowler, C. 1994. Unnatural selection: Technology, politics, and plant evolution. Gordon and Breach Science Publ., Yverdon, Switzerland. Fowler, C. 1998. Rights and responsibilities: Linking conservation, utilization, and sharing of benefits of plant genetic resources. Intellectual property rights. III. Global genetic resources: Access and property rights. Crop Sci. Soc. Am., Am. Soc. Agron., Madison. Fowler, C. 2000. The Plant Patent Act of 1930: A sociological history of its creation. J. U.S. Patent and Trademark Office Soc. 82:621–644. Fretz, T. A., and D. R. MacKenzie. 2002. The public university, intellectual property and agricultural R&D. In: Max Rothschild and Scott Newman (eds.), Intellectual property rights in animal breeding and genetics. CAB Int., Oxon, UK. Frey, K. J. 1996. National plant breeding study. I. Human and financial resources devoted to plant breeding research and development in the United States in 1994. Iowa Agr. Home Econ. Special Rep. 98, Iowa State Univ., Ames. Frey, K. J. 1997. National plant breeding study. II. National plan for promoting breeding programs for minor crops in the U.S. Iowa Agr. Home Econ. Special Rep. 100, Iowa State Univ., Ames. Frey, K. J. 1998. National plant breeding study. III. National plan for genepool enrichment of U.S. crops. Iowa Agr. Home Econ. Special Rep. 101, Iowa State Univ., Ames. Frey, K. J. 2000. National plant breeding study. IV. Future priorities for plant breeding. Iowa Agr. Home Econ. Special Rep. 102, Iowa State Univ., Ames.

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Fuglie, K. O., and D. E. Schimmelpfennig. 2000. Public–private collaboration in agricultural research. Iowa State Univ. Press, Ames. George, S. 1977. How the other half dies: The real reasons for world hunger. Allanheld Osmun, Montclair, NJ. Goodman, M. 2002. New sources of germplasm: Lines, transgenes, and breeders. In: J. Martinez (ed.), Simposio El Fitoramiento ante los Avances Científicos y Tecnológicos (2 Sept.), Saltillo, Mexico. GRAIN. 1995. Towards a biodiversity community rights regime. Genetic Resources Action Int., Barcelona. GRAIN. 2002. Biopiracy by another name? A Critique of the FAO-CGIAR Trusteeship System. Seedling (Oct.) www.grain.org/seedling/seed-02-10-2-en.cfm. Griffin, K. 1972. The green revolution: An economic analysis. United Nations Research Institute for Social Development, Geneva. Haughton, C. 1979. Green immigrants. Harcourt Brace Jovanovich, New York. Heisey, P., Srinivasan, C., and C. Thirtle. 2001. Public sector plant breeding in a privatizing world. Agr. Inform. Bul. 772. Resource Economics Division, Economic Research Service, U.S. Department of Agriculture, Washington, DC. Heller, M., and R. Eisenberg. 1998. Can patents deter innovation? The anticommons in biomedical research. Science 280:698–701. Hightower, J. 1973. Hard tomatoes, hard times. Schenkman Publ., Cambridge. ISNAR (International Service for National Agricultural Research). 2003. Group Awarded $15 Million for Work on Strategies and Policies. Press Release. (June 9) Jaffé, A. 2000. The U.S. Patent System in transition: Policy innovation and the innovation process. Res. Policy 29. Jondle, R. 2003. How to apply for a patent related to a naturally occurring gene. HortScience 38:142 (Abstr.). Kloppenburg, J. 1988. First the seed: The political economy of plant biotechnology, 1492–2000. Cambridge Univ. Press, Cambridge. Knudsen, M. K., R. L. Lower, and R. Jones. 2000. State agricultural stations and intellectual property rights. p. 199–215. In: K. O. Fuglie and D. E. Schimmelpfennig (eds.), Public-private collaboration in agricultural research. Iowa State Univ. Press, Ames. Lappé, F., J. Collins, and C. Fowler. 1977. Food first: Beyond the myth of scarcity. Houghton Mifflin, New York. Lesser, W., and M. Mutschler. 2002. Lessons from the patenting of plants. In: M. Rothschild and S. Newman (eds.), Intellectual property rights in animal breeding and genetics. CAB Int., Oxon, UK. Michaels, P. 2003. Is Europe returning to the dark ages? Cato Institute. (15 February). http://www.cato.org/dailys/02-15-03.html Mooney, P. 1979. Seeds of the earth: Private or public resource? Canadian Council for Int. Co-operation and the Int. Coalition for Development Action, Ottawa. National Academy of Sciences (NAS). 1987. Introduction of recombinant DNA-engineered organisms into the environment: Key issues. National Academy Press, Washington, DC. Naylor, R., W. Falcon, R. Goodman, M. Jahn, T. Sengooba, H. Tefera, and R. Nelson. 2004. Biotechnology in the developing world: A case for increased investments in orphan crops. Food Policy (forthcoming). Palacios, X. F. 1998. Contribution to the estimation of countries’ interdependence in the area of plant genetic resources. Background Study Paper 7, Rev. 1. FAO Commission on Genetic Resources for Food and Agriculture, Rome. Palmer, I. 1972a. Food and the new agricultural technology. United Nations Research Institute for Social Development, Geneva.

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Palmer, I. 1972b. Science and agricultural production. United Nations Research Institute for Social Development, Geneva. Pardey, P., and N. Beintema. 2001. Slow magic: Agricultural R & D a century after Mendel. IFPRI Food Policy Statement 36. International Food Policy Research Institute, Washington, DC. Pinstrup-Andersen, P., and M. Cohen. 2000. Biotechnology and the CGIAR. Conf. sustainable agriculture in the next millennium: The impact of modern biotechnology on developing countries. Brussels, 28–31 May. Plucknett, D., N. Smith, J. T. Williams, and M. Anishetty. 1987. Gene banks and the world’s food. Princeton Univ. Press, Princeton, NJ. Price, S. C. 1999. Public and private plant breeding. Nature Biotechnol. 17(10):938. Rader, R. 2000. Trends in biotechnology patenting. Am. Chem. Soc. Natl. Meeting. Washington, DC. Ragan, W. 1926. Nomenclature of the apple: A catalogue of the known varieties referred to in American publications from 1804 to 1904. Bureau of Plant Industry, Bul. 56. Government Printing Office, Washington, DC. Resnik, D. 2003. A biotechnology patent pool: An idea whose time has come? J. Philo. Sci. Law 3(Jan.). http://www.psljournal.com/archives/papers/biotechPatent.cfm Rothschild, M., and S. Newman. 2002. Intellectual property rights in animal breeding and genetics. CAB Int., Oxon, UK. Ryder, E. J. 2003. Nature’s gifts: Are they intellectual property? HortScience 38:742 (Abstr.). Smith, S. 1997. Farmers’ rights and benefit sharing. Background Paper for the Intellectual Property Rights III Symp. Washington (June 4–6). Stover, J. 1975. History of the Illinois Central Railroad. Macmillan Publ., New York. Taylor, M. 2003. Rethinking US leadership in food biotechnology. Nat. Biotechnol. 21(8). Taylor, W. 1901. The influence of refrigeration on the fruit industry. Yearbook of Agriculture, 1900. U.S. Government Printing Office/USDA, Washington, DC. Tracy, W. F. 2004. Proceedings of the Seeds and Breeds Summit, Washington, DC. September 8–9, 2003. Published by the Rural Advancement–USA, Pittsboro, NC. In press. Tripp, R. (ed.). 1997. New seed and old laws: Regulatory reform and the diversification of national seed systems. Intermediate Technology Publications, London. USDA. 1918. Seed reporter. 2(2) (August 10). Bureau of Markets. Government Printing Office, Washington, DC. USDA/USTR (United States Department of Agriculture, United States Trade Representative). 2003. U.S. and cooperating countries file WTO case against EU moratorium on biotech foods and crops. Press Release. Washington, DC (May 13). Winner, L. 1987. Autonomous technology: Technics-out-of-control as a theme in political thought. MIT Press, Cambridge. Wolf, H., and R. Wolf. 1936. Rubber: A story of glory and greed. Covici Friede, New York.

3 Doubled Haploids in Genetics and Plant Breeding* Brian P. Forster and William T.B. Thomas Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK

I. INTRODUCTION II. DOUBLED HAPLOID TECHNOLOGY III. DOUBLED HAPLOID POPULATIONS IN GENETICS A. Classic Studies B. Early Studies on Doubled Haploid Populations C. Bulked Segregant Analysis D. Genetic Maps E. Mapping of Quantitative Traits F. Genomics IV. DOUBLED HAPLOIDS IN BREEDING A. Early Breeding Attempts B. Elite Crossing C. Comparison of Breeding Methods D. Cultivar Releases E. Backcross Conversion V. PROSPECTS LITERATURE CITED

I. INTRODUCTION Doubled haploids (DHs) are produced from haploids. Haploid cells occur naturally in the gametophytic phases of higher plants, in their ovules and pollen. It is the cells of these tissues that are the main targets for doubled haploidy. By manipulating the environment of the gametic cells it is possible to divert development to produce embryos rather than mature pollen grains or ovules. The microspores of developing pollen grains are *Acknowledgments: The Scottish Crop Research Institute receives grant-in-aid from the Scottish Executive Environment and Rural Affairs Department. The authors are also grateful to members of the EU COST Action 851 “Gametic cells and molecular breeding for crop improvement” for valuable comment. Plant Breeding Reviews, Volume 25. Edited by Jules Janick. ISBN 0471666939 © 2005 John Wiley & Sons, Inc. 57

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of particular interest since plants generally produce pollen to excess. Microspore embryogenesis therefore has the potential to generate several hundred DH plants from a single anther. Induced or spontaneous chromosome doubling of haploid embryogenic cells produces doubled haploid plants. These are completely homozygous, true breeding, and consequently of great importance in genetics and plant breeding. The relevance of doubled haploids (DHs) to plant breeding has increased markedly in recent years owing to the expansion of application to over 200 species and the development of more reliable protocols. Doubled haploids can be exploited not only to produce new cultivars, but also to construct genetic maps, locate genes of agronomic and economic importance, identify markers for trait selection, and to increase plant breeding efficiency. The benefits accrue from the efficiency of DH technology in producing completely homozygous lines, vital resources in plant breeding and genetics. The potential of doubled haploidy has been the subject of various reviews (Choo et al. 1985; Dunwell 1985; Kasha et al. 1989; Pickering and Devaux 1992; Touraev et al. 2001; Maluszynski et al. 2003a). The recent rapid expansion of doubled haploid technology, which now includes over 200 plant species (Maluszynski et al. 2003b) sets the stage for greater DH deployment in a range of species and disciplines. We describe the exploitation of DHs from classic genetic studies to contemporary plant breeding and attempt to gauge current and future impact.

II. DOUBLED HAPLOID TECHNOLOGY Doubled haploids can be produced via in vivo and in vitro systems. Haploid embryos are produced in vivo by parthenogenesis, pseudogamy, or chromosome elimination after wide crossing. The haploid embryo is rescued, cultured, and chromosome-doubling produces doubled haploids. The in vitro methods include gynogenesis (ovary and flower culture) and androgenesis (anther and microspore culture). Methods can be complex and exacting. Critical stages include: growing conditions, pre-treatment of floral parts (before and after collection of material for culture), physiology of donor plants, genotype, media composition, incubation, regeneration, and chromosome doubling (Kasha and Reinbergs 1981; Pickering and Devaux 1992; Jain et al. 1996). Androgenesis, where available, is the preferred method (Sopory and Munshi 1996). In 1922, Blakeslee et al. reported the finding of a haploid plant in Jimson weed, Datura stramonium. It took another 40 years to develop laboratory methods for the routine production of Datura haploids via embryogenesis in cultured anthers (Guha and Maheswari 1964, 1966). This pioneering work led to the testing of many species, but responses to the protocols used have been extremely varied across the range of

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species. Tobacco, rapeseed, and barley are currently among the most responsive species, and although these are considered model species for DH production, response is further dependent on genotype. Legumes remain a particularly difficult group. There has been a progressive trend in reclassification from recalcitrant to responsive to highly responsive. Barley, for example, was considered recalcitrant to androgenetic protocols before the patented incorporation of maltose in anther/microspore medium (Hunter 1987), which paved the way to large-scale commercial application. A typical scheme for barley anther culture is given in Fig. 3.1. Legumes have been a notoriously difficult group, but in 1994 Ormerod and Caligari reported successful anther and microspore culture of lupin. Potato moved from recalcitrant to responsive in anther culture (Rokka et al. 1996) and, more recently, improved microspore culture methods have been described for wheat (Liu et al. 2002). Problems in doubled haploid production vary among species. For most species androgenesis is the preferred route, but for some (e.g., onion), the only practical method is gynogenesis. For large trees there is the practical problem of controlling the donor plant environment and materials for culture (flower buds) are restricted to outdoor collections during spring months. A frequent problem in cereals and other grasses is genotype dependency; many genotypes regenerate high frequencies of albino plants from cultured tissues (e.g., Holme et al. 1999). The production of doubled haploids using wide crossing can circumvent many of these problems. In barley (Hordeum vulgare), haploids can be produced by wide crossing with the related species H. bulbosum, fertilization is effected, but during the early stages of seed development the H. bulbosum chromosomes are eliminated leaving a haploid embryo. The embryos can be rescued and cultured, and plants derived from them artificially doubled (Devaux 2003; Hayes et al. 2003). The bulbosum technique has been very successful in producing DH cultivars (the majority of barley cultivars, 74/115, listed in the web-site http://www.scri.sari.ac.uk/assoc/cost851/Default.htm have been produced by this method). Wide crossing is also common in haploid/doubled haploid production in bread wheat (Inagaki 2003), macaroni wheat (Jauhar 2003), and triticale (We ¸dzony 2003); in these protocols maize is commonly used as a pollinator. Pollination with maize is also successful in oat haploid production (Rines 2003). There are, however, disadvantages in wide crossing: it is inefficient when compared with a responsive anther/microspore method, more crossing is required, specialized growing conditions are needed to grow both the target (female) and inducer (pollinator) species, haploid embryos must be rescued and cultured before they die, and at some stage artificial chromosome doubling (often involving toxic chemicals) is required. Table 3.1 lists species in which doubled haploidy is an active area in European research and breeding. We have tried to order the species

60

B. FORSTER AND W. THOMAS

Doubled haploid production in a barley cross via anther culture

A

2

1 B

3

4

2

1

3

C

4 D 1

1

2 2

3 Fig. 3.1. Doubled haploid production in barley via anther culture. There are four basic components in the production of a barley DH population. A. Crossing: typically the DH population is derived from a cross; this can be done at any generation. Crossing normally consists of emasculation of the female parent (1), selection and preparation of a male parent (dehiscing ear, 2), controlled pollination (3), and protection (4). B. Donor plant production: In order to save time, developing seed from crosses can be extracted from ears (1) and immature embryos excised and cultured (2). The progeny is grown out in controlled environment conditions (3) that maximize the harvest of anthers containing responsive microspores. Spikes are harvested when developing microspores are at the uni-nucleate stage (4). C. Anther culture: anthers (or ears) are pre-treated by either cold or starvation treatments (1) and then transferred to specialized media to induce embryogenesis of the microspores (2). The green shoots that arise are transferred to another medium to promote root growth (3). D. Bulking of DH lines: rooted plantlets are transferred to pots, usually in a glasshouse (1), where they are grown on to maturity. In barley, 60–90% of plants are spontaneously doubled and the production of excess plants precludes the need for chemicalinduced doubling, though this is required in other species. Sufficient seed can be produced in the glasshouse for field trial evaluation in plots in the next generation (2).

Table 3.1.

Current status of doubled haploid production in some target species.

Species Common name (Latin name) Tobacco (Nicotiana tabacum) Rapeseed (Brassica napus) Cauliflower, broccoli, Brussels sprouts, cabbage, etc. (Brassica oleracea) Barley (Hordeum vulgare) Wheat (Triticum aestivum) Maize (Zea mays) Triticale (×Triticosecale) Eggplant (Solanum melongena) Rice (Oryza sativa) Pepper (Capsicum annuum) Cucumber (Cucumis sativus) Onion (Allium cepa) 61

Production capacity (no. plants produced)

Cultivars developed via doubled haploidy

>10,000

Yes

Genotype

>10,000

Yes

>10,000

Yes

Genotype, germination of DH embryos, polyploids produced Genotype, germination of DH embryos, polyploids produced

>10,000

Yes

>10,000

Yes

>10,000

?

Anther culture, microspore culture, wide crossing Anther culture

>10,000

?

>5,000

Yes, F1 hybrids

Anther culture, haploid somaclone Anther culture

>1,000

Yes

>1,000

Yes, F1 hybrids

Genotype, embryo germination

Gynogenesis

>1,000

Yes, F1 hybrids

Genotype, inbreeding depression

Flower culture, gynogenesis

>1,000

No

Genotype, inbreeding depression, low fertility, chromosome doubling

Common methods Microspore culture, anther culture Microspore culture, anther culture Microspore culture, anther culture

Anther culture, microspore culture, wide crossing Anther culture, microspore culture, wide crossing Inducer lines

Major problems

Genotype, albinism, laboratory specificity Genotype, chromosome doubling, albinism Chromosome doubling Genotype, albinism, chromosome doubling Chromosome doubling, genotype Genotype

(continued )

62

Table 3.1.

Continued

Species Common name (Latin name) Turnip rape (Brassica rapa) Mustard (Brassica juncea) Ethiopian mustard (Brassica. carinata) Rye (Secale cereale) Oat (Avena sativa) Ryegrass (Lolium perenne) Potato (Solanum tuberosum) Sugar beet (Beta vulgaris) Asparagus (Asparagus officinalis) Citrus/clementine (Citrus spp.) Flax (Linum usitatissimum) Apple (Malus spp.) Cork Oak (Quercus suber) Aspen (Populus spp.)

Production capacity (no. plants produced)

Cultivars developed via doubled haploidy

Microspore culture, anther culture Microspore culture, anther culture Microspore culture, anther culture Anther culture

>1,000

Yes

Genotype

>1,000

Yes

Genotype

?

No

Genotype, albinism

<1,000

No

Anther culture

<1,000

No

Genotype, embryo production, low regeneration Genotype, low regeneration

Anther culture

<1,000

No

<1,000 dihaploids

No

Gynogenesis

?

Yes, F1 hybrids

Anther culture, crossed with supermale Anther culture, gynogenesis

?

Yes

>200

No

?

No

Genotype, seasonal, physiology of donor Genotype

10–100

No

Many

?

No

Seasonal

?

No

Seasonal

Common methods

Anther culture, wide crossing

Anther culture, microspore culture Anther culture, microspore culture, in situ Anther culture Anther culture

Major problems

Genotype, albinism, donor plant quality Genetics, donor plant quality Genotype, chromosome doubling, low fertility, mixaploidy Low fertility

3. DOUBLED HAPLOIDS IN GENETICS AND PLANT BREEDING

63

from the most responsive (top) to most difficult (bottom), though this is somewhat subjective and changing. Doubled haploid techniques have now been applied to over 250 species, from Acontium carmichaeli to Zingiber officinale (Maluszynski et al. 2003b). Prior to the 1980s only a handful of species were reported, but over 100 were added during the 1980s, and the rate of new species additions has been sustained in the last decade (Maluszynski et al. 2003b); one of the most recent additions is pear (Bouvier et al. 2003). Much of the expansion in DH technology has been due to empirical improvements in techniques and culture media, but the application of model protocols (particularly of tobacco, rapeseed, and barley) to other species has been a significant factor. III. DOUBLED HAPLOID POPULATIONS IN GENETICS A. Classical Studies Although contemporary genetics of DHs concentrates on the analysis of DH populations, there are classic examples in which haploidy and doubled haploidy have been exploited in opening up new areas of genetic research. Two notable examples, in contrasting crops, are wheat (a seed propagated inbreeder) and potato (a vegetatively propagated outbreeder). In the 1930s Earnest R. Sears began studying chromosomal aberrations in progeny of a haploid of bread wheat, Triticum aestivum. This pioneering work generated the first wheat aneuploids and culminated in the production of a range of genetic stocks for each of wheat’s 21 chromosomes: nullisomics, monosomics, telosomics, trisomics, tetrasomics, substitution, addition lines, and so on (Sears 1944; 1953; 1954). The development of wheat aneuploid stocks paved the way for the genetic dissection of the genomes and chromosomes of wheat and its related species (reviewed by Law et al. 1987). Furthermore, cytogenetic manipulations of the aneuploids provided a means of introgressing genes from related species, including wild relatives, for important traits (Gale and Miller 1987). Breeders have taken up these lines and have incorporated alien disease and pest resistance into new wheat cultivars. Potato has been a model species for ploidy manipulations in polyploid, vegetatively propagated crops for both genetics and breeding. Potato genetics is complicated by tetraploid inheritance and heterozygosity. In the early 1960s Chase introduced the concept of “analytical breeding” in which dihaploids are produced from tetraploid potato lines (Chase 1963). Recombination in the dihaploids allows genetic studies to be carried out at a simplified, diploid, level. Additionally, the dihaploids can be inter-crossed, or

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crossed with related diploid species, the latter case providing a route for alien introgression (Peloquin and Ortiz 1992). Progeny testing is used to select lines having the captured and desired genetic diversity and the tetraploid nature of the crop can be restored through chromosome doubling. This approach has been tested in a range of vegetatively propagated crops, e.g., potato, sweet potato, cassava, plantain/banana, blackberry, blueberry, and strawberry (reviewed by Ortiz 2003). B. Early Studies on Doubled Haploid Populations Today DH populations are used routinely in mapping genes, but there is little evidence of such studies prior to 1990. As DH populations became easier to produce they became more amenable to genetic studies (Snape and Simpson 1981). One of the earliest reports was the use of a small DH population to map the dwarfing gene (ari-e.GP) to chromosome 5H of barley (Thomas et al. 1984). In another study a small (44 DH) population was used to study the segregation of a range of markers in barley (Schon et al. 1990). The ability to produce doubled haploids after a single round of recombination—i.e., from an F1—provided an opportunity to compare doubled haploid populations with other random inbred populations derived by pedigree inbreeding or single seed descent (SSD). If the association was due to linkage, then the amount of the genetic variation for the trait associated with the major gene should decrease with each round of recombination (Snape and Simpson 1981). There will therefore be a difference between the amount detected in the F1DH population and the pedigree inbred or SSD population. This approach was used in studies of associations of some major genes found in commercial cultivars and has almost exclusively been applied in barley. In cases in which DH populations segregated for single major genes, it was possible to find associations with a range of phenotypes, including economically important ones. This was done by searching for significant differences between the two major phenotype means for a trait of interest, e.g., semi-dwarf (sdw 1) versus tall stature (Sdw 1) on yield. In this way, significant pleiotropic effects were found for several major genes, but predominantly developmental and disease resistance genes, on agronomic, yield, and quality traits. Again the approach has been used almost exclusively for barley (e.g., Choo et al. 2001; Jui et al. 1997; Kjaer et al. 1990, 1991; Powell et al. 1985a,b, 1990, 1992; Thomas et al. 1991), although in wheat the Glu and Rht2 genes were associated with agronomic traits using this method (Inagaki and Egawa 1994).

3. DOUBLED HAPLOIDS IN GENETICS AND PLANT BREEDING

65

C. Bulked Segregant Analysis Bulked segregant analysis (BSA) was described by (Michelmore et al. 1991) and is a relatively simple method used in detecting marker/trait associations in segregating populations. In BSA a population is screened for a trait, and individuals at the two extreme ends of the distribution formed into two contrasting bulks. Comparisons are then made between the two bulks for the presence and absence of genetic markers. Since the bulks are expected to contrast for alleles contributing positive and negative effects, any marker polymorphisms between the two bulks is indicative of linkage. The technique is dependent upon accurate phenotyping and DH populations have a particular advantage here in that they can be repeatedly tested. Additionally, genotypic data can be built up on the DH lines of interest, and DH populations can be deployed to map the trait/marker. If markers of known map location are used, genetic locations can be quickly identified, but this would be relatively fortuitous, as single locus markers are much less efficient than poly locus markers in BSA. Markers with high multiplex ratios such as randomly amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), or their derivatives are generally the marker systems of choice in BSA. Since these markers have wide and dense genome coverage they allow rapid detection of marker trait associations. The marker can then be used directly by breeders in marker-assisted selection (MAS) schemes, in which the need for genetic mapping can be dispensed with. BSA has proved most successful for traits controlled by single genes, particularly disease-resistance genes in which large DH populations are available. Table 3.2 gives marker/trait associations detected using BSA in conjunction with DH populations. The method has been applied mostly to rapeseed and barley, but examples for wheat, rice, pepper, and tobacco are also given.

D. Genetic Maps Genetic maps are important for several reasons. They aid our understanding of the structure and organization of genomes from which evolution patterns and syntenic relationships between species can be deduced. Genetic maps also provide a framework for the mapping of genes of interest and estimating the magnitude of their effects and aid our understanding of genotype/phenotype associations. This has practical implications for plant breeders, as the identification and location

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B. FORSTER AND W. THOMAS

Table 3.2. Marker/trait associations detected using bulked segregant analysis and DH populations. Crop Barley

Trait Milling energy Resistance to scald Resistance to loose smut Resistance to BaMMV and BaYMV Resistance to BaMMV and BaYMV Resistance to powdery mildew Resistance to net blotch Resistance to leaf rust Resistance to stem rust Resistance to covered smut

Markers

Reference

RAPD RAPD, RFLP SSR

Chalmers et al. 1993 Barua et al. 1993 Li et al. 2001

RAPD

Weyen et al. 1996

RAPD

Ordon et al. 1995

RAPD

Manninen et al. 1997

RAPD

Molnar et al. 2000

RAPD RAPD, RFLP SCAR

Borovkova et al. 1997 Borovkova et al. 1995 Ardiel et al. 2002

Mustard

Resistance to white rust

RAPD

Prabhu et al. 1998

Rapeseed

Seed coat color Linoleic acid desaturation Erucic acid Resistance to blackleg Linolenic and erucic acid Linolenic acid content T-DNA Insertion Seed color

RAPD, AFLP RAPD

Somers et al. 2001 Somers et al. 1998

RAPD RAPD, RFLP RAPD

Jourdren et al. 1996b Mayerhofer et al. 1997 Rajcan et al. 1999

RAPD RAPD RFLP

Jourdren et al. 1996a Baranger et al. 1997 Vandeynze et al. 1995

Pepper

Resistance to root-knot nematode

RAPD, AFLP

Djian-Caporalino et al. 2001

Rice

Resistance to RYMV

RFLP

Ghesquiere et al. 1997

Tobacco

Resistance to root-knot nematode Androgenesis Dough quality Resistance to root lesion nematode

RAPD

Yi et al. 1998

AFLP Range of markers AFLP

Torp et al. 2001 Cornish et al. 2001 William et al. 2002

Wheat

of genes of economic importance provides a basis for their manipulation in producing the best recombinants. Early genetic mapping studies in plants were severely constrained by the numbers of easily identifiable single gene phenotypes and the abil-

3. DOUBLED HAPLOIDS IN GENETICS AND PLANT BREEDING

67

ity to assign genes to chromosomes, usually by cytological markers. Recombination maps developed as more genes and genetic markers became available, but before the 1980s these were confined to morphological mutants, major disease-resistance genes, and some protein and isozyme markers and were comprised of relatively few genes. Data from studies using different mapping populations, typically F2 and F3 families from simple crosses, were collated and composite maps generated, e.g., for barley (Wettstein-Knowles, 1992). Considerable progress was made in this manner but the maps were of limited value, as many of the markers were deleterious mutants not found in commercially relevant germplasm. Doubled haploid populations have become standard resources in genetic mapping for species in which DHs are readily available. Doubled haploid populations are ideal for genetic mapping. Doubled haploidy is the fastest route to homozygosity and it is possible to produce a DH population, extract DNA, screen with markers, and produce a genetic map within two years of the initial cross regardless of the species, provided sufficient DHs can be produced. Even for plants with relatively rapid life cycles such as barley this is comparable to the production and sampling of an F2 or BC1 population and faster than a pedigree inbred or SSD population. For inbreeding species the DH populations can be conveniently stored and distributed as seed, and regrown repeatedly. General-purpose genetic maps have a marker density of about one marker per 5 centi-Morgans and often incorporate a range of molecular markers. They currently have a development time of 2–3 years, though high throughput genotyping systems are expected to speed this up considerably. Map construction is relatively easy using a DH population derived from a hybrid of two homozygous parents as the expected segregation ratio is simple (1:1) and is that of a backcross (Snape 1976). However, since large numbers of genetic markers are involved, computer software is required to construct whole genome maps, e.g., GMENDEL, MAPMAKER, and MAPMANAGER. The synergy between molecular markers systems and DHs provided new impetus in genetic mapping. Because DH populations were readily available, barley was among the first plant species to undergo a genome wide scan using molecular markers (Heun et al. 1991; Graner et al. 1991). DH populations have now been used to produce genetic maps of barley, rapeseed, rice, wheat, and pepper. Software programs such as JOINMAP are also available to construct consensus maps of a species by merging mapping data which share common markers, e.g., for barley (Langridge et al. 1995; Qi et al. 1996), and more recently pepper (Lefebvre et al. 2002). In addition to the use of DHs as mapping populations in their own right, doubled haploidy is also used to produce homozygous parental lines in the development of mapping populations. This has been par-

68

B. FORSTER AND W. THOMAS

ticularly relevant in heterozygous species where genetic analysis can be simplified by providing at least one homozygous (DH) parental line. The strategy has been used to develop high-density molecular marker maps in perennial ryegrass (Lolium perenne) (Bert 1999; Jones et al. 2002) and in the cabbage/cauliflower/Brussels sprouts/broccoli complex of Brassica oleracea (Sebastian et al. 2002). E. Mapping Quantitative Traits Many genes with small, but cumulative, effects control most economically important traits such as yield and quality. As described above, early work in locating such genes was limited to associations with major genes and then with molecular markers in BSA studies. Although the potential for DH populations in quantitative genetics has been understood for some time (Snape et al. 1984; Snape and Simpson 1986), it was the advent of molecular marker maps that provided the impetus for their use in identifying loci controlling quantitative traits. Quantitative trait locus (QTL) analysis is particularly effective with DH populations. Since QTL effects can be small and influenced by the environment, accurate phenotyping requires replication; since DHs are homozygous and true breeding, they can be easily bulked and assessed in multiple sites and seasons in replicated trials. For annual species it is possible to cross, produce an F1-derived DH population, bulk DH seed, and phenotype for target traits in multi-site replicated trials in 3 years. This timescale cannot be achieved with any other population type, making the investment in deploying DH well worthwhile. The development of molecular marker maps in DH populations led to a rapid deployment in QTL studies. For instance, the first QTL studies in barley (Heun 1992; Hayes et al. 1993) were published within about a year of their respective marker maps (Heun et al. 1991; Kleinhofs et al. 1993). Several software packages are available for QTL mapping. Some, such as MQTL (Tinker and Mather 1995), are suited to the detection of crossover QTL × Environment interactions, whereas PLABQTL (Bohn et al. 1999) and QTL Cartographer (Basten et al. 1994) are better suited to identifying QTLs with consistent effects over several environments, and MapQTL (Jansen et al. 1995) has advantages for mapping trait data from just one environment. QTL analysis not only provides a general location for genes controlling a trait, but also provides an estimate of the magnitude (negative and positive) of their effects and that of the environment. Table 3.3 gives a list of published traits mapped using QTL analysis on DH populations. The table lists 130 traits mapped in nine species, but is not exhaustive, as many data are not published. For example, although

3. DOUBLED HAPLOIDS IN GENETICS AND PLANT BREEDING

69

over 50 traits are listed in Table 3.3 for barley, we are aware of over 100 QTL studies; in one barley cross alone over 60 traits have been mapped (Derkado × B83-12/21/5, Thomas unpublished data). Over 20 DH populations have been used in QTL analysis in barley compared to only three for maize, which reflects the ease of DH production in barley compared to maize in which other types of populations are exploited. The data in Table 3.3 have been drawn from a total of 57 DH mapping populations over nine species; the vast majority (23) are from barley. Barley has certainly been the dominant species in QTL detection using DHs, and it is expected that other crops will catch up as more DH populations are produced, mapped, and phenotyped; for instance, many of the wheat studies have publication dates from 2000. Despite the wealth of information on quantitative traits and the availability of published procedures (e.g., backcross conversion of a QTL for stripe rust resistance in barley, see Section D), there has been little take up of QTL data by plant breeders. This is mainly due to worries related to QTL × Environment interactions and effects of genetic background. There is now a trend away from these anonymous effects and toward functional marker maps (Section E), which have more promise for exploitation. F. Genomics Although QTL analysis has generated a vast amount of information on gene locations and the magnitude of effects on many traits, the identification of the genes involved has remained elusive. This is due to the poor resolution of QTL analysis; the confidence interval of a QTL can be massive, in the order of 30 cM (Kearsey and Farquhar 1998), and precludes gene identification through map-based cloning. Other methods are therefore required to get at these genes. One method in refining QTL analysis is through the production of recombinant chromosome substitution lines (RCSLs, Paterson et al. 1990), or stepped aligned recombinant inbred lines (STAIRS, Kearsey 2002). Here backcrossing is carried out until a desired level of recombination has occurred and genetic markers are used to detect desired recombinant chromosome substitution lines in the target region, which can be fixed by doubled haploidy (Thomas et al. 2000). In this manner both coarse and fine-focus mapping can be developed, which can then lead to gene identification. A major driving force in gene identification is the worldwide initiative in genomics, now being applied to several crops. Expressed sequence tags (ESTs) are one means of searching for candidate genes. ESTs can be mapped and their position relative to QTLs determined and their physical chromosomal location identified via BAC libraries. DH

70

Table 3.3. List of traits detected by QTL analysis of DH populations. Data presented are an under-estimate, as many QTL studies have not been formally published. Details of the crosses, markers, and references are given in Forster and Thomas (2003). Crop (No. DH populations used) Barley (23)

Cauliflower, broccoli, cabbage, etc. (4)

Flax (1) Maize (3)

Trait Yield

Quality

Yield, yield components, thousand grain weight

Hardness, dormancy, germination, extract, malt enzymes, grain and straw nitrogen and phosphorus, soluble nitrogen ratio fermentability, wort β-glucan, viscosity, starch, endosperm texture

Agronomic

Developmental

Biotic stress

Grain size, grain shape, specific weight, brackling, lodging, shattering

Habit, leaf number, heading, photoperiod response, vernalization response, height, ear morphology, maturity

Mildew, scald, leaf, stripe, leaf rust, cereal cyst nematode, fusarium head blight, net blotch, rice blast, barley yellow dwarf virus, spot blotch, bacterial leaf streak

Germination, vigor, leaf, flower bud and stem development, flowering time

Club root

Abiotic stress Boron, salt, winter hardiness, deep sowing

Other Tissue culture, crossing with wheat, callus and shoot growth

Agrobacterium transformation efficiency, adventitious root production

Fusarium wilt Androgenesis

Mustard

Seed quality, oil and fatty acid content

Pepper (2)

Cucumber mosaic virus, PVY, Phytophora capisci

Rapeseed (9)

Linolenic acid, erucic acid, glucosinolates

Blackleg, leaf spot, club root, TuYV

Rice (8)

Yield, yield components, tiller number

Aroma, paste, viscosity

Shoot biomass, number of panicles, grain size and shape, specific weight

Root morphology, root penetration, internode lengths lengths, heading, height, morphological index

Rice blast, bacterial blight, sheath blight, RYMV, brown plant hopper

Cold tolerance, drought, salt, metal ions, cell membrane stability

Domestication, anther culture

Wheat (5)

Milling, yield, yield components

Grain hardness, bread making quality, starch, flour color, arabinoxylans, protein, dormancy, a-amylase, dough, noodle color

Seed dormancy, vigor, competition, ear morphology

Coleoptile length, heading, height, photoperiod response, vernalization response, maturity

Stripe rust, fusarium head blight

Drought induced ABA, boron

Anther culture, crossability with rye

71

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B. FORSTER AND W. THOMAS

populations play a vital role in facilitating this approach. For example, the barley Igri × Franka DH population has been used to integrate translocation break-points on the genetic map and provided genome-wide comparisons of genetic and physical map distances in barley, highlighting large areas of low recombination (Kunzel et al., 2000). In rice, ESTs have been found to be co-located with major genes and QTLs for resistance to rice blast, bacterial blight, and sheath blight in the IR64 × Azucena DH map (Wang et al. 2001). By anchoring the various BAC libraries (that have been developed across the world for a range of crops), candidate genes can be targeted with some precision. Ultimately, such strategies will lead to tools for allele mining and therefore a targeted means of exploiting germplasm resources for crop improvement.

IV. DOUBLED HAPLOIDS IN BREEDING The rapid attainment of homozygosity at any generation is probably the most valuable feature of doubled haploidy for plant breeding. Other general benefits include, in some cases, the development of large numbers of homozygous lines, efficient genetical analysis, and the development of markers for trait selection (particularly valuable for recessive traits). More specific benefits include the possibility of seed propagation as an alternative to vegetative multiplication in ornamentals, and in species such as trees in which long life cycles and inbreeding depression preclude traditional breeding methods, doubled haploidy provides new alternatives. Despite proven and theoretical benefits of doubled haploidy, deployment in breeding programs must be practicable, cost efficient, satisfy breeding objectives, and produce marketable cultivars. Although doubled haploidy is useful in fixing rare alleles, overuse may reduce genetic variation in breeding germplasm in which genetic diversity may be better preserved in heterozygous lines. The nature of the crop species therefore determines the extent of DH deployment. Rapeseed is an interesting crop to consider in this respect, as it can be bred to produce pure and population cultivars. Heterozygous cultivars have the advantages of being relatively cheap to produce and have high yield stability over a number of environments. Pure lines, however, are better able to satisfy strict distinctiveness, uniformity, and stability (DUS) regulations required of cultivars in some countries. In addition, several important traits in rapeseed are controlled by alleles rare in non-selected material (e.g., restorer genes to cytoplasmic male sterility, specialty oils, herbicide tolerance, and blackleg resistance), which can be fixed and manip-

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73

ulated in DH lines. Several factors therefore must be considered before the incorporation of doubled haploidy in a plant breeding program. A. Early Breeding Attempts The potential of haploids in plant breeding was realized long before routine methods in haploid/doubled haploid production became available (Blakeslee and Belling 1924). Some of the early breeding attempts with haploids, therefore, struggled with a technology gap, which persists today for some species. Maize was among the first species in which haploids were utilized in breeding (Chase 1974). The classic work of Chase provides salutary lessons in weighing the pros and cons in utilizing haploids in a commercially viable plant breeding program. In 1946 Sherret S. Chase began developing haploids in maize using a spontaneous parthenogenesis system, and in 1948 produced the first maize doubled haploid. The first step, the production of haploid plants, was very inefficient, as haploids occurred at very low frequencies (1–20 per thousand seedlings). The method became more practicable with the use of genetic markers, which allowed more efficient selection of haploid seedlings by tissue coloration (Chase 1949). Furthermore, the frequency of haploids was critically dependent on the genotype, the environment, and the production of healthy seedlings and plants. The second step, the production of doubled haploid lines, was in turn dependent upon (1) spontaneous or induced (colchicine) doubling to create sectors of male and female fertility (most haploid plants were seed sterile), and (2) the presence of sufficient pollen for repeated self-pollinations. Several hundred doubled haploid plants were painstakingly produced by this method. These were compared against standard inbred lines for agronomic performance in field trials with some encouraging results. Selected DH lines were subsequently crossed with other lines to produce commercially successful hybrids, e.g., DeKalb 640. Despite these successes, maize breeders were reluctant to take up haploid/doubled haploid technology. Although doubled haploidy saves time, the early maize methods, based on parthenogenesis, were difficult, exacting, and relatively inefficient, especially as inbreeding in maize can easily produce high numbers of homozygous lines, i.e., high numbers out-weigh the benefits of speed. Haploid/doubled haploid methods in maize now included in vitro gynogenesis and in vitro androgenesis as well as parthenogenesis. The new protocols of anther and isolated microspore culture (Zheng et al. 2003; Barnabas 2003) offer the greatest potential in combining increased efficiency, reliability, and low cost with speed in maize doubled haploid production. A major breakthrough has been the incorporation of responsive

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genes from Chinese sources (Kuo et al. 1978) into commercial germplasm. These developments offer a new impetus in the application of DH technology to maize breeding. B. Elite Crossing Traditional pedigree inbreeding (PI) programs are slow and can take 10–15 years to produce a cultivar. Another major negative feature of PI is its inefficiency in selection in early generations. Because all individuals in early generations are unique and heterozygous, there can be no replication and selection is carried out in non-crop conditions. Consequently there is a bias toward dominant traits, and non-competitive individuals are rejected. Furthermore, selection for quantitative traits cannot be undertaken until lines approach homozygosity and sufficient seed is available for replicated field trials. One way of accelerating the development of homozygous lines is through the use of single seed descent (SSD, e.g., Brim 1966) in which life cycle and hence generation times are minimized. However, SSD also suffers from time delays and competitive interactions among plants. Doubled haploidy not only offers an opportunity to speed up traditional breeding methods, but allows greater flexibility in that it can be applied at any generation, allowing rapid response to changing market demands. In annual inbred crops such as spring barley and rapeseed, it is possible to conduct field trials of DH progeny from an F1 within two years of making the original cross without recourse to shuttle breeding (the bulking of lines in other countries during off seasons). The DH lines are normally bulked in glasshouse conditions in which selection can be practiced for some traits, such as disease resistance, so that field trialing is applied to the more promising lines. Field selection of DH lines is more reliable than partial inbred lines of PI because the rapid bulking of DH lines provides early replicated field trialing of completely homozygous lines in which dominance effects are not an issue. C. Comparison of Breeding Methods Snape (1976) compared the theoretical properties of DH and SSD populations and concluded that there was no difference between them provided there were no linkage effects. In SSD there is a greater opportunity for recombination (standard practice can involve 5 or more rounds of meiosis), and here the frequency of desired genotypes can be higher than in DHs derived from an F1 (one round of meiosis). Breeders can therefore decide whether to preserve linkage blocks (F1DH) or break them up by applying doubled haploidy at later generations (including those of

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SSD and PI). In some crosses it may be possible to produce sufficient DH lines in which to select rare recombinants, but at the moment such high response rates are limited to a few breeding companies. Low DH productivity has been a factor limiting selection in plant breeding. In crosspollinated species there is the alternative of creating favorable gene frequencies in recurrent selection schemes; in such cases the change to doubled haploidy is unwarranted. DH, PI, and/or SSD populations have been compared in a number of studies, but principally inbreeding cereals (reviewed by Choo et al. 1982; Forster and Thomas 2003). All studies conclude that the adoption of doubled haploidy does not lead to any bias of genotypes in populations, and random DHs were even found to be comparable to selected lines produced by PI (Friedt et al. 1986; Winzeler et al. 1987). Therefore, the replacement of PI by doubled haploidy cannot be ruled out on genetic grounds. Most commercial breeders, however, continue to rely on PI or variations of PI with possible incorporation of SSD, though there are examples in which breeders have converted to DH programs. The principal reason not to adopt doubled haploidy is cost. For meaningful DH production, it is necessary to hire one or two trained persons to grow up donor plants, harvest and pre-treat material, and perform all the various tissue culture procedures. These are critical components to DH production requiring proficiency in tissue culture and top-quality growth facilities for donor plants. The investment involved can be prohibitive and sometimes risky; DH protocols are not always transferable and some breeders have tried and failed to generate sufficient DH lines. To be competitive, DH numbers need to be similar to those of an F3 population. Shuttle breeding of spring crops provides an alternative method of speeding up breeding and it is possible to enter lines into official trials from a PI program at the same time as those from DH programs. Also, for some rapid cycling species such as pea, in which three generations can be easily produced per annum by SSD, there is less pressure to adopt DH systems. Although DH lines are homozygous, out crossing can occur even in inbreeding species and therefore stock production requires the same rigor as in other breeding programs. The decision to convert to DH production cannot be taken lightly and the advantages of working with fixed inbred lines needs to be weighed against these provisos. D. Cultivar Releases Kasha et al. (1989) reviewed doubled haploids in cereal improvement; today doubled haploidy features in cultivar production of many crop species. Uniformity is a general requirement of cultivated lines in most species, which can be easily obtained through DH production.

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There are various ways in which DHs can be used in cultivar production. The DH lines themselves can be released directly as cultivars, they may be used as parents in hybrid cultivar production or more indirectly in the creation of breeders lines and in germplasm conservation. The barley cultivar “Mingo” was the first major DH crop cultivar; it was produced and grown in Canada in the late 1970s (Ho and Jones 1980). Doubled haploidy now features in the production of many crops. Barley has over 100 direct DH cultivars (Thomas et al. 2003) and a similar number of rice cultivars have involved doubled haploidy in their breeding (Li 1992; Gosal et al. 1997). Over 150 named DH cultivars are listed by Thomas et al. (2003), which is updated on the Web site http://www.scri.sari.ac.uk/assoc/cost851/Default.htm. According to published information, there are currently around 300 DH derived cultivars in 12 species worldwide, but this is likely to be a gross underestimate, as information from some countries is scant and many breeders are reluctant to reveal their production methods. It is also not clear how many of these releases have been adopted on a large scale. Table 3.4 summarizes the various DH methods used in production of cultivated lines in various crops (for more details see Thomas et al. 2003). The method of choice varies from species to species and between workers. Androgenetic methods, particularly microspore culture, are preferred because they can be highly efficient; however, they also suffer from genotypic dependency, so wide crossing is also used. Anther and microspore culture also have the advantage in that they can generate high numbers of spontaneously doubled plants. In wide crossing, Table 3.4.

Estimated numbers of cultivated lines produced by doubled haploidy.

Crop

Estimated number of cultivars

Methods deployed

Asparagus Barley Mustard Eggplant Melon Pepper Rapeseed Rice Swede Tobacco Triticale Wheat

>10 >100 <20 <10 <10 >10 >50 >100 <10 >10 <10 >20

Female × DH supermale, DH female × DH supermale Anther culture, microspore culture, wide crossing Microspore culture F1 from DH parent(s) F1 from DH parent(s) F1 from DH parent(s) Microspore culture, spontaneous, DH parental line Anther culture Anther culture Microspore culture, anther culture Anther culture Anther culture, wide crossing

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haploids are produced that must be doubled using hazardous chemicals such as colchicine. In some species, however, none of these methods works, but haploid plants can be obtained via gynogenesis, e.g., from ovule and ovary culture (Mukhambetzhanov 1997). Gynogenesis is important in onion, sugar beet, melon, cucumber, pear, gerbera, and ornamental species such as rose, petunia, and Spathiphyllum. Many of these species suffer from inbreeding depression, and in onion strong inbreeding depression of DH lines often necessitates the restoration of fertility before lines can enter a breeding program. However, in sugar beet, melon, and cucumber, F1 hybrid cultivars have been produced from DH parental lines. Gynogenesis is not as efficient as androgenesis and in citrus and cotton gynogenesis has been superceded by microspore methods. This trend is likely to continue in other species once difficulties in androgenesis are overcome. Doubled haploidy has been extremely effective in cultivar production in barley, rice, and rapeseed, but even here there are strong genotypic effects that limit application. Cultivar production in these and other major crops is highly competitive and market shares of new DH cultivars can be relatively modest. A notable exception is the blacklegresistant rapeseed (canola) DH cultivar ‘Quantum’ that captured 30% of the acreage in Western Canada in 1995 (Stringham et al. 1995). The relevance of DHs to plant breeding has increased markedly in recent years owing to the development of protocols for over 250 species (Maluszynski et al. 2003b). Although major agricultural crops have driven the application of DHs, doubled haploid technology is set to have a major impact in other species. Doubled haploidy already plays an important role in hybrid cultivar production of vegetables, and the potential for ornamental production is being vigorously examined. Some of the greatest gains can be envisaged in high-value plants, particularly those that have received little attention from breeders (Schulte 2000). For example, DHs are being developed in the medicinal herb Valeriana officinalis to select lines with high pharmacological activity. Hybrids of V. officinalis DH lines are expected to exhibit heterosis, have increased biomass, and contain large amounts of high-quality product, enabling standardized manufacturing of the sedative drug, valium. Another exciting development is that fertile homozygous DH lines can be produced in species that have self-incompatibility systems and/or suffer from inbreeding depression, such as rye (Immonen and Anttila 1996) and many forage grasses (Nitzsche 1970), providing new opportunities in breeding (Tenhola et al. 2000; and Humphreys et al., respectively 2000). Anther culture in forage grasses has generated novel and interesting genotypes. Androgenic plants derived from Lolium multiflorum

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× Festuca arundinacea and F. pratensis × L. multiflorum hybrids showed interspecific genomic re-arrangements, rare gene combinations, and tolerance to freezing and drought, all of which are of commercial and therefore breeding interest (Humphreys et al. 1998; Zare et al. 1999; Lesniewska et al. 2001; Zare et al. 2002). E. Backcross Conversion In backcross conversion genes are introgressed from a donor cultivar or related species into a recipient elite line through repeated backcrossing. A problem in this technique is being able to identify the lines carrying the trait of interest at each generation. The problem is particularly acute if the trait of interest is recessive, as it will be present only in a heterozygous condition after each backcross. Early attempts at backcross conversion therefore targeted easily recognizable phenotypes such as disease resistance. Even so, the process is time consuming, especially in which linkage drag of deleterious genes persists over several generations. For example, the mildew resistance gene, MlLa, from Hordeum laevigatum was first deployed in the cultivars ‘Vada’ and ‘Minerva’ in the 1960s. However, cultivars carrying the gene had poor malting quality and it was not until the release of ‘Doublet’ some twenty years later that the association was broken (Swanston 1987; Qi et al. 1998). The development of genetic markers now provides an easier method for selection based on genotype rather than phenotype (Melchinger 1990), and is particularly effective when used in combination with doubled haploidy. In marker-assisted backcross conversion, a recipient parent is crossed with a donor line and the hybrid (F1) backcrossed to the recipient. The resulting generation (BC1) is backcrossed and the process repeated until desired genotypes are produced. The combination of molecular markers and doubled haploidy provides a shortcut (Fig. 3.2). Genotyping is performed at various BC generations with both specific markers for the target donor trait and fingerprinting (e.g., with AFLPs) for the recipient genetic background, and selected lines backcrossed. Once desired genotypes are produced they can be made homozygous and doubled haploidy is the quickest method. The amount of backcrossing required will depend on the ease of breaking down linkage blocks. Chen et al. (1994) used marker-assisted backcross conversion with doubled haploidy of BC1 individuals to select stripe rust resistant lines in barley. However, an AFLP survey of the BC1DH lines showed they had on average more than the theoretical 75% recurrent genome content (Toojinda et al. 1998). This provides a further opportunity for increasing efficiency, as it is possible to select lines from the BC1DH that are equivalent to a BC3 and further backcrossing, or crossing with another line can reduce the donor genome con-

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Scheme for backcross conversion Recipient Parent

Donor Parent F1

BC1

BC2 BC3

Selfing

BCn Doubled Haploids & Marker assisted Selection

Backcross Inbred line Fig. 3.2. A typical scheme for backcross conversion is shown whereby the genome of the donor parent is reduced (theoretically halved) at each generation. Selection for the trait is carried out at each generation and backcrossing of the selected lines is repeated until a desired level of the recipient genome is recovered along with the introgressed segment, at which point selected lines are selfed. Several rounds of backcrossing are required in traditional backcrossing; however, the process can be shortened by doubled haploid production in early generations. Success will depend on the number of DH lines produced, because essentially the rounds of meiotic recombination are compensated for by DH population size. Reproduced by kind permission of Kluwer Academic Publishers, from Thomas et al. (2003).

tent further still (Tanksley et al. 1989). New genes, including transgenes, can be rapidly introduced into elite material using this combination of backcrossing, marker-assisted selection, and doubled haploidy.

V. PROSPECTS Technological advances have now provided DH protocols for most plant genera. The number of species amenable to doubled haploidy has reached a staggering 250 in just a few decades. Response efficiency has also improved with a gradual removal of species from the recalcitrant category.

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The wide and varied range of species that can be manipulated using doubled haploidy opens up new opportunities. Doubled haploidy is an established biotechnology used in genetic mapping, gene discovery, and breeding that can now be applied to an increasing number of species. The production of DH mapping populations is becoming routine, and in some species it is common practice to produce individual mapping populations for specific traits and thereby identify markers for use in breeding. We do not expect DH production to replace traditional breeding methods; rather it will provide greater efficiency and new options. The application of doubled haploidy, even in the most responsive species, is restricted by genotype dependency and there is a challenge to develop more genotype independent methods. Response to doubled haploidy is known to be under genetic control and in rolling plant breeding programs responsive genotypes can be self-perpetuating, but care will be needed to prevent erosion of the breeder’s gene pool. The most important considerations for breeders are: investment in good plant production facilities, tissue culture facilities and skilled technical support, and the availability of cheap, efficient, genotype independent protocols. Doubled haploidy continues to be an active research area and the benefits from DH production are set to increase. Doubled haploidy has great potential in the production of transgenic crops. Microspore embryogenesis provides an ideal system whereby a single cell (the microspore) can be targeted for transformation and manipulated to produce an embryo and eventually a DH plant homozygous for the transgene. Transgenes can also be manipulated and selected for in DH breeding programs in the same manner as any other gene or marker, e.g., in backcross conversion. It may also be possible to develop screens for particular genotypes in vitro so that only desired DH plants are generated. More DH cultivars are anticipated in a wider number of crops and novel applications are inevitable.

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4 Biochemistry and Genetics of Flower Color R. J. Griesbach Floral and Nursery Plants Research Unit, U.S. National Arboretum, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705-2350

I. INTRODUCTION II. FLAVONOID CHEMISTRY A. Chemical Structure B. Co-pigmentation C. Effect of pH and Metals III. ANTHOCYANIN BIOSYNTHESIS A. Structural Genes B. Regulatory Genes IV. MENDELIAN INHERITANCE A. Classical Breeding B. Gene Tagging C. Gene Silencing V. TRANSGENE TECHNOLOGY LITERATURE CITED

I. INTRODUCTION Flower color is economically very important. Floricultural crops are worth approximately US $50 billion worldwide (Horn 2002) and one of the most important characteristics of these plants is their flower color.

Plant Breeding Reviews, Volume 25. Edited by Jules Janick. ISBN 0471666939 © 2005 John Wiley & Sons, Inc. 89

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Four different pigments—chlorophylls, carotenoids, flavonoids, and betalains—are responsible for most flower colors. Chlorophyll and carotenoid pigments are chemically related and located within plastids that are found in the cytoplasm of the cell. These pigments are responsible for green, yellow, and orange colors. Flavonoid and betalain pigments are located within the cellular vacuole and are responsible for red through blue colors. Betalains and flavonoids are not chemically related and are mutually exclusive, never being found together. The betalains are very rare and only found within a few plant families (Stafford 1994). Many flowers contain a mixture of flavonoids and carotenoids. A wide array of different colors can be created by mixing and matching these pigments. For example, the red color of Sophronitis (Matsui and Nakamura 1988) and Phalaenopsis (Griesbach 1984) is the result of mixing orange carotenoids with magenta flavonoids. Each type of pigment is the result of a different sequence of biochemical reactions. The production of each pigment is independent of the other pigments. In most cases, a defect in the flavonoid pathway has no effect on the carotenoid and chlorophyll pathways. This can be clearly seen in the green mutants of Sarracenia L. where the red flavonoids that are normally present in the leaves and flowers are absent (Sheridan and Mills 1998). The chlorophyll and carotenoids, however, are unaffected; therefore, leaves are green and flowers yellow. Carotenoid free mutants have been very important in breeding. For example, the rosea mutant of Hemerocallis fulva L. was used extensively in breeding to create the first red and pink flowered cultivars (Stout 1942). Hemerocallis fulva. fm. rosea is a carotenoid free mutant with rose colored flowers (Munsell 2.5YR 5/10) as compared to the orange colored flowers (Munsell 7.5R 7/14) of the wild type (Griesbach and Batdorf 1995). Carotenoid free mutants have also been used in breeding to create pink hybrids of Gerbera Cass. (Asen 1984; Valadon and Mummery 1967), Disa L. (Volgelpoel 1986), Dendranthema Kitam. (Teynor et al. 1989), and Tulipa L. (Nieuwhof et al. 1989). Classical breeding, as well as transgene technology, can be used to create novel flower colors. However, a thorough understanding of the chemistry, biochemistry, and genetics of flower color is necessary for creating flowers in an endless array of colors. Very little information is available on betalain chemistry, biochemistry, or genetics (Steiner et al. 1999). The carotenoid biosynthetic pathway has recently been elucidated; however, most of the information is on fruit pigmentation (Cunningham and Gant 2002). On the other hand, a significant amount of information is available for the flavonoid biosynthetic pathway.

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II. FLAVONOID CHEMISTRY A. Chemical Structure The flavonoids can be artificially subdivided into two groups, the anthocyanins and the co-pigments. The co-pigments typically fall into one of two classes (flavonols and flavones), while the anthocyanins are more restricted in their chemical structure. The chemical structure of anthocyanins consists of a 3-ring skeleton with sugar and acyl moieties attached at specific locations (Fig. 4.1). The anthocyanins minus their sugars and acyl moieties are called anthocyanidins. There are six major anthocyanidins (pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin), three major flavonols (kaemperferol, myricetin, and quercetin), and three major flavones (apigenin, tricetin, and luteolin). In nature, the anthocyanins and co-pigments are glycosylated with one or more sugars (Harborne 1967). The anthocyanins are most commonly glycosylated at the 3, 5, or both hydroxyl positions on the ring, while the co-pigments are most commonly glycosylated at the 3, 7, or both positions. The most common sugars are glucose, galactose, and rhamnose. In some plants, the anthocyanidins and co-pigments can be acylated with one or more cinnamic acids. The most common cinnamic acids are coumaric, ferulic, malonic, and caffeic acids.

B. Co-pigmentation In vitro under acidic conditions (pH ≤ 6.0), co-pigments are colorless to light yellow, while anthocyanins are red to blue. At near neutral pH, anthocyanins are not very stable and are nearly colorless (Asen 1975 and 1976). Addition of co-pigments to anthocyanins increases both the stability and intensity of the color (Robinson and Robinson 1931). For example, a solution containing 10 mM of cyanidin 3,5-diglucoside, and 30 mM of quercetin 3-glucoside had an absorbance eight times greater than a solution containing only the anthocyanin (Asen et al. 1972). This effect is called co-pigmentation. Within the cell, anthocyanins and co-pigments have been shown to be bound together in a chemical complex within the vacuole. In Commelina communis L., the anthocyanin/co-pigment complex contains six molecules each of an anthocyanin and co-pigment surrounding two magnesium ions (Kondo et al. 1992). To date, this is the only anthocyanin/ co-pigment complex in which the exact structure has been elucidated through X-ray crystallography. In the anthocyanin/co-pigment complex,

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A

R1 3’

O HO

OH

7 5’ 3 5

R2

OH

OH

B

R1 3’

O HO

OH

7 5’ 3 5

OH

OH

R2

|| O

C

R1 3’

O HO

OH

7 5’ 3 5

OH

R2

|| O

Fig. 4.1. Flavonoid 3-ring skeleton for anthocyanins (A), flavonols (B), and flavones (C). The major anthocyanins are pelargonidin (R1 and R2 = H), delphinidin (R1 and R2 = OH), malvidin (R1 and R2 = CH3), cyanidin (R1 = OH and R2 = H), peonidin (R1 = CH3 and R2 = H), and petunidin (R1 = CH3 and R2 = OH). The major flavonols are kaemperferol (R1 and R2 = H), myricetin (R1 and R2 = OH), and quercetin (R1 = OH and R2 = H). The major flavones are apigenin (R1 and R2 = H), tricetin (R1 and R2 = OH), and luteolin (R1 = OH and R2 = H).

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hydrophobic interactions between all the 3-ring skeletons prevent the anthocyanin molecule from becoming hydrated. When hydrated, the anthocyanin molecule loses its color. As the strength of the binding between the anthocyanin and co-pigment increases, the complex becomes more stable and more intensely pigmented (Brouillard 1988). Quantum mechanic models are being developed to predict color based upon the strength of binding (Torskangerpoll et al. 1999). The most recent models are not able to predict the exact color of a complex, but are accurate enough to predict the relative color of different complexes. As the co-pigment concentration increases at a constant anthocyanin concentration, the absorbance of the anthocyanin/co-pigment complex increases sigmoidally. The co-pigmentation constant (Kc) is defined as the co-pigment equivalents required to yield half-maximal absorbance. C-glycosylflavone co-pigments form a stronger complex (lower Kc) than flavonol co-pigments (Asen et al. 1972). Cyanidin 3,5-diglucoside when complexed with the C-glycosylflavone, 6-C-glucosylapigenin, had an absorbance at its visible λmax that was 1.3-fold higher than when complexed with the flavonol, quercetin 3-glucoside, at the same molar ratio. Besides being more stable and more intensely pigmented, an anthocyanin/co-pigment complex that contains C-glycosylflavones was bluer in color than one containing flavonols. The visible λmax for the 6C-glucosylapigenin complex was 537 nm, while the visible λmax for quercetin 3-glucoside complex was 527 nm. Anthocyanin/co-pigment complexes containing acylated anthocyanins have a lower Kc than those containing nonacylated anthocyanins (Giusti et al. 1999; Honda and Saito 2002; Hoshino et al. 1980). When complexed with 6-C-glucosyl-4’-glucosyl-7-O-methyl apigenin, the Kc for malvidin 3-p-coumaroyl-glucosyl-5-glucoside was five-fold lower than for malvidin 3,5-diglucoside. Even the isomeric configuration of the acyl residue can make a difference, with the trans-p-coumaric acid configuration having a lower Kc than the cis-configuration (George et al. 2001). In addition, the longer the length of the side chains (i.e., glycosyl-acyl-), the lower the Kc (Yoshida et al. 2000). Delphinidin, with two caffeoyl moieties, had a two-fold higher absorbance at its visible λmax than the corresponding delphinidin with one caffeoyl residue. C. Effect of pH and Metals It is generally assumed that red flowers contain predominantly cyanidin and blue flowers mostly delphinidin. Although this is usually true, there are many exceptions. For example, the red flower color of Petunia exserta Stehman is the result of delphinidin, while the pigment in red

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flowered Petunia × hybrida Vilm. cultivars is cyanidin (Ando et. al. 2000). Similarly, flowers that contain cyanidin can be either red as in Rosa L. hybrids (Asen et al. 1971) or blue as in Meconopsis grandis Prain. (Takeda et al. 1996). Differences in vacuolar pH can result in flowers containing the same anthocyanin having different coloration (Stewart et al. 1975; Yoshida et al. 1995). As the pH becomes more alkaline, the color of a specific anthocyanin/co-pigment complex becomes bluer. All the anthocyanins except pelargonidin have the capability of producing blue flowers (Asen 1976). Anthocyanins with an ortho-dihydroxyl system (cyanidin, delphinidin, and petunidin) can form stable complexes with certain metals (Bayer et al. 1966; Toyama-Kato et al. 2003). The most stable complexes are formed with molybdenum, iron, tin, aluminum, titanium, chromium, uranium, and lead. The pH can greatly affect the color of these metal complexes (Asen et al. 1969). Between pH 3.0 and 3.5, a change of less than 0.1 unit can change the color from red to blue. At other pHs, the change is not as dramatic. This is the reason why many species that have evolved to grow on low pH mine spoils have blue flowers (Hale et al. 2002).

III. ANTHOCYANIN BIOSYNTHESIS A. Structural Genes The anthocyanin biosynthetic pathway has been studied extensively in Petunia (Fig. 4.2). All of the enzymes and their corresponding genes have been elucidated in this genus (Holton and Cornish 1995; Mol et al. 1998; Winkel-Shirley 2001). The earliest studies identified genes that were involved in the inheritance of flower color. As biochemical data became available, these genes were assigned specific functions (Wiering and deVlaming 1984). The nomenclature for genes and enzymes is distinct. Gene abbreviations are represented in italics and only the first letter of the abbreviation is capitalized (i.e., Chs), while enzymes are not italicized and all letters are capitalized (i.e., CHS). Since many of the anthocyanin genes were named before their functions were identified, their nomenclature is not straightforward (i.e., the gene encoding DFR is An6 not Dfr). In Petunia, three enzymes (CHS, CHI, and F3H) are responsible for creating the flavonoid skeleton (Fig. 4.1 and 4.2). The first enzyme (chalcone synthase, CHS) is encoded by Chs and condenses three acetate units from malonyl-CoA and p-coumaroyl-CoA into tetrahydroxychal-

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p-coumaroyl-CoA + 3 malonyl-CoA CHS / Chs hydroxychalcone CHI / Chi naringenin F3H / An3 F3’H / Ht1 dihydrokaempferol DFR / An6 [leucopelargonidin] ANS / An17 + 3GT

F3’,5’H / Hf1 dihydroquercetin DFR / An6 [leucocyanidin] ANS / An17 + 3GT

dihydromyricetin DFR / An6 [leucodelphinidin] ANS / An17 + 3GT

pelargonidin-3-glucoside 3RT / Rt

cyanidin-3-glucoside 3RT / Rt

delphinidin-3-glucoside 3RT / Rt

pelargonidin-3-rutinoside

cyanidin-3-rutinoside AAT / Gf

delphinidin-3-rutinoside AAT / Gf

cyanidin-3-acyl-rutinoside 5GT

delphinidin-3-acyl-rutinoside 5GT

cyanidin-3-acyl-rutinoside5-glucoside 3’AMT / Mt

delphinidin-3-acyl-rutinoside5-glucoside 3’AMT / Mt1

peonidin-3-acyl-rutinoside5-glucoside

petunidin-3-acyl-rutinoside5-glucoside 5’AMT / Mf malvidin-3-acyl-rutinoside5-glucoside

Fig. 4.2. Anthocyanin biosynthetic pathway in Petunia × hybrida with corresponding enzymes and genes (modified from Holton and Cornish, 1995; Winkel-Shirley, 2001).

cone. There are eight complete (ChsA, B, D, F, G, H, J, and L) and four incomplete (ChsC, E, I, and K) Chs genes per haploid genome (Koes et al. 1987 and 1989). ChsA is the only gene transcribed to a significant extent in flower tissue. In leaf tissue, ChsA is silenced due to the methylation of one of its EcoRII sites (O’Dell et al. 1999). In floral tissue this site is not methylated. Each complete Chs gene consists of two exons separated by an intron of variable size and sequence (Koes et al. 1989). Incomplete Chs genes do not contain introns. The second enzyme (chalcone flavanone isomerase, CHI) is encoded by Chi and isomerizes tetrahydroxychalcone to naringenin. There are

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two Chi genes (ChiA and ChiB) per haploid genome (van Tunen et al. 1988). ChiA is expressed in all floral tissue and contains no introns, whereas ChiB is only expressed in anthers and contains three introns. The Po mutation is the result of a mutation in the regulatory region of ChiA abolishing promoter activity in anthers but not in the corolla (van Tunen et al. 1991). The final enzyme involved in creating the flavonoid skeleton (flavanone 3-hydroxylase, F3H) is encoded by An3 and is a 2-oxoglutaratedependent dioxygenase that converts naringenin into dihydrokaempferol with the release of carbon dioxide and succinate (Britsch 1990). An3 has been cloned and the active site of the enzyme determined to be at the serine molecule in position 290 (Lukacin et al. 2000). In corolla cells, three different genes (Ht1, Hf1, and Hf2) are responsible for hydroxylating the flavonoid ring to create additional dihydroflavonols (Stotz et al. 1985). Ht genes encode cytochrome P450-dependent monooxygenases (F3'H) that hydroxylate the carbon at the 3' position (Brugliera et al. 1999). While Hf genes encode cytochrome P450-dependent monooxygenases (F3'5'H) that hydroxylate the carbon at the 5' position instead of the 3' position (de Vetten et al. 1999; Shimada et al. 2001), the 5' hydroxylase requires the presence of an additional protein (cytochrome b5) encoded by DifF. Cytochrome b5 acts as the electron donor between NADPH and cytochrome P450-dependent monooxygenase. Conversion of dihydroflavonols into anthocyanins requires the concerted action of three enzymes (Nakajima et al. 2001; Saito et al. 1999; Turnbull et al. 2000). This is a very complex step in the pathway and involves two different reactions—reduction of the double-bonded oxygen on the carbon at the 4-position and glucosylation of the hydroxyl group at the 3-position. The first enzyme (dihydroflavonol reductase, DFR) is encoded by An6 and catalyzes the conversion of dihydroflavonols to leucoanthocyanins (Huitts et al. 1994). An6 contains five introns. Besides An6, there are two other dihydroflavonol reductase genes (Beld et al. 1989). The second enzyme in this complex reaction (anthocyanidin synthase, ANS) is encoded by An17 and is a 2oxoglutarate-dependent oxygenase that converts leucoanthocyanins into 3-flaven-2,3-diols (Weiss et al. 1993). The last enzyme in the reaction (UDP-glucose : anthocyanin 3-O-glucosyltransferase, 3GT) creates the anthocyanin-3-glucoside (Kho et al. 1978). This enzyme exhibits a wide range of activity in glucosylating flavonols, as well as anthocyanins, but not dihydroflavonols (Yamazaki et al. 2002). The 3-O-glucosyltransferase gene in Petunia has been cloned and exists in two copies (Yamazaki et al. 2002). It has not yet been determined if these genes have previously been identified and given an abbreviation.

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Dihydroflavovols can also be converted into flavonol glycosides. Flavonol synthase (FLS) is a 2-oxoglutarate-dependent oxygenase and is encoded by Fl (Holton et al. 1993). FLS has a greater Km for dihydrokaempferol and dihydroquercetin than for dihydromyricetin (Gerats et al. 1982; Forkman and Ruhnau 1987). In addition, FLS has a greater Km for dihydrokaempferol and dihydroquercetin than does the An6 encoded dihydroflavonol reductase. Therefore in Fl+ genotypes, quercetin glycosides accumulate at the expense of cyanidin-based anthocyanins. To a lesser extent, myricetin glycosides accumulate at the expense of delphinidin-based anthocyanins in Fl+ genotypes. The 3-glucosyl anthocyanin is the substrate for the Rt encoded enzyme (3RT) that adds a rhamnose to the glucose at the 3 position to create a rutinoside (Brugliera et al. 1994; Kroon et al. 1994). The 3-rutinoside is now the substrate for the Gf encoded enzyme (AAT) that acylates the 3rutinoside with either caffeic acid or coumaric acid (Griesbach et al. 1991; Jonsson et al. 1984a; Slimestad et al. 1999). Once the acyl group is attached, UDP-glucose : anthocyanin 5-O-glucosyltransferase (5GT) adds a glucose at the 5 position (Jonsson et al. 1984a). The 5-Oglucosyltransferase gene in Petunia has been cloned and exists in two copies (Yamazaki et al. 2002). This enzyme exhibits a strict substrate specificity for the anthocyanin 3-acylrutinoside. It has not yet been determined if these genes have previously been identified and given an abbreviation. The last steps in the pathway involve the methylation of the acylated rutinoside. There are four different anthocyanin-O-methyltransferase (AMT) genes in Petunia (Mt1, Mt2, Mf1, and Mf2) (Jonsson et al. 1983). Each gene controls a distinct and independent enzyme that is capable of methylating both the 3' and 5' positions on the anthocyanin molecule. All methylation steps can occur if only one of the genes is expressed; however, the relative ratio of the different methylated anthocyanins depends upon which gene is expressed. For example, the four enzymes differ in their in vitro efficiency in methylating delphinidin: Mf1 (175 pkat⋅mg protein–1), Mf2 (100 pkat⋅mg protein–1), Mt1 (60 pkat⋅mg protein–1), and Mt2 (30 pkat⋅mg protein–1). If one or more of the Mt genes is expressed and all four of the Mf genes are not expressed, then 3'methylated anthocyanins (peonidin and petunidin) accumulate as the major product. If at least one of the Mf genes is expressed, then the 3',5' methylated anthocyanin (malvidin) accumulates as the major product. The Mf1 encoded enzyme has a higher Km (8 µM) for petunidin than the Mf2 encoded enzyme (21 µM), thereby producing a higher ratio of malvidin to petunidin. Through substrate inhibition, high concentrations of delphinidin reduce the amount of malvidin produced, but not the amount

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of petunidin produced. In addition, a dosage effect appears to operate, such that increasing the number of Mf +/Mt + genes results in a higher relative concentration of malvidin. High concentrations of petunidin, coupled with low concentrations of delphinidin, promotes malvidin synthesis ( Jonsson et al. 1984b). The last gene in the pathway is An9, which encodes a type I glutathione S-transferase (GSTI; Alfenito et al. 1998). GST binds to the anthocyanin and functions in transport into the vacuole (Mueller et al. 2000). Mutants lacking a functional An9 have white flowers. B. Regulatory Genes Besides the structural genes that encode enzymes, there are regulatory genes that encoded proteins that control the expression of the structural genes. In Zea mays L, there are two regulatory gene families (R and C1) that control the expression of anthocyanin structural genes (Dooner et al. 1991). The R family consists of the R and B genes that encode a protein with a N-terminal region rich in acidic amino acids and a C-terminal region with a basic helix-loop-helix (bHLH) structure characteristic of MYC oncoprotein transcription factors. The C1 family consists of the C1 and Pl genes that encode a protein with an N-terminal region rich in basic amino acids and a C-terminal region rich in acidic amino acids characteristic of MYB oncoprotein transcription factors. The MYB gene family regulates the structural genes upstream of Dfr in the biosynthetic pathway and the MYC gene family regulates Dfr and the genes downstream (Grotewold et al. 1994). Each gene family contains other members that have arisen by gene duplication (Hanson et al. 2000; Zhang et al. 2000). In Zea mays L., anthocyanin expression requires the co-expression of at least one gene from each family (Bradley et al. 1998). The promoter for the structural gene encoding dihydroflavonol reductase has two binding sites for the MYB transcription factor and at least one independent binding site for the MYC transcription factor (Sainz et al. 1997). Tissue specific expression is defined by the diversity among the alleles, each of which regulates expression in a different tissue in a different manner (Dooner et al. 1991). For example, the combination R/C1 induces pigmentation in the kernel, while the B/Pl combination induces pigmentation in mature tissues. In Petunia, at least four regulatory genes (An1, An2, An4, and An11) control the expression of anthocyanin structural genes (Quattrocchio et al. 1993). An1 encodes a MYC transcription factor that is active in all parts of the flower (Spelt et al. 2000). An2 and An4 encode MYB tran-

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scription factors (Quattrocchio et al. 1999). An2 is only active within the limb, while An4 is only active within the anthers. An11 encodes a regulatory protein with five tryptophan and aspartic acid (WD) repeat units that is active in all parts of the flower (de Vetten et al. 1997). An1, An2, An4, and An11 regulate Dfr and the downstream genes in the biosynthetic pathway (Quarttrocchio et al. 1998). Another set of genes, which have not yet been identified, is responsible for regulating genes upstream of Dfr. Myc-type anthocyanin regulatory genes have also been identified: Delila in Antirrhinum majus L. (Goodrich et al. 1992), Gmyc1 in Gerbera jamesonii Bolus. (Elomaa et al. 1998), and Tt8 in Arabidopsis thalliana L. (Nesi et al. 2000). All three genes regulate the expression of Dfr and not Chs. Myb anthocyanin regulatory genes have been identified in Antirrhinum (Rosea; Schwinn et al. 2001) and Arabidopsis (Pap1; Borevitz et al. 2000). In Petunia, the Myc and Myb regulatory genes operate in a complex regulatory hierarchy that is still not completely understood. An11 encodes a cytoplasmic protein that acts upstream of An2 (de Vetten et al. 1997). An11 appears to link cellular and/or environmental signals with transcription of An2. Although white flowers are produced in an2– genotypes and pigmented flowers in An2+ genotypes, the intensity of pigmentation is determined by the interaction of An2 with at least two other genes (Griesbach 2002). An2, however, does not directly regulate the transcription of a structural gene. An2 and An4 control the expression of An1 that then directly activates the transcription of the structural genes within the limb and tube, respectively (Spelt et al. 2000). Besides regulating anthocyanin biosynthesis, An1, An2, and An11 also control vacuolar pH and seed coat morphology (Mol et al. 1998). An1 (previously studied as Ph6) regulates the expression of Ph1 and Ph2 independently of the anthocyanin structural genes (Griesbach 1998). The bHLH domain in An1 is required for the regulation of vacuolar pH and seed coat morphology, but not for DFR expression (Spelt et al. 2002). When mutant An1 alleles lacking the bHLH domain are over-expressed, near-normal levels of anthocyanin biosynthesis occur without vacuolar acidification or normal seed coat development. Studies with transgenic plants have shown that the same class of regulatory genes controls different structural genes in different plants. In Petunia, the MYB transcription factors regulate the expression of Dfr and not Chs, while in Zea mays they regulate Chs and not Dfr expression (Quattrochio et al. 1998). The Rosea gene from Antirrhinum increases the anthocyanin concentration of vegetative tissue when expressed in Petunia and of floral tissue when expressed in Eustoma grandiflorum

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Grise. (Davies et al. 2002). Similarly, the Lc gene (an allele of the R locus) from Zea increased the anthocyanin concentration of vegetative and floral tissue when expressed in Petunia, but had no effect in Eustoma or Pelargonium × domesticum Bailey (Bradley et al. 1999). The Delila (Myc) gene from Antirrhinum increased anthocyanin concentration when expressed in Nicotiana, but not when expressed in Arabidopsis (Mooney et al. 1995). This data suggests that the regulatory genes are conserved between species and that divergent expression is based upon target gene promoter evolution. The MYB proteins are so highly conserved that a single amino acid substitution can switch from plant specific to animal specific binding (Solano et al. 1997).

IV. MENDELIAN INHERITANCE A. Classical Breeding Many of the first genetic studies were on flower color (Olby 2000), specifically in orchids (Cattleya Lindl.), sweet pea (Lathyrus odoratus L.), and garden stock (Matthiola incana R.Br.). Two of the rediscovers of Mendel, Correns (1900) and Tschermack (1912), studied the inheritance of flower color in Matthiola. Four genes (B, C, R, and V ) were identified that affected color. Both C and R were essential for pigmentation. B converted red color into blue and V was required for pure bright color. Further studies by Saunders (1928), identified three additional genes (A, A', and W) and a number of other genes responsible for diluting the color. Both A and A', as well as R and C, were all required for pigmentation. W resulted in carotenoid pigmentation. In Lathyrus, six genes (A1, D2, F1, G1, G2, and G3) were identified that affected flower color (Bateson et al. 1905 and 1911; Punnett 1925). Both F1 and G1 were essential for pigmentation. A1 converted red color into purple and D2 was required for pure bright color. G3 was responsible for dark undiluted color. G2 resulted in carotenoid pigmentation. In Cattleya species, white flowered mutants were separated into two classes—true alba with absolutely no pigmentation and false alba (more properly called albescent) with very slight pigmentation (Hurst 1905). A three gene model (R, C, and D) was proposed, whereby an absence of either an R and C allele results in alba flowers and an absence of a D allele results in albescent flowers (Hurst 1913). Plants with a R- C- Dgenotype would have a pigmented phenotype; while plants with an Rcc or rr C- genoytpe would have an alba phenotype; and plants with an R- C- dd genotype would have a albescent phenotype. This model was confirmed by others using additional mutants (Curtis and Duncan 1942;

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Mehlquist 1958). A number of additional genes (P, S, T, U, V, W, X, Y1, and Y2) have been proposed to explain the differences in specific colors (Harper 1976; Mehlquist 1958; Storey 1946). These studies created an interest in determining the inheritance of flower color in many species (Crane and Lawrence 1934; Emsweller et al. 1937; Paris et al. 1960). Based upon a through review of the literature, a general model for flower color inheritance was proposed (Paris et al. 1960). In this model, five genes (W, Iv, B, P, and Dil) could explain nearly all the possible flower colors in most plants. For example, W- genotypes results in pigmented flowers, while ww genotypes results in white flowers. B- or P- genotypes result in purple flowers, while bb and pp genotypes result in blue and red flowers, respectively. Dilgenotypes result in dark colors, while dil dil genotypes result in pale colors. Iv- genotypes result in non-ivory (pure bright) colored flowers, while iv iv genotypes result in ivory flowers. This model closely follows what is now known about the biochemistry of flower color. Current research attempts to determine the specific function of genes previously identified through Mendelian genetics. For example, in Cattleya, biochemical studies demonstrated that plants with an RR cc DD genotype accumulate flavonols, while plants with an rr CC DD genotype accumulate intermediates upstream of chalcone (Harper 1976). This data suggests that R encodes CHS and C encodes DFR. Further studies using in situ genetic complementation demonstrated that the albescent phenotype may be the result of a defective regulatory gene (Griesbach and Klein 1993). B. Gene Tagging In order to truly identify the function of a gene, it is necessary to isolate or clone it. One novel way of identifying and cloning genes involves the use of transposable or controlling elements. Transposable elements are responsible for many variegated flowers (i.e., flowers with flecks of a different color; Bianchi et al. 1978). When a controlling element inserts itself into a gene, the gene becomes non-functional. These elements are unstable and become excised. When a transposable element is excised, the wild type function is usually restored. In a variegated flower, the background tissue expresses the mutant phenotype (transposon inserted into the gene), while the sector tissue expresses the restored wild type phenotype. Transposable elements have been most thoroughly studied in maize. Three major families of transposable elements (Activator-Dissociation (Ac-Ds), Mutator (Mu), and Suppressor-Mutator (Spm)) have been defined (Fincham and Sastry 1974). The members of each transposable element

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family can be placed into one of two groups. Autonomous elements can transpose without the help of additional genes; while, nonautonomous elements require an autonomous member of the family for transposition. In the Ac-Ds family, Ac transposes autonomously and Ds transposition requires trans-activation by Ac (Federoff 1989). The DNA sequence of Ds is identical to that of Ac except for a 200 bp central deletion. Ac is 4565 bp with an 11bp imperfect terminal inverted repeat and encodes a transposase that is sufficient for transposition. Upon Ac insertion, a 8 bp duplication is created in the target gene. In the presence of an active Ac, Ds becomes demethylated, allowing it to be transcribed. The Ds transcription production can either cause transposition or chromosome breakage. The broken chromosome usually mends itself by forming a Ushaped dicentric chromosome with two centromeres. This unusual chromosome leads to the breakage-fusion-bridge cycle. During this cycle, the U-chromosome breaks during mitosis at a random point between the two centromeres. The resulting daughter cells will either contain gene duplication(s) or deletion(s). Unlike the Ac element, the Spm element encodes at least two different functions that are derived by alternative splicing. Both products are required for transposition (Masson et al. 1989). Spm can exist in one of three forms (stable inactive called cryptic, unstable active called programable, and stable active) (Banks et al. 1988). There are two phases in control of Spm activation. During the setting phase, the element’s activity is determined (active vs. inactive). During the program phase, the element’s degree of stability is determined. Active elements are transcribed, while inactive elements are untranscribed as the result of upstream methylation. When an active Spm is introduced into a genome containing a cryptic element, the cryptic element is partially demethylated but remains transcriptionally inactive. When an active Spm is introduced into a genome containing a programable element, the programable element is extensively demethylated, allowing it to be transcribed. Upon Spm insertion, a 3 bp duplication is created in the target gene. Genes in which a transposable element has inserted itself are referred to as being “tagged” (Feldmann and Meyerhoff 1991; Gerats et al. 1989; Jayaram and Peterson 1991). Tagged genes are very useful in genetic and biochemical studies, for within the same tissue one has both the mutant and wild type phenotype in isogenic cells. This makes it possible to identify new genes, determine gene function, and physically isolate a gene. Nearly all of the Zea mays anthocyanin structural and regulatory genes were identified via gene tagging (Jayaram and Peterson 1991). Two transposable elements (dTph1 and Ps1) have been described in Petunia. The dTph1 element is 284 bp, a member of the Ac-Ds family, and trans-activated by the Act1 gene (Gerats et al. 1990; Huits et al.

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1995). There are between 2 and 25 copies of dTph1 within the genome. The Ps1 element is 9900 bp and a member of the Spm family (Snowden and Napoli 1998). There are between 2 and 4 copies of Ps1 within the genome. Through classical breeding, dTph1 was used to tag 40 alleles of seven genes (van Houwelingen et al. 1998). The seven genes affected were An3 (encodes F3H), An11 (encodes an anthocyanin regulatory gene), Alf (causes aberrant leaf and flower morphology), Nam (causes no apical meristem), Ph4 (vacuolar pH gene), Ph3 (vacuolar pH gene), and Ph7 (vacuolar pH gene). One of these genes (Ph7) was not previously described. Four transposable elements (Tam1, Tam2, Tam3, and Tam4) have been described in Antirrhinum (Sommer et al. 1988). Tam1, Tam2, and Tam4 belong to the Spm family (Krebbers et al. 1987), while Tam3 belongs to the Ac-Ds family (Carpenter et al. 1987). In one plant, there were 40 copies of Tam3 within its genome (Kishima et al. 1999), while in another plant, only eight copies were detected (Kitamura et al. 2001). These transposable elements have been used to identify anthocyanin structural (Luo et al. 1991) and regulatory (Noda et al. 1994) genes. Transgenic technology has been used to introduce the Zea mays transposable element Ac into a number of species (Hehl and Baker 1990). In Petunia, one of the resulting transgenic plants had Ac inserted into the Ph6 gene controlling vacuolar pH (Chuck et al. 1993). In Petunia, the pH of floral tissues ranged between 5.2 and 6.5 units (deVlaming et al. 1983). Five genes (Ph1, Ph2, Ph3, Ph4, and Ph5) were identified that controlled the pH. In the presence of dominant alleles of a Ph gene, the pH is lowered. The genes are co-dominantly inherited, with each allele reducing the pH by about 0.4 unit (Griesbach 1998). The Ph genes encode tonoplast Na+/H+ exchanger proteins (Reuveni et al. 2001; Yamaguchi et al. 2001). Further classical genetic studies with Ph6-tagged plants demonstrated that Ph6 not only controlled pH, but also regulated anthocyanin production (Griesbach 1998). It was suggested from these studies that Ph6 was a regulatory gene, which was subsequently shown to be an allele of the An1 regulatory gene (Spelt et al. 2002). C. Gene Silencing Knowing the function of a gene can greatly help in breeding. This is easily shown in the development of white-flowered cultivars. There are many ways in which a white-flowered mutation can arise, the most obvious being a non-functional enzyme in the biosynthetic pathway. These mutations are quite commonly found in nature. Another path to white flowers can be through a non-functional regulatory gene. Because flavonoid regulatory genes can also affect other traits, mutation in these

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genes may have a selective disadvantage and are not commonly found. A non-functional gene behaves as a typical Mendelian recessive trait, always resulting in pure white flowers and typically referred to as having an alba phenotype. Not all white flowers have an alba phenotype. For example, some Gladiolus cultivars have pure white petals with a small amount of pigmentation in the stamens and pollen (personnel observation). These cultivars are referred to as having an albescent phenotype. While an alba phenotype is recessively inherited, an albescent phenotype is usually dominantly inherited. The molecular basis for this dominance can be quite complex. In maize, two different mechanisms have been identified. In the first mechanism, the protein encoded by the C1 regulatory gene was modified such that it could bind to a structural gene’s promoter but could not activate transcription (Goff et al. 1991). The defective protein out-competed the normal protein in binding. In the second mechanism, the C2 encoded CHS contained multiple copies of a rearrangement (Wienand et al. 1991). The duplication initiated a process called gene silencing. There are two mechanisms for gene silencing (Fagard and Vaucheret 2000). In transcriptional gene silencing (TGS), there is a decrease in mRNA synthesis because of DNA methylation (Vaucheret and Fagard 2001). In post-transcriptional gene silencing (PTGS), there is a decrease in steady state mRNA accumulation because of sequence specific degradation (Sijen et al. 2001). In the PTGS model (Metzlaff 2002; Tijsterman et al. 2002), a transgene produces an aberrant RNA that is the template for a doublestranded RNA (dsRNA). An endonuclease called “dicer” then digests the dsRNA into siRNAs (small interfering RNAs). The siRNAs are subsequently transferred to another enzyme complex called RISC for RNA Induced Silencing Complex containing a different endonuclease than dicer. The siRNA, which is complementary to the target RNA, guides the endonuclease to the target mRNA that is then digested. Genes that are silenced are not always uniformly turned off. Many different factors can affect the degree of silencing (Meyer et al. 1992). Since gene silencing is not stable, the color of albescent mutants can vary pending upon the health of the plant and the environment. For example, in albescent Gladiolus and Dendranthema, high light intensity and low temperature can increase pigmentation (personnel observation), while the environment has no effect on the color of alba phenotypes. This is the reason why alba phenotypes have a higher commercial value. Successful breeding programs have concentrated on developing alba cultivars. Fifty years ago, most pure white-flowered Gladiolus cultivars had an albescent phenotype; while modern white Gladiolus have an alba phenotype.

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V. TRANSGENE TECHNOLOGY One of the first practical examples of plant genetic engineering involved the development of a novel flower color in Petunia through the engineering of dihydroflavonol reductase (DFR). DFR from Petunia has a low substrate specificity for dihydrokaempferol; therefore, pelargonidin is rarely found (Huitts et al. 1994). The experimental goal was to increase the production of pelargonidin in Petunia by introducing foreign Dfr genes with a greater substrate specificity for dihydrokaempferol. Two leaky ht – hf1– mf1– mutants (RL01 and W80) and one leaky ht – hf1– hf2 – mutant (Skr4 × Sw63) were used as parent plants in transformation. Mutants RL01 and W80 both produced one tenth the normal amount of total anthocyanin. The anthocyanin profile was 28% delphinidin, 63% cyanidin, and 9% pelargonidin (Griesbach 1993; Johnson et al. 1999). Mutant Skr4 × Sw63 also produced one tenth the normal amount of total anthocyanin, but had a different anthocyanin profile of 24% pelargonidin, 7% peonidin, 4% petunidin, and 65% malvidin (Tanaka et al. 1995). When Dfr from Cymbidium Sw. was expressed in W80, there was no change in flower color (Johnson et al. 1999). Therefore, in Petunia, the DFR from Cymbidium did not recognize dihydrokaempferol as a substrate. This is not unexpected, since only cyanidin and peonidin are found in Cymbidium (Tatsuzawa et al. 1996). When Dfr from Zea (A1) was expressed in RL01, there was a ten-fold increase in the total amount of anthocyanin as well as an increase in the relative amount of pelargonidin from 9% to 55% (Griesbach 1993; Meyer et al. 1987). In the untransformed RL01, there was nearly four times more kaemperferol than quercetin, while in the transformed RL01 there was nearly an equal concentration of both flavonols. Therefore, in Petunia, DFR from Zea recognized both dihydroquercetin and dihydrokaempferol as a substrate. This is not unexpected, since in Zea mays pelargonidin is only produced in the absence of dihydroquercetin. When Dfr from Gerbera (Gdfr) was expressed in RL01, the total amount of anthocyanin and the relative amount of pelargonidin did not differ significantly from RL01 plants expressing A1 (Elomaa et al. 1995). Therefore, in Petunia, DFR from Gerbera recognized both dihydroquercetin and dihydrokaempferol as a substrate. This is unexpected, since in Gerbera both pelargonidin and cyanidin are readily produced depending upon what precursor is present (Asen 1984). One would have expected a higher concentration of pelargonidin in the transgenic plants. When Dfr from Rosa was expressed in Skr4 × Sw63, there was a ten-fold increase in the total amount of anthocyanin, as well as an increase in the relative amount of pelargonidin from 24% to 97% (Tanaka et al. 1995). Therefore, in Petunia, DFR from Rosa recognized both dihydroquercetin

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and dihydrokaempferol. This is unexpected, since in Rosa only a small amount of pelargonidin is produced even when significant amounts of dihydrokaempferol are present (Asen 1982). One would have expected a lower concentration of pelargonidin in the transgenic plants. In order to identify the DNA sequence leading to substrate specificity, chimeric Dfr genes were constructed and introduced into P. × hybrida ‘W80’ (Johnson et al. 2001). It was determined that the substrate binding region was between amino acids 132 and 158, with amino acid 134 critical in substrate specificity. A switch from asparagine to leucine at position 134 caused a change in substrate preference from dihydroquercetin to dihydrokaempferol. This series of experiments clearly demonstrates that the endogenous expression of a gene may not reflect its transgenic expression. Besides the Km, other factors can also influence its expression. For example, it is well known that the flow at a particular step in a biosynthetic pathway depends upon the concentration of all the metabolite intermediates both upstream and downstream of that step (Keightley 1989). In addition, transcription and translation rates, mRNA stability, and protein turnover can also influence gene expression. Another factor that can influence gene expression is gene silencing. When grown in the greenhouse, Petunia RL01 plants expressing A1 from Zea produced solid orange flowers, while plants grown outdoors produced weakly pigmented flowers with reduced levels of A1 expression (Meyer et al. 1992). Reduced expression (gene silencing) was due to DNA methylation within either the 35S promoter in the A1 construct or in A1 itself (Meyer and Heidmann 1994). Unlike plants expressing A1, plants expressing Gdfr from Gerbera very rarely produced weakly pigmented flowers with reduced levels of transgene expression (Elomaa et al. 1995). In those rare plants with reduced expression, only the 35S promoter was methylated. Gdfr itself was never methylated. It was suggested that the increase in the stability of Gdfr expression over A1 expression was due to the lower GC content (39%) of Gdfr than A1 (60%). Dicotyledonous plants have a lower GC content than monocotyledonous plants. Transgene silencing can be induced by differences in the GC content between the foreign and host DNAs (Fagard and Vaucheret 2000). The silenced phenotype was dependent upon the orientation of the transgene relative to the promoter. Petunia plants expressing a chalcone synthase (Chs) transgene in the same orientation as the endogenous gene (co-suppression) produced flowers with reduced pigmentation at the junctions between adjacent petals (Napoli et al. 1990). Petunia plants expressing a chalcone synthase (Chs) transgene in the opposite orientation as the endogenous gene (anti-sense suppression) produced a different phenotype with white petal margins and reduced pigmentation throughout the flower (van der Krol et al. 1988). Through breeding, it

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was possible to combine both phenotypes in a single plant (Que et al. 1998). It was suggested that sense and anti-sense suppression occurred in different cellular compartments or at different times in development. Gene silencing has also been used to create bluer flowers. When Dfr from Torrenia fournieri Lind was inactivated by the introduction of an antisense copy of the gene, the concentration of anthocyanins (the 3,5diglucosides of cyanidin, peonidin, delphinidin, petunidin, malvidin) decreased by 86% and the concentration of flavones (luteolin 7glucoside, luteolin 7-glucuronide, and apigenin 7-glucuronide) increased by more than 150% (Aida et al. 2000). This change in the anthocyanin/co-pigment ratio resulted in a shift of the visible λmax from 540 to 570 nm and a significant change in color. Gene silencing has also been used to do the opposite—decrease copigment concentration and create redder flowers. When Fls from Nicotiana (Holton et al. 1993), Eustoma (Nielsen et al. 2002), Torenia (Ueyama et al. 2002), and Petunia (Davies et al. 2002) was inactivated by the introduction of antisense copies of the gene, the concentration of co-pigments decreased and the concentration of anthocyanins increased. This change in the anthocyanin/co-pigment ratio resulted in redder flower colors. Blue flowers have also been created by the expression of foreign genes encoding flavonoid-3',5'-hydroxylase (F3'5'H). This enzyme is the key step in the switch in the biosynthesis from cyanidin to delphinidin. A unique electron donor (cytochrome b5) is also required for this step in the pathway. If all the other factors influencing flower color (i.e., pH, copigmentation, and intramolecular interactions) are appropriate, then a switch from cyanidin to delphinidin could lead to blue flowers. Transgenic Dianthus caryophyllus L. plants expressing the Petunia F3'5'H and cytochrome b5 genes produced flowers with a very unique deep purpleblue color (Brugliera et al. 2000). When the Petunia F3'5'H was expressed in the absence of the cytochrome b5 gene, the flower color remained unchanged. This research resulted in the release by Florigene of the first genetically engineered plants with novel flower colors that have been commercially successful (Dianthus ‘Moondust’™, ‘Moonshadow’™, ‘Moonvista’™, and ‘Moonlite’™). Although significant effort has been directed toward creating a blue rose, the blue rose remains elusive. One of the reasons for the lack of success has been that the researchers have assumed that if a rose expressed delphinidin, then it would be blue. Thus, all efforts have focused on introducing a transgene(s) for delphinidin production. Depending upon the relative Kms of the competing enzymes for the various substrate, the expression of transgene may not reflect its endogenous expression (Keightley 1989). For example, a Petunia gene which results in delphinidin production in Petunia does not necessarily result in delphinidin production in rose. Even if a rose produced delphinidin, it still would not be true-

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blue. The co-pigment and pH are not appropriate for blue color (Asen et al. 1971). In order to create a true blue rose, transgenes for both co-pigment and pH need to be introduced, besides a delphinidin transgene(s). The promise of being able to create custom colored flowers is becoming reality. As more is learned about the basic chemistry and genetics of flower color, it is becoming easier to modify colors in specific directions. We still cannot create the perfect sky-blue flower, but we sure can create a flower that is significantly bluer. LITERATURE CITED Aida, R., et al. 2000. Copigmentation gives bluer flowers on transgenic plants with antisense dihydroflavonol-4-reductase. Plant Science 160:49–56. Alfenito, M. R., et al. 1998. Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferase. Plant Cell 10:1135–1149. Ando, T., et al. 2000. Differences in the floral anthocyanin content of red petunias and P. exserta. Phytochemistry 54:495–501. Asen, S., et al. 1969. Absorption spectra and color of aluminium-cyanidin 3-glucoside complexes as influenced by pH. Phytochemistry 8:653–659. Asen, S., et al. 1971. Affect of pH and concentration of the anthocyanin-flavonol copigment complex on the color of ‘Better Times’ roses. J. Am. Soc. Hort. Sci. 96:770–773. Asen, S., et al. 1972. Co-pigmentation of anthocyanins in plant tissues and its affect on color. Phytochemistry 11:1139–1144. Asen, S. 1975. Factors affecting flower color. Acta Hort. 41:57–68. Asen, S. 1976. Known factors responsible for the infinite flower color variations. Acta Hort. 63:217–223. Asen, S. 1982. Identification of flavonoid chemical marker in roses and their HPLC resolution and quantitation for cultivar identification. J. Am. Soc. Hort. Sci. 107:744–750. Asen, S. 1984. HPLC analysis of flavonoid chemical markers in petals from Gerbera flowers as an adjunct for cultivar and germplasm identification. Phytochemistry 23:2523–2526. Banks, J. A., et al. 1988. Molecular mechanisms in the developmental regulation of the maize suppressor-mutator transposable element. Genes & Dev. 2:1364–1380. Bateson, W., et al. 1905. Experimental studies on the physiology of heredity. Rep. Evol. Comm. Roy. Soc. Bateson, W., and R. C. Punnett. 1911. On gametic series involving reduplication of certain terms. J. Genet. 1:293–302. Bayer, E., et al. 1966. Complex formation and flower colors. Angew. Chem. Int. Edit. 5:791–798. Beld, M., et al. 1989. Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes. Plant Mol. Biol. 13:491–502. Bianchi, F., et al. 1978. Regulation of gene action in Petunia hybrida: unstable alleles of a gene for flower color. Theor. Appl. Genet. 53:157–167. Borevitz, J. O., et al. 2000. Activation tagging identifies a conserved MYB regulatory gene of phenylpropanoid biosynthesis. Plant Cell 12:2383–2393. Bradley, J. M., et al. 1998. The maize Lc regulatory gene up-regulates the flavonoid biosynthetic pathway of Petunia. Plant J. 13:381–392. Bradley, J. M., et al. 1999. Flower pattern stability in genetically modified lisianthus under commercial growing conditions. New Zealand J. Crop Sci. 28:175–184. Britsch, L. 1990. Purification of flavanone 3-hydroxylase from Petunia hybrida: antibody preparation and characterization of a hemogenetically defined mutant. Arch. Biochem. Biophys. 276:348–354.

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Teynor, T. M., et al. 1989. Inheritance of flower color in Dendranthema grandiflora using cultivars and inbreds. I. Plastid pigmentation. Euphytica 42:199–207. Tijsterman, M., et al. 2002. The genetics of RNA silencing. Annu. Rev. Genet. 36:489–519. Torskangerpoll, K., et al. 1999. Color and substitution pattern in anthocyanidins. A combined quantum chemical-chemomtrical study. Spectrochim. Acta. 55:761–771. Toyama-Kato, Y., et al. 2003. Analysis of metal elements of hydrangea sepals at various growing stages by ICP-AES. Biochem. Engineer. J. 14:237–241. Tschermack, E. 1912. Bastardierungsversuche an Levkojen, Erbsen, und Bohnen mit Rucksicht auf die Facktorenlehre. Z.I.A.V. 7:81–234. Turnbull, J. J., et al. 2000. Are anthocyanidins the immediate products of anthocyanidin synthase? Chem. Commun. 2000:473–474. Ueyama, Y., et al. 2002. Molecular and biochemical characterization of torenia flavonoid 3'-hydroxylase and flavone synthase II and modification of flower color by modulating the expression of these genes. Plant Sci. 163:253–263. Valadon, L. R. G., and R. S. Mummery. 1967. Carotenoids of certain compositae flowers. Phytochemistry 6:983–988. van der Krol, A., et al. 1988. An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature 333:866–869. van Houwelingen, A., et al. 1998. Analysis of flower pigmentation mutants generated by random transposon mutagenesis in Petunia hybrida. Plant J. 13:39–50. van Tunen, A. J., et al. 1988. Cloning of the two chalcone flavone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes. EMBO J. 7:1257–1263. van Tunen, A. J., et al. 1991. Regulation and manipulation of flavonoid gene expression in anthers of Petunia; the molecular basis of the Po mutation. Plant Cell 3:39–48. Vaucheret, H., and M. Fagard. 2001. Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet. 17:29–35. Vogelpoel, L. 1986. Flower colour—an appreciation. S. Afr. Orchid J. 17:109–113. Weiss, D., et al. 1993. The Petunia homologue of the Antirrhinum majus Candi and Zea mays A2 flavonoid genes; homology to flavanone 3-hydroxylase and ethylene forming enzyme. Plant Mol. Biol. 22:893–897. Wienand, U., et al. 1991. Molecular analysis of the dominant inhibitory C2 allele C2-Idf. Maize Genet. Coop. Newslett 65:51. Wiering, H., and P. deVlaming. 1984. Genetics of flower color and pollen colours. p. 49–75. In: K. C. Sink (ed.), Petunia. Springer-Verlag, Berlin. Winkel-Shirley, B. 2001. Flavonoid biosynthesis: a colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 126:485–493. Yamaguchi, T., et al. 2001. Genes encoding the vacuolar Na+/H+ exchanger and flower coloration. Plant Cell Physiol. 42:451–461. Yamazaki, M., et al. 2002. Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning, biochemical properties and developmental regulated expression. Plant Mol. Biol. 8:401–411. Yoshida, K., et al. 1995. Cause of blue petal colour. Nature 373:291. Yoshida, K., et al. 2000. Contribution of each caffeoyl residue of the pigment molecule of gentiodelphin to blue color development. Phytochemistry 54:85–92. Zhang, P., et al. 2000. A segmental gene duplication generated differentially expressed myb-homologous genes in maize. Plant Cell 12:2311–2322.

5 The Influence of Mitochondrial Genetics on Crop Breeding Strategies Sally A. Mackenzie Plant Science Initiative, University of Nebraska Lincoln, Nebraska 68588-0660

I. INTRODUCTION II. STRUCTURE OF THE MITOCHONDRIAL GENOME IN PLANTS III. CYTOPLASMIC MALE STERILITY A. Nuclear-Mitochondrial Interactions Underlying Male Fertility Restoration B. The Nature of Cytoplasmic Instability and Its Nuclear Influence IV. OCCURRENCE AND DEVELOPMENT IMPLICATIONS OF NUCLEARCYTOPLASMIC INCOMPATIBILITY V. SOME IMPLICATIONS OF CYTOPLASMIC GENETICS FOR THE PLANT BREEDER A. Cytoplasmic Male Sterility B. Cytoplasmic Diversity and Crop Evolution C. Emerging Directions for Plant Mitochondrial Research and Breeding Strategies LITERATURE CITED

I. INTRODUCTION For well over 50 years, plant breeders have known that the cytoplasm can have a marked influence on the outcome of particular crosses. Michaelis (1954) published a virtual treatise on the significance of cytoplasmic inheritance, using Epilobium as his model, that demonstrated a dramatic influence of the maternal parent on crossing behavior and, through an intricate analysis of nuclear substitution lines, Michaelis postulated that the cytoplasmic genotype (“plasmon”) could influence post-pollination development of interspecific hybrids. Present-day breeders of most crop species can recount examples, both documented and undocumented, of

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striking differences observed from reciprocal crosses. Some unilateral incompatibilities are known to result from pre-zygotic, stigmatic crossing barriers that are unassociated with cytoplasmic gene effects. However, more than 14 plant genera have been identified within the literature to show asymmetric, post-zygotic crossing failures as a likely consequence of cytoplasmic-nuclear interactions (Tiffin et al. 2001). In spite of the extensive documentation of cytoplasmic influence on crossing behavior, early investigations of cytoplasmic genetics in plants were relatively slow to provide meaningful insight. Attempts at detailed investigation of plant cytoplasmic-nuclear interactions must contend with the technical difficulties of isolating and manipulating plant organellar genomes, and of analyzing cytoplasmic traits within highly dynamic, heteroplasmic (mixed) populations of organelles. The past few years have seen important progress in our understanding of plant organelles, with the availability of genome sequence information and the emergence of more tractable genetic systems, often utilizing cytoplasmic male sterility mutations. In this review, I will not attempt to survey the many aspects of cytoplasmic inheritance in crops, but will concentrate on those recent advances that are most likely to influence a plant breeder’s approach to crop management. With the development of complete mitochondrial DNA sequence information for at least five plant species, Arabidopsis, rice, rapeseed, liverwort, and beet (Oda et al. 1992; Unseld et al. 1997; Kubo et al. 2000; Notsu et al. 2002; Handa 2003), it has become clear that the genetic information contained within organellar genomes is quite limited but essential to plant growth and development. The 367-kb mitochondrial genome of Arabidopsis encodes 57 genes, of which only 27 are protein coding, mostly for components of the energy transduction apparatus (Unseld et al. 1997). Mitochondrial gene collections appear to be fairly conserved throughout the Plant Kingdom, and cytoplasmic genetic variation, though common, produces relatively few distinct traits of agricultural importance. Perhaps the three most important cytoplasmic traits of interest across a spectrum of plant species are green-white leaf variegation (Tilney-Bassett 1991), valued by the ornamental horticulture industry, cytoplasmic male sterility (Schnable and Wise 1998), important for hybrid seed production, and unilateral crossing barriers, likely imposing constraints on hybridization strategies in some plant species (Tiffin et al. 2001). The relatively small number of distinct cytoplasmic traits that are now utilized agriculturally should not be interpreted as lack of breeder opportunity for exploitation of cytoplasmic genetic variation. A detailed review of the literature reveals a host of additional plant traits that are influenced by cytoplasmic factors, including plant disease resistance, components

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of yield, quality and combining ability, and tissue culture performance (Frei et al. 2003). Moreover, the targeted modulation of particular mitochondrial genes has resulted in intriguing new opportunities to alter plant metabolism (Mooney et al. 2002), starch biosynthesis (Jenner et al. 2001), and cold and oxidative stress (Breusegem et al. 1999).

II. STRUCTURE OF THE MITOCHONDRIAL GENOME IN PLANTS Although generally well conserved in coding capacity, the plant mitochondrial genome is known to vary dramatically in size across plant species, ranging from about 200 to over 2000 kb (reviewed by Palmer 1990; Wolstenholme and Fauron 1995). Remarkably, some of this size disparity is accounted for by the apparent accumulation of integrated sequences of nuclear, plastid, and viral origin (Unseld et al. 1997; Marienfeld et al. 1999). Mitochondrial DNA maintenance functions are not yet well understood in plants. Although physical mapping studies have suggested a circular genome structure, electron microscopic and pulsed-field gel electrophoresis studies have shown the genome to be largely comprised of linear molecules, some larger than predicted genome size (Backert et al. 1996; Bendich 1993, 1996; Oldenburg and Bendich 1996, 2001). Evidence suggests that the plant mitochondrial genome may replicate by a rolling circle mechanism that includes recombination-mediated replication initiation processes similar to the T4 phage (Backert and Borner 2000). Interestingly, the mitochondrion appears to share much of its DNA maintenance machinery with the plastid, and proteins associated with these processes are commonly dual-targeted (Elo et al. 2003). Essentially all of the genes involved in mitochondrial DNA replication, recombination, and mismatch repair are nuclear encoded. A curious observation was made during the recent localization of these components within the Arabidopsis nuclear genome sequence. It was found that the largest proportion of mitochondrial DNA and RNA maintenance loci yet identified are clustered on a single chromosome with a second, smaller gene cluster at a second genomic site (Elo et al. 2003). A similar pattern of gene clustering was noticed in the rice genome. The mitochondrial genome displays maternal inheritance in the majority of plant species, yet nuclear mitochondrial maintenance genes are biparentally contributed. Therefore, it has been suggested that a clustered organization for nuclear genes involved in these processes might provide the ability to selectively silence or co-regulate the paternally contributed loci. Such regulation might be important during the

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essential stages of embryogenesis at which nuclear-mitochondrial compatibility is established to allow for the massive increases in mitochondrial DNA replication that occur early in seed germination (Zlatanova et al. 1987). Certain complexity derives from a significant degree of recombination that seems to characterize nearly all plant mitochondrial genomes. This recombination activity appears to take two forms (Fig. 5.1). Highfrequency homologous recombination occurs at repeated sequences

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Fig. 5.1. Genomic rearrangements distinguishing the plant mitochondrial genome. I. Homologous intra- and intermolecular recombination occurs at repeated sequences that, when in direct orientation, can produce equilibrium of non-recombinant and recombinant subgenomic molecules. A–D designate distinct flanking sequences. II. Intragenic ectopic recombination can occur to produce sequence chimeras. What is shown is the simplest scenario; often these chimeric sequences are derived from multiple recombination and/or insertion events (Schnable and Wise 1998). The final population of molecules often does not include all parental and recombinant forms. III. Stoichiometric shifting represents a nuclear-directed process that can modulate the relative copy number of particular recombinant molecules within the genome, often reducing them to one copy per every 100–200 cells of the plant. The shifting mechanism is not yet understood.

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within the mitochondrial genome that, when in inverted orientation, produce sizeable genomic inversions. DNA exchanges at repeated sequences in direct orientation permit subdivision of the genome to subgenomic molecules, each containing only a portion of the genetic information. Such recombination results in a highly redundant genome configuration, and allows the retention of mitochondrial genes in multiple genomic environments (Fauron et al. 1995). Although the complexity of these genomes has been known for many years, it is not yet clear whether the resulting individual subgenomic DNA molecules are retained by a process of constant recombination, or whether they might acquire the ability to replicate autonomously. A second type of recombination activity has been important to mitochondrial genome evolution in higher plants. Low-frequency nonhomologous recombination has been documented in a wide spectrum of plant species. Recombination often occurs within gene sequences, resulting in unusual gene sequence chimeras. Oddly, the majority of sequence chimeras that have been reported in plant mitochondria appear to be expressed, giving rise not only to transcripts, but also to aberrant translation products in many cases. Nonhomologous recombination activity appears to occur at low frequency throughout the mitochondrial genome, and has been documented at sites sharing as few as 4–7 nucleotides of homology (Andre et al. 1992; Marienfeld et al. 1997). This activity may account for the mitochondrial genome variation that commonly arises in cell suspension cultures, alloplasmic lines, and protoplast fusion experiments. The resulting gene chimeras are often associated with cytoplasmic male sterility (CMS) and abnormal growth mutants (Newton 1995). In fact, alloplasmy, which arises by recurrent backcrossing to effect interspecific nuclear-cytoplasmic substitutions, has produced numerous examples of CMS (Kaul 1988).

III. CYTOPLASMIC MALE STERILITY CMS is a maternally inherited form of anther dysfunction or pollen sterility that is conditioned by mutations within the mitochondrial genome. Maternally inherited male sterility has been observed in members of nearly every plant family, and the phenotypes can range from premature tapetal degeneration (Warmke and Lee 1977) to incomplete cytokinesis (Abad et al. 1995) to gametophytic abortion (GabayLaughnan et al. 1995; Pring et al. 1999). Likewise, the mitochondrial genetic lesions conditioning the CMS phenotype differ in each case studied. What is common among nearly all cases of CMS examined in

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detail is the association of male sterility with mitochondrial gene chimeras that, almost without exception, encode aberrant proteins with a hydrophobic amino terminus or at least one membrane-spanning domain (Schnable and Wise 1998). The unusual sterility-associated rearrangements can produce proteins capable of conferring new properties to the mitochondrial membrane (Dewey et al. 1988) or to the developing pollen itself (Abad et al. 1995). Detailed analysis of CMSassociated genetic lesions has been accomplished in a number of species, including CMS-T maize (Dewey et al. 1986), CMS-S maize (Zabala et al. 1997), three Brassica cytoplasms (Singh and Brown 1991; Bonhomme et al. 1992; L’Homme and Brown 1993; L’Homme et al. 1997), common bean (Chase and Ortega 1992; Johns et al. 1992), petunia (Young and Hanson 1987), sorghum (Tang et al. 1996), tobacco (Gutierres et al. 1997), rice (Iwabuchi et al. 1993), and sunflower (Laver et al. 1991). Two features of a CMS system are essential for viable hybrid seed production strategies. The mutant cytoplasm must be amenable to stable maintenance on a wide array of genetic backgrounds, and a nuclear genotype that fully suppresses the cytoplasmic mutation in the F1 generation must be available. Partial restoration of fertility in the F1 generation directly impacts the grower’s yield, while instability of the cytoplasm complicates the breeder’s ability to recapture a uniformly male-sterile population. Given the tremendous yield potential of hybrids, coupled with the value of the male sterility trait in preventing pollen escape in transgenic crops (Hails 2000), further research into fertility restoration mechanisms as well as cytoplasmic reversion has been warranted. A. Nuclear-Mitochondrial Interactions Underlying Male Fertility Restoration A fascinating aspect of the CMS phenomenon is the widespread demonstration that male sterility-inducing mitochondrial mutations can be suppressed by nuclear gene action. Nuclear fertility restorer genes are generally dominant in action. The most useful, breeder-friendly systems involve a single nuclear gene that restores normal pollen production within the F1 generation. Such restorer systems exist for most of the current CMS types implemented in hybrid seed production. CMS-T maize, sorghum, sunflower, and onion systems are exceptions, with two dominant nuclear genes required to fully restore normal pollen production. In most cases, DNA marker systems can be used to map the fertility restorer loci and facilitate their efficient transfer to new inbred genotypes (Wise and Schnable 1994; Zhang et al. 1997).

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The mechanisms underlying fertility restoration are of particular interest. From the perspective of the molecular biologist, this area of investigation has been important to our understanding of the nature of nuclear-mitochondrial genetic interaction, and to the breeder’s benefit, the identification of fertility restorer mechanisms provides powerful strategies to search for new restoration loci. The first restorer of fertility (Rf) gene to be cloned and characterized was the Rf2 locus that participates in restoring fertility to CMS-T maize. Rf2 was found to encode an active aldehyde dehydrogenase (Cui et al. 1996; Liu et al. 2001), but presence of the restorer allele does not appear to directly influence expression of the mitochondrial sterility-associated T-urf13 sequence. Furthermore, the Rf2 allele is widely present in maize germplasm, implying its essential role in other plant functions in addition to fertility restoration (Schnable and Wise 1994). In this regard, it has been postulated to serve as a “compensatory restorer,” to counteract the metabolic disorders that arise with the expression of the T-urf13 sequence within the tapetal layer (Liu et al. 2001). One envisions that in this two-gene restorer system, the Rf1 allele mediates the accumulation of novel T-urf13 transcripts, reducing but not completely eliminating gene expression, while the product of Rf2 modulates the cellular influence of the TURF13 protein that accumulates. The role of Rf2 is distinct from that of other characterized fertility restorers in its indirect interaction with the mitochondrial sterility sequence. The majority of the fertility restorers studied in detail appear to directly influence expression of the mitochondrial lesions. The gene modulation most often occurs post-transcriptionally, altering the levels and/or species of transcripts, or post-translationally to effect more rapid turnover of the sterility-inducing protein. The most common restorer influences on mitochondrial transcript stability appear to affect transcript processing and/or editing, such as in maize CMS-T by Rf1 (Wise et al. 1996), sorghum (Pring et al. 1998), rice (Iwabuchi et al. 1993), and oilseed rape (Li et al. 1998; Brown 1999). Over the past two years, what is perhaps the most important advance in this field was made with the cloning of a fertility restorer gene in petunia, followed a year later by radish. CMS in petunia is associated with expression of a 1200-bp chimeric open reading frame designated pcf (petunia CMS-associated fused). This gene chimera is comprised of the 5' sequence of atp9 fused to part of the first and second exons of the coxII gene, together with unidentified sequences (urfS). In the presence of Rf, the accumulation of pcf-derived transcripts is reduced, as is the accumulation of the 25-kDa PCF protein (Hanson et al. 1999). Map-based cloning of the gene was carried out, permitting the identification within

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the restorer gene product of 14 repeats of a 35-amino acid pentatricopeptide repeat (PPR) motif (Bentolila et al. 2002). PPR protein genes comprise an unusually large gene family of over 200 members in Arabidopsis, with most predicted to encode proteins that are organellar targeting (Small and Peeters 2000). These unusual proteins are postulated to be involved in protein and RNA binding, given their helical-repeat structural features, and to possibly mediate the intricate, gene-specific RNA processing functions that occur in plant mitochondrial transcripts (Small and Peeters 2000). Following the identification of the petunia gene, map-based cloning of fertility restorer genes for CMS kosena radish (Koizuka et al. 2003) and Ogura radish (Brown et al. 2003; Desloire et al. 2003). Interestingly, this restorer gene is also predicted to encode a PPR protein. However, in this case the restorer protein is thought to inhibit translation (Koizuka et al. 2000) and may even bind directly to the sterility-associated orf125 protein (Koizuka et al. 1998). Given the number of examples of fertility restorers that appear to directly influence post-transcriptional processing and translation of CMS-associated sequences, it appears likely that PPR protein genes will comprise a number of restorer loci (Wise and Pring 2002). Although details of PPR protein-mediated suppression of mitochondrial gene expression are not yet defined, the plant breeding community may be able to capitalize on this recent advance. Various strategies will likely emerge to survey a plant genome for PPR protein-encoding genes, allowing the identification of candidate fertility restorers (Wise and Pring 2002). Comparative analyses utilizing the available Arabidopsis and rice genomic sequence databases should greatly facilitate these efforts. B. The Nature of Cytoplasmic Instability and Its Nuclear Influence Several years ago, a second form of fertility restoration was reported that differed in its genetic behavior from what had been reported in other crops. This second type of fertility restoration, identified in common bean (Phaseolus vulgaris), results in apparently permanent, nonsegregating fertility associated with mitochondrial genome rearrangement (Mackenzie and Bassett 1987; Mackenzie et al. 1988; Mackenzie and Chase 1990). Remarkably, the mitochondrial genomic rearrangement results in the elimination of the sterility-associated sequence from most plant cells. Although the discovery of a single dominant nuclear gene effect that directs reproducible mitochondrial DNA alteration was unprecedented at the time, the nature of the mitochondrial rearrangement was not.

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Plant mitochondrial genomes, in their commonly multipartite, interrecombining configurations, are prone to a phenomenon referred to as stoichiometric shifting. Stoichiometric shifting was first detailed by Small et al. (1987) as a process by which the plant mitochondrial population arrives at a stable condition of heteroplasmy, or cytoplasmic heterogeneity. Due, in part, to the prevalent redundancy in mitochondrial coding capacity, the mitochondrial genome configuration can vary cell to cell, and tissue to tissue, during the development of a plant. Relative copy numbers for various portions of the mitochondrial genome differ dramatically, with some subgenomic mitochondrial DNA molecules estimated at as low as 1 copy per every 200 cells (Arrieta-Montiel et al. 2001). These low copy molecules are apparently silent but are stably maintained through gametogenesis. The up and down modulation of copy number for particular portions of the mitochondrial genome, a nuclear-controlled process, provides the plant with the capacity to influence its own cytoplasmic genotype, and that of its progeny. Mitochondrial substoichiometric intermediates, termed sublimons, have been reported in a wide variety of plant families (Mackenzie and McIntosh 1999), and the copy number shifting process has been demonstrated to occur as a consequence of plant tissue culture (Vitart et al. 1992; Kanazawa et al. 1994), protoplast fusions (Sakai and Imamura 1993; Bellaoui et al. 1998), and wide hybridization (Tsunewaki 1993). Although mitochondrial genome configuration is shown to be influenced by nuclear genotype in other plant systems (Newton and Coe 1986) and is likely widespread, the specific control of mitochondrial substoichiometric shifting by a single nuclear locus has been documented in only two plant systems to date. In CMS common bean, the nuclear gene Fr was shown to effect fertility restoration by directing the elimination of the portion of the mitochondrial genome that encompasses the male sterility determinant pvs-orf239 from most plant cells (Mackenzie and Chase 1990). The 239amino acid protein ORF239, when present, can accumulate in developing microsporocytes to produce a condition of incomplete cytokinesis (Johns et al. 1992; Chase and Ortega 1992). Pollination of the CMS line with the fertility restorer (FrFr) results in only incomplete restoration in the first generation, a condition termed semi-sterility, with full fertility restoration segregating in the F2 generation. Given the influence of the Fr gene on mitochondrial genome configuration, one presumes that the semi-sterile condition arises as an intermediate, heteroplasmic condition while the mitochondria undergo cytoplasmic sorting. In Arabidopsis, mutation of the single dominant nuclear gene chloroplast mutator (CHM) results in the copy number amplification of a mitochondrial gene chimera that produces green-white leaf variegation

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(Martinez-Zapater et al. 1992; Sakamoto et al. 1996). The effect of chm in causing leaf variegation is non-segregating in following generations regardless of CHM genotype, similar to the effect of Fr in bean. In bean and Arabidopsis, expression of the dominant form of the genes Fr or CHM directs elimination of the mutant chimeric mitochondrial sequence from most plant cells, and loss of Fr or CHM function permits the reamplification of the mitochondrial mutation. These features of Fr and CHM behavior suggest that their nuclear gene products somehow act to suppress copy number of the mitochondrial gene chimeras or, alternatively, suppress the recombination activity that gives rise to such molecules. The striking similarity in behavior of Fr and CHM also implies a conservation of this function across plant families. The numerous reports of mitochondrial substoichiometric forms in plants suggest that this type of nuclear-mitochondrial interplay occurs widely throughout the Plant Kingdom. Following the discovery of sublimons within the mitochondrial genome, debate has continued regarding the likely means by which these unusual genomic forms are maintained during plant development. In some instances in which a chimeric sequence was found on a sublimon, a portion of the rearranged gene was comprised of unique sequences not found elsewhere within the genome, or one of the predicted reciprocal products of recombination was absent. These observations, and detailed analysis of the parental forms predicted to give rise to the observed recombinant, led investigators to the conclusion that such present-day molecular forms are not likely maintained by a recurring process of recombination (Atlan and Couvet 1993; Albert et al. 1996). Thus, several reports initially postulated their retention by autonomous replication (Janska and Mackenzie 1993; Kanazawa et al. 1994). However, an alternative explanation was offered early in the debate by Lonsdale et al. (1988), who postulated that the entirety of the mitochondrial genomic information, referred to as the “master” form, was not likely present uniformly in all cells of the plant. In fact, later observations showed that mitochondrial DNA content and organization appears to differ in meristematic tissues of the plant (Fujie et al. 1993, 1994), lending impetus to Lonsdale’s theory. It is possible that substoichiometric forms are, in fact, generated by de novo recombination events (Bellaoui et al. 1998) that occur within a limited cell type in the plant, with subsequent distribution to vegetative cells occurring under stochastic cytoplasmic sorting processes. As a means of investigating the nature of the substoichiometric shifting event, the CHM locus was identified using a map-based cloning strategy. A locus comprised of 22 exons, the CHM gene encodes a prod-

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uct that resembles the E. coli MutS component of the mismatch repair apparatus (Abdelnoor et al. 2003). The MSH1 product is predicted to be organellar targeting. These observations are especially intriguing because the mismatch repair apparatus of yeast has been shown to influence not only the repair of nucleotide mismatches, but the suppression of nonhomologous recombination (Chen and Jinks-Robertson 1999; Harfe and Jinks-Robertson 2000; Welz-Voegele et al. 2002). Upon discovery of the CHM sequence, the gene was renamed AtMSH1 in keeping with mismatch repair nomenclature, and its mutation was postulated to result in enhanced nonhomologous recombination activity to give rise to novel mitochondrial DNA molecular forms. The identification of MSH1 in plants is likely to be of significance to plant breeding efforts in two important regards. Nonhomologous recombination activity is thought to be the means by which CMS-associated gene chimeras arise. Consequently, the cloning and characterization of MSH1 may facilitate experimental strategies to induce new CMS mutations. This opportunity would be of particular interest in crops in which no natural source of CMS has yet been identified for hybrid seed production. Moreover, substoichiometric shifting appears to be the process underlying spontaneous reversion to fertility in unstable CMS lines (Bonhomme et al. 1991; Janska et al. 1998; Bellaoui et al. 1998; Chowdhury and Smith 1988). Cytoplasmic instability is a common problem for many CMS sources when in combination with particular nuclear backgrounds (McVetty 1998). Consequently, it may now be feasible to assess MSH1 allelic variation as a means of selecting genotypes with enhanced cytoplasmic stability.

IV. OCCURRENCE AND DEVELOPMENTAL IMPLICATIONS OF NUCLEAR-CYTOPLASMIC INCOMPATIBILITY Nuclear-cytoplasmic incompatibility in plants can arise by various means to produce a range of interesting phenotypes. One common crossing strategy to effect nuclear-cytoplasmic incompatibility substitutes nuclear genotype from one species onto the cytoplasm of another using a recurrent backcrossing strategy to create alloplasmy. This process can result in mitochondrial genomic rearrangements and cytoplasmic male sterility (Kaul 1988). In carrot, tobacco, wheat, and Plantago, male sterility is accompanied by aberrant floral morphologies (VanDamme 1983; Bonnett et al. 1991; Kitagawa et al. 1994; Farbos et al. 2001; Murai et al. 2002). In the case of tobacco, subsequent transgenic manipulation of established anther development pathways in the male-sterile line can

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restore pollen development (Bereterbide et al. 2002). In carrot, evidence has been found that the induced mitochondrial genomic changes influence expression of genes that are known to direct floral developmental programs (Linke et al. 2003). In maize and Arabidopsis, genetic disruption of nuclear-cytoplasmic signals can result in leaf variegation and, in the case of nonchromosomal stripe mutants of maize, severe stunting of the plant (Newton 1993). It has been suggested that the occurrence of a green-pale green phenotype in association with mitochondrial lesions might reflect the intimate inter-connections of mitochondrial and plastid functions (Roussell et al. 1991). Such organellar inter-dependence has been further substantiated recently with the demonstration of dual targeting to mitochondria and plastids by MSH1 (R. V. Abdelnoor, R. Yule, and S. A. Mackenzie, unpublished) and several other components of the organellar DNA and RNA metabolism apparatus in plants (Elo et al. 2003). An intriguing phenomenon that we are now learning may be associated with nuclear-cytoplasmic incompatibility is the emergence of unilateral crossing barriers. Unilateral crossing barriers can be classified as pre- or post-fertilization in their action. The majority of characterized examples of unilateral incompatibility in plants have been pre-fertilization, effected by expression within the pistil of proteins that preclude pollination by particular genotypes (Hogenboom 1975; deNettancourt 1977). These types of barriers are particularly well characterized in the Solanaceae and Brassicacae (Dhaliwal 1992) but are observed in many other species (Rashid and Peterson 1992). Unilateral post-zygotic crossing barriers are also observed in nature (Tiffin et al. 2001). Such crossing barriers are prevalent but not well characterized genetically. In common bean and Arabidopsis, alterations at Fr or CHM (MSH1) loci to effect mitochondrial substoichiometric shifting produce not only mitochondrial genomic rearrangement with the accompanying pollen or leaf phenotypic changes, but an asymmetrical crossing barrier as well (A. Elo, M. Arrieta-Montiel, and S. Mackenzie, unpublished). Gene disruption of the MSH1 locus of Arabidopsis (Col-0) by an EMS-induced mutation or T-DNA insertion produces the characteristic chm mutant phenotype (Abdelnoor et al. 2003). This phenotype is readily transferred by pollinating wildtype Col-0 ecotype with the derived mutant, producing variegation and mitochondrial genomic rearrangement in approximately a quarter of the resulting F2 progeny. This is the case with all chm mutants tested as chm1-1 and chm1-2 (Redei 1973), and chm1-3 (MartinezZapater et al. 1992). However, attempts to pollinate the chm mutant with wildtype Col-0 pollen can result in poorly developed, prematurely yel-

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lowing siliques, reduced seed set, a 50% reduction in seed germination, and production of severely stunted, malformed plants from germinating seed (Sakamoto et al. 1996; A. Elo and S. Mackenzie, unpublished). In common bean, a similar phenomenon is observed. A line that has undergone mitochondrial genomic shifting (pvs-orf239 substoichiometric) serves as an efficient pollinator to fertile accession line G08063 (Pvs-orf239 amplified), resulting in nearly 100% seed set. Efforts to carry out the reciprocal cross result in pod abortion and poor seed set (M. Arrieta-Montiel and S. Mackenzie, unpublished.). In both Arabidopsis and bean, the crossing barrier dissipates with substitution of the wildtype allele at the MSH1 or Fr locus, implying that the barrier arises as a consequence of disrupted nuclear-cytoplasmic interactions. V. SOME IMPLICATIONS OF CYTOPLASMIC GENETICS FOR THE PLANT BREEDER A. Cytoplasmic Male Sterility Cytoplasmic male sterility has, to date, remained the single most valuable cytoplasmic trait from a plant breeding perspective. However, the CMS trait has not been available in natural populations of several crop species for which it has been sought, including soybean and tomato. With the increasing utility of transgenic traits for crop enhancement, the demand for multiple sources of stable male sterility in a variety of crops will likely increase. This is because implementation of CMS to the production of transgenic crops provides the opportunity to constrain transgenic pollen transfer to neighboring areas (Hails 2000), and facilitates the management of the proprietary transgene. Several plant breeding strategies have been reported for yieldenhancing hybrid development utilizing CMS. For most plant species, breeding efforts with CMS are focused primarily on the incorporation of a broad diversity of genotypes for hybrid production, and the inclusion of DNA marker-based approaches to follow cytotype (Bach et al. 2002; Engelke et al. 2003) or restorers. In crops where a stable CMS source has been available for several years, more advanced breeding strategies are sought for further yield enhancement. In maize, recent studies suggest that the CMS trait can provide a yield advantage (Stamp et al. 2000). Performance was generally higher in lines carrying the CMS cytotype when isonuclear hybrids were tested with and without CMS. With modification to hybrid planting schemes that encourage cross-pollination of the hybrid, this advantage can be

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further increased by exploiting a xenia effect (Weingartner et al. 2002). The “Plus-Hybrid” effect capitalizes on the advantage conferred by the CMS cytoplasm in combination with the xenia effect that comes from pollination with a genetically dissimilar pollen source. To achieve this enhanced performance, the system blends a non-restored CMS-hybrid with an unrelated fertile hybrid as pollinator. In a predominantly selfpollinating species like rape, introduction of CMS in the development of synthetic varieties can provide enhanced heterozygosity to the population by greatly reducing self-pollination (Atlin 1995). In millet, in which CMS has been used widely in hybrid production, and at least eight distinct male sterility-inducing cytoplasms exist, it has been feasible to evaluate the influence of cytoplasm on plant productivity. The combination of various nuclear-cytoplasm types in CMS millet breeding produces a significant range of phenotypic variation, with particular influence on general and specific combining ability (Virk and Brar 1993). Consequently, a variety of CMS sources may prove valuable for optimizing the selected phenotype and hybrid. To pursue the development of multiple CMS sources in a given crop, strategies exist to enhance mitochondrial recombination. These include protoplast fusion and development of interspecific alloplasmic genotypes by recurrent backcrossing. Cytoplasmic male-sterile mutants have arisen by both approaches. In fact, alloplasmic substitution is the only means to date for inducing CMS in crops for which no natural source has been identified, such as tomato (Melchers et al. 1992) and soybean (Sun et al. 1997; Smith et al. 2002). The recent cloning of MSH1, and the demonstration of significant sequence conservation for this gene across a variety of plant species, offers another approach for CMS induction in crops of interest. Anti-sense or RNA interference (RNAi)-mediated suppression of MSH1 expression, as a means of enhancing mitochondrial recombination activity, could prove an effective strategy for producing novel CMS mutations. A common problem for the practical implementation of CMS in a breeding and hybrid seed production program is cytoplasmic instability, the spontaneous reversion to fertility. This phenomenon generally involves production of a variable number of seed on otherwise malesterile plants, often manifested as sectoring. Generally, reversion is a cytoplasmic event, although both cytoplasmic and nuclear instability have been described in the CMS-S cytoplasm of maize (Laughnan and Gabay-Laughnan 1983; Gabay-Laughnan et al. 1995). When the cytoplasmic event has been limited to the pollen, the revertant seed produced will give rise to male sterile plants, a relatively minor problem for hybrid seed production. When the reversion event occurs early enough

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in development to encompass both ovule and pollen production, the revertant seed gives rise to male fertile plants, complicating stable maintenance of the male sterile line. In pearl millet, with multiple distinct male sterile cytoplasms, the majority of these CMS types are not implemented to any significant extent due to instability (Rai et al. 1996). Cytoplasmic instability has also hindered deployment of CMS in other crops (McVetty 1998). The frequency of spontaneous reversion is influenced by nuclear genotype. Therefore, opportunities for the breeder to screen for nuclear genotypes conducive to cytoplasmic stability are essential to effective utilization of CMS for hybrid seed production. Efforts to genetically map nuclear genes contributing to cytoplasmic instability have not been described to date, although the recently cloned MSH1 gene likely represents one of the nuclear genes contributing to the reversion process. To address the problem of reversion, most breeders have attempted to identify nuclear genotypes that enhance cytoplasmic stability by recurrent backcrossing regimes. An alternative strategy would be to screen for lines in which the mitochondrial male sterility determinant is present in a different genomic environment. In some plant species, including maize, common bean, sunflower, and radish, the CMS determinant is prevalent within a wide diversity of undomesticated or domesticated germplasm and different forms of CMS exist within a species (Manicacci et al. 1997; Arrieta-Montiel et al. 2001; Ducos et al. 2001; Yamagishi and Terachi, 2001; Horn 2002;). In common bean, it was shown that screening for variation in the mitochondrial genomic environment immediately surrounding the CMS determinant can identify lines in which reversion-associated substoichiometric shifting does not occur (ArrietaMontiel et al. 2001). B. Cytoplasmic Diversity and Crop Evolution Although cytoplasmic mutations can arise as a consequence of interspecific hybridization and protoplast fusion manipulations, most CMS sources are naturally arising. Cytoplasmic male sterility has likely played an important role in the evolution of gynodioecious species (Frank 1989; Budar and Pelletier 2001; Budar et al. 2003) and may have facilitated outcrossing during the evolution and domestication of selfpollinating plant species. Efforts to trace the origin of CMS cytoplasms in various crop species often reveals broad distribution of the male sterility determinant in undomesticated populations. The presence of a CMS-inducing cytoplasm within natural populations is important to female:hermaphrodite equilibria maintained by natural gynodioecious populations (McCauley

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et al. 2000; Olson and McCauley 2002; Budar et al. 2003). In common bean, studies of crop domestication have suggested that sporadic outcrossing likely occurred during the process of domestication (Papa and Gepts 2003). This observation might predict a role for the male sterility trait, and perhaps accounts for the high degree of sequence conservation observed for the pvs-orf239 sterility-associated sequence in wild selections (Hervieu et al. 1993; Arrieta-Montiel et al. 2001). With more detailed study of the evolution of nuclear fertility restorer genes, a pattern may be emerging. For many cases in which multiple fertility restorer genes have been identified and mapped, the genes appear to be linked. This is the case in Brassica (Jean et al. 1997), cotton for restorers D8 and D2-2 (Zhang and Stewart 2001), in common bean, in which restorers Fr and Fr2 are linked (Jia et al. 1997), CMS-T maize for Rf8 and Rf* (Dill et al. 1997), and in CMS-S cytoplasm maize, in which several of the spontaneously arising restorers have been linked to restorer Rf3 (Gabay-Laughnan et al. 1995). In petunia and Brassica, in which fertility restorer genes have been cloned, duplication of the gene sequence has been observed in the region (Bentolila et al. 2002; Koizuka et al. 2003; Brown et al. 2003). One explanation for the widely observed linkage of fertility restorers might simply be gene duplication. However, nuclear genes that are involved in mitochondrial DNA and RNA metabolism functions have been found to cluster within the genome of Arabidopsis and rice (Elo et al. 2003). Therefore, it may also be that genes of related organellar function cluster within the genome as a means to facilitate their coordinate regulation. C. Emerging Directions for Plant Mitochondrial Research and Breeding Strategies The recent identification and cloning of genes that regulate CMSassociated mitochondrial gene expression and mitochondrial genome configuration opens the field of plant mitochondrial genetics to important avenues for the transgenic manipulation of CMS in crops. To date, research in these areas has remained hampered by the unavailability of a reliable mitochondrial transformation system. Nevertheless, several interesting approaches exist for modulating mitochondrial functions in plants via nuclear gene manipulation. The discovery of a large number of PPR protein genes in Arabidopsis that are predicted to encode organellar proteins represents a significant breakthrough. Several of these are likely to be potential sources of fertility restoration for newly emerging CMS systems. The transgenic induction of cytoplasmic male sterility may now be feasible with the

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identification of MSH1 in plants. It has already been shown that the disruption of MSH1 expression in Arabidopsis directs a precise and reproducible substoichiometric shifting event (Abelnoor et al. 2003). It remains to be seen what effect MSH1 manipulation will have in other plant species. Advances in our ability to target transgenic proteins to the organellar compartments of the cell will also provide new avenues for the manipulation of organellar functions. Moreover, recent advances in the manipulation of gene expression within isolated mitochondria (Farre and Araya 2001), and in the introduction of intact mitochondrial genomes to E. coli (Yoon and Doob 2003) provide enticing glimpses of some of the technologies likely to be implemented in the future to facilitate our understanding of organellar genome functions.

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6 Genetic and Cytoplasmic-Nuclear Male Sterility in Sorghum* Belum V. S. Reddy and S. Ramesh Global Theme—Crop Improvement, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India Rodomiro Ortiz International Institute of Tropical Agriculture (IITA), Eastern and Southern Africa Regional Center (ESARC), Plot 7, Bandali Rise, Bugolobi, Kampala, Uganda

I. INTRODUCTION II. GENETIC MALE STERILITY (GMS) III. CYTOPLASMIC-NUCLEAR MALE STERILITY (CMS) A. Origin of CMS Systems B. Induction of CMS Systems C. Inheritance of Fertility Restoration D. Diversity Assessment 1. Classical Method 2. Molecular Markers IV. MOLECULAR CHARACTERIZATION OF CYTOPLASMS V. DNA POLYMORPHISM AND MAPPING RESTORER GENES VI. FACTORS INFLUENCING CMS SYSTEMS USE A. Stability of CMS Systems B. Effect of CMS Systems on Economic Traits C. Restorer Gene Frequency D. Cytoplasm Effects on Heterosis for Economic Traits

*The financial grant from the Dr. Surinder (Suri) M. Sehgal Foundation, India, to undertake this research is gratefully acknowledged. Plant Breeding Reviews, Volume 25. Edited by Jules Janick. ISBN 0471666939 © 2005 John Wiley & Sons, Inc. 139

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VII. DIVERSIFICATION OF CMS SYSTEMS A. Germplasm Base and Traits B. Other Cytoplasms C. Information Management and Knowledge Sharing through the Website D. Effect of Genetic Background E. Seed Parents’ Purity VIII. HETEROSIS AND HYBRID DEVELOPMENT IX. CONCLUSION LITERATURE CITED

I. INTRODUCTION Sorghum [Sorghum bicolor (L.) Moench, Poaceae] is the fifth most important cereal crop in the world after wheat, rice, maize, and barley. It is cultivated in the semi-arid tropics in 86 countries (FAO 1998). de Wet and his colleagues suggested that the cultivated sorghum had a diverse origin and probably arose from S. verticilliflorum, which is found in sorghum cultivated areas (House 1985) and that the domestication began around 3000 BCE (Doggett 1965). The cultivated sorghum taxa have been classified into five basic races (bicolor, caudatum, durra, guinea, and kafir), and 10 hybrid races (e.g., bicolor-caudatum) by Harlan and de Wet (1972). The inflorescence of sorghum is a panicle consisting of racemes with one or several spikelets, which are either sessile or pedicellate. The sessile florets are bisexual, while the pedicellate are staminate and may rarely have a rudimentary ovary. The seed or caryopsis is usually the product of self-fertilization in sessile florets. However, there is 5 to 15% of out-crossing depending on the wind direction, nature of genotype, and humidity (House 1985). Discovery of genetic male sterility (GMS) and cytoplasmic-nuclear male sterility (CMS) facilitated the application of recurrent selection procedures and hybrid cultivar development methods, respectively, in sorghum improvement. In the following sections, the discovery, inheritance, and utilization of GMS and CMS in sorghum improvement are discussed.

II. GENETIC MALE STERILITY (GMS) Genetic male sterility (GMS) in sorghum is imparted by (1) absence or degeneration of pollen grains, (2) lack of viable pollen, or (3) indehiscent anthers. In the first two situations, the anthers are small, scaly, and whitish, while the indehiscent anthers look normal, yellowish, and

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plumpy (Reddy 1997a). Thus, genetic male sterility in sorghum is expressed in many ways. Several sources of genetic male sterility have been reported from both India and the United States, and in all cases it was shown that a recessive allele in homozygous condition designated with a series of alleles such as ms1 to ms7 and all confer male sterility (Table 6.1). El’konin (2000) reported a dominant GMS mutation induced in sorghum tissue culture. The goal of most population improvement programs across the globe is to accumulate favorable alleles for the traits of interest, while maintaining as much genetic diversity as possible. The recurrent selection methods used for such purposes require extensive hybridization. Because sorghum is a self-pollinated crop, there was relatively little effort in population improvement in sorghum. However, the discovery of GMS and the advantage of various mating systems and reciprocal recurrent selection methods in exploiting additive (A) and A × A and other epistatic genetic variation (Comstock and Robinson 1952; Eberhardt 1972), led many breeders to adopt population improvement methods (Maunder 1972; Doggett 1972) in the 1960s. In sorghum, both ms3 and ms7 alleles induced male sterility have been extensively used in population improvement, as they are stable across locations and seasons (Reddy and Stenhouse 1994; Murthy and Rao 1997). The Ethiopian cultivar “Melka-mash” ensued from a population improvement program (Murthy and Rao 1997). In Nigeria, the national sorghum breeding program developed six random mating populations between 1963 and 1978 using ms7 gene (Obilana and El-Rouby 1980). Only three of these (B composite, Y composite, and YZC composite) were used

Table 6.1. Genetic male sterility genes, their designated symbols and mechanism of sterility. Source: Adapted from Rooney (2000). Gene symbol ms1 ms2 ms3 ms4 ms5 ms6 ms7 al

Mechanism

Reference

Normal pollen is dominant over aborted or empty pollen cells ″ ″ ″ ″ Empty pollen cells Aborted pollen Micro anthers without pollen Empty pollen cells Antherless stamens

Ayyangar and Ponnaiya (1937) Stephens (1937) Webster (1965) Ayyangar (1942) Barabas (1962) Barabas (1962) Andrews and Webster (1971) Karper and Stephens (1936)

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as base populations for grain yield improvement, while a fourth, MSRC (modified Striga resistant composite), was used in recurrent selection for Striga resistance. After three cycles of mass selection in these populations, a gain of 38% and 40.4% for grain yield were observed in B and Y composites, respectively (Lukhele and Obilana 1980; Obilana and El-Rouby 1980). In Southern Africa, the four random mating populations developed jointly by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) and the National Agricultural Research Systems (NARS) using the ms7 gene provided broad genetic-based gene pools from which NARS of the Southern African Development Community (SADC) could breed improved lines and new cultivars using recurrent selection (Obilana 1989). Two random mating broad genetic-based sudan grass populations (NP34 and NP35) and two grain sorghum populations (NP36 and NP37) developed co-operatively by the United States Department of Agriculture–Agricultural Research Services (USDA–ARS) and the Nebraska Agricultural Research Division with the ms3 gene were released in 1989 (Gorz et al. 1990a; Gorz et al. 1990b). NP34 is a good source of agronomically desirable R-lines and hydrocyanic acid potential (HCN-P), whereas NP35 is a good source of low-dhurrin. Both NP36 and NP37 were selected for reduced dhurrin content and they are valuable sources for A- and R-lines of grain sorghum. NP37 also possesses the brown midrib-6 gene and therefore may be useful in producing sorghum-sudangrass or forage sorghum hybrids with brown mid-rib trait. Although population improvement programs are not the most common in sorghum breeding, they are an important source of genetic variation and improved traits (Rooney and Smith 2000). In GMS-facilitated population improvement programs, the breeder must take care to ensure that the alleles that cause male sterility are not eliminated. Sorghum improvement at ICRISAT (Patancheru, Andhra Pradesh, India) was initiated in 1972 with population improvement using male sterility induced by the ms3 gene following recurrent selection procedures to breed for wider adaptability. By 1980, the emphasis was shifted to specific adaptation and several specific disease and pest-resistant gene pools with ms3/ms7 male-sterile genes following half-sib/s1/s2 testing procedures. In the 1990s trait-specific gene pools improvement using ms3 and ms7 genes through simple mass selection alternated with recombination methods became the cornerstone of developing diverse breeding materials (Reddy et al. 2003). A total of 19 populations were developed at ICRISAT using ms3 and ms7 genes into which 501 diverse germplasm accessions were introgressed (Table 6.2). At ICRISAT, both ms3 and ms7 alleles are being maintained in different bulks.

Table 6.2.

No.

Details of the sorghum breeding populations developed and maintained by ICRISAT at Patancheru (Andhra Pradesh, India).

Name of population

Gene

Original population

Traits/lines introgressed

Present cycle

Suggested use

Expected products

1

ICSP HTz

ms3

US/B C6

Sudangrass lines, early maturing lines, tillering lines, good grain characteristics, sweet stalk lines, DM, AN, LB, resistant lines, tall maturity & tillering

C4

Tillering ability with high biomass & sweetness

Cvs. & restorers

2

ICSP LGz population (rainy season)

ms3

US/B C6

Large grain lines, early maturing lines, short height, shoot pest populations, large grain lines

C2

Large grain & high yielding

Cvs. & restorers

3

ICSP LGz population (post-rainy season)

ms3

US/B C6

M 35-1, SPV 1359, M 35-1-19, M 35-1-36, NTJ 2 & others

C1

Large grain & high yielding

Cvs. & restorers

4

ICSP Bz (rainy season)

ms3

US/B C6

QL3, 286B, DM, midge resistant lines, SF & SB resistant lines, large grain durra lines, M 35-1, stay-green B-lines, SF B-lines

C3

Source for new cytoplasmic male-sterile lines

Large grain, dwarf Blines for rainy season

5

ICSP Bz (post-rainy season)

ms3

US/B C6

OL3, 296B, DM, midge resistant, SF & SB resistant lines, large grain durra lines, M 35-1, stay-green B-lines, SF B-lines

C0

Source for new cytoplasmic male-sterile lines

Large grain, dwarf Blines for rainy season

6

Fast Lane Ry

ms3

Nebraska, USA

White pearly grain, short height, photosensitivity

C3

High yielding rainy season

Cvs. & restorers

7

Fast Lane By

Ms3

Nebraska, USA

White pearly grain, short height, photosensitivity

C3

High yielding rainy season

Maintainer lines

143 (continued )

144

Table 6.2.

No.

Continued

Name of population

Gene

Original population

Traits/lines introgressed

Present cycle

Suggested use

Expected products

8

US/Ry

Ms3

Purdue & Nebraska, USA

Grain yield & quality, agronomic desirability, resistance to leaf diseases, charcoal rot & molds, & pests (shoot fly, SB & midge), late maturity, white pearly grain, photoinsensitivity, short height

C6

High yielding rainy season

Cvs. & restorers

9

US/B y

ms3

Purdue & Nebraska, USA

Grain yield & quality, agronomic desirability, resistance to leaf diseases, charcoal rot & molds, & pests (shoot fly, SB & midge), late maturity, white pearly grain, short height, photo-insensitivity

C6

High yielding rainy season

Cvs. & B-lines

10

Serere elite y

ms3

EA Pop lines

White pearly grain, short height, photoinsensitivity, good grain quality & grain color

C1

High yielding rainy season

Red/brown Cvs. & restorers

11

Tropicaly conv.

ms3

Puerto Rico & Serere R/S Pop

White pearly grain, short height, photoinsensitivity, good grain quality & grain color

C3

High yielding rainy season

Cvs. & restorers

12

Good grain y

ms3

Good grain, grain pop & red flinty pop

GPR 370 & other Indian bred lines

C1

High yielding, short stature, early, with good grain quality

Post-rainy season, photosensitive

13

West African y early

ms7

WABC & Bulk Y, Nigeria

Grain yield & quality, agronomic desirability, resistance to leaf diseases, charcoal rot & molds, & pests (SF, SB & midge), photoperiod sensitive & pest resistant lines

C4

Early seeding vigor

Post-rainy cvs. & restorers

14

Indian diallely

ms3 & ms7

Diallel crosses world collection

High yielding tan bred lines from different programs including Indian program

C0

A broad cytoplasmic base for male-sterile lines & restorers

Rainy season cvs. & restorers

15

Indiany synthetic

ms3 & ms7

Indian lines

CK 60B, pioneer lines, 2Kx from East Africa

C1

A broad cytoplasmic base for male-sterile lines & restorers

Rainy season cvs. & restorers

16

RS/By

ms3

Developed by Doggett at Serere

White pearly grain, elimination of photosensitivity, stabilization of plant height (reduced), grain color, grain yield & quality, agronomic desirability, resistance to leaf diseases, charcoal rot & molds, & pests (SF, SB & midge)

C6

A broad cytoplasmic base for male-sterile lines & restorers

Rainy season cvs. & maintainers

17

RS/Ry

ms3

Developed by Doggett at Serere

White pearly grain, elimination of photosensitivity, stabilization of plant height (reduced), grain color grain yield & quality, agronomic desirability, resistance to leaf diseases, charcoal rot, & molds, & pests (SF, SB & midge), also, resistance to Striga, grain mold, shoot fly, SB & midge

C6

A broad cytoplasmic base for male-sterile lines & restorers

Rainy season cvs. & restorers

18

ICSP 2B/Ry MFR

ms3

RS/B, RS/R, US/B, US/R

Improved grain yield, agronomic desirability, resistance to SF, SB, midge, good seedling emergence

C1

High yielding restorers

Post-rainy season Blines/R-lines

19

Grain moldy population

ms3

US/B C6

High yielding cultivars, mold resistant parents

C1

Resistance to GM

Cvs. & restorers

z

Mass selection with recombination used Half-sib, S1 or S2 methods with recombination used DM = downy mildew; AN = anthracnose; LB = leaf blight; SF = shoot fly; SB = stem borer; GM = grain mold y

145

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III. CYTOPLASMIC-NUCLEAR MALE STERILITY Cytoplasmic-nuclear male sterility (CMS) has been used extensively to exploit heterosis in hybrids development on a large scale for commercial cultivation since the 1960s. In the pre-hybrid era of the early 1960s, the average sorghum productivity (t ha–1) was 0.49 in India, 0.66 in China, 0.76 in sub-Saharan Africa, 1.48 in Australia, and 2.8 in the USA. In North and Central America, where commercial hybrids were exploited, there was a 40% increase in productivity from the early 1960s to the early 1990s. A similar trend was noticed globally. The productivity increases were 47% in China and 50% in India from the early 1960s to the early 1990s. However, it remained static at 0.79 t ha–1 in Africa from the 1960s to the early 1990s (FAO 1960–1996), and this may be attributed to nonexploitation of hybrids for commercial cultivation in Africa. A. Origin of CMS Systems CMS is a physiological abnormality, resulting from a disharmonious interaction between the cytoplasmic factors (now widely identified as mitochondrial genes) and nuclear genes leading to the production of degenerated or non-viable pollen grains or non-dehiscent anthers with or without functional pollen grains. Understandably, this disharmonious interaction is likely to be more pronounced in populations incorporating divergent sources of cytoplasm and nuclear genes (Reddy et al. 2003). Sorghum is no exception to this. For example, the A1 CMS source in sorghum was identified in the F2 population of cross Double Dwarf Yellow Sooner Milo × Texas Blackhull kafir by Stephens and Holland (1954), in which the milo inbred belongs to the durra race from the Sudan and Ethiopia border (Duncan et al. 1991), and the kafir inbred from Eastern Africa (House 1985). In the F2 generation, 25% of malesterile plants were observed from this cross if milo was the female parent. The male-sterile segregants from this cross produce male-sterile hybrids if crossed with the kafir parents and fully fertile hybrids if crossed with the milo parent. Thus, it was recognized that kafir could be used as a maintainer source of cytoplasmic-genetic male sterility. Since the progeny received the cytoplasm from the female, it was hypothesized that the milo parent had a male sterility-inducing cytoplasm and dominant nuclear genes for the restoration of pollen fertility, whereas the Combine kafir parent contained a normal (fertile) cytoplasm but recessive nuclear genes for fertility restoration. All progenies of the milo × kafir cross contained milo (sterility-inducing) cytoplasm, but individuals that also inherited the homozygous recessive genes from the kafir parent were male sterile. The male-sterile plants in the milo ×

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147

Combine kafir cross were used as females in repeated backcrossing with kafir as the male parent. At the end of seven backcrosses, the entire genome of kafir was transferred into the milo cytoplasm. This resulted in two morphologically similar versions of the Combine kafir (CK 60) parent: a male-sterile Combine kafir (CK 60A) and a male-fertile Combine kafir (CK 60B). The male-sterile lines are thus designated as A-lines and their maintainer lines as B-lines. B. Induction of CMS Systems In Russia, L. A. El’konin and his colleagues used tissue culture to induce male sterility and fertility restoration. A new CMS source, called Atc 1, different from milo cytoplasm was developed from callus cultures of milo 145. When Atc 1 mutants were crossed with milo, restorers resulted in sterile hybrids (El’konin 1995). El’konin et al. (1995) also showed a partial restoration of male fertility in 3 of the 20 regenerants from panicle fragments of a CMS F2 plant of the cross A1 Savatovskoya-3 × 3752 callus culture. Moreover, the induced fertility was retained even after eight generations of selfing. It is not known, however, whether the induced CMS or fertility restoration is different from non-milo CMS systems. C. Inheritance of Fertility Restoration The inheritance of fertility restoration is dependent on the specific combinations of cytoplasms and nuclei. Fertility restoration is controlled by a single gene in some combinations (e.g., A1 cytoplasm) but is controlled by two or more genes when the same nuclear genotype interacts with a different cytoplasm (Schertz 1994). Analysis of F2 segregating progenies with A1 cytoplasm revealed that a single gene was responsible for fertility restoration of A1 male-sterile cytoplasm (Murthy 1986; Murthy and Gangadhar 1990). Other research on A1 cytoplasm concluded that one or two genes (Qian 1990) or even 1 to 3 genes (Lonkar and Borikar 1994) are involved in fertility restoration. However, at least three genes control the fertility restoration of A2 cytoplasm (Murthy 1986). In another study, F2 progenies with A2 cytoplasm showed a 9:7 ratio, indicating that two complementary genes (both Msc1 and Msc2) are needed for fertility restoration in A2 (Murthy and Gangadhar 1990). Lonkar and Borikar (1994) indicated that 2 to 4 genes are necessary, but three genes were more optimal for the fertility restoration in A2 cytoplasm in backcross generations. Research at ICRISAT showed that the frequency of recovery of fertile plants was lower for A3 than A2, A4, and A1 cytoplasm, which indicated that more genes are involved in fertility restoration on A3 than in the other systems (Reddy and Prasad

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Rao 1992). El’konin et al. (1996) concluded that the fertility restoration in sorghum is controlled by an interaction of two complementary dominant genes in 9E cytoplasm. In their study, the tester line, KVV 114, was a restorer for 9(ET) × 398 and a maintainer for 9E milo 10, indicating that one or more dominant inhibitor genes in milo 10 suppressed the action of restorer gene of KVV 114. Further, a novel and unusual phenomenon of gradual restoration of male fertility was observed in subsequent backcross generations of A4 and 9E cytoplasms in sorghum (El’konin et al. 1998). This research clearly suggests that at least two genes are needed for fertility restoration on A1 and three on A2 cytoplasm. Fertility restoration in T-cytoplasm of maize is controlled by dominant alleles at two unlinked and complementary nuclear encoded genes (Rf1 and Rf2) (Schnable and Wise 1994). However, additional restorer genes and duplicated loci complicate their analysis and identification in maize (Sisco 1991). Hence, it may be easier to find restorers on A1 than A2 CMS systems in sorghum owing to the number of genes involved in fertility restoration. D. Diversity Assessment The milo CMS system has been extensively used in developing the hybrids for commercial cultivation in America, China, Australia, and India. Nearly all the hybrids released so far and that are widely grown have milo (A1) cytoplasm (Moran and Rooney 2003; Reddy and Stenhouse 1994). Cytoplasmic diversity may be assessed through the restoration pattern in testcrosses and anther morphology (classical method) or by using molecular markers. 1. Classical Method. Schertz and Pring (1982) summarized various cytoplasm sources with respect to the restoration pattern of 42 lines from India, 24 from the USA, and one from Africa. Some of the cytoplasms were similar in reaction considering their restoration pattern. For example, Schertz and Pring (1982) and U. R. Murthy (1996, pers. commun.) indicated that cytoplasms of G1 (G1-S, ms G1, G1-G, G1A) are analogous to IS 1112C of the USA. The more comprehensive classification of cytoplasm sources is provided in Table 6.3. Over the years, many of these cytoplasm sources were either lost or not widely available. The most commonly available ones include: A1 (milo source), A2 (IS 12662C or TAM 428), and A3 (IS 1112C) of U.S. origin, A4 (Guntur, VZM, and Maldandi) of Indian origin, and 9E (a selection made in 9E) from Ghana. These cytoplasms were grouped on the basis of fertility restoration patterns. Reddy and Stenhouse (1994) reported the identification of minimum differential testers for A1 to A4 cytoplasms as:

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TAM 428B (A2) gives fertile F1s only on A1 cytoplasm; IS 84B (A4-Maldandi) gives fertile F1s on A1 and A2 cytoplasms; IS 5767R (A4-Maldandi) gives fertile F1s on all cytoplasms, except A3; and CK 60B (A1) gives male-sterile F1s on all cytoplasms. Based on pollen development and anther morphology, these A1 to A4 (Guntur, VZM, Maldandi) and 9E cytoplasms were further subdivided into two distinct groups: (1) those with small anthers but without fertile pollen that degenerates during microsporogenesis (A1, A2, A5, and A6), and (2) those with large non-dehiscent anthers that may contain some viable

Table 6.3. Sources of cytoplasmic-nuclear male sterility in sorghum. Source: Adapted from Schertz (1994). Cytoplasm fertility groupz

z y

Source Identity

Race y

Origin — India Sudan Burkina Faso Nigeria Sudan India India Sudan

A1

Milo IS 6771C IS 2266C IS 6705C IS 7502C IS 3579C IS 8232C IS 1116C IS 7007C

D G-C D G G C (K-C)-C G G

A2

IS 1262C IS 2573C IS 2816C

G C C

A3

IS 1112C IS 12565C IS 6882C

D-(D-B) C K-C

A4

IS 7920C

G

Nigeria

9E

IS 7218 IS 112603C

G

Nigeria Nigeria

Nigeria Sudan Zimbabwe India Sudan USA

A5

IS 7506C

B

Nigeria

A6

IS 1056C IS 2801C IS 3063C

D D D

India Zimbabwe Ethiopia

Type member for each fertility group D = durra, G = guinea, C = caudatum, B = bicolor, K = kafir

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pollen (A3, A4, and 9E) (Schertz et al. 1989). A1 to A4 CMS cytoplasms are being maintained at Patancheru (Andhra Pradesh, India) by ICRISAT. The lack of differential restoration patterns, however, does not provide conclusive evidence that the CMS sources involved are necessarily similar. It is possible that the pollinator parents used in developing the testcrosses were not adequate in number and diverse enough to pick up the CMS differences. It is also important in such field studies that testcrosses to be evaluated are made on isonuclear A-lines to ensure that genotypic differences of the female parents are not confounded with their cytoplasmic differences in determining fertility restoration of testcrosses. 2. Molecular Markers. Conventional breeding cytoplasms in various female parents are differentiated through the pattern of male sterility or restoration response in the testcrosses of various female lines. Other approaches to determine diversity among cytoplasms include the use of restriction fragment length polymorphism (RFLP) as molecular markers (Schertz et al. 1997). Cytoplasmic factors associated with male sterility have been shown to be encoded by the mitochondrial genome (Hanson and Conde 1985). Using maize and pearl millet mitochondrial (mt) DNA specific probes, RFLP of mtDNA showed the difference between A1 to A6 cytoplasms (Sivaramakrishnan et al. 1997). A4 and 9E were distinguished by RFLP analysis (Xu et al. 1995), and their cytoplasms included an abnormal form of the mitochondrial gene Cox 1 (Bailey-Serres et al. 1986a; Bailey-Serres et al. 1986b; Pring et al. 1995). Moreover, these cytoplasms also share several mtDNA RFLP that distinguish them from all other Indian and US cytoplasms examined to-date, including polymorphism of the gene atp9 (Schertz et al. 1997). Similarly, the restriction analysis of mtDNA of six male-sterile lines from Kansas State University using several endonucleases revealed two subgroups, with the patterns of KS 34, KS 38 and KS 39 corresponding to that of milo, as represented by CK 60 male-sterile; and KS 35, KS 36, and KS 37 being distinct both from milo and the other male-sterile lines (Schertz and Pring 1982).

IV. MOLECULAR CHARACTERIZATION OF CYTOPLASMS As a consequence of the 1970 epidemic of southern corn leaf blight, CMS-T cytoplasm is no longer widely used in commercial maize hybrids (Kishan and Borikar 1988; Wise et al. 1999). Cytoplasmic diversification in the new cultivars is therefore important. It is relatively easy to assess diversity with molecular markers. Changes in the mitochondrial genome are known to be responsible for male sterility in sorghum (Pring et al. 1993; Sivaramakrishnan et al. 1997; Tang et al. 1996a), e.g. mitochon-

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drial DNA of CMS line IS 1112C showed unusual configurations. Although initially a cell type-specific loss of atp6 RNA editing was reported to occur (Howad and Kempken 1997), e.g. in anthers of an A3T × 398 male-sterile sorghum line, more recent research did not confirm this finding (Pring and Tang 2001). It seems that a transcript processing internal to a mitochondrial open reading frame may be correlated with fertility restoration in male-sterile sorghum (Tang et al. 1996b). The incompatibility in nuclear cytoplasmic interactions leading to aberrant microgametogenesis in sorghum may be explained in terms of incompatible subunits being synthesized by the mitochondria and nucleus for a multi-subunit complex of the mitochondrial membrane such as ATP synthase (Sane et al. 1994). Further, aberrant microgametogenesis in sorghum CMS line IS 1112C occurs very late in pollen maturation and the restoration of pollen fertility is conferred by two genes (Rf3 and Rf4). Rf3, which is tightly linked to Mmt1 that confers transcript processing on orf25, represents the transcript processing activity, or it is tightly linked to the processing activity, for orf107. This chimeric mitochondrial open reading frame is specific to IS 1112C and results in fertility restoration (Tang et al. 1998; Pring et al. 1998; Tang et al. 1999). In maize, some genes, especially ATPase, are disrupted by genetic changes, which produce otherwise normal plants, and cause pollen sterility. The hybrids derived from such sterile plants using the pollen of restorer plants are fertile; e.g., Rf1 affects the expression of maize mitochondrial T-urf13 and encodes the 13kDa sterility protein URF13. Considering the importance of mitochondrial genome in male sterility, CMS lines have been studied in a number of crops. Pedigree and RFLP-based analyses indicate that seven independent rf2 alleles for Rf2 locus produce a functional product necessary for pollen fertility restoration in Texas cytoplasm (CMS-T). Molecular markers flanking the rf1 and rf2 loci were used to decipher segregating patterns in progenies (Schnable and Wise 1994).

V. DNA POLYMORPHISM AND MAPPING RESTORER GENES Fertile plants from S. versicolor, S. almum, S. halepense, and Sorghastrum nutans (yellow Indian grass) each possess a 3.8 kb DNA fragment, which differed from CMS lines containing A1, A2, and A3 cytoplasms with a 3.7 kb DNA fragment. A 165bp deletion located in the middle of the RNA polymerase β-subunit, which was encoded by the gene rpoC2, occurred in the CMS lines (Chen et al. 1993). A1 and A2 cytoplasms produced similar patterns with Hind III restriction enzyme, while restriction

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fragments ensuing from EcoRI and Pst I showed identical patterns in A1, A2, A3 and A4 cytoplasms (Thin et al. 1993). Genetic similarity and co-ancestry coefficient in sorghum suggested that RFLP might help to quantify the degree of relatedness in sorghum germplasm (Ahnert et al. 1996). A total of 276 out of 326 patterns of RFLP bands were common to both R- and B-lines, whereas 32 and 18 bands were unique to R- and B-lines, respectively. Cluster analysis further revealed that R-lines could be in two main groups (fertile and zerazera), while B-lines lie within different sub-clusters. RFLP and expression pattern of mitochondrial genes indicated that the cytoplasms classified tentatively as Indian A4 types were distinct from the American A4 and A1 types. Although the geographical origin of cytoplasms was identical to each other, they are distinguished from each other based on RFLP analyses for atp 6, atp 9, and rrn 18. The three A4 cytoplasms also differed from their maintainers in the location of nad 3, rps 12, and atp A. The differences in the pattern of expression of atp A between all the CMS and their respective maintainers was also observed (Sane et al. 1996). The molecular differences observed within the A4 cytoplasmic group also provide an explanation for the inconsistency in fertility restoration behavior with a definite set of testers (Sivaramakrishnan et al. 1997). In maize, RFLP analyses were used to localize the restorer genes for CMS-C and Rf4, demonstrating that a single dominant restorer gene for CMS-C was in chromosome 8, approximately 2 cM from the RFLP marker locus NPI 114A (Sisco 1991). The Rf3 allele of the nuclear gene rf3 gametophytically restores male fertility with the S-type of CMS. The rf3 locus is on the long arm of maize chromosome two (2L). Using 2L RFLP and three-point mapping analysis, it was shown that the rf3 locus is located at 4.3 cM distal to the whp locus, and 6.4 cM proximal to the bnl17.14 locus. This information was used in combination with RFLP on two additional maize chromosomes to show that Rf3/rf3 CMSS plants may aberrantly transmit the non-restoring allele, rf3, through the male gametophytes (Kamps and Chase 1997). Recently the A1 and A3 restorer genes Rf1 and Rf4, respectively, were mapped in sorghum (Klein et al. 2000; Wen et al. 2002).

VI. FACTORS INFLUENCING CMS SYSTEMS USE Although numerous CMS sources have been found, all are not commercially useful. There are various factors that determine CMS options. These include stability of male sterility, effect of male sterile cytoplasm on agronomic traits, restorer gene frequency in germplasm, and the availability of commercially viable heterosis.

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A. Stability of CMS Systems The instability of male sterility in A-lines increases the roguing of pollen shedders in seed production plots, which results in increased seed production cost. Such an unstable CMS system also reduces breeding efficiency, as backcross progenies that may be fully sterile initially may not remain as such in subsequent generations, leading to their rejection. Male sterility stability influences also the cost and quality of hybrid seed production. Ideally, a commercial male sterile line should neither shed pollen nor should set seed when selfed, regardless of the location and the season, which appears to be seldom feasible. For example, several A-lines based on A1 CMS systems in sorghum are extensively used to breed hybrids, which are planted in millions of hectares in India alone. Most of these A-lines, however, produce a low frequency (< 1%) of pollen shedders, depending on the environment (Reddy et al. 2003). Thus, stability of male sterility across environments is an important criterion in the utilization of CMS systems for commercial production of hybrids. Several workers reported the role of temperature on the expression of male sterility and restoration in sorghum (Downes and Marshall 1971; Li et al, 1981), which affects some cytoplasms more than others (Schertz et al. 1997). Restoration may be poor when night temperature falls below 10°C just before flowering during the post-rainy season. Also, the male sterility in CMS lines breaks down when the day temperature rises above 42°C before flowering (Reddy and Stenhouse 1994). This finding evidently increases the need to screen the CMS lines for the absence of seed setting under bag to ensure stability of male sterility in areas where the temperature rises above 42°C before flowering. The hybrids need to be screened in areas where night temperatures are low (below 10°C) for seed setting under bags to identify stable fertility restorers. While comparing seed setting in A1, A2, A3, and A4 male-sterile lines upon selfing during the summer (> 42°C) at Bhavanisagar (Tamil Nadu, India), Reddy and Stenhouse (1994, 1996) reported that A1 was more stable for maintaining male sterility than others, whereas A3 stability was greater than that of A2 and A4, and that of A2 better than that of A4. The tapetum was intact and pollen was sterile in A2 male-sterile lines in winter (< 10°C), while partial or complete degeneration of tapetum and pollen grains were fertile in summer (> 42°C), indicating the unstable nature of male sterility in the A2 CMS system (Devi and Murthy 1993). At Patancheru, the low temperature-induced female sterility in A1 CMS female lines was similar to the line 296A, which indicated that sterility may be reduced significantly by using their non-parental single cross F1 male-sterile lines (Reddy 1992). In the sorghum breeding program at ICRISAT, the frequency of maintainer lines observed in A1 (Table 6.4)

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Table 6.4. Maintainers and restoration frequency in sorghum A1 cytoplasm in rainy and post-rainy seasons at Patancheru (Andhra Pradesh, India). Frequency Season (2002)

A1 line

Total tested

Maintainers

Restorers

Rainy

ICSA 56 ICSA 84 ICSA 101 CK 60 A Total

75 66 87 49 277

0.71 0.83 0.84 0.55 0.75

0.29 0.17 0.16 0.45 0.25

Post-rainy

ICSA 1 ICSA 9 ICSA 101 ICSA 88005 Total

39 39 200 21 299

0.62 0.87 0.95 0.90 0.89

0.38 0.13 0.06 0.10 0.11

and A2 CMS systems (Table 6.5) was higher in the post-rainy season (< 10°C) than in the rainy season (Reddy et al. 2003). However, Indian researchers have reported higher fertility restoration in the A2 CMS system in the post-rainy season than in the rainy season (U.R. Murthy, pers. comm.). Thus, stability of the expression of male sterility may vary with the temperature as well as type of cytoplasm. Research involving the same CMS lines in both seasons may provide a better understanding of the stability of different CMS systems in different seasons. Table 6.5. Maintainers and restoration frequency in sorghum A2 cytoplasm in rainy and post-rainy seasons at Patancheru (Andhra Pradesh, India). Frequency A2 line

Total tested

Maintainers

Restorers

Rainy 1999

MR 750 ICSA 94003 Total

130 140 270

0.62 0.63 0.62

0.38 0.37 0.38

Post-rainy 1999

MR 750 ICSA 88004 ICSA 94001

19 110 20

0.47 0.45 0.85

0.53 0.55 0.15

Post-rainy 2000

ICSA 38 ICSA 743 ICSA 88001 Total

133 72 34 388

0.97 1.00 0.85 0.79

0.03 0.00 0.15 0.21

Season

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B. Effect of CMS Systems on Economic Traits The observed frequency of segregation for tall and dwarf plants in crosses of two dwarf isocytoplasmic lines carrying A1 cytoplasm and two tall tropical landraces (IS 2317 and IS 35613) confirmed that height was controlled by four recessive non-linked genes (Murthy 1986). However, in crosses between dwarf isocytoplasmic lines of A2 cytoplasm and two landraces, the segregation pattern of dwarf and tall deviated significantly from the four gene theory, indicating an effect of the A2 cytoplasm on plant height. A comparative assessment of five pairs of sorghum iso-nuclear A1 and A2 CMS lines in Mexico revealed that CMS did not have any effect on days to flowering (Table 6.6) (Williams-Alanis and Rodriguez-Herrera 1992). Similarly, considerable variation was observed at the ICRISAT sorghum breeding program between the available male-sterile lines and maintainer lines in the A1 CMS system for flowering. In the early group, a few A-lines tended to be late by a day or two, but in the medium and late maturity groups, A-lines tended to be significantly late in flowering, and there was a tendency of increased delay in flowering in A-lines with the increased maturity period (Table 6.7). B-lines had more open panicles than those from A-lines. Rodriguez-Herrera et al. (1993) also reported a delay in flowering of A- lines (A2) compared to their maintainer (B) counterparts. Spikelet damage and adult emergence of midges was significantly lower on midge-resistant B-lines (PM 7061 and PM 7068) than their corresponding A-lines, and vice versa in the midge-susceptible parental lines (296A and ICSA 42) (Sharma et al. 1994; Sharma 2001). At Patancheru, the maintainer lines (B) flowered early by one or two days and had more open panicles than those of their A-lines. Further, A1 cytoplasm was more susceptible to shoot fly than the maintainer line cytoplasm, while the reverse was true for stem borer resistance (Reddy Table 6.6. Days to anthesis in sorghum iso-nuclear CMS lines A1 and A2 at three planting dates in Mexico. Source: Williams-Alanis and Rodriguez-Herrera (1992). 20 February

8 March

23 March

Lines

A1

A2

A1

A2

A1

A2

LRB-1104A LRB-1102A LRB-1110A LRB-1106A E-15A Means Significance

84 83 84 79 87 83 NS

83 82 85 82 86 83

80 80 79 78 81 79 NS

79 76 82 80 79 79

76 74 78 73 76 75 NS

77 75 76 76 78 76

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Table 6.7. Frequency of sorghum male sterile and maintainer lines differing in days to 50% flowering at Patancheru (Andhra Pradesh, India).

Difference in days (A-B)

Early maturity group (< 67 days)

Medium maturity group (67–74 days)

Late maturity group (>74 days)

–2 –1 0 1 2 3 Total number tested

0.00 0.00 0.74 0.24 0.03 0.00 34

0.02 0.08 0.41 0.40 0.09 0.00 108

0.04 0.08 0.31 0.35 0.19 0.04 26

et al. 2003). This finding has significance in developing shoot fly and stem borer resistant hybrids. Evaluation of five pairs of sorghum isonuclear A1 and A2 CMS lines in four locations of Tamaulipas (Mexico), viz. Rio Bravo (irrigated), El Tapo (drought), El Canelo (drought), and Guelatao (drought), during the fall summer season of 1992 indicated significant differences between A1 and A2 CMS lines for grain yield only in drought conditions (RodriguezHerrera et al. 1993) (Table 6.8). However, no significant differences were Table 6.8. Economic traits as influenced by iso-nuclear A1 and A2 sorghum CMS lines evaluated in four locations in Tamaulipas (Mexico). Source: Rodriguez-Herrera et al. (1993).

Grain yieldz (t ha–1)

Days to floweringz

Plant heightz (cm)

Panicle lengthz (cm)

Panicle exertionz (cm)

Rio Bravo A1 A2

NS NS

82 ab 84 a

NS NS

30 ab 33 a

NS NS

El Tapon A1 A2

2.3 b 2.1 b

75 b 78 a

137 b 135 b

NS NS

15 ab 11 b

El Canelo A1 A2

1.3 b 1.5 a

83 a 83 a

NS NS

27 b 30 a

NS NS

Guelatao A1 A2

0.34 a 0.25 b

82 ab 83 a

NS NS

NS NS

NS NS

Location Isogenic lines

z

Means followed by same letter at each location are not significantly different; NS = non-significant.

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Table 6.9. Grain yield and agronomic characteristics of sorghum isonuclear hybrids (in A1 and A2 CMS backgrounds) evaluated in ten environments in Northern Mexico. Source: Williams-Alanis et al. (1993). Characteristics Grain yield (kg ha–1) Days to flowering Plant height (m) Panicle length (cm) Panicle exertion (cm) z

A1

A2

CV (%)

4195 az 76 b 1.4 a 30 a 17 a

4210 a 77 a 1.4 a 30 a 17 a

23 4 7 15 33

Means in rows followed by same letter are not significantly different

found between A1 and A2 CMS lines for plant height, panicle length, and panicle exertion. In yet another study using 32 isonuclear A1 and A2 CMS line-based hybrids evaluated in 10 environments in Northern Mexico during the fall–winter season of 1990, 1991, and 1993 under irrigated and dry conditions, Williams-Alanis et al. (1993) reported an absence of significant differences between A1 and A2 CMS lines-based hybrids for grain yield, plant height, panicle length, and panicle exertion (Table 6.9). Evaluation of two sets of 36 hybrids obtained by crossing two different sets of six A1 and A2 isonuclear CMS lines with common three dual restorers at Patancheru during the post-rainy season of 2001 and the rainy season of 2002 indicated an absence of significant differences between A1 and A2 CMS systems for mean performance for traits such as days to 50% flowering, plant height and grain yield, lodging resistance and aphid resistance (Tables 6.10 and 6.11). Although hybrids based on Table 6.10. Effect of A1 and A2 CMS systems on mean performance for grain yield and other economic traits in sorghum during post-rainy season of 2001 at Patancheru (Andhra Pradesh, India). Trait

A1

A2

Difference

Significance

Days to 50% flowering Plant height (m) Grain yield (t ha–1) Plant agronomic performancez Lodging resistancey Aphid resistancex Seed set under open pollination Seed set upon selfing

69.20 2.11 7.01 2.41 1.98 3.11 92.04 84.63

69.37 2.10 6.80 2.65 2.04 3.09 91.20 78.80

–0.17 0.01 0.21 –0.24 0.06 0.09 0.84 5.83

NS NS NS * NS NS * *

* and NS indicate significant at P = 0.05 and non-significant 1–5 scale where 1 = good and 5 = poor y 1–5 scale where 1 = less than 10% plants lodged and 5 = more than 80% plants lodged x 1–5 scale where 1 = leaf free from aphids damage and 5 = more than 60% leaf area damaged z

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Table 6.11. Effect of A1 and A2 CMS systems on mean performance for sorghum grain yield and other economic traits in the rainy season of 2002 at Patancheru (Andhra Pradesh, India). Trait

A1

A2

Difference

Significance

Days to 50% flowering Plant height (m) Plant agronomic performancez Grain yield (t ha–1)

68.44 2.58 1.65 5.71

68.59 2.55 1.78 5.72

–0.15 0.05 –0.13 0.01

NS NS * NS

*and NS indicate significant at P = 0.05 and non-significant 1–5 scale where 1 = good and 5 = poor

z

A2 cytoplasm showed superior plant agronomic performance, A1 based hybrids excelled in seed set under open pollination as well as selfing. A comparative evaluation of A1 and A3 cytoplasm-based iso-nuclear sorghum-sudan grass hybrids at the University of Nebraska field laboratory, Ithaca during 1990 and 1991 by Pedersen and Toy (1997) revealed that cytoplasm had no effect on days 50% flowering, plant height, dry matter of forage yield, in vitro dry matter digestibility and protein content (Table 6.12). However, while fertility restoration was equivalent in A1- and A3-based hybrids, it was significantly lower in a few A3-based hybrids. Recently, by evaluating a set of 12 isonuclear hybrids each in A1, A2, and A3 cytoplasmic background at Weslaco and the Texas Agricultural Experimental Station farm located near College Station (Texas) during 1998 and 1999, Moran and Rooney (2003) reported that A1, A2, and A3 cytoplasms had no effects on plant height and had minimal practical effect on days to anthesis (Table 6.13). However, grain yield in A3 cytoplasmic background was significantly reduced as compared with A1 Table 6.12. Effect of A1 and A3 cytoplasms on forage traits in sorghum-sudan grass hybrids evaluated during 1990 and 1991 at Univ. of Nebraska. Source: Pedersen and Toy (1997).

Cytoplasm source A1 A3 Significancez z

Days to 50% flowering

Plant height (m)

Dry matter yield (t ha–1)

In vitro dry matter digestibility yield (t ha–1)

Cut 1

Cut 2

Cut 1

Cut 2

Cut 1

Cut 2

Cut 1

Cut 2

72 72 NS

1.77 1.79 NS

2.16 2.13 NS

3.52 3.50 NS

5.85 5.97 NS

637 635 NS

602 607 NS

12.2 12.3 NS

9.4 9.5 NS

NS indicates non-significant

Crude protein (%)

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Table 6.13. Effect of A1, A2, and A3 cytoplasms on average grain yield, days to anthesis, and plant height in iso-nuclear grain sorghum hybrids evaluated during 1998 and 1999 at Weslaco and the Texas Agricultural Experimental Station farm. Source: Moran and Rooney (2003). Cytoplasm source Trait Average grain yield (t ha–1) Days to anthesis Plant height (m) z

A1

A2

A3

5.01 az 70 a 1.44 a

4.92 a 70 a 1.43 a

4.74 b 71 b 1.42 b

Means followed by same letter are not significantly different

and A2 cytoplasm-based hybrids. Although the specific reason for the reduced yield of A3 hybrids is not known, seed set data indicated that it was not associated with fertility restoration. C. Restorer Gene Frequency The availability of restorers determines the extent of the use of various CMS systems in hybrid seed production. Scheuring and Miller (1978) reported a frequency of 0.62 restorers and 0.23 maintainers on milo (A1) cytoplasm in the world collection of 3,507 sorghum accessions. The work carried out at ICRISAT showed a restoration frequency of 0.9 on A1, 0.5 on A2, 0.1 on A3, and 0.3 on A4, when 48 germplasm lines were test crossed onto A1, A2, A3, and A4 CMS systems (Reddy et al. 2003). Senthil et al. (1998) found that the frequency of restoration was 0.15 on A1, 0.04 on A2, 0.01 on A3, and 0.03 on A4 CMS systems. Both results suggest that the restorer frequency is highest on A1, and lowest on A3 CMS system. Hence, considering the restoration frequency, A1 CMS system provides the widest possible choice in selecting restorers. D. Cytoplasm Effects on Heterosis for Economic Traits Even if all the requirements are met in a CMS system, the existence of economically viable heterosis ultimately determines the use of a CMS system. In sorghum, as indicated earlier, the A1 cytoplasm is more stable than other alternative cytoplasms. The frequency of restorer genes for A1 CMS is higher than with others. The heterosis estimates reported for grain yield using A1 CMS system vary. For example, results from the Indian National Program Testing showed that the standard heterosis with A1 CMS system for grain yield ranged from 18 to 31% in the rainy season, and from 19 to 29% in the post-rainy season in the years 1999

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and 2000. The heterobeltiosis (or highest parent heterosis) estimates for the same period ranged from 15 to 26% in the rainy season and 1.5 to 11% in the post-rainy season (Reddy et al. 2003). Siddiq et al. (1993) reported that heterobeltiosis was 38% for grain yield in the rainy season. Similar studies with alternate CMS systems are limited. Senthil et al. (1998) also reported that the A1 CMS system produced a higher number of heterotic combinations than the A2, A3 or A4 system. Kishan and Borikar (1989a) observed that A2-based hybrids had larger grains and higher yields than A1- and A4-based hybrids. Based on testing of 15 hybrids derived from three isonuclear male-sterile lines and five common restorers, the A4-based hybrids were inferior to others for grain yield in the rainy season. However, another report indicated that A4-based hybrids had higher grain yield and larger grain size than A1 hybrids during the post-rainy season study (Kishan and Borikar 1989b). The favorable and higher frequency of mid-parent heterosis observed in the landrace-based hybrids for several of the post-rainy season traits such as grain and fodder yields in the Advanced Hybrid Trials (AHT) and Landrace-based Advanced Hybrids and Parents Trials (LRAHPT) at Patancheru and Nandyal (India) during the post-rainy season (Table 6.14) indicates the potential for exploitation in the field for post-rainy season adaptation. Evaluation of two sets of 36 hybrids obtained by crossing two different sets of six A1 and A2 isonuclear CMS lines and three dual common restorers at Patancheru during the post-rainy season of 2001 and the rainy season of 2002 indicated an absence of significant differences between A1 and A2 CMS systems for mean heterosis (%) for any of the traits (Tables 6.15 and 6.16).

Table 6.14. Standard heterosis of landrace-based sorghum hybrids over M 35-1 (check) in landrace advanced hybrid trials at two locations during post-rainy season at Patancheru (Andhra Pradesh, India). Hybrids showing standard heterosis (%)

Range of standard heterosis (%)

Location

Year

Grain yield

Fodder yield

Grain yield

Fodder yield

Advanced Hybrid Trial (AHT)

ICRISAT, Patancheru, India

1993

79

45

3.8–49.2

0.4–40

Landrace Advanced Hybrids and Parents Trial (LRAHPT)

Nandyal, India

1995

100

97

23.2–81.3

0.3–118

Trial

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Table 6.15. Effect of A1 and A2 CMS systems on mean heterosis for grain yield and other economic traits in sorghum during post-rainy season of 2001 at Patancheru (Andhra Pradesh, India). Mean heterosis (%) Trait

A1

A2

Difference

Significancew

Days to 50% flowering Plant height (cm) Grain yield (t ha–1) Plant agronomic performancez Lodging resistancey Aphid resistancex Seed set under open pollination Seed set upon selfing

–6.67 47.18 31.78 –1.62 62.57 –6.22 4.05 9.04

–6.45 46.61 27.87 7.88 67.93 –6.34 3.11 1.99

0.22 –0.58 –3.90 9.51 5.37 –0.12 –0.93 –7.05

NS NS NS NS NS NS NS NS

z

1–5 scale where 1 = good and 5 = poor 1–5 scale where 1 = < 10% plants lodged and 5 = > 80% plants lodged x 1–5 scale where 1 = leaf free from aphids damage and 5 = > 60% leaf area damaged w NS indicates non-significant y

It may be advantageous to use the A2 CMS system among the alternate cytoplasms available after considering the restoration frequency, development of high-yielding male-sterile lines, and hybrid performance when using A1 restorers. However, the A2 CMS system is not popular, as the anthers in A2 male-steriles, unlike the A1 male-steriles, mimic the fertile or maintainer lines and lead to complex monitoring of the purity of hybrid seed production. Extensive research is underway at ICRISAT and among Indian researchers for the development of A2 cytoplasmbased hybrids. Based on A2 CMS systems, the hybrid ‘Zinza No. 2’ was released in China for commercial cultivation. This hybrid is now grown Table 6.16. Effect of A1 and A2 CMS systems on mean heterosis for grain yield and other economic traits in sorghum during rainy season of 2002 at Patachenru (Andhra Pradesh, India). Mean heterosis (%) Trait

A1

Days to 50% flowering Plant height (m) Plant agronomic performancez Grain yield (t ha–1)

–3.27 42.46 0.72 43.79

z y

1–5 scale where 1 = good and 5 = poor NS indicates non-significant

A2 –3.071 41.23 10.26 44.49

Difference

Significancey

0.20 –1.23 9.55 0.70

NS NS NS NS

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in an area of 200,000 ha, accounting for one sixth of total sorghum area in China (Liu Qing Shan et al. 2000).

VII. DIVERSIFICATION OF CMS SYSTEMS Most of the sorghum hybrids worldwide depend on the A1 CMS system, which may lead to cytoplasm uniformity among hybrids for this crop (Moran and Rooney 2003; Reddy and Stenhouse 1994). This uniformity may increase the risk of outbreak or quick transmission of cytoplasmically inherited susceptibility to pests or diseases, as reported in maize with Helminthosporium leaf blight, which was linked to T-cytoplasm (Kishan and Borikar 1988) and pearl millet downy mildew disease epidemic (C. T. Hash 1998, pers. commun.). There are no reports of such an occurrence of cytoplasm-linked diseases or pests in sorghum. However, cytoplasmic diversity should be kept in this crop to avoid such hazards that may arise in future. In addition to causing uniformity of cytoplasm in the hybrids, the use of milo (A1) cytoplasm-based hybrids restricts the nuclear diversity (Schertz and Pring 1982). With the introduction of additional cytoplasmicnuclear male sterility systems, new parental combinations should be possible. Hence, new and alternate cytoplasms have been sought with additional nuclear diversity, and are studied in several countries (Rao 1972; Nagur and Menon 1974; Tripathi 1979; Quinby 1981; Schertz and Pring 1982; Rao et al. 1984), based on restoration patterns in testcrosses made with common pollinators. The frequency of maintainer genes in a diverse range of improved populations and breeding lines has a direct bearing on the success of genetic diversification of A-lines. Conversely, the frequency of restorers for several established CMS sources directly influences the use of such diversified male steriles in grain hybrid development. In addition, as already discussed in previous sections, the factors influencing the use of alternate CMS systems that include stability of their male sterility, their effect on economic traits, and extent of heterosis for economic traits, should be analyzed to make a rational judgment about CMS diversification. Often such analyses are not carried out, leaving new useful CMS systems unused or underused. A. Germplasm Base and Traits Although the need to diversify the cytoplasm base of seed parents has been recognized, research at ICRISAT showed that the kafir-based crosses with CK 60B produced higher frequency of B-lines than the

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caudatum-based B-lines with A1 CMS, which essentially derives its male sterility maintainer genes from kafir. This finding, coupled with the reduced frequency of restoration and the difficulties in distinguishing the fertiles from male steriles among alternate CMS systems at field level, forced many programs to depend mostly on the A1 CMS system for the production of hybrids (Moran and Rooney 2003). There are four major national programs (USA, India, China, and Australia) and an international program (ICRISAT) engaged in diversifying the base of the male steriles and hybrids. All the national programs depend mostly on kafir-milo based male-sterile lines. The U.S. program diversified its kafir-milo (durra) male-sterile system by crossing with other kafir or bicolor lines in red- or brown-grain color background up to the mid1980s (Schertz et al. 1997). The U.S.-based program influenced the sorghum breeding programs in China and Australia. Later on, the U.S.based program further diversified its female parent base by crossing the kafir-bicolor B-lines with kauras (durra-caudatum) and other caudatum lines of African origin (Duncan et al. 1991; Schertz et al. 1997). This approach led to the development of a series of white-grain color malesterile lines (e.g., Tx 623A, Tx 624A). The Indian program introduced CK 60A (milo-kafir) from the USA in the early 1960s, and developed 2219A (kafir-shallu), 2077A, 3675A, and 3677A among other kafir types in the late 1960s. Later on, male steriles were diversified by further selection and identification of maintainers in the crosses of the kafir Blines with Indian durras, which are usually restorers on the milo-kafir system (Rao 1972). The most significant heterotic female line developed from such a program is 296A. However, this line was reported to be temperature-sensitive and seed production based on this line, therefore, became difficult due to low temperature-induced female sterility. The national programs diversified their male-sterile line base mostly for grain yield or the yield components such as grain number, grain size, etc. (Rao 1972; Duncan et al. 1991). Little effort was directed toward breeding male-sterile lines for resistance to abiotic and biotic stresses during the 1970s and 1980s (J. W. Stenhouse, pers. comm.). During late 1970s, ICRISAT began the development of high-yielding male-sterile lines on the A1 CMS system in white-grain background by extensively using kafir-caudatum-guinea crosses, and selecting whitegrain male-sterile lines for grain yield. This program resulted in the development of a series of male steriles such as ICSA 1 to ICSA 110 and ICSA 88001 to ICSA 94013. In the late 1980s, ICRISAT made a major shift in its emphasis on the improvement of A1 CMS male-sterile lines for resistance to various abiotic and biotic yield constraints. Considering the high correlation between per se performance of lines and hybrids (Rao 1972), ICRISAT followed simultaneous selection, test-crossing,

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B. REDDY, S. RAMESH, AND R. ORTIZ Table 6.17. Trait-specific resistant male sterile lines developed by ICRISAT sorghum breeding program at Patancheru (Andhra Pradesh, India). Trait

Lines

Downy mildew Anthracnose Leaf blight Rust Grain mold Shoot fly Stem borer

ICSA 201 to ICSA 259 ICSA 260 to ICSA 295 ICSA 296 to ICSA 328 ICSA 329 to ICSA 350 ICSA 351 to ICSA 408 ICSA 409 to ICSA 463 ICSA 464 to ICSA 474 ICSA 475 to ICSA 487 in post-rainy season ICSA 488 to ICSA 545 ICSA 546 to ICSA 565 in rainy season ICSA 566 to ICSA 599 ICSA 600 to ICSA 614 ICSA 615 to ICSA 637 ICSA 638 to ICSA 670 ICSA 671 to ICSA 674 ICSA 675 to ICSA 68

Midge Head bug Striga Acid soils Early maturity Bold grain Tillering Stay green

and backcrossing methods to convert the maintainer lines selected for resistance to individual stresses and grain yield, under specific screenings in trait-specific breeding populations (Reddy et al. 1992). As a result, several trait-specific resistant male-sterile lines were developed (Table 6.17) Each ICRISAT breeding population involved different source materials, namely guinea for grain mold, durra for shoot fly, stem borer, and Striga, and caudatum-guinea for stay-green lines. B. Other Cytoplasms Breeding programs in the USA also developed a few high-yielding A2 and A3 CMS lines (Duncan et al. 1991). ICRISAT has converted some of the high-yielding A1 CMS restorers into A2 (ICSA 688 to ICSA 738), A3 (ICSA 739 to ICSA 755), and A4 (ICSA 756 to ICSA 767) CMS lines. Most of these lines were developed from kafir-durra-caudatum crosses. C. Information Management and Knowledge Sharing through the ICRISAT Website The development, characteristics, and pedigrees of the above malesterile lines improved at ICRISAT are described fully in the ICRISAT web page, which can be accessed under crop at http://www.icrisat .org/text/research/grep/homepage.htm (Mahalakshmi et al. 2002). Infor-

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mation about the performance of individual lines for traits of interest can be displayed, and further information on parents used in the breeding program can be obtained by clicking on the icon for the parent, which is linked to the germplasm passport data. Seeds of both breeding lines can be obtained by accepting the material transfer agreement on line and placing the request. Other information available in the sorghum web page includes gene bank germplasm and its score collection, crop diseases, pest, parasitic weeds, and plant nutritional disorders. The on-line system from the main crop page allows a query using the common or scientific name for a pest. There is also a two-module on-line learning system on sorghum that is based on sorghum practices ensuing from ICRISAT training manuals on breeding and seed technology in sorghum. D. Effect of Genetic Background Nuclear genetic background also has profound influence on male sterility. The fertility maintenance patterns differ from cross to cross, although the parents involved in such crosses by themselves are maintainers. Work at ICRISAT showed that the frequency of non-maintainers in B × B crosses ranged from 0 to 25%. For example, all the progenies were male-sterility maintainers in a cross of ICSB 554 × ICSB 79. On the other hand, in a cross like ICSB 583 × ICSB 64, nearly 20% of the progenies failed to maintain male sterility. The extent of the influence of genetic background thus determines the effectiveness of diversification of malesterile lines. At ICRISAT, it is also observed that the maintainer gene frequency among the progenies of B × B crosses depends on the tester (male-sterile line) used, indicating the role of the “residual” cytoplasm factors accumulated in the converted male-sterile lines. It is useful to use, as much as possible, the original CMS source. For example, to diversify A1 CMS lines, it may be desirable to convert to female the original male-sterile line CK 60A. E. Seed Parents’ Purity Maintaining the purity in hybrid parents is important because impurity may arise due to mechanical admixture of the parents among themselves or with other lines. Impurity can also arise due to occurrence of pollen shedders in A-line, which depends on the CMS systems and the seed production season. The pollen shedding revertants may be due to mutation occurring either in cytoplasm or nuclear genes. The cytoplasm fertile revertants can be pulled out at flowering before they contribute to seed setting to maintain the purity of seed. If a nuclear gene mutation is the cause of reversion to male fertility, A-line seed purity can be

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maintained by making plant-to-plant crosses between A- and B-line progenies for two successive generations.

VIII. HETEROSIS AND HYBRID DEVELOPMENT Restorer and male-sterile lines should be as divergent as possible to gain maximum advantage in heterosis (Allard 1960). There is a considerable body of evidence to suggest that per se performance of the parents is highly correlated with hybrid performance (Rao and Rana 1982). Besides, other characters such as flowering behavior, pollen quantity (in R-lines), seed setting (in A-lines), and relative plant height of A- and Rlines also influence the hybrid seed production. A-lines with <50% of seed setting under open pollination are usually rejected. The restorers should be taller than male steriles usually by 0.1 to 0.8m, and possess good pollen shedding ability (Reddy 1997b). Milo (A1) cytoplasm male-sterile lines and restorer lines are usually based on kafir and caudatum races (Reddy and Prasada Rao 1993). In the Indian program, caudatum race (particularly IS 3541 from Sudan) has been thoroughly exploited not only for developing restorers but many of the released hybrids can be traced to caudatum source parents (AICSIP 1999). Research was undertaken by ICRISAT at Patancheru involving a complete set of hybrids made by crossing five representative lines from each of the sorghum landraces guinea, bicolor, caudatum, durra, and kafir onto six common male-sterile lines to assess the relative magnitude of hybrid performance and heterosis in various landrace groups. Hybrids with landraces such as guinea followed by bicolor in the post-rainy season, and guinea followed by caudatum in the rainy season, were the highest yielders. However, the heterosis was greater with restorers of caudatum in the rainy season, and of guinea in the post-rainy season. Therefore, considering mean performance and heterosis for grain yield, further gains in sorghum hybrid yields can be realized by exploiting guinea race in restorer line development (Reddy and Prasada Rao 1993). CMS has been used in sorghum population improvement to a limited extent. Dr. O. J. Webster at the University of Nebraska developed the first random mating population of sorghum using CMS in 1960. This population improvement was continued by Dr. Paul Nordquist at the same University (Murthy and Rao 1997). In Africa, Doggett and Jowett (1964) developed improved populations using the gene msc1. In India, a random mating population was developed by Rao and his coworkers using an indigenous CMS source (Murthy and Rao 1997).

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IX. CONCLUSION The milo cytoplasm male-sterility system still remains the most widely used because the hybrids based on this cytoplasm produce sufficient heterosis (20–30%) over the best available pure lines in sorghum. In spite of A2 cytoplasm being as promising as A1 cytoplasm for either mean performance or heterosis for economic traits such as grain yield, days to 50% flowering, or plant height, the A2 CMS system is not popular because the anthers in A2 male-steriles, unlike the A1 male-steriles, mimic the fertile or maintainer lines and lead to complex monitoring of the purity of hybrid seed production. Although diverse male-sterile cytoplasms are available, alternate sources are not useful for sorghum hybrids primarily because the frequency of restorer genes is low, and male steriles cannot be readily distinguished from male fertiles. There is therefore a need to search for a more useful form of male sterility, yet different from milo (A1). Milo restorers need to be diversified in guinea background to further enhance the yield advantage in hybrid development. Restorer frequency is very low on other cytoplasms. Hence, there is a need to identify and breed for high-yielding non-milo cytoplasm restorers. Limited work has been carried out on the inheritance of fertility restoration, but it has involved distinct genetic backgrounds. The use of isonuclear lines will provide a better means to determine the inheritance of fertility restoration. Similarly, the work on the identification of molecular markers will hasten the process of directed transfer of restorer genes. Although milo (A1) male-sterile lines are diversified for resistance to biotic and abiotic stresses at ICRISAT, further breeding for grain yield potential and bold grain, and superior agronomic performance among them is needed.

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Reddy, Belum V. S., K. David Nicodemus, G. Sreeramulu, and J. W. Stenhouse. 1992. Diversification of sorghum seed parents at ICRISAT center. 22nd Ann. AICSIP (All India Coordinated Sorghum Improvement Project) Workshop, April 2–4, 1992. Gujarat Agr. Univ., Surat, Gujarat, India. Reddy, Belum V. S., and K. E. Prasada Rao. 1993. Genetic divergence and heterosis. p. 48–49. In: ICRISAT Annu. Rep. 1993. ICRISAT, Patancheru, Andhra Pradesh, India. Reddy, Belum V. S., and J. W. Stenhouse. 1994. Sorghum improvement for the semi-arid tropic region: past, current and future research thrusts in Asia. Punjabrao Krishi Vidyapeeth Res. J. 18:155–170. Reddy, Belum V. S., and J. W. Stenhouse. 1996. Improving postrainy-season sorghum, a case for landrace hybrids approach. 26th AICSIP (All India Coordinated Sorghum Improvement Project) Workshop, April 18–20, 1996. Pantnagar, U.P., India. Reddy, Belum V. S., K. N. Rai, N. P. Sarma, IshKumar, and K. B. Saxena. 2003. Cytoplasmic-nuclear male sterility: Origin, evaluation, and utilization in hybrid development. In: H. K. Jain, and M. C. Kharkwal (eds.), Plant breeding: Mendelian to molecular approaches. Narosa Publ. House Pvt. Ltd., New Delhi, India. (In press). Rodriguez-Herrera, R., H. Torres-Montalvo, and H. Williams-Alanis. 1993. Comparative performance of maintainer and male-sterile sorghum iso-genic lines in A1 and A2 cytoplasms. Sorghum Newsl. 34:50. Rooney, W. L. 2000. Genetics and cytogenetics. p. 261–307. In: C. W. Smith and R. A. Frederiksen (eds.), Sorghum: Origin, history, technology and production. Wiley, New York. Rooney, W. L and C. W. Smith. 2000. Techniques for developing new cultivars. P. 329–347. In: C. W. Smith and R. A. Frederiksen, (eds.), Sorghum: Origin, history, technology and production. Wiley, New York. Sane, A. P., P. Nath, and P. V. Sane. 1994. Mitochondrial ATP synthase genes may be implicated in cytoplasmic male sterility in Sorghum bicolor. J. Biosci. 19:43–55. Sane, A. P., P. Nath, and P. V. Sane. 1996. Cytoplasmic male sterility in sorghum, organization and expression of mitochondrial genes in Indian CMS cytoplasms. J. Genet. 75:151–159. Schertz, K. F., and D. R. Pring. 1982. Cytoplasmic sterility systems in sorghum. p. 373–383. In: N. G. P. Rao and L. R. House (eds.), Sorghum in the eighties. ICRISAT, Patancheru, Andhra Pradesh, India. Schertz, K. F., A. Sotomayor-Rios, and S. Torraco-Cardona. 1989. Cytoplasmic-nuclear male-sterility opportunities in breeding and genetics. Proc. Grain Sorghum Res. & Util. Conference 16:175–186. Schertz, K. F. 1994. Male-sterility in sorghum: Its characteristics and importance. p. 35–37. In: J. R. Witcombe and R. R. Duncan (eds.), Use of molecular markers in sorghum and pearl millet breeding for developing countries. Proc. Int. Conf. Genet. Improvement Overseas Development Administration (ODA) Plant Sci. Res. Conf., 29 March–1 April 1993, Norwich, UK, ODA, UK. Schertz, K. F., S. Sivaramakrishnan, W. W. Hanna, J. Mullet, Yi Sun, U. R. Murthy, D. R., Pring, K. N. Rai, and Belum V. S. Reddy. 1997. Alternate cytoplasms and apomixis of sorghum and pearl millet. p. 213–223. In: Proc. Intl. Conf. Genet. Improvement of Sorghum & Pearl Millet, Texas A&M Univ., Lubbock. Scheuring, J. F., and F. R. Miller. 1978. Fertility restorers and sterility maintainers to the milo-kafir genetic cytoplasmic male sterility system in the sorghum world collection, Misc. Publ., Texas A&M Agr. Expt. Sta. 1367. College Station, Texas. Schnable, P. S., and R. P. Wise. 1994. Recovery of heritable, transposon-induced, mutant alleles of the rf2 nuclear restorer of T-cytoplasm maize. Genetics 136:1171–1185. Senthil, N., P. Ramasamy, and A. K. F. Khan. 1998. Fertility restoration and heterosis involving different cytoplasms in sorghum (Sorghum bicolor (L.) Moench) hybrids. J. Genet. Breed. 52:339–342.

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Sharma, H. C., P. Vidyasagar, C. V. Abraham, and K. F. Nwanze. 1994. Effect of cytoplasmic male-sterility in sorghum on host plant interaction with sorghum midge, Contarinia sorghicola. Euphytica 74:35–39. Sharma, H. C. 2001. Cytoplasmic male-sterility and source of pollen influence the expression of resistance to sorghum midge Stenodiplosis sorghicola. Euphitica. 122:391–395. Siddiq, M. A., I. A. Madrap, and S. S. Ambekar. 1993. Heterosis studies in sorghum. p. 123–124. In: Abstracts Nat. Symp. Plant Breeding Strategies for India 2000 and Beyond, 25–27 Dec. 1993. Indian Soc. Genetics and Plant Breeding, Indian Agricultural Research Institute (IARI), New Delhi, India. Sisco, P. H. 1991. Duplications complicate genetic mapping of Rf4, a restorer gene for CMSC cytoplasmic male-sterility in corn. Crop Sci. 31:1263–1266. Sivaramakrishnan, S., K. Seetha, and Belum V. S. Reddy. 1997. Characterization of the A4 cytoplasmic male-sterile lines of sorghum using RFLP of mt DNA. Euphytica 93:301–305. Stephens, J. C. 1937. Male sterility in sorghum: Its possible utilization in production of hybrid seed. J. Am. Soc. Agron. 29:690–696. Stephens, J. C., and P. F. Holland, 1954. Cytoplasmic male sterility for hybrid sorghum seed production. Agron. J. 46:20–23. Tang, H. V., R. Y. Chang, and D. R. Pring. 1998. Co-segregation of single genes associated with fertility restoration and transcript processing of sorghum mitochondrial orf 107 and urf 209. Genetics 150:383–391. Tang, H. V., W. Chen, and D. R. Pring. 1999. Mitochondrial orf 107 transcription, editing, and nucleolytic cleavage conferred by the gene Rf3 are expressed in sorghum pollen. Sexual Plant Reprod. 12:53–59. Tang, H. V., D. R. Pring, F. R. Muza, and B. Yan. 1996a. Sorghum mitochondrial orf25 and a related chimeric configuration of a male-sterile sorghum. Curr. Genet. 29:285–274. Tang, H. V., D. R. Pring, L. C. Shaw, F. A. Salazar, F. R. Muza, B. Yan, and K. F. Schertz. 1996b. Transcript processing internal to a mitochondrial open reading frame is correlated with fertility restoration in male-sterile sorghum. Plant J. 10:123–133. Thin, T., D. Z. Skinner, S. Muthukrishnan, and G. H. Liang, 1993. Chloroplast DNA restriction endonuclease fragment patterns of male sterility inducing cytoplasms. Sorghum Newsl. 34:32. Tripathi, D. P. 1979. Characterization of diverse cytoplasmic genetic male steriles in sorghum (Sorghum bicolor (L.) Moench). Ph.D. thesis, Indian Agr. Res. Inst., New Delhi. Webster, O. J. 1965. Genetic studies in Sorghum vulgare (Pers.). Crop Sci. 5:207–210. Wen, Y., H. V. Tang, W. Chen, R. Chang, D. R. Pring, P. E. Klein, K. Childs, and R. R. Klein. 2002. Development and mapping of AFLP markers linked to the sorghum fertility restorer rf4. Theor. Appl. Genet. 104:577–585. Williams-Alanis, H., and R. Rodriguez-Herrera. 1992. Cytoplasmic-genic male-sterility effect in flowering of sorghum iso-genic lines. Sorghum Newsl. 33:17. Williams-Alanis, H., R. Rodriguez-Herrera., J. Aguirre-Rodriguez, and H. Torres-Montalvo. 1993. Comparative performance of iso-genic sorghum hybrids in A1 and A2 cytoplasms. II. Yield and agronomic characteristics. Sorghum Newsl. 34:51. Wise, R. P., K. Gobelman Werner, D. Pei, C. L. Dill, and P. S. Schnable. 1999. Mitochondrial transcript processing and restoration of male fertility in T-cytoplasm maize. J. Hered. 90:380–385. Xu, G. W., Y. X. Cui, K. F. Schertz, and G. E. Hart. 1995. Isolation of mitochondrial DNA sequences that distinguish male-sterility-inducing cytoplasms in Sorghum bicolor (L.) Moench. Theor. Appl. Genet. 90:1180–1187.

7 Improving Drought Tolerance in Maize T. Barker, H. Campos, M. Cooper, D. Dolan, G. Edmeades, J. Habben, J. Schussler, D. Wright, and C. Zinselmeier Pioneer Hi-Bred International, Inc., 7300 NW 62nd Ave., Johnston, IA 50131

I. INTRODUCTION A. Definition of Drought B. Importance of Drought in Maize C. Scope of Review II. PHYSIOLOGY OF THE RESPONSE OF MAIZE UNDER DROUGHT A. Yield Potential Spillovers B. Crop Duration: Escape or Tolerance? C. Stress-sensitive Periods in the Life of the Crop D. Tolerance 1. Seedling Establishment 2. Vegetative Tolerance 3. Reproductive Processes 4. Osmotic Adjustment 5. Growth Regulators as Signals 6. Visual and Functional Staygreen 7. Remobilization of Stem Reserves 8. General Stress Tolerance E. Relative Importance of Secondary Traits in Selection for Drought Tolerance III. EXPERIMENTAL METHODS A. Target Environments and Environmental Characterization B. Experimental Design and Analysis 1. Extraneous Variation 2. Spatial Variation C. Describing, Understanding, and Managing G*E in Drought Trials D. Hybrid Characterization: Measures of Drought Tolerance 1. Need for Managed Stress Approach 2. Execution of Managed Stress Approach 3. Application of Managed Stress Approaches IV. APPLIED BREEDING METHODS A. Breeding Goals 1. Long-term Plant Breeding Reviews, Volume 25. Edited by Jules Janick. ISBN 0471666939 © 2005 John Wiley & Sons, Inc. 173

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2. Medium-term 3. Short-term B. Inheritance of Tolerance C. Selection of Parental Germplasm 1. Populations and Improved Germplasm 2. Identification of Superior Parents 3. Breeding Values D. Criteria for Progeny Selection E. Methods of Selection 1. Index Selection 2. Recurrent Selection 3. Pedigree Selection 4. Divergent Selection F. Introgression of Novel Variation V. MOLECULAR BREEDING A. Linkage Mapping B. QTL Identification C. Marker Assisted Selection (MAS) D. Summary of Results from QTL/MAS Studies on Drought Tolerance E. QTL Analysis as an Aid to Identify Candidate Genes F. QTL Mendelization and Positional Cloning G. HAPPY Mapping H. Bulk Segregant Analysis I. Association Analysis VI. CONCLUSIONS LITERATURE CITED

LIST OF ABBREVIATIONS ABA AFLP ASI BLUP BSA CER CIM CIMMYT EGR ERA GY LD MAS MET OA

abscisic acid amplified fragment length polymorphisms anthesis-silking interval best linear unbiased predictors bulked segregant analysis carbon exchange ratio composite interval mapping International Maize and Wheat Improvement Center ear growth rate era of release hybrid trial grain yield linkage disequilibrium marker assisted selection multi-environment trials osmotic adjustment

7. IMPROVING DROUGHT TOLERANCE IN MAIZE

PCR PHI QTL RAPD RCB RFLP RIL RUE SNP SOD SSR TPE

175

polymerase chain reaction Pioneer Hi-Bred International, Inc. quantitative trait loci randomly amplified polymorphic DNA randomized complete block random fragment length polymorphisms recombinant inbred line radiation use efficiency single nucleotide polymorphisms superoxide dismutase simple sequence repeats target population of environments

I. INTRODUCTION A. Definition of Drought In the broadest sense, drought is defined as “a prolonged period of dry weather; lack of rain,” or “a prolonged or serious shortage or deficiency” (Webster’s New World Dictionary of American English, 3rd Edition, 1988). For maize research purposes, the definition of drought may be refined to specify “a period of limited moisture availability resulting in yield loss.” A more precise definition of agricultural drought is stress occurring when plant-available water in the soil drops below 65% of the total accessible by the crop and atmospheric demand is such that significant degrees of leaf rolling are evident. Because the crop cannot photosynthesize when leaves are rolled, these events reduce productivity. Numerous reports in the literature give further details regarding the definition and characterization of drought stress and its components. Useful references on the improvement of drought tolerance are available for general aspects of plant breeding (Blum 1988; Ludlow and Muchow 1990; Mitra 2001), breeding crops other than maize (Myers et al. 1986; Nguyen et al. 1997; Araus et al. 2002; Richards et al. 2002), and molecular approaches to improving drought tolerance (Lebreton et al. 1995; Quarrie 1996; Frova et al. 1999; Bruce et al. 2002; Price et al. 2002; Tuberosa et al. 2002a,b). The only available comprehensive review of practical issues in maize breeding for drought tolerance is that of Bänziger et al. (2000), which summarizes much of the extant literature and describes practical applications of drought tolerance breeding based on CIMMYT’s (International Maize and Wheat Improvement Center) work with tropical maize. To accurately convey the nature of the stress imposed on maize, it is useful to specify three characteristics of the water deficit: timing,

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duration, and intensity. Timing refers to the phenological phase or phases during which stress occurs. Establishment, vegetative growth prior to flowering, flowering, and grain fill are the four periods covering the life cycle of maize, each with differing degrees of sensitivity to drought and with differing consequences for yield. The general effects of water deficit can be measured in many ways, but for the purposes of this review they will usually be defined as reduction in grain or silage yield compared to an unstressed control. Many factors such as competition, high temperatures and vapor pressure deficit, crop nutrition, and pests may contribute to yield reductions in the field, and each of these can modulate the crop’s response to drought. B. Importance of Drought in Maize Worldwide, drought is the most pervasive limitation to the achievement of yield potential in maize (Edmeades et al. 2001). Estimates of annual regional losses to maize crops vary from zero to 30% in the United States in the summer of 1988 (FAOSTAT 2003) to as high as a 60% loss in 1993 in southern and eastern Africa (Rosen and Scott 1992). Average annual global losses range from 15% in temperate zones to 17% in the tropics, as estimated by empirical methods (Edmeades et al. 2000b). A precise measurement of yield losses worldwide is not possible due to the range of occurrences from within individual fields to regional in extent, and the range of severity from slight to catastrophic. Losses are greatest in parts of the world where soils and weather patterns are less favorable than the U.S. Corn Belt, which was named for its long-term suitability for growing maize at a relatively low level of risk for crop failure. There is a more subtle impact of moisture stress on yields and yield stability. Transient drought stress may limit yield even when visual stress symptoms are not evident and the drought is not widespread or responsible for crop failure. It is common throughout the U.S. Corn Belt, for example, to experience periods with no rainfall lasting a few days to several weeks at various times during the growing season. This may limit the top-end yield potential of the crop, especially if the stress occurs during a susceptible growth period such as pollination. Variation in moisture stress imposed on a high and increasing mean yield may partly explain the increased variation in average maize yields in the United States over the last 20 years (Fig. 7.1). Average loss data only portray one aspect of the picture, however. In any given year, areas that are relatively close to each other can experience different degrees of stress due to spatial variation in rainfall and in soil texture. This is often acute in the tropics where large convectional storms account for much of the annual precipitation.

7. IMPROVING DROUGHT TOLERANCE IN MAIZE

177

8.8

1994

7.6

b = 1.52

6.3 Yield (t/ha)

1993

5.0

b = 2.85

Open Pollinated

3.8 2.5

1983 1970 1974

1988

Single Cross Hybrids

b = 1.17

b = -0.67

1.2

1936 1934

1947

Double Cross Hybrids 1995

1975

1955

1935

1920

0

United States Department of Agriculture

Fig. 7.1.

Historical and realized U.S. maize yields since 1920.

Within a given field of maize, variation in soil depth and texture may induce stress unevenly across the field, allowing portions to appear unstressed while others suffer severe stress. Kitchen et al. (1999) documented a maize field where yields varied from a low of 3.4 t/ha in sandy or compacted portions of the field to a high of 11.3 t/ha in areas with deep soil or where runoff may have ponded. Variation in water stress within a field of even greater magnitude has been reported on tropical soils (Bouma et al. 1997). Since a single cultivar is normally grown, it needs to withstand a wide range of water stresses without sacrificing yield in the better areas of the field. Creating more drought tolerant maize germplasm remains a major challenge. It is also an important opportunity to help address world food needs at a time when human population continues to rise and agricultural land area currently under cultivation is diminishing. However, we recognize the limits of plant breeding and genetic variation to close the gap between rainfed and well-watered yields. Water-conserving growing practices with limited or complete irrigation play a critical part, but are limited in many parts of the world due to water availability or economic reasons for non-adoption. Genetics solutions, although not complete, can be more easily packaged and promoted than agronomic practices and dependence on inputs, and are therefore more readily adopted.

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C. Scope of Review There have been no reviews published on breeding temperate maize for drought tolerance, particularly from the commercial perspective. Our focus in this review is on developing methods and germplasm for the commercial maize seed industry. A focus on commercial applications is appropriate since much of the research investment currently allocated to maize improvement worldwide is in the private sector. The seed industry also has the means to efficiently distribute improved maize genetics to most major maize growing areas of the world. However, since much of the historical research done on maize is public, and most of today’s commercial research is proprietary, the review extends well beyond private sources. Inferences will be drawn from work on openpollinated cultivars of maize, as well as on other crops. Four broad areas of research are addressed in this review: physiology of responses to water deficits, experimental methods designed to expose genetic variation for drought tolerance, applied breeding methods, and emerging molecular methods used to enhance breeding. This paper is not intended to be a comprehensive review of the extensive literature on physiological responses to drought, experimental design, breeding methodology, or molecular biology as these might relate to drought. The focus is on issues and opportunities for applied maize research aimed at improved drought tolerance in commercial hybrids.

II. PHYSIOLOGY OF THE RESPONSE OF MAIZE UNDER DROUGHT A. Yield Potential Spillovers Yield potential is the single most important trait in maize breeding, and selection in temperate maize has led to consistent increases in yield in non-limiting conditions over the past 70 years (Castleberry et al. 1984; Duvick 1997). Selection based on multilocation testing results has also led to an increase in grain yield under drought stress that occurs at or near flowering (Tollenaar and Wu 1999; Edmeades et al. 2003). These improvements may also be due to the widespread use of self-pollination during inbred line development and the use of high plant densities in breeding nurseries and early generation evaluation. Richards (1992) concluded that yield potential is the most important trait when breeding for wheat performance under saline conditions. Heterosis, or the degree to which a hybrid outyields its inbred parents, is itself a source of stress tolerance (Blum 1997), and maize hybrids usually outyield

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179

open-pollinated cultivars over a wide range of stress intensities (G. Edmeades, unpubl. data, 1998). If yield potential strongly affects performance under stress, is it necessary to test under stress conditions? The answer depends on the severity of stresses encountered in the target population of environments (TPE) (Comstock 1977), since the degree with which yield under stress is predicted by yield under unstressed conditions depends on the intensity of that stress. The dependence of the phenotypic correlation of grain yields under well-watered conditions (GYWW) and under stress (GYSTR) on stress severity is shown (Fig. 7.2) for a number of sets of elite hybrids. The fitted regression suggests that variation in GYSTR accounted for by variation in GYWW falls to 10% (R2 = 0.10; r = 0.32) when GYSTR is reduced by 50% of GYWW. Under moderate stress (GYSTR/GYWW = 0.75), R2 = 45%, and it approaches 100% as GYSTR approaches GYWW. Exceptions in this study were a historical hybrid series (referred to hereafter as the “ERA” hybrid set) that showed large differences in performance due to selection, and the ranking for yield was more consistent here as GYSTR fell. Thus, when considered over the full history of the progress made by a breeding program, we expect and observe a stronger correlation

Phenotypic correlation (GYWW, GYSTR)

1.0

Y = 0.39 + 0.74X R2 = 0.89*, N = 5

■ Current hybrids ▲ ERA hybrids

0.8 0.6 0.4

Y = –0.39 + 1.41X R2 = 0.44***, N = 37

0.2 0.0

–0.2 0.25

0.45

0.65

0.85

GYSTR/GYWW Fig. 7.2. Relationship between the phenotypic correlation of yield of temperate hybrids under stressed (GYSTR) and well-watered (GYWW) conditions vs. the degree to which the stress reduced yield below its well-watered level. ERA hybrids are a series of hybrids released between 1953 and 2001. The remainder consists of 15 sets of 10–36 modern elite hybrids evaluated under 2–3 water levels. Stress was imposed near flowering or early grain filling, and only rarely was terminal in nature.

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of the differences in performance of hybrids for a range of the target environments (Duvick et al. 2004). A similar relationship between yield under nitrogen stress and unstressed yields was determined by Bänziger et al. (1997a) among progenies of tropical populations. In that study r(GYunstressed, GYstr) fell to 0.4 when N stress reduced yields by 50%. This suggests that yield potential is a reasonable predictor of grain yield under moderate stress levels (GYSTR/GYWW = 0.7–0.9), but as stress becomes more severe genetic mechanisms directly related to stress adaptation become increasingly important as a source of yield stability. For this reason Bänziger and co-workers (1997) concluded that when stressed yields are less than 60–70% of potential, it is more efficient to select for improved yields under stress environments. If the TPE includes a significant proportion of severely stressed sites, yield potential should be combined with specific secondary traits that conserve and stabilize yield under that level of stress. If negative genetic correlations exist between yield in unstressed and stressed environments, this would indicate a need to breed for each environment separately. Fortunately for temperate maize, such negative genetic correlations are rare. B. Crop Duration: Escape or Tolerance? A good match between the crop cycle duration and rainfall supply is essential to ensure stable grain production. In tropical environments where the length of the crop cycle is demarcated by the onset or cessation of rains, the successful matching of crop duration to the variable length of the rainy season is an important means of stabilizing production. Where Mediterranean-type climates occur with a winter rainfall peak and dry summers, summer crop growth in the absence of irrigation must be matched to water supply via stored soil water and spring rainfall. For most of the temperate maize growing areas, however, the frostfree growth period usually determines optimal crop duration. If a predictable mid-season dry period is common (e.g., southern Africa and Argentina) then an early-maturing hybrid may complete flowering before stress reduces kernel set and yield, and thus escape its effects. In wet years this could result in lost yield potential since later maturing hybrids normally outyield those with shorter crop duration. Edmeades et al. (2000a) advocated hybrids with a maturity suited to the wettest 20% of years in the TPE but with increased drought tolerance so that improvements in yield potential and stability would occur together. The complex nature of maturity, yield potential and their interactions with yield-stabilizing traits has recently been illustrated in silico by Chapman et al. (2003). They simulated changes in sorghum yield and

7. IMPROVING DROUGHT TOLERANCE IN MAIZE

181

gene frequency for traits contributing to drought tolerance in a recurrent selection program using 108 years of data from Queensland, Australia, where severe droughts are common. For the individual traits maturity, osmotic adjustment (OA), staygreen and transpiration efficiency they assumed simple additive-effect inheritance models. Grain yield was modeled as an outcome of interactions between traits as they influenced crop growth, development and yield accumulation within a range of environments differing in water availability. Populations were then subjected to cycles of yield selection using a simulated breeding program. As expected, severe terminal drought stress favored earliness. Performance under mid-season and moderate terminal-stress environments, however, was enhanced by higher transpiration efficiency, the higher yield potential of later maturing genotypes, and yield-stabilizing effects of improved OA and staygreen. Traits were selected and fixed in the breeding population at different rates (maturity > transpiration efficiency, OA > staygreen), depending on the types of environments to which progenies were exposed over the breeding program cycles. C. Stress-sensitive Periods in the Life of the Crop In older maize germplasm not subject to a long history of improvement, yield is reduced two to three times more when water deficits coincide with flowering compared with other growth stages. A drought-sensitive period from a few days before silking to about 25 days after has been identified, and water deficits during this time can sharply reduce grain number per plant (Claasen and Shaw 1970; Shaw 1977; Herrero and Johnson 1981; Grant et al. 1989; NeSmith and Ritchie 1992). Sensitivity during this period may be greater in maize than other cereals due to its monoecious habit and its relatively synchronous development of many florets on a single ear. Factors affecting kernel set under drought are reviewed in Section III D. In modern hybrids, considerable improvement has occurred in yield under stress at flowering. In a recent study 18 ERA hybrids released between 1953 and 2001 (approximately 3 hybrids per decade) were subjected to a period of approximately 50 days without irrigation in a rain-free environment. Genetic gain per year during this period was greatest under unstressed conditions (211 kg ha–1 yr–1) and declined by 41% under flowering stress and by 77% under late grain fill stress (Table 7.1). Data show that gains in tolerance of grain yield to flowering stress have outpaced those for stress tolerance during the latter half of grain filling. This was accompanied by systematic reductions in the anthesis-silking interval (ASI) with relatively little change in staygreen or in weight per kernel under grain filling and terminal stress (Fig. 7.3). Thus, susceptibility to stress at flowering has kept pace with increases

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Table 7.1. Yield reduction and genetic gain observed in 18 hybrids that were released between 1953 and 2001, when exposed to a stress imposed at different growth stages. Data are from two trials conducted in a dryland environment, 2001–2003. Period of major drought stress

Variable Stress started (°C days) Stress relieved (°C days) 50% anthesis (°C days) Mean yield (t ha–1) Percent of control Gain (kg ha–1 yr–1) 2 R

Control

663 15.91 100.0 211 0.93***

Early grain fill

Mid grain fill

Late grain fill

Terminal

257

354

456

583

769

772

861

956

1103

1283

681

676

670

662

660

Flowering

6.91 43.4 124 0.47**

4.95

4.73

6.26

10.98

31.1

29.7

39.3

69.0

92 0.42**

91 0.60***

48 0.71***

77 0.83***

in yield potential, while performance under late-season stress has not improved as rapidly as yield potential. Elite hybrids show considerable variation in drought susceptibility at flowering and in ASI, but the best of these show little yield loss when drought coincides with flowering (Bruce et al. 2002). D. Tolerance 1. Seedling Establishment. Adequate stands are critical to high yields. Plant establishment may be severely reduced by drought when a dry spell is encountered after initial planting rains. Research in tropical maize directed toward improving seedling establishment under drought stress showed only modest increases in survival under water deficit (Bänziger et al. 1997b). The authors concluded that selection for improved survival and biomass production under post-emergence drought stress is difficult since environmental variation is high in field screens, and because natural selection may have exploited much of the naturally occurring genetic variation for these traits. Drought during establishment is a rare occurrence in temperate production zones, and selection for seedling tolerance therefore is not a priority in Corn Belt germplasm.

7. IMPROVING DROUGHT TOLERANCE IN MAIZE

183

85 65

Flowering stress Slope = NS (R2 = 0.01)

45

50 25 0 –25

25 1950

7

5

1970

1980

1990

Well-watered Slope = 0.120 yr–1 (R2 = 0.62***)

0.45

Flowering stress Slope = 0.035 yr–1 (R2 = 0.51***)

3

1 1950

Filling, terminal Slope = 0.022 yr–1 (R2 = 0.10 NS) 1960

1970

1980

1990

No flowering stress Slope = –0.87°Cd yr–1 (R2 = 0.73***)

–50 1950

2000

2000

Weight per kernel (g)

Stay green visual score

9

1960

Flowering stress Slope = –1.50°Cd yr–1 (R2 = 0.66***)

75 ASI (°Cd)

Yield as percent of well-watered

100 Filling and terminal stress: Slope = –0.53% yr–1 (R2 = 0.65***)

1960

1970

1980

1990

2000

Well-watered/flowering Slope = 0.0024g yr–1 (R2 = 0.64***)

0.40 0.35 Filling/terminal stress Slope = 0.0004 g yr–1 (R2 = 0.17 NS)

0.30 0.25 0.20 0.15 1950

1960

1970

1980

1990

2000

Year of release of hybrid

Fig. 7.3. Grain yield of hybrids exposed to grain filling/terminal and flowering drought stress, expressed as a percent of the well-watered control, and anthesis-silking interval (ASI), visual stay green score, and weight per kernel vs. year of release. The 18 hybrids, a subset of the ERA hybrid set released from 1953 to present, were evaluated in Chile in 2003.

2. Vegetative Tolerance. Maize is relatively tolerant of stresses that occur after establishment but prior to flowering. At this stage cell expansion is very sensitive to plant turgor (Hsiao et al. 1970) and the primary stress effects are reductions in leaf area and plant height since leaves are expanding and stalks are elongating. Internode elongation patterns are a sensitive stress-timing diagnostic since any reduction in internode length is usually permanent. Poor husk cover is another byproduct of drought effects on cell expansion. Prophyll elongation occurs up to a week earlier than ear elongation. If a water deficit is relieved following the peak of husk growth, the ear may grow beyond the husk tip, creating an easy entry point for diseases and insects. This can negatively impact grain yield data quality under water stress. Drought prior to flowering reduces plant biomass because intercepted radiation is less and because radiation use efficiency (RUE) declines with stomatal closure and reduced gas exchange. A 20% reduction in RUE

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and a 7% reduction of intercepted radiation were reported in tropical maize subject to a severe flowering stress (Bolaños and Edmeades 1993a), although the effects on radiation interception became cumulatively greater during grain filling. Earl and Davis (2003) reported a 5–20% reduction in intercepted radiation but a 21–61% reduction in RUE under severe stress imposed in two successive seasons. There is also a concomitant reduction in leaf chlorophyll (Chaves et al. 2003). Yellowing of young leaves and a 15–25% reduction in chlorophyll levels are often observed in vegetative drought-stressed plants (G. Edmeades unpubl. data, 2002). Pre-flowering drought tolerance reflects a capacity to generate and maintain a large flux of current assimilate to the developing ear. Leaf rolling is commonly observed during the vegetative period and contributes to decreased light interception. Its occurrence varies greatly among genotypes and has usually been selected against in breeding programs that target improved drought tolerance (Bolaños and Edmeades 1996). Rolling typically begins mid-day a few days after first observing reduced plant height in water stressed plots. It reflects plant water status, leaf morphology and the presence of bulliform cells. However, the role of leaf rolling is being reconsidered because it is observed more frequently in newer hybrids than in older ERA hybrids (Fig. 7.4). Apparently, elite hybrids can reduce radiation interception and water use by leaf rolling to conserve water yet generate sufficient assimilate flux to the ear for adequate kernel set. Proclivity to roll is undoubtedly aided by the more upright leaf habit of hybrids released since 1970 (M. Cooper, unpubl. data, 2003). Prior to flowering, maize roots increase extensively in length, number and dry weight, and their capacity to capture water normally reaches a maximum near flowering (Mengel and Barber 1974). Excess or too little soil resistance to root growth appear to send signals, perhaps abscisic acid (ABA), that reduce top growth and directly affect yield (Passioura 2002). Root length density (cm length cm–3 soil) decreases exponentially with depth in the soil in most maize cultivars (J. Bolaños, unpubl. data, 1989; Gregory 1994). Intense rooting near the soil surface is of limited value to the plant under prolonged water stress, and is less necessary for nutrient capture in modern production systems because fertilizer is placed close to the plant. Recurrent selection for eight cycles in one tropical maize population exposed to severe drought stress at flowering resulted in a 35% root biomass reduction in the upper 0.5 m of soil (Bolaños et al. 1993) even though grain yield under flowering stress increased by 40% (Bolaños and Edmeades 1993a). Richards (1991) also reported improved stress tolerance in wheat when roots were reduced

7. IMPROVING DROUGHT TOLERANCE IN MAIZE

Flower, early fill and midfill stages Slope = –0.035 yr–1 (R2 = 0.79***)

40 Canopy temperture (°C)

Leaf rolling score

8

7

6

5

4 1950

1960

1970

1980

1990

2000

185

Flower, early fill and midfill stages Slope = –0.027°C yr–1 (R2 = 0.48**)

35

30

Well-watered control Slope = –0.010°C yr–1 (R2 = 0.17 NS)

25 1950

1960

1970

1980

1990

2000

Year of hybrid release

Fig. 7.4. Visual leaf rolling score (1 = severely rolled; 9 = completely unrolled) and canopy temperature observed on three occasions shortly after solar noon in 18 ERA hybrids grown under three water stress treatments imposed at different development stages prior to or shortly after flowering, Chile 2003. Canopy temperatures were observed one day prior to relieving the stress.

in the top 30 cm of soil. Limited data suggest that selection for high stable yield in temperate maize has led to deeper rooting and conserved water use, possibly aided by increased leaf rolling under stress. Increased capacity to access soil water is supported by a significant trend in lower leaf temperature in more recent hybrids of the ERA set grown under stress (Fig. 7.4). In drying soils, even root distribution with depth without an increase in root biomass would be desirable. There is little evidence that a further increase in rooting intensity over 1 cm cm–3 will improve water uptake (Ludlow and Muchow 1990), although maximum N and P uptake may call for root length densities of 2 to 5 cm cm–3. Increasing fine root numbers with depth seems fully justified (Boyer 1996) provided water is present at those depths. This is supported by recent results from simulation in wheat (King et al. 2003). Since measuring roots is difficult in a breeding program, several researchers have used electrical capacitance measurements for rapid root dimensions assessment. Although electrical capacitance and total root volume are positively correlated (van Beem et al. 1998), the measurement does not assess root distribution (CIMMYT, unpubl. data, 1998). 3. Reproductive Processes Partitioning. Breeding impact on maize reproductive processes has been significant. Duvick (1992, 1997), Castleberry et al. (1984), and Russell (1985) have documented improved yield potential over time in both public and private lines. Improved hybrids have also exhibited altered

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partitioning. A clear example of this change can be seen in ERA hybrid data (Duvick 1997; Duvick et al. 2004). For example, there has been a shift in partitioning away from tassel development (as measured by decreased tassel size) and toward increased ear growth (as measured by increased ear number per plant and decreased ASI). Other examples include: (1) drought tolerant CIMMYT populations partition more assimilate to ears than drought susceptible populations (Edmeades et al. 1993; Lafitte and Edmeades 1995); (2) barrenness associated with high-density planting is due to a shift in partitioning away from the ear and toward the stalk (Edmeades et al. 1999); and (3) improved yield under drought stress can be achieved by partitioning stem-infused sucrose to reproductive structures (Zinselmeier et al. 1995). Therefore, tassel, root, and stalk tissue may be perceived as competing structures vying for assimilate with the immature ear near anthesis. Anthesis-to-silking Interval. Grain yield per plant is closely associated with kernel number per plant, especially when the crop is subjected to stress around anthesis. Bolaños and Edmeades (1996) observed genetic correlations between grain yield and kernels per plant of 0.86 to 0.88 under severe flowering drought stress. Otegui et al. (1995) reported similar results. Utilizing managed drought stress locations, Pioneer Hi-Bred International, Inc. (PHI) elite commercial and pre-commercial maize hybrids have been evaluated for flowering stress tolerance. Results have shown that although shortened relative to older genetics, ASI in newer hybrids can be lengthened in susceptible genotypes under stress resulting in significant yield losses. Given the ease of measuring ASI and the association between a short ASI and increased grain yield under stress (Edmeades et al. 2000b), this trait is routinely measured in precommercial hybrid evaluations. Pre-commercial hybrids that are screened in managed drought stress testing sites and advanced to commercial status may be less likely to have a long ASI when subjected to flowering drought stress in grower fields than other commercial hybrids that were not evaluated under managed drought stress conditions. Ear Growth Rate (EGR). Kernel number per plant is positively correlated to plant and ear growth rates around anthesis (Tollenaar et al. 1992). Kernel set increases and ASI is shortened as plant and ear growth are increased near flowering. Kernel set is increased as source is increased during the critical flowering period (Schoper et al. 1982; Zinselmeier et al. 2002) and when source is decreased during this time, kernel set decreases (Early et al. 1967; Borrás et al. 2004). Genotypic variation exists for EGR at a given source level, and gains in EGR and kernel set may be made. Ribaut et al. (CIMMYT, unpubl.

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data) have shown that ASI and EGR are highly correlated with grain yield in a tropical recombinant inbred line (RIL) population subjected to flowering drought stress. At PHI, selections were made for rapid and slow EGR at flowering under irrigated nursery conditions in a B73*MO17 syn 4 RIL population and then evaluated for flowering drought tolerance under managed drought stress conditions (Zinselmeier, unpubl. data, 2002). Yield levels were reduced approximately 50% by the flowering drought stress treatment (Fig. 7.5). Mean yield of the rapid EGR selections under drought stress was greater than that of the slow EGR selections (p = 0.05). Grain yield was highly correlated with barrenness (data not shown), suggesting that selection for EGR improved drought tolerance by enhancing partitioning to the ear. Although more difficult to quantify than ASI, EGR is a trait associated with stress tolerance that may be useful in some screening programs or in attempts to understand the biology of stress tolerance. Lizaso et al. (2003) recently used the coincidence of pollen and emerged silks to successfully model kernel set at low pollen concentrations typical of stressed fields. In an open-pollinated population, asynchrony in pollen supply and silk emergence may be relatively unlikely since there is a wide range of flowering dates in the field, but asynchrony may be an issue in a large field of a uniform single cross hybrid. Maintaining the partitioning of assimilate to the ear ensures adequate ear growth and allows synchronous floral development of the staminate and pistillate flowers (short ASI) and appears to be important for adequate kernel set in maize. Given the importance of ear growth, gene

Full Irrigation

Stress 9.4

12.6 LSD0.05 = 12

a

LSD0.05 = 26.6

a

Yield (t/ha)

9.4

c 6.3

c

Yield (t/ha)

b 6.3 b c

3.1

c

3.1

0

0 Check

Fast Parent Material Type

Slow

Check

Fast Parent Material Type

Slow

Fig. 7.5. Relationship between ear growth rate (EGR) and yield from B73*Mol7 recombinant inbred lines. Tails (n = 10) were selected under irrigated nursery conditions over 3 years for ear growth rate at anthesis, classified as a Fast or Slow EGR entry and evaluated for yield under fully irrigated and flowering drought stress conditions, Chile, 2001. Performance of parents and elite inbred checks are included for comparison.

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expression profiling of immature maize ears subjected to drought stress at flowering has been conducted to determine pathways and genes associated with reduced ear growth. Initial trials were conducted in greenhouse/growth chamber conditions where the rooting volume was restricted (20-L volume pot). Water deficits sufficient to completely inhibit photosynthesis were achieved within five days and expression profiling of immature ears showed large changes in gene expression (Sun et al. 1999; Zinselmeier et al. 2002; Yu and Setter 2003). Classic “stress responsive” genes were identified as differentially expressed under drought stress, including the ABA pathway (rab17/dehydrin3 and rab 15), cell cycle (cyclin D, Cks1, MCM5, and CDK) and sugar metabolism (glu1 and hexose-transporter). Initially, these studies generated enthusiasm for the combination of expression profiling, physiology, and natural genetic variation for drought tolerance. However, when similar studies were conducted in the field where root volume was essentially unrestricted and the kinetics of stress was different (four to five weeks to completely inhibit photosynthesis instead of five days), a different story emerged. Habben (2001) showed that many fewer genes showed differential expression when stress was applied gradually (Table 7.2). Using rab17 as an example, classically drought responsive genes showed no differential expression when stress was imposed slowly under field conditions (Table 7.3). Genes showing differential expression under short-term stress were usually not a subset of the genes showing differential expression under long-term stress in the field. These results led us to conclude that field environments are unique from greenhouse/growth chamber environments and will require a re-examination of the metabolism that constitutes the physiology. Essential to this reexamination will be the use of new tools such as expression profiling and metabolic profiling to measure stress effects on gene expression at the cellular or organ level within diverse genotypes at multiple develTable 7.2. Effect of stress kinetics on differential gene expression of immature maize ears at anthesis. Greenhouse experiments were conducted with buckets and the transient water stress was sufficient to inhibit photosynthesis within 5 days after withholding water (i.e., Rapid stress kinetics). Field experiments required >5 weeks to deplete soil moisture sufficiently to inhibit photosynthesis (Slow stress kinetics). Stress category

Stress kinetics

% genes changed

Shading Drought (bucket) Density Drought (field)

Rapid Rapid Slow Slow

18 27 1 <1–2

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Table 7.3. The effect of stress kinetics on the differential expression of rab17 in maize tissues. Greenhouse experiments were conducted with buckets and the transient water stress was sufficient to inhibit photosynthesis within 5 days after withholding water (i.e., Rapid stress kinetics). Field experiments required >5 weeks to deplete soil moisture sufficiently to inhibit photosynthesis (Slow stress kinetics). Stress category

Stress kinetics

Organ

Fold increase

Drought (bucket) Drought (field) Drought (bucket) Drought (field) Drought (bucket) Drought (field)

Rapid Slow Rapid Slow Rapid Slow

Seed-pedicel Seed-pedicel Pedicel Pedicel Leaf Leaf

11 NSz 71 NS 134 NS

NS = not significant at p ≤ 0.1

z

opmental stages. Genetic transformation will also be critical in confirming the effect of these genes and pathways. Silk Growth. Maize silks are sensitive to changes in plant water status (Tatum and Kehr 1951; Herrero and Johnson 1981) and their growth pattern under stress is important in determining kernel set. Mechanistically, silk growth patterns may affect kernel set directly if silk exertion is not synchronous with pollen shed. Does this trait impact current elite genetics under flowering drought stress? Examining silk exertion relative to pollen shed of maize hybrids grown under flowering drought stress suggests that this trait shows significant variation in elite genetics (J. Schussler, unpubl. data). When considering a genetic solution to the problem of kernel set, one needs to ask whether the addition of kernels under water limiting conditions is wise. If a transient stress at flowering is expected, we believe that additional kernel set will improve yield given the plants’ ability to fill these additional kernels after the reduction of stress. Schussler et al. (2002) demonstrated that this concept is applicable to some germplasm grown under high plant-density stress. However, if a terminal stress is expected, this strategy will likely not improve yield. Silk growth patterns may also alter kernel set indirectly. Carcova and Otegui (2001) demonstrated that basal kernels may suppress kernel tip development in some genetic backgrounds and the suppression may be sufficient to prevent these tip kernels from being harvestable. Consequently, a breeding or transgenic strategy to enhance yield stability may be to speed silk exertion of tip silks relative to basal silk exertion, leading to more synchronous pollination of basal and tip spikelets.

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Kernel Set. Water deficit at flowering can increase the frequency of abnormal embryo sac development and kernel abortion (Moss and Downey 1971; Damptney and Aspinall 1976; Westgate and Boyer 1986). Abortive mechanisms may occur as early as 2–3 days after pollination (Westgate and Boyer 1986). The lag phase of kernel development (usually defined as 0–14 days after pollination) is considered key in determining successful kernel set. Critical physiological, genetic, and cytological events occur during this period and are not reversible. Although the extent of natural variation for some of these key processes is not known, genetic solutions may be achievable. For example, manipulation of genes that control the cell cycle may govern the extent to which endosperm cells endoreduplicate (Setter and Flannigan 2001; Yu and Setter 2003). 4. Osmotic Adjustment. Capacity to accumulate osmotically active solutes (e.g., organic acids, sugars, ions) in the cell generates a gradient in water potential from cell to soil that increases cell turgor in the presence of available water. In turn, this maintains meristematic activity and leaf area expansion while delaying foliar senescence. OA has been associated with stable hybrid maize grain yields across moderately dry environments in Argentina (Lemcoff et al. 1998), but appears to have less impact under severe stress (Bolaños and Edmeades 1991). Maize OA is modest (0.4 Mpa) relative to rice, wheat, and sorghum (> 1 Mpa) (Ludlow and Muchow 1990; Nguyen et al. 1997). This mechanism may serve as a stabilizer of growth only under moderate and transient stresses. Serraj and Sinclair (2002) suggest that the principal value of OA lies in maintaining root growth in dry soils. It is likely that selection for stability under multi-environment testing has already exploited much of the useful genetic variation for OA in temperate maize. Further increases in OA under drought in roots will likely be from transgenes linked to stress inducible, root-preferred promoters. 5. Growth Regulators as Signals. Phytohormone concentrations vary with stress level and can have profound growth effects. ABA concentration, often considered a growth regulator fostering survival rather than productivity, increases significantly under stress, leading to root growth maintenance, stomatal closure, leaf shedding, dormancy and possibly tip kernel abortion in maize (Ober and Setter 1992; Jones and Setter 2000; Setter et al. 2001; Ober and Sharp 2003). Improved yield under stress has also been associated with reduced ABA leaf concentrations in tropical maize (Mugo et al. 2000). Recent research suggests that ABA may function to limit ethylene production since ethylene increases under

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stress, inhibiting root growth and building up in rolled leaves (Sharp 2002). Heat stress reduces cytokinin concentrations in developing kernels (Jones and Setter 2000), and water stress likely has similar effects. Enhanced cytokinin biosynthesis by over-expression of transgenes driven by stress-inducible promoters in the roots could provide a positive signal to stimulate both root and shoot growth, provided the translocation of cytokinin to the developing grain did not induce undesirable side effects such as precocious germination on the ear. 6. Visual and Functional Staygreen. Drought accelerates leaf senescence, especially in the lower leaves. These leaves have a relatively minor role in assimilation in a normal canopy but serve as a source of remobilized assimilates and nutrients for younger leaves and developing grain. Breeding programs usually report delayed senescence as visual staygreen scores. Greenness per se does not guarantee continued C assimilation, and the distinction between visual staygreen and functional staygreen (i.e., high carbon exchange ratio, CER) is critically important (Thomas and Howarth 2000). Staygreen, however, has been associated with improved performance under drought (Borrell et al. 2000, 2001) and N stress (Bänziger and Lafitte 1997; Bänziger et al. 2002). There has been significant improvement in staygreen scores with selection in temperate germplasm evaluated under well-watered conditions (Fig. 7.3). Gain, however, was much less under grain filling and terminal stresses, a difference that is reflected also in small changes in weight per kernel under these stresses. Heterosis delays leaf death under drought in maize (CIMMYT, unpubl. data). Since N cannot be taken up readily from dry soil, a large grain sink may also accelerate leaf senescence. For a leaf area index of 4, up to 30% of the N in foliar biomass would be required if yields under drought stress increased by 0.8 t ha–1 and leaves were the only source of N for grain growth (Chapman and Edmeades 1999). Attention must be paid to slowing the decline in functional staygreen (best measured as CER) with age under the highly photo-oxidizing conditions that prevail in drought-stressed canopies exposed to high radiation levels. Modern hybrids grown under high density or drought had a reduced rate of decline in staygreen when compared with a hybrid released 30 years previously (Tollenaar 1992; Nissanka et al. 1997; Valentinuz 2002). Exploration of antioxidant enzymes such as superoxide dismutase (SOD) to control the concentration of active oxygen species (O2 and H2O2 mainly) and the metabolism of antioxidants ascorbate and glutathione remains a research priority. Over-expression of

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SOD has been reported to increase chilling tolerance in maize and drought tolerance in wheat (Noctor and Foyer 1998). Furthermore, there is evidence that specific metabolites such as glycinebetaine serve as protectants for enzymes and membranes against deleterious desiccation and photoxidation effects (Makela et al. 1996). Late embryogenesis abundant proteins increase sharply in tissues during desiccation or chilling, are upregulated by ABA, and are thought to stabilize plasma membrane structure (Campbell and Close 1997). However, formation of these compounds may represent an emergency response by the plant under severe drought conditions that rarely occur in a temperate production environment. While the small increase in CER provided by protectants in plants exposed to flowering stress may prevent barrenness in some cases, it is likely to be of limited value under severe terminal drought stress. 7. Remobilization of Stem Reserves. This is generally considered to be of greater importance in other cereals, such as wheat, than in maize, although in both species the process helps maintain a reasonably constant rate of grain filling under stress. Blum (1998) identified genetic variability for remobilization in wheat by applying chemical defoliants midway through grain filling (Blum 1998). A larger flux of remobilized assimilate might be expected from taller vs. shorter genotypes, but taller maize varieties have not proven more stable in yield under drought than their shorter counterparts (Fischer et al. 1983; Edmeades and Lafitte 1993). Stem reserves are much less effective at maintaining kernel set at flowering than in stabilizing filling rate (Schussler and Westgate 1991a,b, 1994, 1995; Blum 1997). Finally, capacity to remobilize stem reserves is likely to induce stem lodging in temperate maize, and any effort to screen for this trait must be done concurrently with evaluation of lodging susceptibility. 8. General Stress Tolerance. The strong correlation between kernel numbers and grain yield under any stress that affects photosynthesis per plant during flowering suggests that improvement for drought tolerance may increase tolerance to a number of other stresses. Drought tolerance has been positively correlated with high plant density-stress tolerance (Dow et al. 1984). There have been concomitant increases in plant densitystress and drought tolerance in temperate hybrids released over the past 50 years (Edmeades et al. 2003). Grain yield improvement and ASI reduction under drought through selection were accompanied by yield increases and reduced ASI under low N in four populations (Bänziger et al. 1999) and under high plant density (Mugo et al. 2003). As the stress

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level intensifies, general stress tolerance may become less important and tolerance mechanisms specific to that stress may become more important. Until a severe stress intensity is reached, however, selection for rapid ear growth rate using ASI and barrenness as external indicators should confer broad tolerance to an array of abiotic stresses that have their primary effect through reduced plant assimilation during flowering and grain fill (Edmeades et al. 2000b). We contend that carefully controlled water deficits generated in a rain-free environment provide the most effective means to display genetic variation for these traits. E. Relative Importance of Secondary Traits in Selection for Drought Tolerance Traits described in this section emphasize underlying mechanisms that confer stable grain yield, allowing us to dissect this complex trait and identify the key control points for further physiological and genetic analysis. Secondary traits may also enhance direct selection, allowing the breeder to develop an ideotype for specific environments that differ in drought stress timing and intensity (Rasmusson 1991; Chapman et al. 2003). There is a well-developed body of theory surrounding the use of secondary traits in breeding (Falconer and MacKay 1996). Genetic gain in the primary trait (GYSTR) is greater from indirect selection for a secondary trait such as ASI, than from direct selection when [hGY < |rG × hST|] where hGY and hST are the square roots of heritability for the primary and secondary traits, and rG is the genetic correlation between them (Bänziger and Lafitte 1997). It is rare to satisfy this condition under drought except where stress reduces GYSTR by 80% or more and its heritability falls sharply (Bolaños and Edmeades 1996). There are a few cases, however, where gain from selection for yield alone has been compared with gains from selection for [yield + secondary traits]. In an evaluation of a series of progeny trials conducted within a recurrent selection scheme for tolerance to low N, Bänziger and Lafitte (1997) reported that gain for GYSTR was increased by 14% when secondary traits were included during selection. Secondary traits have maximum utility in selection when they are (1) genetically associated with grain yield under stress, (2) highly heritable, (3) cheap, fast to measure and nondestructive, (4) stable over the measurement period, (5) observed at or before flowering so that undesirable parents are not crossed, (6) not associated with yield loss under unstressed conditions, and (7) represent an actual measurement rather than a subjective score (Edmeades et al. 2000a). Based on current knowledge (see Bolaños and Edmeades 1996) only GYSTR, ASI, barrenness, kernels per ear and staygreen are candidates for

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inclusion in such a selection index. While other secondary traits discussed may have potential in a selection index, most are not currently considered because they do not meet, or have not been evaluated for, the above criteria.

III. EXPERIMENTAL METHODS A. Target Environments and Environmental Characterization Maize breeding programs are designed to change the genotype and phenotype for multiple traits. However, the genetic basis for most important traits and their contributions to grain yield within the context of a TPE is not yet well understood. Lacking detailed knowledge of the gene-tophenotype relationships, it is difficult to predict many of the new trait phenotypes resulting from genotypes created over multiple breeding cycles. Consequently, maize breeding programs, regardless of strategy, rely heavily on direct evaluation of the new genotypes for their trait phenotypes within multi-environment trials (METs) that are expected to represent target production conditions. To predict and achieve high realized genetic gain for yield and yield stability, METs should be conducted with low experimental error and with sampling of environments and management conditions that predict the expected performance of the genotypes within key target environments, or more broadly in the TPE (Comstock 1977; DeLacy et al. 1996). With regard to experimental error, we are concerned with micro-environmental variation and other sources of withinexperiment measurement effects that impact experimental precision. Environment sampling controls the effects of genotype-by-environment (G*E) interactions that impact the accuracy of the differences observed in the METs relative to true values in the target environments and TPE. Conducting METs to achieve high levels of precision and accuracy is particularly challenging for drought-affected environments where experimental errors are often high and heterogeneous, and G*E interactions are common and poorly understood. Experimental error and G*E together contribute to low heritability and low realized genetic gain for yield and other performance-related traits discussed in Section II. Many authors have discussed issues regarding trial precision and accuracy when testing genotypes that have already been created (e.g., DeLacy et al. 1996; Smith et al. 2002a,b). Comstock (1977, 1996) articulated these points relative to the creation of new genotypes by the breeding process. Predicting performance of current genotypes and the capacity to create new genotypes are both important considerations in

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the design and implementation of a successful breeding program (see Section IV C and D). For commercial hybrid maize, we create and test both new inbreds with superior breeding value and new hybrids with superior performance for grain yield and drought tolerance component traits (Section IV A). Here we consider some general aspects of G*E interaction and experimental error and implications for the design and analysis of METs to predict the performance of maize genotypes in a drought-affected TPE. This is done within the context of a framework for evaluating and improving conventional maize breeding strategy (Section IV) and considering the opportunities to use molecular technologies to enhance the breeding strategy (Section V). For important quantitative traits, the phenotype is many levels of organization removed from that of allelic variation at the genome level. However, defining a breeding target requires joint consideration of the genetic changes created by breeding strategies and the effects of these changes on phenotypic performance. Technology only recently advanced to the level required to examine the detailed genetic architecture of traits and breeding strategy impact extending from the genome through to the phenotypic variation of quantitative traits. In practice hybrids are grown across a range of environmental conditions within a geographically defined target domain. At the phenotypic level, this is represented as a linear statistical model: pijk = m + ej + gi + (ge)ij + eijk

[1]

where pijk is the trait phenotypic value for measurement k taken on genotype i in environment j, m is the grand mean over all observations, e j is the marginal effect of environment j, gi is the marginal effect of genotype i, (ge)ij is the genotype-by-environment interaction effect for genotype i in environment j, and eijk is the residual effect for observation k on genotype i in environment j, often referred to collectively as experimental error. Note that in this equation and the following discussion, random effects are indicated with an underline. Nyquist (1991) and Holland et al. (2002) discussed alternative forms of this linear model. From equation [1] we see that predicting the outcomes of selection among genotypes and differences among a given set of genotypes within a target environment or the TPE, i.e., the differences in gi + (ge)ij effects, is complicated by the presence of G*E interactions and experimental errors. To consider the impact of these effects on breeding, we can extend the statistical model from the genotypic to the genic (gene action) level. For example, in relation to equation [1] we can represent this extension of the linear model as:

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(

pijk = µ + e j + α q + ηq

)

i

(

+ (αe)qj + (ηe)qj

)

ij

+ ε ijk

[2]

where we have partitioned genotypic effects gi into an additive (a q) and non-additive (hq) components for the alleles of gene (or quantitative trait locus, QTL) q possessed by genotype i. Similarly, we have partitioned the G*E interaction effect (ge)ij into an additive-by-environment effect (a e)qj and a non-additive-by-environment effect (he)qj for the alleles of gene q possessed by genotype i in environment j. Explicit examples of this extension of G*E interaction analysis from the genotypic to the QTL level are discussed by van Eeuwijk et al. (2002). From the above discussion, we recognize that predicting differences in genotypic performance or selection response requires an understanding of the genetic architecture of the traits, the multi-genic composition of the genotypes tested, and the types of environments and their frequencies of occurrence within the TPE (Cooper and Hammer 1996; Schlichting and Pigliucci 1998; Cooper 1999; Cooper and Podlich 2002; van Eeuwijk et al. 2002). Selection pressure directs desired changes in allele frequency in the germplasm pool and the creation of specific gene combinations. From a population improvement perspective, the objective is to increase the frequency of “favorable” alleles across the segregating loci that contribute to the genetic variation for the target traits (Chapman et al. 2003). At the theoretical level these changes in allele frequency may be considered as trajectories of genetic change within the genetic space available to the breeding program. This provides a quantifiable framework for modeling how effectively breeding programs achieve their objectives (Cooper et al. 2002). A major research challenge for breeding programs today is to combine molecular and phenotypic information to understand and efficiently achieve preferred genetic trajectories. The TPE may be international, national, local, or in some cases a series of non-contiguous regions with a common environmental constraint, e.g., drought, disease incidence, or acid soils. Comstock (1977) introduced the concept of a TPE to describe the range of environmental conditions encountered over time within the target region of a breeding program. This concept can be extended to quantify the TPE in terms of the major types of environments and their frequencies of occurrence within the defined target region (Cooper and Podlich 1999; Chapman et al. 2000a,b,c; Löffler et al. 2003). Environment classes may recognize the role of abiotic and biotic stresses in generating the important genotypeby-environment interactions that complicate selection (Chapman et al. 2002, 2003). Combining a characterization of the TPE with an understanding of the trait genetic architecture provides a basis for evaluating the merits of alternative selection (Cooper and Podlich 1999; Podlich et al. 1999) and breeding strategies (Podlich and Cooper 1998, 1999).

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Selection pressure is applied assuming that there is a positive association between phenotypic performance and the value of the alleles segregating for the trait. As the complexity of the target genotypeenvironment system increases, e.g., in the presence of single or multiple abiotic stresses, the relationship between the phenotype and genotype becomes more complex. At the genic level, G*E interactions can change the value of alleles across different environmental conditions. Interdependencies between traits contributing to adaptive responses under stress conditions can result in epistatic interactions among genes such that the relative value of alternative alleles at one locus is conditioned by the allelic conformation of other loci (Holland 2001; Chapman et al. 2002). Therefore, the breeder’s ability to direct changes in allele frequency is highly conditional upon how phenotypic determinations are made. Values of selected genotypes within a large and diverse TPE are determined by a MET sample of the TPE. These MET samples can vary and their representativeness of the TPE can significantly influence genetic trajectories achieved by a breeding program (Cooper and Podlich 1999). Much work has been done on the influence of test environment on selection response. However, most breeding programs do not quantify the relationship between the conditions imposed in the MET test environments and those that make up the TPE. Breeding programs generally utilize environments that are readily available at a limited set of research stations. In the worse case scenario, research station environments may be atypical of on-farm conditions within the TPE and result in selection and genetic trajectories that do not optimize performance in the TPE (Cooper and Podlich 1999). One strategy to avoid these limitations is to utilize a large number of “randomly sampled” environments representative of the TPE. While this strategy will work (e.g., Duvick et al. 2004), it does not enable a knowledge-based breeding strategy by improving our understanding of the relationships between allelic variation and phenotypic performance in the TPE. An alternative strategy is to establish a set of well-characterized managed environments to represent the key types of environments within the TPE (Fischer et al. 1983, 1989; Cooper et al. 1995; Fukai et al. 1999). Given that drought-resistant genotypes must perform well in both stressed and non-stressed environments, and that the TPE will encompass non-stressed environments, we increase the likelihood that the trials cover such a range of environments by strategically manipulating available water throughout the growing season (see Section III B). As a prelude to the discussion of G*E interactions, experimental errors, experimental design and statistical analysis in maize breeding experiments, we give an overview, without derivation, of the key components

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of a prediction framework for studying selection response in a TPE. See details in DeLacy et al. (1996). A basic prediction equation to estimate expected selection response is: 2

∆GM = iM hM sp(M)

[3]

where ∆GM is the predicted selection response based on the conditions sampled in the MET, iM is the standardized selection differential applied 2 on the phenotypes measured in the MET, hM is the heritability in the MET, and sp(M) is the standard deviation of the phenotype in the MET. Here we have used a subscript M to indicate that the j = 1,…, J environments sampled in the MET are taken to be a random sample of environments representing the distribution of environmental types that constitute the TPE. We may consider this as a basic equation in the sense that it is often used to predict expected improvements in broad adapta2 tion within the context of the TPE. Here the heritability component (hM ) is related to the model given in equation [1]. The line-mean heritability can be estimated in several ways (Nyquist 1991; Holland et al. 2002) by using appropriate estimates of variance components based on the specified linear model and its alternative forms. For example, for a given 2 set of genotypes created by the breeding program, line-mean hM(1) is defined as:

σ g2

2 hM (1) =

σ 2g

σ 2ge σ 2ε + + J JK

[4]

2

where hM (1) is a measure of degree of genotypic discrimination (or repeatability of the differences among the reference set of genotypes), s g2, s 2ge and s 2e are the genotypic, genotype-by-environment interaction and residual variance components, respectively, defined in relation to their respective effects included in equation [1], J is the number of environments sampled in the MET, and K is the number of replicates per envi2 ronment. Narrow-sense heritability (hM (2)) can be defined at the gene-effect level as:

σ α2

h2M (2) =

σ 2g

σ 2ge σ 2ε + + J JK

[5]

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where s a2 is the additive component of genetic variance defined in relation to the additive effect in equation [2] and is a component of the total genotypic variance. Narrow sense heritability predicts the expected transmission of average gene effects across generations in a defined reference population. The purpose for introducing equations [3], [4], and [5] is to emphasize the critical influence that both G*E interaction and experimental error sources of variation have on the breeder’s capacity to predict differences among genotypes and therefore the selection response based on the results of J environments sampled in METs. 2 Throughout the following we consider the hM as a general case, recognizing key issues that impact predicted selection response at both the genic and genotypic levels, but without giving the explicit derivations for both cases represented by equations [1]–[4]. Following Falconer (1952) and later developments given in Falconer and MacKay (1996), an alternative form of the basic selection response equation has been given where response is the result of indirect selection. We are interested in the indirect response in the TPE based on selection in the MET, which is written as DGT|M such that: DGT|M = iMhThMrg(TM)sp(T)

[6]

where, iM is the standardized selection differential applied to the phenotypic data in the MET, hT and hM are the square root of heritability in the TPE and MET, respectively, rg(TM) is the genetic correlation between trait performance measurements in the TPE and MET, and sp(T) is the standard deviation of trait phenotypic measurements in the TPE. This representation of indirect selection response demonstrates its relationship to direct selection response and indicates its value within the quantitative framework for examining the effects of G*E interactions and experimental errors on predicting performance and selection response in target environments. A specific application of indirect selection occurs when selecting in managed environments of the MET to achieve a response to selection within target production environments of the TPE. Equation [6] allows explicit consideration of G*E interaction effects on the genetic correlation of trait performance measurements between pairs of environments or following classification of environment-types between pairs of sets of environment types (e.g., flowering water deficit, grain-filling water deficit, well-watered conditions) by estimation and inspection of rg(TM) (Cooper and DeLacy 1994; van Eeuwijk et al. 2001; Smith et al. 2002a). Equations [3] and [6] are similar since the genetic variance component from a multi-environment analysis of variance

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represents an average of all of the genetic covariances between all possible pairs of environments that constitute the MET (Cooper and DeLacy 1994; Cooper et al. 1996). Therefore, with the appropriate model assumptions, equation [6] constitutes an expanded form of equation [3] whenever there is specific interest in the impact of G*E interactions on genetic correlations between the environments, and the conditions they represent, sampled in a MET. The impact of interactions on genotypic performance can be examined by a range of graphical tools developed within the broader family of pattern analysis methods. Using the framework discussed above, we now turn our attention to a more detailed consideration of G*E interactions and experimental errors in maize drought experiments. B. Experimental Design and Analysis Management to achieve a uniform plant water deficit across the experimental area is critical to optimize the design of drought METs. Analysis methods cannot compensate for poorly managed trials. However, there are several useful design and analysis techniques given the types of variation and non-uniformity that commonly occur even in the bestmanaged trials. 1. Extraneous Variation. Variation associated with experimental management and measurement techniques can be random or systematic; for example, systematic variation occurs when experiment operations are aligned with the rows or columns of a field trial. This is often referred to as extraneous variation (Gilmour et al. 1997). To illustrate this, Fig. 7.6A shows results of a trial where yield trends caused by the water management regime are apparent. Irrigation was supplied via drip tape for each column and charged from a pressurized supply manifold located at the bottom of the graphic. Due to frictional loss of water pressure in drip tapes along the columns, water delivery was slightly higher closer to the manifold. In water deficit treatments where plants were forced to use previously applied water during growth, variation in pre-stress irrigation supply resulted in differential plant water deficits down the length of the columns leading to consistently lower yields at the field end furthest from the irrigation supply (top of the graphic). There were also differences between the columns: the left side of Fig. 7.6A indicates a lower yielding section of the field in comparison to the right side. This gradient was likely due to differences in water supply among drip tapes or to gradients in soil water holding capacity. Such row and column effects are not as noticeable in the experiment shown in Fig. 7.6B.

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Fig. 7.6. Level plots of the raw yield data (tons per hectare) for two experiments, where darker colors indicate higher yielding plots. (A) An example where yield trends are introduced due to the water management regime; (B) An example where no trend is present.

Randomized complete block (RCB) designs help control differences between replicates by randomizing genotypes assigned to plots within each complete block. Other experimental designs better cope with extraneous variation (see Basford et al. 1996). A common alternative design for plant breeding is the resolvable alpha design (Patterson and Williams 1976). These are formed by dividing each complete replicate into sets of smaller blocks to adjust for heterogeneity between the smaller blocks. The designs can be created by the commercially available software, Alpha+ (Williams and Talbot 1993). This software also can be used to create row-column designs, which utilize two sets of smaller blocks in each complete replicate: one set that is aligned with the columns of the experiment and one that is aligned with the rows (Nguyen and Williams 1993; John and Williams 1995; Basford et al. 1996). Row-column designs

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are often appropriate for plant breeding programs since many activities such as planting, harvesting, and applications of fertilizer, herbicide, and pesticide are aligned with rows or columns of the experiment and can cause extraneous variation aligned with these factors. 2. Spatial Variation. In addition to extraneous variation, experiments may exhibit spatial variation with global or local trends where phenotypic measurements are related to position in the field (Gleeson and Cullis 1987; Cullis and Gleeson 1991; Cressie 1993; Gilmour et al. 1998; Smith et al. 2002a). For a spatially correlated process, the correlation between phenotypic values is higher for adjacent plots than for plots far apart. Smith et al. (2002a) describe how global spatial trends can be accounted for by including appropriate model terms and appropriate covariance structures for the residual error to accommodate a local (or natural) stationary trend (Gilmour et al. 1997). A common model is the autoregressive covariance structure for residuals in the rows and columns (AR1*AR1) of an experiment (or with AR1 on only rows or columns), which expresses the covariance between plot residuals as a function of the number of rows and columns separating the plots (see Smith et al. 2002a for details). Variograms indicate the correlation of residuals for plots that are separated by an arbitrary number of rows and columns. The variogram for an independent process is constant for plots separated by any number of rows and columns above zero; such an ideal variogram was shown in Smith et al. (2002a). Analyses of the two experiments in Fig. 7.6 are shown in Table 7.4 as examples to illustrate concepts utilizing data from our work with drought treatments. This is not intended to be an exhaustive treatment; the references provided in the preceding paragraphs are appropriate to review for more detail. Table 7.4 displays results from alternative models: (i) RCB analysis, (ii) RCB analysis with random row and column effects, and (iii) RCB analysis with random row and column effects and the AR1*AR1 covariance structure for the residuals. Note that the models for data in the second experiment (Fig. 7.6B) do not include an effect for replication since this was not found to be a significant source of variation in preliminary investigations. For each model, the deviance and variance component values are shown in addition to the autoregressive coefficients. The deviance can be used to compare models that have the same fixed effects structure and nested random effects. For example, with a change in two degrees of freedom, a difference in deviance of 5.99 (95th percentile of Chi-Square distribution with 2 degrees of freedom) between models (i) and (ii) or between models (ii) and (iii) would be considered a

Table 7.4. Several analyses of the data in Fig. 7.6A and 7.6B are shown. Results from (i) an RCB analysis, (ii) an RCB analysis with random row and column effects, and (iii) an RCB analysis with random row and column effects and the AR1 * AR1 covariance structure for the residuals. The models shown in (iv-a) and (iv-b) are the final analyses of those considered. Ind = independent errors; AR1 * AR1 = autoregressive covariance among row and column residuals.

Data Fig. 7.6A

Fig. 7.6B

Model

Error

(i) Rep + Entry

ind

(ii) Rep + Row + Col + Entry

ind

(iii) Rep + Row + Col + Entry

AR1 * AR1

(iv-a) Rep + Col + Entry

AR1 * AR1

(i) Entry

ind

(ii) Entry + Row + Col

ind

(iii) Entry + Row + Col

AR1 * AR1

(iv-b) Entry + Col

ind

Deviance (df)

Vg (S.E.)

Ve (S.E.)

1839.05 (220) 1762.28 (218) 1755.11 (216) 1755.20 (217)

318.0 (115.0) 307.3 (90.5) 267.5 (82.5) 267.6 (82.3)

1065.0 (117.0) 565.9 (67.7) 687.8 (104.5) 718.2 (89.7)

1023.92 (165) 1013.48 (163) 1012.73 (161) 1013.51 (163)

168.56 (42.81) 173.60 (43.23) 170.39 (42.55) 173.92 (43.29)

99.10 (12.49) 83.69 (11.78) 81.50 (11.50) 84.53 (10.92)

R: Vrow (S.E.) C: Vcol (S.E.)

Autoregressive coefficients

NA

NA

R: 123.9 (61.1) C: 407.3 (229.8) R: 23.7 (61.1) C: 387.0 (227.2)

NA

C: 383.7 (226.2)

R: 0.21 (0.08) C: 0.24 (0.11) R: 0.22 (0.08) C: 0.27 (0.08)

NA

NA

R: 0.85 (4.85) C: 16.73 (12.35) R: 3.49 (5.41) C: 16.29 (11.90)

NA

C: 16.71 (12.36)

R: –0.05 (0.11) C: –0.11 (0.13) NA

203

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significant improvement. For the data in Fig. 7.6A, the deviance indicates that model (iii) would be chosen among the three considered. This is not surprising given the column variance component and the two autoregressive parameters, which are both more than twice their standard errors. However, the variance component for the row factor in model (iii) is smaller than its standard error and it should be omitted from the statistical model. Results for this model are shown as model (iva) for the data from Fig. 7.6A. For the data used in Fig. 7.6B, the deviances indicate that model (ii) would be the best choice among the first three considered, but again the row factor should be omitted from the model, i.e., as in model (iv-b). While model selection can be based on quantitative measures (Table 7.4), the variogram is useful to evaluate and select alternative models. Figs. 7.7A and 7.7B show variograms corresponding to the first model (i) and final models (iv-a, iv-b) for each experiment. In Fig. 7.7A (i), differences among the columns of the experiment are reflected by the pat-

A

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Fig. 7.7. Variograms corresponding to the first model (i) and final models (iv-a, iv-b) for the yield data displayed graphically in Figures 7.6A and 7.6B.

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tern in the plot factor. This trend is removed in the variogram (Fig. 7.7A (iv-a)) corresponding to the model (iv-a) and is much closer to the “ideal” variogram that is flat at all row and/or column lags above zero. Fig. 7.7B (i) and (iv-b) display variograms for the first and final models applied to the data in Figure 7.6B. In this case the variogram in Fig. 7.7B (iv-b) corresponds to the best choice from among the four models, although the difference between the two variograms is not nearly as striking as for Fig. 7.7A. Fig. 7.8 contains scatter plots showing the impact that improved experimental analysis has on the best linear unbiased predictors (BLUPs) of genotype yield for these data. Figs. 7.8A and 7.8B are graphs of the BLUPs from the final models in Table 7.4 (models (iv-a) and (iv-b)) versus the BLUPs based on the initial models (model (i)) from data shown in Fig. 7.6A and 7.6B, respectively. From Fig. 7.8, it is evident that there has been a greater impact on the adjusted means under model (iv-a) using the data from Fig. 7.6A. These examples are intended to show an application to drought research where experimental design and analysis helped reduce experimental error. Given that extraneous variation and spatial trends are common in drought experiments and drought-affected METs, maize breeders will be assisted by combining the use of incomplete block experimental designs (e.g., alpha and row-column designs) and appropriate spatial analysis methodology to adjust the raw data for sources of extraneous variation and global and local trends. Ultimately this will A

B 17 BLUPs from model (iv-b)

BLUPs from model (iv-a)

6.0 5.5 5.0 4.5 4.0 3.5

16

15

14

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3.5 4.0 4.5 5.0 5.5 BLUPs from model (i)

6.0

14 15 16 BLUPs from model (i)

17

Fig. 7.8. (A) Scatter plot of the yield BLUPs from the final model in Table 7.4 (model (iv-a)) versus the BLUPs based on the initial model (model (i)) using the data shown graphically in Fig. 7.6A. The line y = x, where the two sets of BLUPS would be identical is shown for reference. (B) Scatter plot for the data shown in Fig. 7.6B. It is evident that there has been a greater impact on the adjusted means under model (iv-a) using the data from Fig. 7.6A.

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reduce the influence of experimental error and increase trait heritability estimates (equations [4] and [5]). Increased heritabilities have consequences for the predicted selection response (see Section III A). For example, using equation [5] to compute heritability for data in Fig. 7.6A on a single environment and replicate basis, we estimate 1.26/(1.26 + 4.23) = 0.23 under model (i) and 0.27 under model (iv-a). Bänziger and Lafitte (1997) reported similar analysis effects when conducting selections under low N. Improved experimental analysis increases both heritability and predicted selection response (equation [3]). In the previous examples experiments were designed to achieve maximum gains from the row-column and spatial analyses. However, it is possible to include factors that account for systematic extraneous variation in experiments that were not designed as such through a procedure analogous to the concept of post-blocking. Qiao et al. (2000) used empirical results from a long-term wheat study and Smith et al. (2002b) used theoretical arguments and simulation to show that analysis adjustments to the raw data are expected to improve the correlation between the estimates obtained from analysis of METs and the true values in the target environments and thus the TPE. These findings support the argument that experimenters will generally better understand genotypic effects and G*E interactions using data that has been adjusted for natural and extraneous variation. C. Describing, Understanding, and Managing G*E in Drought Trials Once an optimal analysis is identified for each environment, this information can be incorporated into a multi-environment analysis using appropriate mixed models. In such analyses there are a number of options for modeling the variance-covariance structure between environments (van Eeuwijk et al. 2001). Compound symmetry, also called uniform correlation, is the default variance-covariance structure in many statistical analysis packages. This structure implies that the genetic variance is the same for each environment and that the genetic correlation is the same for each pair of environments. These assumptions are generally not true and rarely appropriate especially when environments are substantially different, such as combinations of well-watered and drought-stressed environments. One alternative to the compound symmetry structure is the unstructured variance-covariance matrix. This allows a different genetic variance for each environment and a different genetic correlation for each pair of environments. However, the variance-covariance matrix has a large number of parameters that can cause difficulty for model fitting. van Eeuwijk et al. (2001) describe a class of mixed multiplicative mod-

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els or factor-analytic models that allow genetic variances and covariances to be dependent on some (unobserved) quantitative descriptors of the environments. This class of models is flexible, and contains fewer parameters than the unstructured variance-covariance matrix. To illustrate multi-environment analysis techniques, we use data from a trial containing 54 hybrids evaluated in two replicates of six different environments. Duvick et al. (2004) have discussed these ERA hybrids within the context of long-term improvements for yield from a commercial breeding program. The objective of our experiment was to study the ERA hybrids under specific drought and density-stress conditions. Instead of relying on a set of randomly sampled environments to capture the diverse and hard-to-predict drought environments, water and density treatments were imposed to help assure that our MET contained the key environments of interest. As key environment types and their frequencies of occurrence in the TPE are better understood, managed stress experiments like this one may reduce some trial-and-error or augment METs that are based on “random” samples of environments from the TPE. In the design of any targeted MET regime, consideration should be given to the genetic correlation between trait performance of genotypes in the managed environments and the production environments in the TPE as emphasized in equation [6]. The six environments in this experiment were combinations of two different density treatments (High Density = 91,000 plants/ha, Low Density = 46,000 plants/ ha) and three different irrigation treatments (well watered, flowering drought stress, and grain filling drought stress). This experiment was designed as a row-column design in each treatment, but initial analyses indicated that there were no significant design effects or spatial correlation (Fig. 7.9). Therefore, all subsequent analyses use a RCB analysis. We have found this to be the exception rather than the rule and recommend the use of improved experimental designs for most drought studies. For example, a row-column design was needed in the second year of this experiment (data not shown). The form of the final model used for the analysis of these data is: yijkl = m + Dj + Wk + (WD)jk + (r/WD)jkl + gi + (gD)ij + (gW)ik + (gWD)ijk + eijkl where yijkl represents the phenotypic data (e.g., yield) for the l th replicate of the i th genotype in density (D) treatment j and water (W) treatment k. Random effects are underlined. Note that we classified hybrids as random effects (Smith et al. 2002a). In practice, consideration should be given to appropriate classification of fixed vs. random effects given the structure and objectives of an experiment. The choice of random hybrid effects is somewhat conservative since the resulting BLUPs of

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High

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Column Fig. 7.9. Level plot of raw yield data (tons per hectare) for experiment with 54 hybrids in six different environments (combination of high density = 91,000 plants/ha and low density = 46,000 plants/ha with flowering stress, filling stress, and well-watered treatments), with darker shades of gray indicating higher-yielding plots.

hybrid means are more “shrunk” back towards an overall mean than if hybrids were fixed effects. Preliminary investigations showed that a uniform correlation structure between environments, which is the default in many software applications, was appropriate for these data. This is due to strong genotypic effects that exist in this set of hybrids spanning the years 1930–2002 and would not be expected for data sets where genotypes were more alike. Fig. 7.10 shows scatter plots and high correlations between the BLUPs for each pair of environments included in this managed stress study. The large genetic variance attributed to improvements in grain yield over time also contributes to higher heritability for yield (0.89). Sophisticated graphical tools have been developed to visualize the genotype-environment matrices of trait data generated from METs, e.g., the biplot (Kroonenberg 1995; DeLacy et al. 1996; Yan and Kang 2003). The biplot in Fig. 7.11A

7. IMPROVING DROUGHT TOLERANCE IN MAIZE

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Fig. 7.10. Scatter plots and correlations between the yield BLUPs for each pair of environments defined and shown in Fig. 7.9. High = High density; Low = Low density; Flower = Flowering drought stress; Fill = Grain filling drought stress; WW = Well-watered control.

contains all 54 ERA hybrids and the primary feature is an increase in average yield in all six environments for later release dates. Fig. 7.11B contains only those hybrids with a release date of 1990 or later. There appears to be one environment that has a particularly low genetic correlation with the other five environments. In this experiment there are several hybrids that perform differently in the high density, grain filling stressed environment than in the other five environments. The reasons for this reduced genetic correlation are a potential subject for further investigation This discussion and example has emphasized appropriate designs and analyses for a MET and graphical interpretation of subsequent results. With this particular data set, we can evaluate how representative the drought MET is of the TPE since these hybrids have been tested extensively in a number of experiments from the TPE (Duvick et al.

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A All Data (1930 – 2002) 33P66_1997

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Fig. 7.11. Biplots of the yield BLUPs for the hybrids from Figure 7.9. (A) Contains all 54 hybrids and it can be seen that the primary feature is an increase in average yield, in all six environments, for later release dates. (B) Contains only those hybrids with a release date of 1990 or later.

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Grain Yield Woodland YldOpt (t/ha)

Fig. 7.12. Scatter plot of the yield BLUPs of each hybrid under optimal density conditions (YldOpt) for each water regime versus the BLUPs obtained in Duvick et al. 2004, which are representative of the TPE. Flowering = Flowering drought stress; Filling = Grain filling drought stress; Well-watered = low stress control. Data from Johnston, Iowa, and Woodland, California.

2004). Fig. 7.12 contains a scatter plot of BLUPs under optimal density conditions for each water regime versus BLUPs obtained under optimal density, i.e., the density under which the hybrids achieved their highest yield, in Duvick et al. (2004), which are representative of the TPE. This comparison suggests that grain yield of the hybrids under managed drought regimes is positively associated with grain yield in target environments of the U.S. Corn Belt. D. Hybrid Characterization: Measures of Drought Tolerance 1. Need for Managed Stress Approach. One challenge in dissecting the basis for genotypic differences in drought tolerance is the identification and management of uniform and reproducible water deficit environments. Historically, the use of many yield test locations within a breeding program was assumed to incorporate a percentage of these environments. In practice, this has proven challenging due to unpredictable rainfall

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patterns in the U.S. Corn Belt and a natural bias to conduct yield trials in areas with high yield potential. An alternative approach to extensive sampling is to develop managed stress locations with carefully designed reproducible levels of timing, duration and intensity of water deficits. This is best accomplished by choosing locations with historically low rainfall during the maize growing season. Ideally, these locations will also possess deep, well-drained soils with uniform water holding capacity in the maize root zone. These factors are critical since small differences in uniformity lead to large differences in plant water deficits under drought stress resulting in unmanageable levels of variability for yield and secondary traits of interest. A dependable high-quality water source is also necessary to control prestress irrigation and “load” soils to specific levels of water holding capacity and to provide adequate irrigation for irrigated control plots. Extreme precision in irrigation application is required, necessitating the use of drip tape or overhead irrigation systems. Knowledge of evapotranspiration rates and hybrid phenology must be combined in order to “stage” plants for specific drought stress levels at specific developmental stages (see Section II C). This can only be accomplished on a routine basis in relatively rare environments where sunshine, temperature, and wind are fairly constant during a growing season and across growing seasons. Preliminary studies to confirm the value of potential sites, such as the example depicted in Figure 7.9, should be conducted for 1–3 years prior to undertaking managed stress studies. 2. Execution of Managed Stress Approach. Drought tolerance can be realized through a variety of putative mechanisms occurring at various plant growth and development stages (see Section II D). Prioritization of drought tolerance trait targets and selection strategies must be dictated by (1) the relative predominance of drought timing and severity in the TPE, and (2) the probability of genetic gain due to utilization of natural variation or candidate transgenes. Flowering Drought Stress. Due to the determinate flowering habit of maize, water deficits occurring during anthesis can be severe (see Section II C and D 3). Ear development and associated silk exertion can be significantly delayed while tassel development and pollen dehiscence are far less affected. Increased ASI under flowering drought stress results in a reduction in the number of pollinated ovaries since some silks do not emerge at all or are delayed beyond the window of pollen availability. Selection for reduced ASI in combination with grain yield has been effective for increasing drought tolerance in maize (see Section II C). In order to characterize hybrid differences for this trait, water must be

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withheld such that the plants utilize most of the stored soil water prior to flowering. An appropriately timed drought stress treatment will result in plants that are shorter, exhibit leaf rolling, lower leaf senescence, and increased ASI and barrenness compared to fully irrigated plants. This results in greater plant-to-plant variability, an indication that some individuals within the population cannot compete with their neighbors. In contrast, hybrids tolerant to flowering drought stress typically maintain more uniform plant-to-plant growth with little barrenness. Other within-ear traits also affect reproductive efficiency. These include the rate of silk exertion, length of silk receptivity under plant water deficits, and amount of post-pollination kernel abortion. These processes establish yield potential during the critical anthesis period. Under managed flowering drought stress conditions, kernel number per ear is highly correlated with grain yield per plant (Fig. 7.13). Data are from individual plants of two different hybrids exposed to different levels of flowering stress. Data at the low end of the scale are from severely stressed plants, data in the middle are from plants receiving a less severe drought stress at flowering, and data at the highest point of the scale are from plants in the fully irrigated control environment. Kernel number per ear increased as stress was minimized.

250

r2 = 0.98, P<0.001 y = –1.1 + 0.32x

Grain Yield Plant–1 (g)

200

150

100

50

0 0

100

200

300 400 Kernels Ear–1

500

600

700

Fig. 7.13. Relationship between kernels per ear and grain yield per plant. Data are from two hybrids grown under full irrigation and various levels of drought stress at flowering, California, 2001.

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In studies of flowering drought stress, irrigation is typically resupplied after anthesis to terminate the transient drought stress. This technique allows the separation of genetic variation for mechanisms conferring flowering drought stress tolerance from other unique mechanisms functioning during grain fill. Transient flowering drought stress typically results in 20–70% yield reductions (Table 7.5). Significant G*E for drought tolerance is usually observed. Interestingly, some hybrids that are relatively tolerant to flowering drought stress are more susceptible to terminal grain filling stress. In general, yield reductions from terminal grain filling stress are less severe compared to those observed under the flowering stress treatment, confirming the critical nature of the flowering stress in limiting yield potential. Grain Filling Drought Stress. Yield reductions observed under flowering stress likely result from weaknesses in reproductive efficiency traits. In contrast, yield reductions from grain filling stress result from alternative mechanisms including maintenance of photosynthesis (functional staygreen), rooting depth, and remobilization of stalk reserves to Table 7.5. Response of ERA hybrids to two types of managed drought stress. Flowering stress was generated by withholding irrigation water from 5 weeks prior to anthesis to two weeks after anthesis. This treatment was then fully irrigated for the remainder of the growing season. In filling stress, water was withheld at flowering until maturity. Data are means of 2 replicate plots. Well watered

Entry 3335 33G26 3394 3379 3563 3362 33R77 34G81 3489 34G13 34B23 3417 33P67 3390 34H31

Year release 1995 1998 1991 1988 1991 1989 2001 1997 1994 2000 1999 1990 1999 1967 2002

Yield % trt (t/ha) mean 16.0 13.7 14.7 13.0 12.9 14.0 16.4 12.2 13.7 13.4 15.3 13.5 13.0 11.0 14.7

137 117 126 111 111 120 141 104 118 115 131 116 112 94 126

Flowering stress

Yield (t/ha) 9.9 8.2 8.6 7.4 7.3 7.9 9.1 6.5 7.3 7.1 7.9 6.9 6.4 5.4 6.9

Yield reduction % trt (%) mean 38 40 42 43 43 43 45 46 47 47 48 49 51 51 53

226 185 195 168 166 179 206 148 166 162 180 157 146 122 155

Filling stress

Yield (t/ha) 7.5 10.8 9.3 7.1 8.8 9.1 10.8 7.9 8.8 8.2 9.3 8.4 8.6 6.9 9.2

Yield reduction % trt (%) mean 54 21 37 45 32 35 33 34 36 39 40 38 34 37 38

108 156 134 102 126 131 156 115 127 119 134 122 125 100 132

7. IMPROVING DROUGHT TOLERANCE IN MAIZE

Well watered

Entry

Year release

33P66 3366 3618 328HYB 3378 3388 3376 Reid YD 3382 340HYB 354 3334 3475 34N44 34M95 307HYB 3529 3377 301B 3301A 3206 3279 329HYB 352HYB 350B 330HYB 3541 3571 322HYB 3517 347HYB Krug YD 3306 344HYB 317OLD 351OLD 354A 336HYB 339HYB

1997 1972 1961 1959 1983 1970 1965 1920 1976 1941 1953 1969 1984 2002 2001 1936 1975 1982 1952 1974 1962 1992 1954 1946 1948 1939 1975 1968 1936 1971 1950 1920 1963 1945 1937 1934 1958 1940 1942

CV (%) Mean LSD (0.05)

Yield % trt (t/ha) mean

215

Flowering stress

Yield (t/ha)

Filling stress

Yield reduction % trt Yield (%) mean (t/ha)

Yield reduction % trt (%) mean

14.8 9.7 7.8 9.3 15.2 10.3 14.4 9.2 11.9 9.6 7.6 11.8 11.8 14.5 13.7 9.3 12.5 14.7 9.3 10.8 10.8 13.1 10.8 6.5 12.4 9.8 10.4 10.8 9.8 9.8 8.4 9.1 11.9 10.9 7.8 7.5 9.6 10.8 8.9

127 83 67 80 131 88 124 79 102 82 66 102 102 124 118 80 107 126 79 93 93 112 93 56 106 85 89 92 84 85 72 78 102 93 68 65 83 92 76

6.6 4.2 3.3 3.9 6.2 4.2 5.7 3.5 4.4 3.5 2.7 4.2 4.2 5.0 4.7 3.1 4.2 4.4 2.7 3.1 3.1 3.5 2.9 1.7 3.1 2.4 2.5 2.6 2.2 2.1 1.8 1.9 2.5 2.3 1.5 1.3 1.4 1.5 1.1

55 57 57 58 59 59 60 62 63 64 65 65 65 66 66 67 67 70 71 71 71 73 74 74 75 76 76 76 77 79 79 79 79 80 81 83 86 86 88

150 95 76 88 142 95 130 79 100 78 61 94 93 112 105 71 94 101 62 72 70 81 65 39 72 55 58 59 50 48 40 43 56 51 34 29 32 35 25

6.2 5.7 6.2 6.2 10.7 6.0 7.0 4.6 8.7 4.2 5.4 8.6 5.9 8.6 10.1 4.5 6.1 8.6 5.1 6.5 6.5 10.0 5.7 5.1 5.7 4.8 7.1 6.6 4.4 7.6 5.2 5.2 6.9 6.1 3.9 5.2 4.3 3.7 5.0

59 40 21 34 29 42 52 51 27 56 30 28 50 41 26 52 51 42 45 39 40 24 48 23 54 51 32 38 55 23 39 44 43 44 50 32 55 66 44

89 83 90 89 155 86 101 66 126 61 77 123 85 124 146 65 89 124 74 95 94 145 82 74 83 70 102 96 64 109 74 74 99 89 56 75 62 53 72

10 11.6 2.3

10 99 20

29 4.4 2.6

16 64 21

29 100 58

13 6.9 1.8

19 40 15

13 100 26

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the grain (see Section II D). Different drought tolerance rankings are often observed for a group of hybrids when comparing their performance in these two drought types. Relative commercial value of improved drought tolerance at these developmental stages is currently being evaluated. When drought stress occurs only during grain fill, plants utilize water rapidly since they have attained normal plant stature prior to flowering. Within a few weeks after initiating grain filling stress treatments, plants begin to exhibit premature leaf senescence and reduced ear growth. Some hybrids fail very rapidly with the entire plant senescing soon afterwards. Kernel number per ear is largely unaffected by this treatment since this yield component is established soon after pollination. In contrast, weight per kernel is usually significantly reduced, reflecting the loss of source activity caused by photosynthesis inhibition during this prolonged drought stress. Significant differences are typically observed for hybrids subjected to grain filling drought. Variation in yield reductions in this treatment is somewhat less than that observed under flowering stress (Table 7.5). Identifying specific mechanisms responsible for this variation is more elusive as well. Possible breeding targets for improving grain filling drought stress tolerance include (1) deeper rooting systems capable of delaying plant water deficits later into filling, (2) maintenance of functional leaf area through control of stress-induced reactive oxygen species, and (3) when those mechanisms are exhausted, improved C and N remobilization from stalks to maintain kernel growth. 3. Application of Managed Stress Approaches. Managed stress studies provide necessary calibration of hybrid responses in that location versus other sites in the TPE. This is challenging, since attempts to compare performance in the managed stress location to non-managed stress locations is subject to variable and random levels of stress at most TPE locations. Nonetheless, estimates of this correlation are needed to validate the resources dedicated to the managed stress program (e.g., Fig. 7.12). Assuming the predictive capability of managed stress studies is confirmed, the next logical application is early-generation screening of new materials in the commercial development pipeline. A dependable screen for drought tolerance early in hybrid development is valuable, similar to screening for diseases, insect resistance, and other biotic factors that have proven extremely valuable in plant breeding programs. Utilizing data from managed drought stress locations should increase the rate of gain for yield, assuming that drought tolerance contributes significantly to yield stability across the TPE.

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The advantage of generating drought tolerance data in managed stress locations logically extends to evaluating transgenic drought tolerance approaches. A reliable phenotypic screen is often the limiting factor in research aimed at identifying functional candidate genes. Finally, managed stress locations for phenotyping drought responses enhance the capacity to dissect physiological and genetic mechanisms of drought tolerance at the molecular level (see Section V). Current genomics technology lessens genotyping as a limiting factor in trait dissection, thus phenotyping quickly becomes the major limiting factor. Precise measurement of individual plant growth under drought stress is a major determinant of the potential success of this complex trait genetic dissection. Identifying QTL associated with drought and using that information to incorporate favorable alleles into new hybrids may be enhanced substantially when phenotypic data produced under drought are precise and reproducible. This represents a significant opportunity in the long term, and certainly complements the current applications of managed stress methodology for enhanced germplasm characterization and conventional selection.

IV. APPLIED BREEDING METHODS A. Breeding Goals Drought tolerance breeding goals must work in concert with the improvement of other traits to ensure success. Starting with a clear definition of purpose and parental material possessing adequate genetic variation (long-term goals), the breeder must develop criteria and progeny selection methods that capture superior genotypes (medium-term goals), and finally characterize the lines and hybrids that have been developed (short-term goals). 1. Long-term. Selection of starting populations is perhaps the single most important decision a breeder makes each year (see Bernardo 2003). Decisions regarding the starting population set the limits on progress that can be made through breeding. This is true whether one starts with an open-pollinated, elite F2, or backcross population. It is true regardless of the purpose of the breeding effort such as elite hybrid development, source population development, understanding trait inheritance, or introgression of novel variation. The long-term impact of breeding efforts initiated in a given season will not be known for perhaps six to eight years. Thus, the importance of careful consideration of long-term goals,

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including choice of parental germplasm (Section IV C), cannot be overstated. Our work is focused on developing hybrids with both improved drought tolerance and improved top-end grain yield. Markets where drought regularly reduces yield may be willing to sacrifice some top-end yield potential to improve yield in drought years. 2. Medium-term. Decisions made during the breeding cycle that impact progeny selection and experimental hybrid performance will impact potential commercialization in the two- to six-year timeframe. Successful implementation of medium-term goals allows the breeder to utilize genetic variation inherent in the starting populations and determines the ultimate value of the breeding effort. Special attention must be paid to criteria for selection, which allows identification of superior progeny for drought responsiveness (see Section IV D). Regardless of the selection methods chosen (Sections IV E and V), progress can only be made if sufficient genetic variation is present and identifiable. 3. Short-term. Decisions made in the current breeding cycle that directly impact hybrid commercialization are short-term goals. These choices are generally data-driven and may be categorized as hybrid characterization and advancement. Genetic variation is relatively fixed, and the breeder’s work is nearly complete. Experimental characterization of the drought response is covered in Section III. Many other factors such as grain quality, standability, insect and disease resistance, maturity, and market demands that may have little to do with the hybrid’s performance under drought also influence the final decision regarding commercialization. Markets with severe drought stress may need a different hybrid than markets with high-end yield potential but a need for yield stability. Concurrent with hybrid advancement and characterization, decisions are required regarding the parental ability of the inbred lines involved, including female yield, pollen shed, tillering, test weight, and seed size. An adequate supply of pure seed is also critical for successful production and reliable supply of the commercial hybrid. Inbred female yield stability including its drought response are important. Although hybrid seed production is usually irrigated, inbreds can experience stress during the growing season. Inbreds are more vulnerable to stress than hybrids, although both respond via the same mechanisms (Section II). For example, slow silk growth and/or kernel tip abortion under drought stress can negatively impact female seed yield. Inbred drought response is not necessarily well correlated with hybrid drought response, and it remains to be proved whether or not per se performance is a useful contributor to improving hybrid performance. How-

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ever, understanding and improving inbred line performance does contribute directly to reduced seed production cost and increased reliability of hybrid supply. Subjecting inbreds to drought stress during their development (medium- and long-term goals) should therefore be considered for improvement of both hybrid drought performance and inbred yield stability. Correlations between inbreds and hybrids for various traits in tropical germplasm are discussed in Edmeades et al. (2000a) and Betrán et al. (2003). B. Inheritance of Tolerance Precipitation, amount and timing of supplemental irrigation, soil conditions, abiotic stresses such as heat and nutrient sufficiency, and biotic stresses can all impact the amount of stress on the maize plant. Physiological mechanisms that control response to external factors and their interactions involve multiple pathways, many of which are well known (Section II). At the same time, recent gene expression studies have shown that there are likely many genes of unknown function associated with drought responses (Zinselmeier et al. 2002; Section II D 3). Given the suite of pathways and array of external factors and all their possible interactions, understanding drought tolerance inheritance and Mendelization of its factors is a daunting challenge. Due to increased error variation, heritability decreases for yield and secondary traits influencing drought response under drought stress as the level of stress increases (Jensen 1971; Jensen and Cavalieri 1983; Blum 1988; Bolaños and Edmeades 1996). For example, Bolaños and Edmeades (1996) reported that grain yield heritability decreased from 0.57 under full irrigation to 0.43 under severe stress that resulted in yields of only 14% of well-watered yield. Table 7.6 shows heritabilities for several additional traits measured under well-watered and severe stress, and their genetic correlations with grain yield in tropical maize. While most secondary traits associated with drought tolerance are quantitatively inherited (Lebreton et al. 1995; Veldbloom and Lee 1996a,b; Tuberosa et al. 1998; Sanguineti et al. 1999; Tuberosa et al. 2002a), there are a few examples of simple inheritance. Gentinetta et al. (1986) suggested that delayed senescence (staygreen) in the maize inbred line Lo87602 may be a simply inherited dominant trait. Studies on staygreen in sorghum have shown small numbers of QTL explaining a large percentage of variation, which suggests a relatively simple level of inheritance and some overlap with known staygreen QTL in maize (Subudhi et al. 2000; Tao et al. 2000; Kebede et al. 2001). In small grains, Lilley et al. (1996), Morgan (1999), and Kim et al. (2000) have reported that

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Table 7.6. Broad sense heritabilities observed under severe drought stress, and genetic correlations between grain yield and selected traits under severe drought stress for S1 progenies drawn from several tropical maize populations. Severe stress yields averaged 14% of well-watered plots. Data from Bolaños and Edmeades 1996.

Variable Grain yield Ears plant–1 Kernels ear–1 Kernels plant–1 Kernel weight Days to anthesis ASI Leaf senescence score

No. trials

Heritability under full irrigation

Heritability under stress

Genotypic correlation with grain yield

9 9 8 8 9 9 8

0.57 ± 0.08 0.45 ± 0.07 0.59 ± 0.07 0.63 ± 0.05 0.66 ± 0.08 0.78 ± 0.06 0.60 ± 0.10

0.43 ± 0.10 0.54 ± 0.08 0.39 ± 0.13 0.47 ± 0.08 0.43 ± 0.14 0.72 ± 0.08 0.51 ± 0.12

0.90 ± 0.14 0.71 ± 0.22 0.86 ± 0.15 0.14 ± 0.17 –0.58 ±0.12 –0.60 ± 0.24

9

0.60 ± 0.08

0.54 ± 0.08

0.14 ± 0.15

osmotic adjustment may be simply inherited. However, it is safe to conclude that inheritance of yield, yield stability, and most secondary traits associated with drought tolerance is complex, involving multiple interacting pathways throughout the plant life cycle. Dissection of the trait into its key components through physiological-genetic investigation is a necessary step toward reducing this complexity. Yield and yield stability have long been the standard means of measuring and improving drought tolerance since they integrate all factors into a single measurable response. Dissection of traits associated with drought response, including yield, shows promise for developing molecular breeding methods (Section V). Currently, however, when compared to their use for other simply inherited traits, molecular methods have not become a truly applied tool in the creation of new commercial drought tolerant hybrids. C. Selection of Parental Germplasm 1. Populations and Improved Germplasm. While unimproved openpollinated maize landraces may provide useful sources of new variation for a given trait, yield drag associated with their narrow adaptation and poor agronomic performance in modern high-input agriculture usually limits their direct use in applied commercial programs. Yield has progressed sufficiently in some populations such as CIMMYT’s Tuxpeño Sequía, La Posta Sequía, and the Drought Tolerant Population (Edmeades et al. 1997a) that they are useful sources of new variation for temperate elite lines, provided care is taken to reduce the impact of their photope-

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riod sensitivity. PHI, for example, has released two commercial hybrids recently that contain a small amount of parentage (less than 12.5%) traceable to CIMMYT populations. Details regarding CIMMYT drought populations are discussed in Bolaños and Edmeades (1993a,b, 1996), Lafitte and Edmeades (1995), Byrne et al. (1995), Ribaut et al. (1996), Chapman et al. (1997a,b), and Edmeades et al. (1997a, 1999). Many large seed companies are international in scope, with active breeding programs established in various regions of the world. Germplasm exchange between these international programs is an important source of variation for drought tolerance. Tropical programs benefit by introgressing elite temperate lines that carry yield potential and flowering stress tolerance. Progeny from this effort in turn can be useful sources of variation for breeders in temperate programs. Broad-based populations constructed from elite lines further recombine favorable alleles and serve as trait source populations for extracting new elite inbreds. Source populations, where the frequency of droughtadaptive alleles has been increased by direct selection, give rise to a higher frequency of drought tolerant inbred lines and topcrosses than do their conventionally improved equivalents (Edmeades et al. 1997b). This is a slow process, requiring both recurrent selection and subsequent line development, and is therefore seldom used in commercial inbred line development. Most commercial programs rely on line development via pedigree or backcross methods from segregating F2 populations. 2. Identification of Superior Parents. There is little documentation of inbred lines that contribute directly to improved drought performance or secondary traits. Table 7.7 shows the few references we were able to identify. Undoubtedly, most commercial maize breeding companies develop lists of inbreds that contribute to improved drought tolerance. PHI, for Table 7.7. Maize inbreds reported to confer improvement in drought yield and/or secondary traits. Inbred Lo876o2 Polj17 Ac7643S5 TL89DRTP1-C5 TL89DTP2-C2 B73 Lo1016 Os420

Trait(s)

Reference

Delayed scenescence Root traits, ABA Yield, short ASI Yield Yield Yield, kernels per plant Yield, root traits Drought tolerance

Gentinetta et al. 1986 Lebreton et al. 1995 Ribaut et al. 1996 Edmeades et al. 1999 Edmeades et al. 1999 Frova et al. 1999 Tuberosa et al. 2002b Sanguineti et al. 1999

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example, maintains a list of both susceptible and tolerant lines from the United States, Europe, South America, Africa, and South/Southeast Asia. Active germplasm exchange among a company’s breeders concerned with improving drought tolerance substantially enhances the elite inbred base for creating improved drought-tolerant hybrids. For example, a PHI hybrid recently released in Indonesia was derived from drought-tolerant lines from programs in the United States and Thailand, combined with locally adapted germplasm from the Philippines (Fig. 7.14). The temperate line from the United States contributed early flowering, heat tolerance, and drought tolerance that were probably quite different from the sources of tolerance present in the Thai line. 3. Breeding Values. By incorporating information from related lines in addition to the performance of a given line’s value in hybrid combinations, breeding values (Falconer and MacKay 1996) are generated that help guide decisions regarding selection of parental material in elite line development. Following the discussion in Section III, to generate meaningful drought breeding values, hybrids must be grown in either managed stress environments or inferences made from random MET sampling of the TPE where the stress level is well documented.

Male

Female Thailand Inbred

Philippine Inbred

×

×

U.S. Inbred

New Thailand Inbred

30N11 Indonesia

Fig. 7.14.

Development of drought-tolerant hybrid from temperate and tropical sources.

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Research structure can lead to a dilemma in identifying superior parents for drought tolerance. Large numbers of locations may be needed to identify the few appropriate locations for inferences to drought tolerance, an expensive and inefficient process. Predicting an inbred’s breeding value becomes more accurate with increasing numbers of testers. Many combinations with a given inbred are evaluated during early testing stages. This reduces the “tester effect” but the number of testing locations is usually low. Later testing stages have large numbers of locations but small numbers of combinations that can bias comparisons between inbreds and narrow the opportunities for selection. Interactions between small plot versus whole field performance should also be considered. We often do not truly understand performance under drought conditions until a hybrid is grown commercially in significant acreage. Unfortunately, by this time use of the hybrid’s parents to create new breeding populations may be diminishing in favor of newer higher-yielding elite inbreds. An alternative is to use a small number of managed stress locations where consistent and uniform drought conditions can be achieved (see Section III D). Inbreds of interest are combined with a common set of testers and evaluated at these locations under both controlled drought stress and full irrigation treatments. While not perfect, comparing the performance between these treatments gives an improved understanding of an inbred’s contribution to yield under drought conditions. Proper characterization improves parent selection for breeding crosses, mapping populations, and any other studies where information on inbred reaction to drought stress is desired. Thus, by combining the results of both the inbred in question and its relatives in breeding values, a more powerful database and inference on parental ability toward conferring drought tolerance is made available. D. Criteria for Progeny Selection Selection of progeny to advance in the drought breeding cycle may be accomplished by evaluating lines per se under stress, evaluating experimental testcrosses under stress, evaluating secondary traits, or some combination of these three processes. Selection based on QTL for yield and secondary traits is also possible under stress or well-watered conditions, and will be discussed in Section V. Another approach is to identify lines conferring good yield stability, thereby indirectly improving drought response (see discussion of yield spillovers, Section II A). Selection of inbred lines under stress has been conducted for many years in commercial programs, whether or not by design (Jensen 1971; Jensen and Cavalieri 1983; Tollenaar and Wu 1999; Tollenaar and Lee

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2002). Shortening ASI over time as illustrated in Fig. 7.15 (Duvick et al. 2004) is an example of drought tolerance improvement via a key secondary trait that occurred as breeders selected maize for improved yield under an array of conditions representing the TPE. Most temperate maize-breeding nurseries experience some stress during pollination, and simply selecting lines that exhibit synchronous pollination shortens ASI. Selfing under drought or density stress also automatically selects for individual plants that have good synchrony. ASI is improved indirectly by advancing high-yielding hybrids due to the correlation between yield and barrenness resulting from poor ASI, especially when grown under high-population densities. While there is some evidence that selecting early-generation material under stress contributes to the subsequent-generation testcross performance under stress (T. Barker, unpubl. data, 2001, 2002), this has not yet been proven effective in commercial hybrid development. Selection for high yield potential and yield stability under stress, particularly intermittent stress or moderate levels of stress, has been effective in improving tolerance to moderate levels of drought stress (Tollenaar and Lee 2002). There have been numerous examples of PHI hybrids that further confirm this assertion. For example, the hybrids 3394 and 3223, both noted for exceptional stability and performance under drought stress, were developed using conventional high yield 14

y = 9.1 – 0.11x (r2 = 0.78) Base = 1930

12

ASI (GDU/10)

10

8

6

4

OPV Double Cross Three Parent Cross Three Parent Modified SC Single Cross (SC) SC-Transgenic

2

0

–2

1920

Fig. 7.15. 2004).

1930

1940

1950

1960 1970 1980 Year of Release

1990

2000

2010

Improvement of ASI over time in a commercial hybrid program (Duvick et al.

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potential methods. Hybrid 3223 performs well under severe stress at flowering, showing good silk growth, short ASI, and a high number of ears per plant while maintaining good yield stability and yield potential under favorable conditions (Campos et al. 2002). However, it is not clear that breeding for yield stability without subjecting early-generation testcrosses to drought stress is the most efficient way to develop hybrids that exhibit both high yield potential and superior performance under drought stress. Studies are underway at PHI to address the question of the relative efficiency of these alternative methods. Currently, the most certain means of improving drought tolerance is to select inbreds that perform well in testcross combination when systematically exposed to drought stress. This is costly and inefficient when testing is done in randomly occurring drought conditions in METs where high numbers of replications and locations are needed to represent the TPE (Cooper and DeLacy 1994; Bruce et al. 2002). A discussion of selection methods used in elite line development for improved drought performance follows. E. Methods of Selection 1. Index Selection. Combining secondary traits with yield in drought trials offers the possibility of increasing the efficiency of identifying superior progeny (see Section II E). If secondary traits correlated with yield but with a heritability that actually increases under stress are identified, these can be used as part of a selection index with grain yield to stabilize the heritability of the index, and thus offset the decline in heritability of yield. This in turn allows one to make efficient gains for yield under stress. This is particularly true if the secondary trait is highly correlated with yield (Bänziger et al. 2000). Currently, barrenness and ASI are secondary traits that meet these criteria, being simple data to collect and evaluate, highly heritable (Bolaños and Edmeades 1996; Ribaut et al. 1996) and highly correlated with drought performance (Chapman and Edmeades 1999; Sari-Gorla et al. 1999). While secondary traits such as ASI and staygreen change with selection for yield under drought stress (Chapman and Edmeades 1999), there is no definitive evidence that index selection increases effectiveness over selecting for yield alone for improving performance under drought. However, the fact that yield gains under drought obtained from multi-environment testing have been significantly less than those under optimal conditions, while those obtained with index selection have been almost identical across a range of yield levels strongly suggests that index selection has resulted in increased selection efficiency under stresses where grain yields have been reduced by more than 50% (Chapman et al. 1997a). This is supported by findings of

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Bänziger and Lafitte (1997), who reported a 14% increase in the heritability of a selection index comprised of yield, ASI, and staygreen under low N compared with the heritability of yield alone. Other secondary traits such as rooting characteristics, ABA synthesis, and leaf rolling appear less useful, either because they lack consistent association with yield under stress, have low heritability, or because of the time and effort required to make the necessary observations. To our knowledge, multitrait selection indices are not widely used among commercial maize breeders. Rather, they select for improved drought tolerance using yield and yield stability. The application of marker-assisted selection (MAS) for yield and drought-adaptive secondary traits may have greater future promise once robust associations are established for key secondary traits. Root trait QTL would be particularly useful due to the known impact on performance under drought stress and the current difficulty in using this trait for selection (Lebreton et al. 1995; Tuberosa et al. 2002b). Index selection could be done using several genotypic traits or a combination of genotypic and phenotypic traits. To date, however, results of markerassisted index selection have not proven effective relative to direct selection for yield performance in the target environment. Some of the current research and the areas where there appears to be potential to improve upon the results of these early studies is discussed further in Section V. 2. Recurrent Selection. Recurrent selection may be used for two applied breeding purposes in the commercial sector, namely enhanced trait development and inbred line extraction. The two occur in the same process. Yield and secondary traits are improved by combining numerous sources of variation and generating large numbers of recombination events (Hallauer and Miranda 1981; Pandey and Gardner 1992). However, in the commercial setting recurrent selection is seldom used for improving drought tolerance in elite inbreds due to the length of time involved. Since inbred line extraction requires pedigree selection following a cycle or more of recurrent selection, this method is slower than inbred line development from elite F2 populations. Time required for inbred line development can be shortened if S1 (rather than full- or halfsib) recurrent selection is employed, and reciprocal recurrent selection can be used if there is a logical organization of the germplasm into heterotic groups and a need to maximize heterosis. In general, recurrent selection schemes have a lower rate of genetic gain than that realized through elite F2 selection, especially given the nature of the product required for commercialization. In commercial programs, recurrent selection has greater application in the development of new sources of tolerance for use in F2 populations, introgression of new variation, and the study of mechanisms involved in drought response.

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3. Pedigree Selection. Line development via pedigree selection from elite F2 populations is the mainstay of commercial maize breeding for drought tolerance as well as most other agronomic traits (Hallauer and Miranda 1981). The key to successful pedigree selection is a combination of the knowledge of superior parents, effective screening of progeny, and careful characterization of testcross performance, all of which are interrelated parts of the same process. Limits to pedigree selection are only encountered by lack of sufficient variation in a given maturity or area of adaptation, or the lack of good data from sufficient numbers of effective managed stress or drought-informative locations. In a sense, commercial pedigree selection programs are a form of recurrent selection with several-year selection cycles and diverse germplasm pools. As shown in Fig. 7.16, breeding populations from contrasting combining ability groups are developed in parallel using the contrasting group’s elite lines as testers. Optimum management of pedigree selection programs for drought tolerance can be achieved by applying stress throughout the breeding cycle. Beginning with known drought-tolerant parents, F2 populations subjected to moderate stress can be screened for floral synchrony. Early Female

Male

Population A

Population B

testers Expt, Hyb Tests

Pedigree programinbred line development

Pedigree programinbred line development

Hybrid testingMETs

Hybrid testingMETs

Hybrid Release

Fig. 7.16.

Pedigree breeding schematic.

Expt, Hyb Tests

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generation testcrosses evaluated under managed stress or multienvironment testing in stress-prone areas helps identify droughtresponsive lines. The use of a susceptible elite tester further helps identify experimental lines that contribute drought tolerance during early generation testing. Resources available for accurate testing are often limiting, but may be offset by careful management, accurate environmental classification for incidence of drought and optimum experimental designs (see Section III and Bänziger et al. 2000). Finally, thorough characterization prior to hybrid release is important in determining how the hybrid performs over a wide range of stress levels. Perhaps the most promising near-term opportunities to improve the rate of genetic progress for drought tolerance in maize hybrids are through the implementation of improved experimental methods as outlined in Section III above. Longer term, the development and introgression of novel variation from various sources (Section IV F) and molecular breeding (Section V) promise sustained improvement. 4. Divergent Selection. Subjecting a given population to contrasting testing regimes effectively “diverges” the population. The best-known example of this method is the Illinois long-term selection experiment, which continues to show divergence of grain oil and protein content after a century of selection (Dudley and Lambert 2004). Divergent selection has also been employed in drought research in forages (Thomas and Evans 1989), wheat (Innes et al. 1984), barley (Tuberosa et al. 1986), and maize (Edmeades et al. 1997c; Landi et al. 2001). The primary benefits of divergent selection are the development of contrasting phenotypes for discovery purposes, and as a means of determining the relative value of individual secondary traits. There have been no studies reported to date that demonstrate the relative efficiency of breeding for high yield potential vs. yield under drought stress in terms of their stability across environments. A significant proportion of the genetic gain for stress response may be obtained through selection in well-watered environments (Byrne et al. 1995; Tollenaar and Lee 2002; Section II A). It is logical to assume that breeding solely for well-watered environments will result in the best performance under those environments, and similarly breeding under welldefined drought stress will result in superior performance under those conditions. However, it remains to be seen which approach will produce the most efficient results in developing commercially viable germplasm that must yield better (e.g., 5%) than commercial check benchmarks under well-watered conditions and at an even higher level of superiority (e.g., 20%) over benchmarks under drought conditions. Perhaps the best situation for the breeder would be to test germplasm

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under both sets of conditions and only retain those that meet some criteria such as the 5%–20% gauge suggested above, and the judicious combination of performance in both should give stable production in both (Rosielle and Hamblin 1981). However, this can be a costly proposition requiring a substantial increase in testing resources. Selection under well-watered conditions has obvious advantages in expression of genetic variation for high yield potential and high heritability. It is an interesting challenge to address the relative efficiency of applying stress during line development in order to gain greater stability under an array of drought stresses, especially considering the significant gap between well-watered yield potential and farmers’ realized yield potential (Tollenaar and Lee 2002). Divergent selection would be a useful tool with which to evaluate this question. F. Introgression of Novel Variation Novel variation may be present in exotic germplasm or created by genetic engineering. Exotic germplasm introgression is slowed by linkage drag associated with low yield potential, poor agronomic performance, and poor adaptation to the target environment. Marker-assisted selection (MAS) holds great promise to efficiently accumulate and introgress genomic regions associated with yield and key secondary traits. Introgression would be accomplished through pedigree selection or marker-assisted backcrossing. Research to identify genes that control important pathways in drought response (Section II) is underway in both commercial and public research. These genes may be introgressed by backcrossing transgenes into existing elite lines, or used directly in more conventional approaches such as tracking the transgene through elite F2 populations and derived progeny. With the application of newer technologies, we are better suited to take advantage of novel variation than we have been previously. Most exciting of all future drought-breeding prospects is the possibility of combining molecular breeding with conventional breeding methods. One approach would be to choose superior F2 parents by identifying desired alleles, followed by selection of individual F2 seeds containing optimum allelic combinations. A similar approach has been suggested based on proven physiological traits by Richards et al. (2002), who point out that such early-generation population truncation would allow field nursery and yield test resources to focus on only those superior genotypes. Below we discuss further the combination of molecular and conventional breeding approaches to create new opportunities for applied drought tolerance breeding.

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V. MOLECULAR BREEDING Understanding the genotype/phenotype relationship is critical to understand the maize genome and to translate such knowledge into the genetic improvement of complex traits such as drought tolerance. The integration of genetic and molecular approaches into improved breeding systems aims to construct a practical genotype/phenotype knowledge bridge. Initially, molecular breeding can increase selection throughput by identifying and managing genes that control relatively simply inherited traits. Longer term, it is particularly well suited to develop improvement strategies for complex quantitative traits since a myriad of genes and genetic interactions contribute toward these traits. Understanding the mechanisms responsible for expressed variation in quantitative traits conferring maize drought tolerance relies upon information at the molecular level. Although the power, speed, and ability to isolate new commercially important genes offered by DNA-based technologies must be fully noted, potential advantages will be realized only through its judicious blending into the genetic framework and appropriate field testing. Since the ultimate test for each genotype is its acceptability by farmers and endmarkets, it is imperative to integrate molecular discoveries into elite genetic backgrounds. Molecular breeding is thus essential for commercial deployment of molecular genetics research aimed at improving maize drought tolerance. Several aspects of the statistical basis of molecular breeding beyond the scope of this review can be found in Liu (1998). Rather, we will review existing approaches to molecular breeding applicable to improving drought tolerance in maize. A. Linkage Mapping Sax (1923) and Thoday (1961), working with beans and Drosophila, respectively, were among the pioneers in using genetic markers to find associations between simply inherited markers and quantitatively inherited traits. For many years QTL analysis was hampered by limited genetic marker availability as well as the pleiotropic effects and recessive nature of mutants. Having in place DNA-based markers developed through recombinant DNA technology such as random fragment length polymorphisms (RFLPs), simple sequence repeats (SSRs), amplified fragment length polymorphisms (AFLPs), randomly amplified polymorphic DNA (RAPDs), and single nucleotide polymorphisms (SNPs) has helped to circumvent earlier limitations since they provide access to an almost unlimited number of genetic markers. Whole genome sequences in species

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like Arabidopsis and rice allow one to select genetic markers of precisely known physical position. Complete genetic maps, i.e., the ordering of loci along linkage groups and the relative recombination distances among them, are currently available for most major crop species and also for many wild species. Once molecular markers are organized into genetic maps, many applications become feasible. B. QTL Identification Using genetic mapping to dissect the inheritance of key complex trait components in a segregating population is a powerful means to distinguish common heredity from casual associations. The merging of QTL analysis and plant physiology also improves our understanding of the plant drought response, offering new avenues for rational and targeted crop improvement (e.g., Ribaut et al. 1997; Chapman et al. 2003). The genetic dissection of any target trait aims to identify the number and genetic positions of QTL involved in its genetic architecture, assess their genetic effects (dominance, additivity, pleiotropic interactions), the proportion of phenotypic variance explained by individual and combined QTL, and the stability of their expression across environments (QTL*E interactions) and genetic backgrounds. It is conducted as a twostep process. First, a segregating population for the target trait is developed based upon clear objectives. Genetic variation within the segregating population is a fundamental requirement in the analysis of quantitatively inherited traits. Although crosses are generally designed between parents polymorphic for the trait under analysis, lack of phenotypic variation among a given set of individuals does not mean there is no genetic variation. It is also important to consider the agronomic value of the mapping population being created and its relationship with elite germplasm at the beginning of any molecular breeding approach. Unsuitable agronomic properties in a cross may render otherwise outstanding QTL discovery unfruitful for product development. In the second step, genetic markers closely linked to the target gene(s) are identified for allelic modification. The simplest approach is single marker analysis. However, this approach has significant disadvantages such as low statistical power and confounding estimates of QTL positions and effects. A more robust approach to QTL mapping is simple interval mapping, which is based upon the joint frequencies of pairs of adjacent markers and a putative QTL flanked by these markers (Lander and Botstein 1989). The availability of software such as MAPMAKER/ QTL has made it a frequently used approach. The resolution and positioning of QTL can be limited, however, since interval mapping is not

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independent for different genomic regions. Therefore, several QTL residing on the same linkage group can produce wide LOD (log of the odds; a LOD score of Z means that the alternative hypothesis is 10z times more likely than the null hypothesis) plots. Furthermore, two QTL linked in coupling phase can be identified as one QTL covering a large chromosomal section, whereas linked QTL in repulsion phase may not be identified at all (Liu 1998). A third approach to QTL identification, CIM (composite interval mapping), combines simple interval mapping and multiple linear regression approaches (Zeng 1994). This method controls residual background genetic variation by using as cofactors markers linked to previously found QTL identified through stepwise regression and fitted into the model. This reduces residual variation throughout the genome and allows a higher QTL resolution and positioning than single interval mapping and also a better handling of linked QTL. Since more variables are included in the model, CIM is more informative and efficient than simple interval mapping. CIM can be implemented with software packages like QTL Cartographer (Basten et al. 1994) or PlabQTL (Utz et al. 2000). It cannot be emphasized enough that using the appropriate field techniques and robust statistical approaches discussed in Section III is critical to identify authentic and robust QTL. This is particularly true for drought tolerance in maize given its complex phenotypic expression. The frequent observation of “ghost” QTL, i.e., those showing unreliable expression, is due in part to inappropriate phenotypic data collection/analysis processes. Although there are powerful and ever-improving methodologies available for QTL identification, some important issues remain, including the actual biological meaning of QTL data, the difficulty of handling epistatic and environmental interactions with currently available statistical tools, and the large confidence intervals generally associated with QTL. While most QTL mapping efforts conducted to date rely on biparental crosses, there are many advantages to QTL mapping in complex pedigrees. Advantages include the display of full QTL variation within the reference population, circumventing the identification of cross-specific QTL, and ability to conduct retrospective analyses, i.e., the ability to use information from pedigrees going back several generations to strengthen current QTL mapping efforts. Linkage-based analyses of QTL in pedigrees proceed through two stages; first, by quantifying “Identity by Descent” probabilities for different genomic regions and, secondly, by analyzing the phenotypic resemblance among pedigree members (Jannink et al. 2001). Combining within- (as in standard biparental cross-based QTL mapping) with across(as in complex pedigree-based QTL mapping) population information

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helps to better understand epistatic relationships between pairs and multiple QTL (Jannink et al. 2001). Haplotype analysis is useful to detect ancestral genomic blocks in parental lines and to model the effects of putative QTL per ancestral genome block (Jansen et al. 2003). When the genetic analysis of drought tolerance in maize is addressed through QTL analysis, phenotyping activities are frequently carried out under stressed environments. It is also necessary to analyze the plant performance under the range of environmental conditions encountered in the TPE, in order to separate the genetic basis of adaptative and constitutive mechanisms of tolerance (see Section II A). C. Marker Assisted Selection (MAS) QTL analysis must be considered as a means rather than an end. The genetic location of QTL only provides a slight fraction of the information available from molecular markers. A significant proportion of research to date does not extend beyond QTL characterization and detection of QTL for a given trait, nor are QTL deployed to develop improved germplasm, novel experimental genetic backgrounds, or commercial products. This trend could be reversed using appropriate MAS strategies to test and utilize the results obtained through QTL analysis. QTL also can be used to trace favorable alleles in the genomic background of target genotypes, ensure the presence of elite alleles at selected loci through repeated cycles of selection, and to identify individual plants carrying favorable alleles in large segregating populations (Dreher et al. 2003). Contributions of MAS to the overall breeding effort need to be thoughtfully assessed on a trait-by-trait basis by including financial issues in its evaluations for applied breeding. For MAS to be more effective than phenotypic selection alone, the following requisites need to be fulfilled: (1) QTL positions are estimated with high precision to choose markers showing a minimum of recombination with the QTL and to resolve linked QTL, (2) estimated QTL effects reflect their true genetic effects and are thus estimated without bias due to genotypic or environmental sampling, and (3) a sufficient proportion of the genotypic variance of the trait under study is explained by the detected QTL (Utz et al. 2000). Generally, MAS is more applicable to traits of medium to low heritability, that are expensive and/or time consuming to evaluate, or that are expressed during late stages of crop development. Given the fixed and variable expenses involved, MAS is more promising for high value traits, although extensive deployment will depend upon the tradeoff between added value and additional expenses. The first requirement for any successful MAS approach to is to understand the genetic basis of QTL regulating a given trait. QTL studies

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related to drought tolerance in maize expressed as grain yield under drought or associated secondary traits are shown in Table 7.8. Once QTL are identified, they may be used to convert otherwise susceptible lines through diverse backcrossing schemes and selecting at non-target loci in every BC generation. Ribaut et al. (2002) discussed the optimal use of markers in backcrossing populations. The utility of markers decreases in advanced backcross generations since recipient alleles fixed (homozygous) at any given non-target locus are no longer included in subsequent DNA analyses. It is important to define as precisely as possible the genetic position of a single QTL to be transferred; otherwise larger linkage drag effects derived from its vicinity may be expected. Alternatively, QTL may be used to truncate initial breeding populations (see Section IV F) or conduct forward selection for favorable alleles. Table 7.8. Published reports on QTL regulating traits associated with drought tolerance in maize. Population

Traits assessed

Reference

RILs derived from B73 * H99

Male and female flowering time, ASI, and plant height

Sari-Gorla et al. 1999

RILs derived from B73 * H99

Ear length, ear weight, kernel weight per ear, kernel number per ear, and 50 kernels weight.

Frova et al. 1999

F2 derived from Polj17 * F-2

Flowering time, stomatal conductance, leaf and xylem ABA content, leaf water relationships and fluorescence, root pulling force, and nodal root number.

Lebreton et al. 1995

F2:3 derived from Mo17 * H99

Male and female flowering, ASI, ear height, and plant height

Veldboom and Lee 1996a

F2:3 derived from Mo17 * H99

Grain yield, kernel weight, ear number per plant, ear length, ear diameter, cob diameter, kernel depth, and kernel rows numbers

Veldboom and Lee 1996b

F2 derived from Ac7643S5 * AC7729/TZSRWS5

Grain yield, ears per plant, kernel number per plot, and kernel weight.

Ribaut et al. 1996

F4 derived from Os420 * IABO78

Bulk leaf ABA concentration, stomatal conductance, leaf temperature, leaf relative water content, ASI, and grain yield

Sanguineti et al. 1999

F4 derived from Os420 * IABO78

Leaf ABA concentration

Tuberosa et al. 1998

F3 derived from Lo964 * Lo1016

Primary root length, primary root diameter, primary root weight, adventitious root weight, and grain yield

Tuberosa et al. 2002b

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Theoretical work suggests that by using appropriate population sizes, multiple QTL can be transferred into a recipient line (Hospital and Charcosset 1997). Recent reports from a detailed marker-based introgression program in maize extending over several years suggest that while several QTL can be transferred and used to create “ideal genotypes,” it is only feasible at this time to affect the phenotypic expression of relatively simple traits like grain moisture at harvest and silking date (Bouchez et al. 2002). More work needs to be done with complex traits like grain yield to attain the same level of results. This suggests that the stringent evaluation of introgressed segments across different genetic backgrounds, their validation, and a thorough analysis of QTL*E and QTL*QTL interactions will play a critical role in the success of MAS. Examples of MAS include drought tolerance traits (Ribaut et al. 2003), and insect resistance, another complex trait (Willcox et al. 2002). In order to better connect the needs of QTL discovery and plant breeding programs and to hasten the development of improved germplasm carrying valuable QTL, MAS systems allowing the simultaneous identification and transfer of favorable QTL alleles from unadapted material to elite lines have been developed, e.g., the Advanced Backcross (Tanksley and Nelson 1996). Although the utility of this approach has been demonstrated primarily in tomato and rice to date, recently Ho et al. (2002) used the Advanced Backcross QTL method to successfully identify and introgress QTL in inbreds of elite maize. They identified QTL for grain yield, grain moisture and plant height, and BC3 testcross entries containing introgressions at two QTL significantly outperformed non-carrier entries. It should also be noted that during this process some of their early candidate QTL did not show the expected advantage. How can QTL studies best be deployed to develop drought tolerant maize? Since the proportion of phenotypic variance explained by individual QTL regulating traits related to drought tolerance is rather small and their contributions are often environment-dependent, it is also necessary to identify QTL for secondary traits that are highly correlated with grain yield under drought conditions. Therefore, one possible MAS strategy for maize drought tolerance is through indices composed of QTL regulating several traits (grain yield plus some secondary traits) as well as actual phenotypic observations (see also Section IV E 1). Another opportunity is the development of MAS strategies to improve root traits associated with drought tolerance. Phenotypic selection of root traits is challenging since the most promising root traits are expressed during later stages of maize development. The tedious scoring of root traits and their plastic-response nature make conventional selection for root traits difficult, thus MAS for root traits associated with drought tolerance could be particularly useful.

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D. Summary of Results from QTL/MAS Studies on Drought Tolerance Key points arising from drought QTL studies (see Table 7.8) are: (1) a larger proportion of explained phenotypic variance and higher heritability values are observed under normal irrigation conditions, (2) individual QTL generally do not explain over 10% of phenotypic variance for grain yield under stress conditions, (3) some regions of chromosome 1 such as bin 1.06 appear promising since they carry QTL related to different traits such as grain yield, drought tolerance index, and some root traits; other interesting chromosome segments are on chromosomes 2 and 10, (4) some QTL for grain yield and secondary traits such as ASI can be detected under both stress and normal irrigation regimes, suggesting a constitutive rather than a stress responsive pattern of gene expression, although most QTL are detected only under one specific irrigation regime, and (5) caution needs to be exerted when results from QTL experiments conducted on inbreds are extrapolated to hybrids. Ribaut et al. (2002) reported successful QTL use through MAS to improve maize drought tolerance. In this study, five genomic regions from the donor line Ac7643 covering approximately 12% of the genome were transferred into the drought susceptible recipient line CML247. A further 7% of the genome lying outside these QTL regions was also transferred. The best genotype among 70 selected BC2F4 by tester hybrids outperformed controls by 2× to 4× under severe drought conditions. No yield reduction was observed under normal irrigation conditions. Why are there so few successful stories about the practical deployment of QTL, either in commercial or academic programs dealing with complex traits? We have mentioned several factors associated with drought stress, the subsequent difficulty in collecting consistent phenotypic data, and the inadequacy of our current knowledge and power of statistical approaches to effectively accommodate QTL * QTL and QTL * E interactions. Taking these factors into consideration and ensuring the active participation of breeders, not only during the final field evaluation step but also across the whole process of trait dissection, should enable us to realize the potential provided by molecular breeding. Finally, we consider that QTL studies will greatly benefit from a closer integration of biological meaning with whole organism level information generated by physiology and modeling approaches since they provide the framework and the context within which QTL will interact under field conditions, be it a farmer’s field or an academic setting (e.g., Chapman et al. 2002).

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E. QTL Analysis as an Aid to Identify Candidate Genes From a purely plant physiology perspective, it has been argued that there are relatively few limiting loci controlling the variation of complex traits like drought tolerance (Prioul et al. 1999). QTL analysis can be valuable to study the regulatory genes involved and further understand the in vivo contribution of a given enzymatic activity/metabolite accumulation to a whole plant process. Also, it can confirm the value of candidate genes, for instance by testing their co-localization with changes in enzymes or metabolites synthesized under specific conditions. This approach has been applied to the study of enzyme activities and substrates that respond to drought stress. The gene Ivr2, encoding a vacuolar isoform of the invertase gene, was shown to map near the position of a QTL regulating vacuolar invertase activity on chromosome 5 (Prioul et al. 1999). Another possibility is to integrate QTL mapping with genomic tools such as transcription profiling to identify novel positional candidate genes for a phenotype of interest whose expression varies between parental lines or is responsive to drought stress. The goal would be to identify a manageable number of candidate genes for follow-up by using an approach that enables the investigator to successfully identify candidate loci associated with the phenotype of interest (Wayne and McIntyre 2002). Candidate genes detected through QTL analyses need to be thoroughly validated before their deployment. Validation and impact on drought response can be achieved through a number of approaches, including: (1) analysis with near-isogenic lines, (2) identifying SNPs in alleles from different individuals well correlated with the trait, and (3) genetic transformation studies aiming to complement mutants susceptible to the trait. As in all biparental studies, the absence of candidate genes matching the genetic position of QTL does not necessarily reflect lack of association between these genetic entities. It rather suggests lack of variation between the parental lines and it is possible that they can be associated in populations derived from other parental genotypes. F. QTL Mendelization and Positional Cloning It is often difficult to assess individual QTL genetic value since traits are subjected to strong environmental factors in traditional drought mapping populations; also sterility may be an issue in wild cross-derived mapping populations. Such difficulties can be overcome by fine mapping QTL as a Mendelian factor (Yano 2000). This is generally achieved using

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recombinant backcrosses that allow recovery of independent plants each heterozygous for a different segment of a donor line. Upon selfing to homozygosity independent introgressed segments are obtained (Zamir 2001; Fig. 7.17). This lessens the immediate epistatic effects related to segregation of non-target genomic regions and thus increases the ability to pinpoint small allelic substitutions. Results can be misleading if they are inferred to the complete genome since context dependency and epistasis may actually render an identified QTL more or less effective than when observed alone. The Mendelization of QTL also provides the starting point for positional cloning. Large populations are needed to iden-

Step

Scheme

Generation

Cross parents

X

F1

First backcross

BC1F1

Second backcross

BC2F1

Third backcross

BC3F1

Selfing

BC3F2

Line 1 Finished backcross substitution lines

Line 2 Line 3 Line 4 Line 5 … : Genotypes eliminated : Recurrent parent : Donor parent

Fig. 7.17.

Production of backcross substitution lines.

BC3F2

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tify informative meioses that can narrow the genetic location of a target QTL to intervals less than 100 kb in length and then allow precise placement on a physical map. In combination with transgenic complementation experiments, the biological significance of cloned QTL can be further analyzed. Individual QTL have been cloned in Arabidopsis, tomato, and rice (Takahashi et al. 2001). G. HAPPY Mapping Alternative mapping approaches have been proposed to map loci that are non-polymorphic yet identical by descent and having poor resolution caused by low recombination rates observed in traditional biparental linkage mapping. One alternative is HAPPY mapping (Thangavelu et al. 2003). Briefly, genomic DNA is sheared, separated through pulse field electrophoresis and then fragments in the range of 300–600 kb are selected. These size-selected DNAs are then subjected to a PCR (polymerase chain reaction)-based preamplification and subsequently to genetic typing using highly multiplexed hemi-nested PCR reactions. Cosegregation patterns between markers reflect the distance between them. Markers less closely linked are more likely to be broken apart during DNA shearing and therefore more likely to segregate. Using this process, the order and distances between markers are determined, and genetic maps are constructed based upon pair wise LOD and θ values. In Arabidopsis, HAPPY mapping was recently used to achieve a physical resolution of about 50 kb based upon genetic markers derived from a chromosome 4 sequence (Thangavelu et al. 2003), thus greatly improving putative candidate gene identification. How can HAPPY mapping enhance genetic analysis of drought in maize? The ability to circumvent polymorphism issues and to better cope with multigene families and duplicated loci (the discrimination power of hemi-nested PCR is more than that of hybridization and non-nested PCR) provides a powerful addition to the repertoire of tools available to conduct genetic analyses in plant genomes. HAPPY mapping could be used to: (1) create detailed maps of gene-rich regions including not only polymorphic markers but also candidate genes and arbitrary DNA sequences, therefore refining available genetic maps and assisting the search for QTL for drought and drought related traits showing value at the field level, (2) provide an independent sequence-tagged-site scaffold to physical maps and so speed up their application as gene discovery tools, and (3) since one of the expected outcomes of most transcription profiling experiments focused on drought related traits would be a number of putative candidate genes showing different degrees of response to drought, HAPPY mapping could also be used for their high-throughput genetic mapping/analysis.

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H. Bulk Segregant Analysis Molecular marker systems availability and/or funding limitations often constrain the capacity to genotype entire mapping populations. In such cases, Bulked Segregant Analysis (BSA) is a practical alternative (Michelmore et al. 1991; Quarrie et al. 1999; Mackay and Caligari 2000). In BSA, pools (bulks) of individuals are grouped based upon their contrasting phenotypic performance. Molecular markers are then run on these extreme phenotypic groups and those markers significantly deviating from the expected 1:1 ratio are inferred to be associated with the genetic factors regulating the target trait. This system can be applied to contrasting segregates from a specific population or to bulks of genetically diverse unrelated individuals. In maize, BSA experiments conducted with composites developed at CIMMYT derived from Tuxpeño Sequía and the Drought Tolerant Population identified several probes associated with drought tolerance (Quarrie et al. 1999). Although BSA can dramatically reduce the number of molecular assays to be conducted, it may not uncover the full array of loci/alleles involved in the regulation of a quantitative trait. Also, it does not provide information on the distance between the markers found and the QTL. I. Association Analysis Linkage disequilibrium (LD) is the correlation of alleles at different loci within a reference population. With appropriate attention to the effects of population structure, LD measures can be used to identify haplotypes transmitted from ancestral genomic regions. A thorough recent account of LD in plants can be found in Flint-Garcia et al. (2003). Association analysis based on LD in diverse populations is an alternative to linkage mapping based on biparental crosses to identify traitmarker relationships and QTL. It can potentially achieve a much higher resolution than linkage mapping since it incorporates information from many historical recombination rounds and scans many alleles at each locus. Association analyses can be applied to a broad range of reference populations and reduce some of the problems of context dependency that has constrained much of the QTL analysis based on biparental crosses. LD relies upon across-population marker-phenotype relationships to identify polymorphisms (e.g., indels or SNPs) associated with phenotypic variation. It is important to take into account the structure of the populations that association studies are based on in order to avoid spurious associations driven by uneven allele frequencies in population sub-structures, bottlenecks in selection, and stratification. Searching for SNPs is more efficient if conducted in untranslated regions due to the incidence of higher levels of polymorphism. SNPs can be developed

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through re-sequencing PCR amplicons with or without pre-screening, electronic discovery in genomic libraries, and electronic discovery in expressed sequence tag libraries (Rafalski 2002). The extent of LD in maize is highly dependent upon the reference population. In elite maize inbreds, it has been found that in some cases such as the Adh1 and y1 loci, LD extends over 100 kb (Rafalski 2002). The most detailed analysis of LD in elite maize germplasm conducted to date shows that the frequency of nucleotide substitutions is almost three times higher in non-coding regions than in coding sequences (Ching et al. 2002). The presence of indels is even more skewed toward noncoding regions. Furthermore, LD between SNP loci extends at least to 500 bp. There are large differences in the extent of LD observed in different populations due to the mutational and recombinational history these populations have gone through in previous generations. Under extended levels of LD, whole genome scanning approaches can be appropriate. However, since a large number of independent tests need to be conducted, issues in association analysis are raised such as the extent of the phenotyping effort required and complex statistical analysis. The statistical corrections needed would result in reduced amounts of variation explained even when using very large sample sizes. However, as pointed out by Rafalski (2002), the use of populations that underwent bottlenecks, such as selection during the development of elite maize germplasm, would be ideal for low-resolution association analysis due to the high LD available in such populations. When the extent of LD is limited, candidate gene–association approaches may be more appropriate, and multiple means can be used to select a set of genes with a better likelihood of success. For example, Thornsberry et al. (2001) showed that several polymorphisms existing in the dwarf8 gene and its promoter were associated with an early flowering phenotype in 92 maize inbred lines. The candidate gene approach can substantially reduce the amount of genotyping required, but more importantly it can reduce the testing of many sites across the genome required in whole genome scanning approaches.

VI. CONCLUSIONS Temperate maize grain yield has undergone long-term yield improvement for its TPE as an outcome of sustained plant breeding efforts (Duvick 1992; Duvick et al. 2004). Drought has always been recognized as an important component of the temperate maize TPE. However, yield improvement did not necessarily focus on drought tolerance as a primary breeding target. Rather, the imposition of drought as a selection regime

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over years within the breeding process occurred like many other environmental conditions since it was sampled within the wide-area testing component of the breeding program. Nevertheless, yield improvements have been realized in both favorable and drought-affected environments (Duvick et al. 2004). Here we pose the question of whether this is the most effective approach for continuing the genetic improvement of drought tolerance. Given advances in the technologies available to maize breeders, we can examine opportunities to improve drought tolerance breeding efficiency by better understanding its components and developing a more targeted breeding effort. Within this review we have discussed these prospects from the perspective of a large commercial breeding program. Working within a commercial breeding program introduces many considerations that do not exist in an academic setting. For our purposes, it is not sufficient to demonstrate that we can dissect the genetic architecture of drought tolerance or that it is possible to achieve progress for drought tolerance. We must demonstrate that research leads to sustainable enhancements of the breeding effort and improvements on the current strategy. Given that the current pedigree breeding strategy is making genetic improvements, we seek to improve on this. Key components we have identified and discussed include: (1) access to sources of genetic variation that are relevant to the elite germplasm pool developed by the breeding program (Section IV C), (2) use of advances in experimental design and analysis (Section III B) to support measurement of trait phenotypes, (3) understanding the importance and genetic-environmental bases of genotype-by-drought interactions for yield (Sections II and III C), (4) strategic use of managed drought environments for screening mapping populations, progeny from elite breeding crosses, and characterization of hybrids (Section III D), and (5) advances in the use of molecular marker and genomic technologies to understand the genetic basis of drought tolerance and the potential for MAS (Section V). Experimental validation of these components of an integrated breeding strategy and an assessment of their benefits and costs will be critical steps in deciding whether to modify the current breeding approach to augment and enhance the genetic gains that have been achieved to date. LITERATURE CITED Araus, J. L., G. A. Slafer, M. P. Reynolds, and C. Royo. 2002. Plant breeding and drought in C3 cereals: what should we breed for? Ann. Bot. 89:925–940. Bänziger, M., and H. R. Lafitte. 1997. Efficiency of secondary traits for improving maize for low-nitrogen target environments. Crop Sci. 37:1110–1117. Bänziger, M., F. J. Betrán, and H. R. Lafitte. 1997a. Efficiency of high-nitrogen selection environments for improving maize for low-nitrogen target environments. Crop Sci. 37:1103–1109.

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8 The Origins of Fruits, Fruit Growing, and Fruit Breeding Jules Janick Department of Horticulture and Landscape Architecture Purdue University 625 Agriculture Mall Drive West Lafayette, Indiana 47907-2010

I. INTRODUCTION A. The Origins of Agriculture B. Origins of Fruit Culture in the Fertile Crescent II. THE HORTICULTURAL ARTS A. Species Selection B. Vegetative Propagation C. Pollination and Fruit Set D. Irrigation E. Pruning and Training F. Processing and Storage III. ORIGIN, DOMESTICATION, AND EARLY CULTURE OF FRUIT CROPS A. Mediterranean Fruits 1. Date Palm 2. Olive 3. Grape 4. Fig 5. Sycomore Fig 6. Pomegranate B. Central Asian Fruits 1. Pome Fruits 2. Stone fruits C. Chinese and Southeastern Asian Fruits 1. Peach 2. Citrus 3. Banana and Plantain 4. Mango

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5. Persimmon 6. Kiwifruit D. American Fruits 1. Strawberry 2. Brambles 3. Vacciniums 4. Pineapple 5. Avocado 6. Papaya IV. GENETIC CHANGES AND CULTURAL FACTORS IN DOMESTICATION A. Mutation as an Agent of Domestication B. Interspecific Hybridization and Polyploidization C. Hybridization and Selection D. Champions E. Lost Fruits F. Fruit Breeding G. Predicting Future Changes LITERATURE CITED

I. INTRODUCTION Crop plants are our greatest heritage from prehistory (Harlan 1992; Diamond 2002). How, where, and when the domestication of crops plants occurred is slowly becoming revealed although not completely understood (Camp et al. 1957; Smartt and Simmonds 1995; Gepts 2004). In some cases, the genetic distance between wild and domestic plants is so great, maize and crucifers, for example, that their origins are obscure. The origins of the ancient grains (wheat, maize, rice, and sorghum) and pulses (sesame and lentil) domesticated in Neolithic times have been the subject of intense interest and the puzzle is being solved with the new evidence based on molecular biology (Gepts 2003). In the late Neolithic and Bronze Ages between 6000 and 3000 BCE, the ancient Mediterranean fruits (date, olive, grape, fig, sycomore fig, and pomegranate) were domesticated (Zohary and Spiegel-Roy 1975). Fruits such as citrus, banana, various pome fruits (apple, pear, quince, medlar) and stone fruits (almond, apricot, cherry, peach, and plum) were domesticated in Central and East Asia and reached the West in antiquity. A number of fruits and nuts were domesticated only in the 19th and 20th centuries (blueberry, blackberry, pecan, and kiwifruit). Some wellknown fruits, although extensively collected, remain to be domesticated, such as lingonberry, various cacti such as pitaya, Brazilnut, and durian. This review will consider the various technologies inherent in the origins of some well-known fruits, emphasizing factors that led to domestication and the genetic changes that ensued.

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A. The Origins of Agriculture The change from food collection to food production requires the domestication of plants and animals. This involves a series of technologies that involves choice of species, bringing them into management or cultivation, genetic alteration brought about by selection—both conscious and unconscious—and the discovery of specific practices (pollination, training, processing) often unique for each crop. Although precise origins are obscure, the first archeological evidence for a developed agriculture is found in Mesopotamia and shortly after in the Nile and Indus Valleys. Evidence suggests a later development in China, Central America, East and West Africa and, perhaps, New Guinea (Diamond 1997, 2002). There is some dispute as to whether the origins of agriculture were completely independent. Those who favor independent invention of agriculture emphasize the adaptability of humans for independent discovery and provide as evidence the domestication of different grains in various parts of the world—wheat in the Mid-East, sorghum in Africa, rice in Asia, and maize in the Americas (Harlan 1992). However, an argument can be made for diffusion based upon the sequential development of agriculture (Carter 1977). Although the diffusion of agricultural information has been ignored in favor of independent development, recent evidence on the domestication of the dog suggests that diffusion of agricultural information may indeed be involved (Leonard et al. 2002; Savolainen et al. 2002). The ancient Neolithic trade in flint suggests diffusion was important to the transfer of ancient technology. It seems doubtful that the complex technology involved in such techniques as fire making, production of weapons like the bow and arrow, ceramic making, and metal working are truly independent discoveries. Evidence of diffusion in the history of agricultural technology includes irrigation technology from Mesopotamia, silkworm technology from China, and horse technology from Central Asia. In the Mid-East, the change from scavenging, collecting, and hunting to agriculture that occurred about 10 to 12 thousand years ago is rather sudden. Taking into consideration the long history of mankind, the term Neolithic Revolution has been coined for this “rapid” transformation. The domestication of crops was preceded by animal domestication, with Nomadism the link between animal agriculture and farming. The first crops domesticated in the Middle East include cereals, such as barley, emmer, and spelt, and pulse crops such as sesame and lentil. Cereal and pulse culture require a number of technologies, including land preparation, planting, harvesting, threshing, and seed storage, that form the basis of crop agriculture. Because the seed is produced following

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genetic recombination, thousands of generations occur in which selection, conscious and unconscious, can bring about large changes. The enormous morphological changes that can occur with selection associated with domestication is wonderfully apparent with the myriad breeds of dog, all shown to be domesticated from the wolf, and the changes in Brassica oleracea (broccoli, Brussels sprouts, cabbage, cauliflower), and Beta vulgaris (table beet, sugar beet, and chard). B. Origins of Fruit Culture in the Fertile Crescent Childe (1958) proposed that a second Neolithic Revolution coinciding with the Bronze Age occurred between 6000 and 3000 BCE involving the change from villages to urban communities. The evolution of urban centers is associated with the development of a settled agriculture. This coincides with the beginning of fruit culture, which involved a long-term commitment to a unique piece of ground. In the case of the date and olive, a fruit orchard can remain productive for over a century. It is fruit culture that bonds humans to a particular piece of land and may be a link associated with the concept of territoriality, the development of citystates, and eventually nationhood. Information on the ancient origins of fruit culture comes from archeological remains of fruit, and from pictorial and literary evidence. The high culture of Mesopotamia and Egypt produced a rich art in which fruit is a common motif. A trove of paintings and sculpture is found in Egyptian tombs and monuments. The Sumerian discovery of writing in the 3rd millennium BCE, and Egyptian writings somewhat later, inaugurated the literary tradition that survives today as a result of the near indestructibility of the baked clay tablets used for cuneiform script, the wide use of stone carving for hieroglyphics, and the preservation of papyrus in desert tombs. Zohary and Spiegel-Roy (1975) proposed that fruit culture, in contrast with mere collection, originated 4000 to 3000 BCE. Although some information before this period is based on archeological remains, much of it is by inference and conjecture. Perhaps the earliest pictorial evidence of fruit growing occurs in a 1 m tall alabaster vessel known as the Uruk vase found in Jemdet Nasr levels at Uruk that date from about 3000 BCE (Fig. 8.1). Uruk (Erech) is on the Euphrates just north of Basra, Iraq. The imagery depicts water at the bottom of the vase, followed by plants (barley and sesame) and domestic animals, and men bearing baskets of fruit with offerings presented to a female, perhaps the Goddess Innana, later known as Istar (Bahrani 2002). Unfortunately, the fruits cannot be identified, but they tend to be large and of various shapes. Predynastic drawings of fruit trees in Egypt depict the date palm (Fig. 8.2).

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Fig. 8.1. The Uruk vase, late 4th millennium BCE, showing attendants bearing fruit in a wedding ceremony, probably between a priest king and the goddess Innana (Istar). Source: Pollock 1999.

Fig. 8.2. Predynastic representation of date palm flanked by gazelle in Egypt, 4000–3000 BCE. Source: Singer 1958, Fig. 67.

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The development of fruit culture in the Fertile Crescent evolved at two loci: the Tigris-Euphrates civilization of Mesopotamia and the Nile valley culture of Egypt. Later infusions of species and technology are from Greece, Persia, Turkey, India, and China. By classical times in Greece and Rome, fruit culture had achieved a sophisticated level, not exceeded for over a millennium.

II. THE HORTICULTURAL ARTS Fruit growing involves a more complicated technology than the cultivation of herbaceous annuals such as cereals or pulse crops. Tree crop culture requires a long-term series of horticultural “craft secrets” more or less unique for each species. These include selection of unique clones, vegetative propagation (use of offshoots, cuttings, grafting), continuous irrigation in dry climates, pruning and training, pollination, harvesting, storage, and processing. The cycle of fruit growing is often a year-round activity, and must involve orchard establishment in anticipation of production, which may only ensue after a number of years. Current additions to the technology of fruit growing include the use of dwarfing rootstocks, growth regulators, disease and pest control, long-term storage, protected cultivation, and biotechnology. Here we examine the Neolithic and Bronze Age origins of technologies essential to fruit growing. A. Species Selection The first cultivated fruits must have been indigenous species that had obvious human value. This is clearly seen in Egypt where the indigenous date palm was the earliest species cultivated, followed by a succession of introduced fruits such as the sycomore fig and pomegranate (Table 8.1). The earliest fruit culture in Mesopotamia included the date and olive (4000 BCE), grape, fig, and pomegranate (3rd millennium BCE). Later fruits introductions, based on literary sources, include the apple, pear, quince, and medlar (Postgate 1987). The small-fruited Malus orientalis and Pyrus syriaca are indigenous to the Fertile Crescent, but these were probably not the forerunners of domesticated apple and pear that were introduced from Western Asia probably via Persia. Contacts between East and West date from as early as 1000 BCE as evidenced by silk strands on Egyptian mummies, but intensified with the incursions of Alexander the Great (356–323 BCE). Thus, by the time of the Greek and Roman eras there was an infusion of Central and East Asian fruits, including the citron and a great variety of stone fruits, including almond, apricot, cherry, peach, and plum. In the Age of Exploration in the late 15th and 16th cen-

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Table 8.1. Evidence for fruit crops in Egypt. Source: Janick (2002a) adapted from Darby et al. (1976).

Fruit crop Date palm Doum palm Sycomore fig Jujube (Christ’s thorn) Fig Grape Hegelig Persea (lebakh) Argun palm Carob Pomegranate Egyptian plum Olive Apple Peach Pear Cherry Citron

Binomial

Earliest record (dynasty or period)

Type of evidence

Phoenix dactylifera Hyphaene thebaica Ficus sycomorus Ziziphus spina-Christi

Pre-dynastic Pre-dynastic Pre-dynastic I (Old Kingdom)

Archeological Archeological Archeological Archeological

Ficus carica Vitis vinifera Balanites aegyptiaca Mimusops shimperi Medemia argun Ceratonia siliqua Punica granatum Cordia myxa Olea europea Malus ×domestica Prunus persica Pyrus communis Prunus avium; P. cerasus Citrus medica

II (Old Kingdom) II (Old Kingdom) III (Old Kingdom) III (Old Kingdom) V (Old Kingdom) XII (Middle Kingdom) XII (Middle Kingdom) XVIII (New Kingdom) XVIII (New Kingdom) XVIII (New Kingdom) Graeco-Roman Graeco-Roman 5 BCE 2nd century CE

Artistic Archeological Archeological Archeological Archeological Archeological Archeological Archeological Archeological Literary Archeological Archeological Literary Literary

turies, a great exchange takes place as fruits of the Americas, including the pineapple, cacao, American species of strawberries, and papaya and the fruit-bearing solanums such as tomato and pepper reach Europe, Asia, and Africa, while East Asian fruits, such as the banana, mango, and persimmon reach the Americas. Some East Asian fruits, such as kiwifruit, are relatively recent introductions, and a great many tropical fruits of both Asia (durian, mangosteen, salaak) and the Americas (passion fruit, sapote) are still not extensively commercialized. B. Vegetative Propagation Although most fruit species can be produced from seed (except, of course, seedless clones), this is usually an inappropriate technique. Most fruit species are highly cross-pollinated (peach is an exception) and therefore highly heterozygous. Thus, open-pollinated seedlings will consist of a highly heterogeneous mixture of fruit types, most of which will be inferior to the selected clone. Furthermore, there is a long juvenile period in trees grown from seed. The Hebrew Bible is full of references to degenerate plants that clearly indicate that the ancients were aware of the hazards of seed-propagated fruit crops. Thus, the basis of most fruit

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cultivation is vegetative propagation of unique phenotypes (elite clones) with subsequent improvement based on sexual recombination of elites. Vegetative propagation is accomplished from offshoots in date, layers in grape, and cuttings in olive and fig and runners in strawberry. Fruit crops that can be easily propagated vegetatively have been considered preadapted for domestication by Zohary and Spiegel-Roy (1975). However, many temperate fruits (apple and pear) do not propagate easily by layers or cuttings and are currently multiplied by graftage. Grafting is ancient (Vöchting 1892; Mendel 1953) and Childe (1958) has speculated that it was known before 3000 BCE. Although both root and shoot grafting occurs naturally (Fig. 8.3), the technology is not obvious and must be considered one of the horticultural craft secrets. We know this because much of the Roman writings on grafting often confuse which species are graft-compatible. Pliny describes a number of ludicrous combinations, such as apple on plane tree, suggesting that he was not writing from real experience. Recently, a cuneiform description of budwood importation for grape has been uncovered from Mari, Mesopotamia (Harris et al. 2002) dated to about 1800 BCE, which confirms Childe’s (1958) speculation on the antiquity of graftage. The next written evidence of grafting comes from the school of Hippocrates (Pseudo-Hippocrates, about 450 BCE) that discusses the graft union (Meyer 1854), but this reference implies that the technique was very much older. There is speculation that grafting was known in China as early as 1560 BCE (Nagy et al. 1977; Hartmann et al. 1997) but the earliest definitive evidence for grafting occurs in the 1st century BCE of the bottle gourd (Lagenaria) in The Book of Fan Sheng, while fruit grafting is referred to in Qi Min Yao Suj, written by Jia Simiao in the 6th century CE (Guangshu Liu, pers. commun.). Grafting is discussed in detail by Theophrastus, and all the Roman agricultural writers, including Cato, Virgil, Columella, and Pliny, describe it in detail. Grafting is accurately pictured in mosaics in the 3rd century CE (Fig. 8.4). Grafting is not specifically mentioned in the Hebrew Bible but is inferred based on Jewish writings [Mishna in Order Zeraim (seeds), tractate Kilaim written ca. 3rd century CE] interpreting prohibitions against mixing of seeds in Leviticus 19:19. Grafting of olive is found in the Christian Bible (Romans 11:17&24), 1st century): And if some of the branches be broken off, and you, being a wild olive tree, were grafted in among them, and with them became a partaker of the root and fatness of the olive tree do not boast against the branches. . . . But if you boast, remember that you do not support the root, but the root supports you. . . . For if you were cut out of the olive tree which is wild by

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nature, and were grafted contrary to nature into a cultivated olive tree, how much more shall these, which are natural branches, be grafted into their own olive tree.

Fig. 8.3.

Natural root grafting in wild apple trees. Source: Dzhangaliev 2003.

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Fig. 8.4. Grafting, fruit harvest, and juice extraction portrayed in Roman mosaic 3rd century CE. Source: St. Roman-en-gal, Vienne, France.

J. JANICK

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The development of graftage must have influenced the movement of temperate fruits, such as the apple, from Central Asia to Europe. Grafting is then a pivotal technology in the history of temperate fruits. Precisely when and where detached scion grafting, which made possible the domestication of a new range of fruit trees, was invented is not clear. Barrie Juniper (pers. commun.) has suggested that the initiation of grafting was outside the area of Mediterranean horticulture and was probably introduced from the east, perhaps Persia. Fruit growing has long been associated with clonal propagation of unique wild seedlings with subsequent evolutionary progress derived from intercrosses of superior clones plus intercrosses with wild races (Zohary and Spiegel-Roy 1975), leading to very high seedling diversity. In a number of species, almond for example, the high diversity of wild clones include segregates that are close to domestic types. This would explain why the ecological adaptation of classic Mediterranean fruits has not exceeded the requirements of their wild ancestors. In most cases, present-day fruit cultivars have undergone far fewer sexual cycles than cereal or pulse crops and some may be only a few generations from wild clones. Thus, many of these crops have not diverged from their progenitors, in contrast to cereals in which selection has operated for thousands of generations. This has been confirmed in apples, where present-day cultivars are not distinct from elite selections obtained from wild stands in Alma Alta, Kazakhstan (Harris et al. 2002; Forsline et al. 2003). C. Pollination and Fruit Set Practically all fruit species are naturally outcrossing with variability maintained by natural barriers to avoid self-fertilization. These include dioecy (carob, date palm, grape, fig, strawberry, kiwifruit, papaya), selfincompatibility (pome and stone fruits), and dichogamy, the uneven maturation and receptivity of pistils and stamens (avocado, lychee). In some fruits, cross-pollination is based on unique adaptation with insects (fig, sycomore fig) or birds (pineapple). In dioecious species, accommodation for pollination is necessary. It would be immediately obvious that staminate clones would be nonfruiting. Mass plantings of vegetatively propagated elite pistillate clones would bear few fruit and require either a limited number of staminate plants, proximity to wild pollenizers, or artificial pollination. This limitation to productivity was solved in various ways, some genetic, some cultural, in different fruit crops. In date palms, early farmers discovered artificial pollination, and this is clearly illustrated in

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Assyrian bas reliefs (Fig. 8.5), with the practice codified in the Laws of Hammurabi, ca. 1750 BCE (Roth 2000): §64. If a man give his orchard to a gardener to pollinate (the date palms), as long as the gardener is in possession of the orchard, he shall give to the owner of the orchard two thirds of the yield of the orchard, and he himself shall take one third. §65 If the gardener does not pollinate the (date palms in the) orchard and thus diminishes the yield, the gardener (shall measure and deliver) a yield of the orchard to (the owner of the orchard in accordance with) his neighbor’s yield.

In fig, the presence of the wild monoecious caprifig that harbored the pollinating blastid fig moth (caprification) was understood as essential for fig production by Theophrastus (371–287 BCE), but later selection for parthenocarpy eliminated this practice. In grape, strawberry, and papaya, domestication exploited mutations from dioecism to hermaphroditism and, in some figs and grapes, parthenocarpy reduced the requirement for pollination altogether. In the sycomore fig, introduced to Egypt without the pollinating fig wasp, artificial wounding to ripen parthenocarpic fruit was the solution. In apple and pear, which are selfincompatible and insect pollinated, the problem was solved with the introduction of bees to facilitate cross-pollination along with inter-

Fig. 8.5.

Date palm pollination depicted in Assyrian bas-reliefs. Source: Paley 1976.

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planting of pollenizers. In recent times, pollenizers for self-incompatible sweet cherry were eliminated by introducing self-compatible mutations. D. Irrigation The civilizations that developed in the arid climates of the Fertile Crescent are dominated by large rivers—the Nile in Egypt and the Tigris and Euphrates in Mesopotamia. In Egypt the regular inundations of the Nile, rising in July until the middle of October, followed by rapid subsidence, permitted a unique horticulture based on basin irrigation (Janick 2002a). The system involved a system of dikes to retain the flood and encourage infiltration into the soil. Earthen banks, parallel to the river together with intersecting banks, created a checkerboard of dike-enclosed areas, between 400 and 1600 ha each. Canals led the water to areas difficult to flood. The flood waters ran through a series of regulated sluices into each basin, flooding the land to a depth of 0.3 to 1.8 m. The water could be held for a month or more; the surplus was drained to a lower level and then returned to canals that emptied into the Nile. The advantage of basin irrigation was that no further irrigation was needed for a winter crop of grain, and the silt, rich in organic matter and phosphates, made fertilization unnecessary. With fruit tree culture, permanent ponds were an important innovation and the ornamental gardens enclosing ponds testify to their widespread use by the wealthy. In addition, shallow wells, 4 to 35 m in depth, were dug to be replaced later by deeper artesian wells up to 380 m deep. The culture of fruit crops demands constant and controlled irrigation during the spring and summer drought. At first, irrigation was carried out manually with pots dipped in the rivers, carried on the shoulders with yokes, and poured into field channels. By the time of the New Kingdom (1500 to 1100 BCE), the shaduf, a balanced counterpoise, became the irrigating mechanism for gardens. Later, water lifting techniques included Archimedes’ screw, the sakieh or chain of pots, and siphons (Fig. 8.6). In Mesopotamia, cultivation in the Tigris-Euphrates flood plain is and always has been dependent on irrigation, and the management of this technology may have been the impetus for the development of nation-states (Pollock 1999). Irrigation started as small-scale projects but eventually increased in complexity and involved centralized control. The creation of state-controlled irrigation led to a strong central authority requiring conscripted service (corvée) for canal maintenance. The Laws of Hammurrabi richly describe a legal system enforced to maintain the integrity of an irrigated agriculture:

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§53 If a man neglects to reinforce the embankment of the irrigation canal of his field and does not reinforce its embankment, and then a breach opens in its embankment and allows the water to carry away the common irrigated area, the man in whose embankment the breach opened shall replace the grain whose loss he caused. §54 If he cannot replace the grain, they shall sell him and his property, and the residents of the common irrigated area whose grain crops the water carried away shall divide the proceeds. §55 If a man open the branch of the canal of irrigation and negligently allows the water to carry away his neighbor’s field, he shall measure and deliver grain in accordance with his neighbor’s yield. §56 If a man opens an irrigation gate and releases waters and thereby he allows the water to carry away whatever work has been done in his neighbor’s field, he shall measure and deliver 3,000 sila of grain per 18 iku of field.

Because of the braiding character of the Euphrates, short canals about 1 km in length could be dug from the numerous river channels and managed by local groups (Pollock 1999). The natural flow of the river and overflow resulted in natural levees, and in the process the riverbed was gradually raised until it flowed above the level of the surrounding land. This made it relatively easy to cut irrigation channels through the natural levee and allow the water to flow by gravity to cultivated fields and gardens. The natural levees with their good drainage were prized for fruit tree cultivation, but irrigation required water lifting technology. The natural vegetation of the alluvial plain provided pasturage for sheep and goats; it was once home to game animals such as jackals, lions, gazelles, onagers, and hyenas, as illustrated in the hunting scenes in Babylonian bas reliefs now hunted to extinction. Long-term irrigation, however, led to unintended consequences and today much of the area is a vast salty waste as a result of salinization. E. Pruning and Training The art of fruit growing is associated with physical techniques to control the shape, size, and direction of plant growth. These include the orientation of the plant in space (training) and judicious removal of plant parts (pruning). In date palm culture the removal of senescing leaves, necessary for both pollination and harvest, is probably the basis of the pruning technique. The dead leaves had a wide variety of uses for shade and basketry. In the case of the grape, vine pruning is essential to control both flowering and yield and to increase fruit quality. Early Babylonian bas reliefs show grapes growing on trees, and the use of arbors

8. THE ORIGINS OF FRUITS, FRUIT GROWING, AND FRUIT BREEDING

A.

B.

C.

E.

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D.

F.

Fig. 8.6. Development of irrigation technology. A. drawing water from pots, ca 1450 BCE, B. irrigation and harvesting in a vegetable garden using yoke, C. irrigation of a date palm orchard by a shaduf, D. date palm with water-storage pond, E. Archimedes screw, F. sakieh, a chain of pots. Source: Singer et al. 1954 (A–D), British Museum (E), FAO (F.)

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and pergolas for grape is well illustrated in late Egyptian paintings (Fig. 8.7). In the Mideast, an ancient training method involved severe pruning whereby the plant was pruned in the fall and mounded with soil over the winter to avoid cold injury.

A.

B. Fig. 8.7. Training of grape. A. Vine climbing a cypress tree in an Assyrian garden. B. Grapes collected from a round arbor from a tomb at Thebes, Egypt, ca 1500 BCE. Source: Singer et al. 1954.

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F. Processing and Storage Most of our important domesticated fruits are delicious in the mature state and indeed this is one of the chief virtues of fruit crops. However, this is not the case with all fruits. The olive, in particular, is bitter and inedible, even in the mature state, and some wild species are toxic. Clearly, the key technology must have been derived from the ameliorating practice of soaking the fruits to make them less bitter. Many primitive societies such as Amerinds with cassava and Australian aborigines with Pandanas (Crib and Crib 1976), independently came to their detoxification techniques (Johns and Kubo 1988). Extraction of oil by pressing the fruit transformed olives into the most widely grown fruit crop in the ancient world. The oil was widely used in cooking and illumination; the flame produced from olive oil has a very high luminosity. Fruit crops grown for their fruit or seed oil also include oilpalm and avocado (used for soap in Brazil). Most fruits have a short life after harvest so that processing is required to have a year-round supply. The perishability of many fruits is one of the limiting factors of commercialization. In the case of the date, the high sugar content of the dried fruit permits extended storage, and dried grapes (raisins) have long been prized for their concentrated sweetness and long storage. Sun drying of many types of Prunus, especially highly sugared plums (prunes) and apricots was facilitated by slicing fruits. The conservation of fruits as jams and preserves was based on the addition of sucrose, a substance unknown until the technology of sugarcane production and processing was developed in the Middle Ages. Some wild fruits, such as lingonberry in Nordic countries have long been collected and stored as preserves. The preservation of fruits by heat (canning) was a 19th century technology developed by Nicolas Appert (1750–1841) as a response to the British blockade of France during the Napoleonic wars. Quick freezing and later freeze drying are 20th century technologies. In more temperate climates, fruit life could be extended by common storage in caves or basements. Caves in Cappadocia (Turkey) maintain a temperature of 12.8°C and are still used to store lemons. The modern transformation of this technique led to refrigerated storage and low oxygen storage (controlled-atmosphere) storage. The transformation of fruit juice to an alcoholic product (wine), along with bread making, is a Neolithic discovery. Beer making probably predated wine making. This leap into Bronze-age biotechnology was facilitated by the ubiquitous presence of yeast spores. Although wine can be made from various fruits, the choice species is grape, probably because

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of its combination of sugars, acids, and tannins. At present, the greatest use of grapes is for wine manufacture. In the East, salting and fermentation technologies were developed as a means of whole fruit storage. A few tropical fruits (e.g., plantain and breadfruit) are staple starch crops and require cooking.

III. ORIGIN, DOMESTICATION, AND EARLY CULTURE OF FRUIT CROPS The origin and changes associated with domestication of fruits in this review are summarized in Table 8.2. A. Mediterranean Fruits 1. Date Palm. The date palm (Phoenix dactilifera, Arecaceae) is a dioecious palm, thought to be indigenous from Northern Africa through the Arabian peninsula to northern India (Goor and Nurock 1968; Zohary and Spiegel-Roy 1975; Smartt and Simmonds 1995). In antiquity, the date palm was esteemed from India through North Africa for its sweet fruit consumed fresh or dried, for its valuable wood and leaves, and for its long life. As a result of its many virtues, the date was transformed into a sacred tree, symbolically referred to in Babylonian and Assyrian iconography. It may have been the first cultivated fruit and was wellestablished in the Middle East during the Bronze Age. The precise origin of the cultivated date is open to conjecture. Vavilov considered the origin of the date to be the mountains of northeastern Africa in Ethiopia and Eritrea but there is evidence that the first cultivation occurred in the Lower Mesopotamian Basin. Archeological evidence places date stones in the Ubaidian horizon (about 4000 BCE) in Eridu, lower Mesopotamia. A predynastic representation of a palm tree (perhaps a date) with gazelles has been dated 4000 to 3000 BCE (Fig. 8.2). The date palm is adapted to long, extremely hot summers with little rain and low humidity from pollination to harvest but requires a source of underground moisture. All cultivated dates set fruit only in dry desert conditions, in contrast to a number of wild species which do not produce suckers and may be adapted to rainy conditions. Since the plant is a monocot and lacks a deep root system, irrigation is essential. An Arab proverb describes the date palm as “its feet in running water and its head in the fire of the sky.” The cultivated date palm is easily propagated from seed and, unlike its wild relative, can be vegetatively propagated from offshoots (suckers)

Table 8.2.

Origin and changes associated with domestication of various fruit crops.

Fruit crop

Species (Chromosome no.)

Reproduction of wild species

Changes associated with domestication

Family

Origin

Mediterranean Fruits Date palm Phoenix dactilifera x =18, 2n=36

Arecaceae

S. Mediterranean basin

Dioecious

Offshoot production, increased fruit size

Fig—common

Ficus carica x=13, 2n=26

Moraceae

E. Mediterranean basin

Gynodioecious

Parthenocarpy

Fig—sycomore

Ficus sycomorus

Moraceae

East

Monoecious

Parthenocarpy

Grape

Vitis vinifera x=19, 2n=38

Vitaceae

W. Asia

Dioecious

Hermaphroditic, increased berry size, parthenocarpy

Olive

Olea europea x=23, 2n=46

Oleaceae

Mediterranean basin

Andromonecious

Increased fruit size, high oil

Pomegranate

Punica granatum

Punicaceae

W. Asia

Hermaphroditic

Increased fruit size, sweetness

Asian & European Fruits Pome Fruits

273

Apple

Malus × domestica x=17, 2n=34, 51

Rosaceae

Central Asia

Hermaphroditic, self-incompatible

Combination of size aroma, loss of astringency, sweetness, parthenocarpy, triploidy

Pear—European

Pyrus communis x=17, 2n=34, 51

Rosaceae

Central Asia

Hermaphroditic, self-incompatible

Combination of size, aroma, loss of astringency, sweetness, parthenocarpy, triploidy

Pyrus—Asian

Pyrus pyrifolia P. bretscheiderii P. ussuriensies x=17, 2n=34, 51

Rosaceae

E. Asia

Hermaphroditic, self-incompatible

Combination of size, aroma, loss of astringency, sweetness, parthenicarpy, triploidy

(continued )

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Table 8.2.

(Continued)

Stone Fruits Almond Apricot Cherry—sweet Cherry—tart Plum—European Plum—Asia Plum—American Peach Vine Fruit Kiwifruit

Prunus dulcis x=8, 2n=16 Prunus armeniaca x=8, 2n=16 Prunus avium x=8, 2n=16 Prunus cerasus x=8, 2n=16, 32 Prunus domestica x=8, 2n=48 Prunus salicina x=8, 2n=16, 32 Prunus Americana x=8, 2n=16 Prunus persica x=8, 2n=16

Rosaceae

S.W. Asia

Rosaceae

Central & E. Asia

Rosaceae Rosaceae

Central Europe & W. Asia W. Asia

Rosaceae

Europe

Rosaceae

Actinidia delicious x=29, 2n=174 A. sinensis x=29, 2n=58

Subtropical & Tropical Fruits Citrus (orange, Citrus spp. mandarin, lemon, x=9, 2n=18 lime, pumello, grapefruit) Mango Mangifera indica x=20, 2n=40

“Sweet” seed, increased kernel size, self-fertility Increase fruit size

China

Hermaphroditic, self-incompatible Hermaphroditic, self-incompatible Hermaphroditic, self-incompatible Hermaphroditic, self-incompatible Hermaphroditic, self-incompatible Hermaphroditic

Rosaceae

North America

Hermaphroditic

Increased fruit size

Rosaceae

China

Hermaphroditic

Freestone, low chill, fuzzless (nectarine), increased size

Actinidiaceae

China

Dioecious

Unchanged

Rutaceae

Southeast Asia, China

Hermaphroditic

Nucellar embryony, interspecific hybridization, parthenocarpy

Anacardiaceae

E. Asia

Hermaphroditic

Nucellar embryony, loss of fibers in fruit

Self-compatibility (recent) Tetraploidy after interspecific hybridization Hexaploid after interspecific hybridization Increased fruit size

Persimmon Asian

Diospyros kaki x=15, 2n=90

Ebenaceae

China

Polygamo-dioecious

Loss of astringency, parthenocarpy

Fragaria × ananassa x=7, 2n=56 Other spp. 2n=14, 28 Rubus idaeus (red) R. occidentalis x=7, 2n=14 Rubus spp. x=7, 2n=28, 35,42,56,84 Vaccinium spp.

Rosaceae

Americas

Dioecism

Hermaphroditic, interspecific hybridization

Rosaceae

Europe, America

Hermaphroditic

Interspecific hybridization, polyploidy

Rosaceae

N. America

Hermaphroditic

Interspecific hybridization, polyploidy, thornlessness

Ericaceae

N. America

Hermaphroditic

Ericaceae

E. United States

Hermaphroditic

Increased fruit size, interspecific hybridization, polyploidy Unchanged

Ericaceae

Circumboreal

Hermaphroditic

Unchanged

Subtropical & Tropical Fruits Avocado Persea americana x=12, 2n=24

Lauraceae

Tropical America

Hermaphroditic, synchronous protogynous dicogamy

High oil, smaller seed size

Papaya

Euphorbiacea

Tropical America

Dioecious

Bromeliaceae

Tropical America

Hermaphroditic

Polygamo-dioecious, reduced fruit size Parthenocarpy, seedlessness

American Fruits Berry Fruits Strawberry

Raspberry

Blackberry

Blueberry Cranberry Lingonberry

Pineapple

Vaccinium macroacarpon Vaccinium vitis-idaea

275

Carica papaya x=9, 2n=18 Ananas comosus x=25, 2n=50

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at the base of the plant (Fig. 8.8). Clearly the production of offshoots is one of the principal characteristics of domesticates. At present, all cultivars can be propagated in this manner and one to several suckers may be removed each year. Offshoots can be planted without roots, but mounding of soil around the base of the mother tree facilitates their rooting. Irrigation is essential to insure survival of offshoots and high productivity. Since the date is dioecious, production of fruit by pistillate clones requires the presence of a source of pollen. In present-day commercial plantations, one male tree to 100 females is sufficient if pollination is performed by hand. In some cases twigs or branches of the male inflorescence from 10 to 15 cm long and bearing 30 to 50 flowers are tied on the female cluster, but this process must be repeated because of variation in time of maturation of pistillate flowers. Pollen has also been distributed by airplanes or helicopters.

Fig. 8.8. The cultivated date palm is vegetatively propagated from offshoots (suckers) at the base of the plant.

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In antiquity, mass plantings of desirable pistillate clones of date palm would have reduced fruitfulness had nonproductive staminate clones not been introduced. The relation between artificial pollination and fruit set was known to early Sumerians and is mentioned in the cuneiform texts of Ur just south of Baghdad in Iraq, ca. 2300 BCE. By the time of Hammurabi the problem of the division of the crop between the landlord and the tenant gardener, who acted as a sharecropper, was defined and penalties for lack of pollination were assessed (see IIC). The practice soon took on religious significance, as illustrated by bas reliefs showing pollination of sacred trees by kings and gods (eagle-headed genii) (Fig. 8.5). The sexual nature of the pollination process was probably known as the date palm became a symbol of fertility. Theophrastus, writing in the 3rd century BCE, clearly mentions the parallels and refers to the pollen bearing plant as male and the fruit-bearing plant as female. Wild date palms often produce small and nonpalatable fruits, while the domesticates are associated with larger size and considerable amounts of sweet pulp. Increased size and quality of the fruit is associated with selection carried out under domestication. Continued clonal propagation of large-fruited dates through offshoots would have increased propagation ability along with fruit size. Genetic improvement ensued from selections of elite wild genotypes, followed by natural intercrosses within elites as well as with wild clones. Since the plant is dioecious, selection must have been confined to pistillate clones. However, selection of male clones may have been facilitated by the presence of metaxenia, the direct effect of pollen on morphology and ripening of the date palm fruit. Thus, selection for fruit characters may have been a method of gamete selection. At the present time, there are over 3000 cultivars, of which 60 are widely grown. Some cultivars have been known for a thousand years. Cultivars have been divided into soft and semi-dry, based on moisture content and an increasing proportion of sucrose, rather than glucose and fructose. Selection has altered fruit quality, storage, suckering, salt tolerance, and spininess. 2. Olive. Olive (Olea europea, Oleaceae) is a slow-growing, long-lived, evergreen tree uniquely adapted to the climate of the Mediterranean basin and considered a defining feature of this climate (Smartt and Simmonds 1995). Indigenous types are still widely found with archeological evidence as far back as 12,000 years ago (Blázquez 1996). The cultivated olive originated about 6000 years ago in Asia Minor, but was generally unknown to the Babylonians and Assyrians, whose source of oil was sesame and walnut. The olive was long known in Syria and the

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Holy Land and was introduced to Egypt from Syria between 3000 and 1800 BCE (Goor and Nurock 1968). By 2500 BCE, the ancient city of Elba in northern Syria had fields containing between 500 and 1000 olive trees, producing and exporting various types of olive oil. According to Hittite texts olives were cultivated in Anatolia and imported to Egypt from the time of Ramses II (1197–1165 BCE), where there is mention of the use of olive oil for illumination and as a skin emollient for cracks and sunburn. The olive moved from Egypt to Carthage in North Africa, reaching Italy in the 7th to 6th century BCE. Olive, along with grape, is the most mentioned fruit in the Hebrew bible. Its importance has permeated the Western world and the olive tree and oil are symbols of beauty, freshness, fertility, wealth, fame, and peace. Greek myths associated with the olive involve Hercules and Athena. The importance of oil is reflected in its widespread use for religious purposes such as consecration ceremonies (anointing) in Judaism and Christianity; the word messiah (Christ) literally means “the anointed one.” Olives were introduced to South America in the 16th century and introduced to Mexico, California, and Australia in the 20th century. In Mediterranean climates, the olive is often grown without irrigation, although yields are low. Moderate pruning is performed to shape trees and to remove unfruitful wood. Harvest is usually carried out by hand, with the time depending on the use (Fig. 8.9) Olives may be vegetatively propagated by cuttings, from buried truncheons (large branches in which incisions are made every 8 to 10 cm), or

Fig. 8.9. Harvesting olives in ancient Greece from a black figure amphora, 520 Source: Singer et al. 1954.

BCE.

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from knobs (ovuli) that occur at the base of the trunk. Theophrastus made the astute observation that basal cuttings root better than terminal ones. Early propagation from seed from unique clones probably increased crop diversity, while the introduction of clonal propagation made it possible to fix unique genotypes. Olives have been propagated from grafting since antiquity (see Vegetative Propagation). Many present-day cultivars are considered to be thousands of years old. There are two processing steps involved in olive utilization. The first is transformation of the bitter fruit to an edible form and the second is oil separation. Somehow, Neolithic humans learned to consume a small, bitter fruit, almost inedible and somewhat poisonous as a result of the phenolic glucoside eleuropein found in various concentrations in all olives. This was accomplished by soaking, a technique still used. Further steps in detoxification included pounding, addition of lye (sodium or potassium hydroxide), or lactic acid fermentation induced by sodium chloride (Colmagro et al. 2001). There is ample evidence that so called “primitive” societies learned to leach a number of foods to reduce their content of bitter substances. Australian aborigines used a combination of soaking and roasting with many native fruits and seeds (Crib and Crib 1974). Later in the Bronze Age, olive processing technology was invented to separate the oil. Olive became the first great industrial crop, grown more for its oil—as a medicinal, for cooking, and for illumination (the light is very bright)—than for direct consumption. The religious practice of anointing probably derived from the medicinal use of olive oil. The ancient golden Menorah with seven arms (Exodus 24, 31–40) is an oil lamp. The earliest process of oil extraction was by crushing the fruit in water and spooning the floating oil. Later improvements were the use of oil presses of various types. Typically water was added and the oil removed by decantation. At present 90% of the world olive crop is pressed for oil that is used for culinary purposes; the remainder is used as table olives, a fermented product. Recently, olive consumption has increased with the recognition of the beneficial properties of oleic acid and the increase in consumption of various types of table olives as a gourmet product. Cultivated olives are characterized by large fruit, large proportion of flesh, and high oil content. At present, olives are grouped as oil types, with fruits containing from 20 to 28% oil used for olive oil production, while types with less oil are for table use, whole or pitted, and for pickling. 3. Grape. Wild grapes of the Old World (Vitis sylvestris, Vitaceae) are indigenous to the south Caspian belt, Turkey, and the Balkans, and were widely distributed in the northern Mediterranean area including

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the Black and Caspian Seas (Zohary and Spiegel-Roy 1975; Smartt and Simmonds 1995; Reisch and Pratt 1996). Wild grapes are dioecious, perennial, forest vines and thrive in cooler and more humid conditions than do olives. Harvest of wild grapes long preceded domestication, as evidenced by carbonized seed in numerous prehistoric sites in Europe. Presence of the wine grape in the Near East is dated as early as the 8th millennia BCE but eastward expansion was limited by lack of winter hardiness and poor adaptation to high summer rainfall. Toward 5000 BCE, and perhaps earlier, the domestic grape, V. vinifera, migrated from Anatolia to Syria and thence to the Holy Land. Signs of Bronze Age domestication are found in Mesopotamia, the Holy Land, Syria, Egypt, and the Aegean. By the 2nd millennium BCE there is evidence of vessels for wine storage as well as of raisins. A number of grape species are found in the New World and these types were domesticated after the European encounter with America (Reisch and Pratt 1996). One American species, V. labrusca, known as the fox grape, was rapidly domesticated, while others were hybridized with V. vinifera to develop Phylloxera-resistant rootstocks. Muscadines (V. rotundifolia), native to the southern United States, were preserved by Native Americans by drying (Olien 2001) but were not cultivated and can be considered a recent domesticate. Grapes are easily propagated vegetatively, permitting extensive plantings of unique clones. When vines are in contact with the soil, rooting often occurs at the nodes (layering). Hardwood cuttings also root easily. At present most grapes are propagated by grafting in order to take advantage of rootstocks resistant to soil-borne diseases and insects. The great genetic change in domestication was the switch from dioecism to hermaphroditism, a mutation in the dominant allele of the male-conferring gene that suppresses development of the gynecium. Other changes associated with domestication include an increase in berry size and sugar content, and selection of various skin colors (white, green, bronze, pink, red, and black). Recently selection of seedlessness has become a key factor for table grapes; seedlessness occurs from both early embryo abortion (stenospermocarpy) or from parthenocarpy (Ledbetter and Ramming 1989). The seedless grape ‘Sultanina’, known as ‘Thompson Seedless’ in the United States, is an ancient Turkish cultivar and still one of the most widely grown for table grapes or wine. The cultivation of grape involves extensive vine training and pruning and in no other fruit crop are these practices more important. The Hebrew Bible is rich in allusions to these practices. The replacement of tree support with arbors or trellises is amply shown in Egyptian iconography (Fig. 8.7). Protection of grapes from birds and thieves is a common

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feature of the early cultivation of wine, and the construction of walls and towers is associated with vineyards in ancient Israel (Walsh 2000). Various techniques were developed for overwintering, including covering sprawling vines with soil. Grapes were preserved in two ways. The simplest was sun drying, and the dried fruit (raisins) were prized for their concentrated sweetness, and long-term storage (Fig. 8.10). The transformation of grape juice to wine occurs almost naturally but requires anaerobic conditions. The fermentation of juice into wine was probably based on beer technology, an older practice. The culture of grapes (viticulture) and the technology of wine making (enology) are common themes in biblical writings, and became infused in Judaism (Walsh 2000) and Christianity, although drunkenness was frowned upon. Wine was the beverage of choice in ancient Greece and Rome. Wine is prohibited in Islam but grapes and raisins are highly prized. At present wine is the major use of grapes, far surpassing table grapes and raisins. 4. Fig. The common fig (Ficus carica, Moraceae) is a gynodioecious species consisting of monoecious wild types (caprifig) and pistillate domesticates (Condit 1947). Figs, borne on small trees, are considered one of the classic fruits of the Mediterranean basin. Signs of fig cultivation are found at various Neolithic and late Neolithic sites (Zohary and Spiegel-Roy 1975), and from Bronze Age sites in the 4th millennium BCE. Domestication was generally contemporary with olive and grape in the Eastern Mediterranean basin; it was mentioned by King Urukagina ca 2900 BCE and became known to the Assyrians as early as 2000 BCE. Egyptian records indicate fig cultivation in Canaan in the 3rd and 2nd millennia BCE (Goor and Nurock 1968) and there are many paintings of fig from antiquity (Fig. 8.11, 8.12). The fig is easily propagated by seed, as well as by cuttings or layers. Propagation by grafting and budding was described by Cato in the 3rd century BCE. The fig is marvelously adapted to insect pollination (Condit 1947; Storey 1975). The fruit is a syconium, a fleshy branch transformed into a hollow receptacle bearing minute flowers on its inner surface and

Fig. 8.10.

Storing raisins, from a tomb at Beni Hasan ca. 1900 BCE. Source: Singer et al. 1954.

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Fig. 8.11. Harvesting figs, from a tomb at Beni Hasan, Egypt, ca. 1900 BCE. Source: Singer et al. 1954.

open to the outside by a small orifice (ostiole). The monoecious caprifig, or goat fig, the name indicating its lack of worth, contains both staminate and short-styled pistillate flowers within the syconium and serves for the multiplication of the parasitic fig wasp (Blastophaga psenes). Pollination is carried out by this tiny (about 1 mm) wasp, an association described by Theophrastus. The insects overwinter in the larval stage in the pistillate flower, pupate in early spring, and emerge as adults within the synconium. The wingless male impregnates the female inside the ovary and then perishes. The winged female exits the synco-

Fig. 8.12.

Wall painting of a basket of figs. Villa at Torre Annunziata. Source: Jashemski 1979.

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nium through the ostiole, where she is covered with pollen from staminate flowers clustering around the orifice. The winged female then migrates to other small figs, deposits an egg in each of a number of pistils and then dies. However, when the female enters the common fig that contains only long-styled pistils that are not adapted to oviposition, the insect perishes, but not before pollination has been accomplished. There are as many as three crops of figs in each season. The first crop, called profichi, are formed on wood of the previous year’s growth. They may contain the wasp or, if uninhabited, are called blanks. The inhabited profichi are usually dark green, firm and plump, while the blanks are yellowish green, ribbed and inclined to be spongy. The next crop, called mammoni, are produced in the axils of the current season’s growth and are often pollinated by wasps from the profichi crop. The last crop is called mamme and remains on the tree during the winter season. Parthenocarpy, the ability of the fruits to grow and mature without fertilization, is of two types, vegetative or stimulatory (Condit 1947; Storey 1975). In vegetative parthenocarpy fruit development occurs without any stimulation (oviposition or pollination). In stimulative parthenocarpy, insect stimulation or fertilization is required. In most caprifigs, the syconium fails to set unless pistillate flowers are stimulated by oviposition with larval development of the wasp. Some caprifigs are partly parthenocarpic, producing seedless figs more or less freely; some are completely parthenocarpic. The domesticated Smyrna-type figs are completely non-parthenocarpic, and thus require pollination; this is often accomplished by introduction of the wasp-bearing caprifig, hence the term caprification. Some cultivated figs are partially parthenocarpic in that the early crop (called brebas) is parthenocarpic but the second crop is non-parthenocarpic. Common type figs such as ‘Mission’, ‘Dottato’, and ‘Brunswick’ are completely parthenocarpic in both crops. Domestication is associated with increase in size of the fruit (syconium), and sugar content, as well as parthenocarpy (Storey 1975). Other characters of figs include various colors (white, amber, red, and purple) in the skin and flesh, increased sugar content, and resistance to splitting when mature. There are many known cultivars; Condit (1955) has described over 600, many of which may be synonyms. ‘Condit’s Adriatic’, now called ‘Conadria’, was the first release from a breeding program, and is characterized by high fresh and processing quality and resistance to splitting. Figs are consumed fresh or dried and are often processed as a paste for pastries or canned. The fruit can be fermented and distilled into alcohol. 5. Sycomore Fig. The sycomore fig (Ficus sycomorus, Moraceae) originated in the savannas of eastern Central Africa where they grow spontaneously

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and reproduce by seed. The tree is easily propagated by cuttings. It was introduced to Egypt in predynastic times, before the 3rd millennium, and became an important cultivated plant in the Early and Middle Kingdoms. Although the fruit was not considered exceptional, the tree was highly prized and held sacred, especially to Hathor, goddess of love, and representations of fruit and tree are found on bas-reliefs and commemorated in song. Fruit and leafy branches were placed in funeral offerings. The wood is decay resistant and esteemed for household utensils, construction, boxes, and coffins. The tree was introduced to the Levant, present-day Israel, Lebanon, and Syria as well as Cyprus. It was well known in Biblical times and became a food for the poor. Flowers are pollinated by the chalcidoid wasp, Ceratosolen arabicus, but the plant was introduced to Egypt without the wasp. Although fruits developed parthenocarpically, they did not enlarge or ripen successfully unless wounded. This fact is found in a famous quote from the Hebrew Bible (Amos 7, 14): I was no prophet, neither was I a prophet’s son but I was a herdman and a “piercer” (mistranslated as “gatherer” in the King James translation) of sycomore fruit. The phrase refers to the practice of ripening the fruit by scraping it (Fig. 8.13). Wounding of the fruit results

A.

B.

C. Fig. 8.13. Induced ripening in sycomore fig. Source: Galil 1968. A. Bas-relief showing a sycomore tree with gashed fruit; B. knives used for gashing; C. gashed sycomore fruit.

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in ripening through the release of ethylene. This remarkable technological discovery was known to Theophrastus, who writes: The sycamore cannot ripen unless it is scraped but the Egyptians scrape it with iron claws, the fruit thus scraped ripens in 4 days. 6. Pomegranate. Pomegranate (Punica granatum, Punicaceae), native to the southern Caspian belt (Iran) and northeast Turkey, is a Bronze Age fruit that has been cultivated for 5000 years (Goor and Nurock 1968; Zohary and Spiegel-Roy 1975). Its presence in Syria, the Holy Land, Egypt, and Mesopotamia indicates domestication. The Latin name Punica refers to Carthage and the fruit was once known as the apple of Carthage. Remains are found in Nimrud, Arad, and Jericho. The pomegranate was introduced into Egypt from Syria about 1600 BCE before the 19th Dynasty during the inflow of Semitic people (Hyksos). The fruit is commonly illustrated in Egyptian art (Fig. 8.14) from the New Kingdom and widely found in Jewish and Greek art. The pomegranate is easily

Fig. 8.14. Offering bearing pomegranates, grapes and flowers, New Kingdom, Egypt. Source: Darby et al. 1977.

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propagated by hardwood and softwood cuttings. Domestication is associated with increased fruit size, and sweetness, and a shift to clonal propagation. Some types have degenerate embryos and are called seedless. In Egypt, juice from the pomegranate was consumed and converted to wine. The rind was prescribed as a medicinal and the flowers were crushed to make a red dye for leather. Based on extensive iconography, the pomegranate seems little changed from antiquity. Peel color varies from bright red to leathery brown. B. Central Asian Fruits 1. Pome Fruits. Pome fruits of the Rosaceae include apple, pear, quince, and medlar, of which the apple is the most important tree fruit of the temperate world. The trees require an annual period of chilling to break dormancy for proper growth, and the seeds require a period of cold stratification before they will germinate, and must be separated from the placental tissue of the fruit to germinate. Apple. The apple is cross-pollinated and most cultivars are selfincompatible. Most cuttings of apple and pear trees do not root from cuttings so that vegetative propagation is difficult without grafting. There are 24 primary species of Malus, distributed in Europe, Central Asia, and Eastern Asia and three in North America (Way et al. 1990). Most of the wild apples are small and bitter. However, the large, sweet-smelling domestic apple (Malus × domestica) clearly originated in Central Asia, specifically Almaty, Kazakstan (Dzhangaliev 2003) and was introduced to the West via Persia in antiquity. Recent explorations in Kazakhstan (Forsline et al. 2003) support Vavilov’s suggestion that Malus sieversii = M. pumila is the major progenitor of the cultivated apple, and this has been confirmed by recent work with molecular markers (Harris et al. 2002). The explorations in Kazakhstan also indicate that elite wild material contains all the characters of modern apples (Fig. 8.15) including size, quality, and various colors from red to yellow to green. Barrie Juniper (pers. commun.), has made the intriguing observation that the selection mechanism for large size may not have been human but rather the result of millions of years of selection for large size by bears endemic to the area who consume large numbers of apples; their droppings provide a unique germination medium. All of the other species of apple appear to be distributed by birds, providing no selection mechanism for large size. Fruits of wild relatives of the domesticated apple occur in the Mideast and Europe and were frequently collected by Neolithic and Bronze Age

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Fig. 8.15. Cultivated seedlings of elite wild apples from Kazakhastan. The two fruits on the bottom are ‘Empire’ and ‘Gala’. Source: Forsline et al. 2003.

farmers. However, it is difficult to know precisely when the larger, sweeter apples of central Asia reached the West because reference to apple (hazur) in the early Sumerian literature may in fact refer to the indigenous, bitter, small-fruited species, Malus orientalis. The earliest archeological evidence (early Dynasty III, about 2200–2100 BCE) of dried apples are rings of small fruits (11 to 18 mm in diameter), possibly threaded on a string, on saucers in Queen Pu-abi’s grave at Ur near present-day Basra, Iraq (Postgate 1987; Renfrew 1987). Apples lose their bitterness when dried (Barrie Juniper, pers. commun.), and if wild M. orientalis were harvested it might have been consumed in this manner. A Sumerian cuneiform literary manuscript, entitled Disputation between the Hoe and the Plow (Vanstiphout 2000), dated about 1900 BCE, refers to gardens of apples. In Anatolia (Turkey), the Hittites, who rose to dominance in the 2nd millennium BCE, referred to 40 apple trees on one estate. Later biblical references to sweet-smelling apples such as in the Song of Songs, written probably no earlier than 1000 BCE, suggest introduction before the 1st millennium BCE. In any case, by the 1st millennium BCE apples were a part of western agriculture. Homer (8th or 9th

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century BCE) refers to apples in the gardens of King Alcinöus, the king of the Phaeacians, a legendary country. Apples, however, were unknown in Egypt until Greco-Roman times. How did the apple reach the west from Central Asia? Because apples and pear do not propagate easily from cuttings, the most likely explanation is the introduction of seed carried in saddle bags by caravans along the trade routes, passing through the fabled cites of Bukara and Samarkand to Persia, perhaps facilitated by seed germination in horse droppings, but propagation from root suckers is another possibility (Fig. 8.16). Another explanation is that grafting technology, dated as early as 3800 years ago (Harris et al. 2002), may have been involved with Persia as an intermediary stop. Persia is clearly the source of many fruits from Central Asia and China. Whatever the precise mechanism, the apple clearly passed through Persia and Greece, and, by the time of the Romans, apple technology including grafting, pruning, storage, and selection of unique adapted clones was perfected. Even the use of specific rootstocks seems to have been discovered (a low growing type is described by Theophrastus) and it is no coincidence that these easily-rooted dwarfing clones are called Paradise, the Persian word for garden. The apple cultivated by the Romans was transported throughout the empire and became naturalized throughout Europe, resulting in thousands of unique types. Possibly some intercrosses with the small, astrin-

Fig. 8.16.

Propagation of wild apples from root suckers. Source: Dzhangaliev 2003.

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gent Malus sylvestris, native to northern Europe, gave rise to cider apples that are still cultivated. Interestingly, a secondary center of diversification was established in the United States in the 18th and 19th centuries as seed from European cider mills was imported, resulting in a new cycle of selection by fruit growers (see IV C). As a result, most of the new cultivars of apple now grown worldwide derive from American and Canadian selections. The technology of apple production led to improved methods of storage in cool areas, first underground storage, later cooling with ice and, finally, refrigeration in modern times. Special methods to control diseases and insect pests, which have always been a problem, were sought but were largely unsuccessful until the 19th century when the fungicidal properties of lime sulfur was discovered and used to control such diseases as apple scab, and lead arsenate was used to control codling moth, a practice that is now illegal. The original apple processing consisted of drying; later juice was extracted and converted to a fermented product called cider, then distilled into a liquor (calvados or apple jack). Apples are now consumed fresh, or cooked, whole or as a sauce, and are especially popular in pastries. Genetic changes in apple include the selection of triploids derived from unreduced gametes, parthenocarpy, mutations involving growth habit, particularly spurry and short internode types, and various forms of pest and disease resistance. Fruit changes include shape, and various quality factors such as flesh firmness, sweetness, acidity, flavor, and shelf and storage life (Janick et. al. 1996). Triploidy seems to confer an advantage to cultivated apples, for about 10–20% of cultivars are triploid yet nonreduction occurs in only about one in a thousand seed. The ability of layered shoots to root is advantageous in rootstocks, which are propagated by mounding soil around the bases of shoots (stooling). Differences in tolerance to many pests are observed in any large collection of apples. Immunity to apple scab has been transferred from Malus floribunda by backcrossing, but the problem of races has also appeared in some areas. This places the durability of this gene (Vf ) in question, although it has held up for many years in the United States (Janick 2002b). Pear. The pear has a story similar to that of the apple except that the domestication of the pear followed two separate paths. Pyrus communis, native to Central Asia, gave rise to the European pears characterized by intense flavors, flesh softening after the climacteric, and a wide range in sizes and shapes. The European pear is described by Homer as one of the fruits in the garden of Alcinöus, and the Roman agricultural writers Cato, Varro, and especially Pliny describe many types (Hedrick 1921).

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In eastern Asia, the crisp, sweet-fleshed fruit of Pyrus pyrifolia, P. bretschneiderii, and P. ussuriensis are round, aromatic, and crispfleshed, and now known either as Asian pears or under the Japanese name nashi (Watkins 1995; Bell et al. 1996). The pear has been known in China for 4000 years (Wang 1990). Systematic breeding of European pear was first carried out by Jean Baptiste Van Mons (1765–1842), a Belgian physician, pharmacist, and physicist and an early apostle of selection in plants. He systematically collected clones of pear, planted (open pollinated) seed of the best material and made selections for eight generations. In an early fruit book, The American Fruit Culture (1863), John J. Thomas (1810–1895) stated that the mean time from seed planting to fruiting in the first cycle was 12 to 15 years, 10 to 12 in the second cycle, 8 to 10 by the third, 6 to 8 by the fourth, and 5 by the fifth. By the 8th generation several fruit trees fruited at the age of four years (emphasis by the author, presumably based on correspondence of Von Mons). This may be the first example of data on long-term selection in plants. Sexual hybridization to produce new pear cultivars (as well as apple, cherry, strawberry, red currant, plum, and nectarine) was first attempted by Thomas Andrew Knight (1759–1838). 2. Stone Fruits. The genus Prunus, Rosaceae, is the source of almond, apricot, cherry (sweet and tart), peach (and nectarine), and plum. With the exception of the almond, which is cultivated for its seed, all are softfleshed temperate fruits known for their delectable flavors. Prunus originated in Central Asia, with secondary centers in Eastern Asia, Europe, and North America (Watkins 1995). The introduction of apricot, cherry, and plum into the Mediterranean basin was associated with the incursion of Alexander the Great (356–323 BCE), who conquered Persia and then continued east through Turkistan, Afghanistan, Pakistan, and northwestern India up to the Indus river. Greek settlements and commercial posts were founded between the Mediterranean and India along the western section of the trade routes, which later were dubbed the Silk Road, and through it passed central Asian as well as east Asian fruits, The Roman names for the stone fruits suggest their presumed origin [peach (persica) from Persia, apricot (armeniaca) from Armenia, cherry (cerasus) from Kerasun on the Black Sea], but it is clear that these locations were clearly way stations from Central and East Asia. The peach originated in China and will be discussed in Section III C 1. Almond. Almonds (Prunus dulcis, Rosaceae, syn. P. amygdalus) grow wild throughout southwest and central Asia, from Turkey and Syria into the Caucasus and into the deserts of Tian-Shan and the Hindu Kush mountains (Zohary and Spiegel-Roy 1975). Based on biblical literature,

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the introduction of the almond may have occurred as early 2000 BCE, and some almond remains have been found in the 18th Dynasty in Egypt (1550 BCE). It does not seem to have been widely cultivated in Egypt until Roman times. It was brought into Greece about 200 to 300 BCE and gradually reached the entire Mediterranean basin. There are two principal types: sweet and bitter. The bitter kernels are due to high levels of the glucoside amygdalin which hydrolizes to benzaldehyde and cyanide when exposed to the enzyme emulsin (Kester and Gradziel 1996). This trait discourages seed predation by birds and rodents and limits its use for human consumption. (The predominantly benzaldehyde flavor of almond extract is derived from bitter almonds). The nonbitter or sweet almond flavor is controlled by a single dominant gene that occurs relatively frequently in wild populations. Domestication involved selection for sweetness and increased kernel size. Two thousand years of continuous culture in the Mediterranean basin concentrated almond into specific regions where well-defined land races evolved, and where almond culture became a low input dryland crop in semiarid areas. Almond types are further separated into hard and soft shell, with the soft-shell types predominating in California and Australia and the hard-shell types being grown primarily in Mediterranean countries. In Arabic countries, the immature fruit is often consumed after slight cooking. The almond has hermaphroditic flowers; self-pollination is avoided by gametic incompatibility but many new cultivars are self-fertile. The tree is easily grown from seed, and seed propagation has been widespread from the beginning of almond cultivation and still exists in Iran, Turkey, Afghanistan, Greece, Crete, and the Balearic Islands. Vegetative propagation is now principally by bud grafting. The introduction of almond cultivation into irrigation production systems has created a high-yielding commodity crop in large areas of California, which is now the major world almond production area. Apricot. The history of apricot (Prunus armeniaca) goes back 5000 years in China with the first attribution to the Emperor Yu (2205–2198 BCE), with other references in 658 BCE; and superior orchards were described in 406–250 BCE (Faust et al. 1998). Grafting of apricots began about 600 CE with defined cultivars developed after this time. There are at least 11 cities in China that contain xing (apricot) as part of their name. The closely related P. mume, Japanese apricot, noted for its early bloom, is especially revered in China for its gnarled branches, profuse early flowering, and fragrance. Painting of this species is a specialized art form. In Central Asia, the apricot appears to have become naturalized in Sogdiana (associated with fabled trading city of Samarkand) as well as

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Armenia, but the lack of wild apricots suggests that Armenia is merely the route through which apricots entered Europe. Alexander the Great brought the apricot to Greece and Epirus (Albania), from whence it reached Italy. Mention by Dioscorides and Columella and Pliny but not by Theophrastus, Cato and Varro, indicates an arrival into Rome by the 1st century CE. Armenia was attacked by Roman legions through Syria in 69–63 BCE, and this may explain its introduction to Rome. Throughout its history, apricot has been noted for delicious flavor, delicate velvety fruit surface, and early flowering and fruiting. The name apricot is a corruption of the Arabic and Greek al-praecox, which means the early fruit. The old spelling “apricock” is retained in English until the 17th century. The fruits are mainly consumed fresh but are also cooked and often stuffed. The high sugar content makes them suitable for drying. In China they were also preserved by salting and smoking. Other uses include fruit leathers, nectars, and liquors. Current breeding efforts are directed toward increasing fruit size, adaptability, hardiness, and processing quality (Layne et al. 1996). Cherry. Cherry consists of three main groups: the sweet cherry (Prunus avium, 2n=16); the tart (sour) cherry (P. cerasus, 2n=32); and the ground cherry (P. fruiticosa, 2n=32). Tart cherries are hybrids between sweet cherry and ground cherry via non-reduced gametes. Natural hybrids between non-reduced gametes of sweet with tart cherries are called duke cherries (2n=32). The origins of the three species overlap and include Central Europe and areas surrounding the Black Sea, with the sweet cherry as far east as central Russia (Fig. 8.17). There is archeological evidence of sweet cherry about 5000 to 4000 BCE in Switzerland, France, Italy, Hungary, Germany, and England. The first description of sweet cherry is by Theophrastus (ca 300 BCE), who named it kerasos, after the town Kerasun in ancient Pontus on the Black Sea, but the town may have been named after the fruit. By Roman times, cherries were a common fruit and are described by Pliny and Virgil, but generally as wild trees (Faust and Surányi 1997). Sweet cherries are usually consumed fresh, but are also used in cold soups, dried, or converted to maraschino cherries, which are often covered with chocolate. Tart cherries are canned or frozen, usually as a filling for pastries and pies, and made into jams and jellies. Dried tart cherries known as chaisins are gaining in popularity. The flowering cherry, P. × yedoensis, is particularly revered in Japan; the flowering cherry trees of Washington D.C. are a 1912 gift from Japan after earlier shipments were destroyed by insect infestations. The appearance of the cherry seems little changed from Roman times. There is diversity in fruit color (from yellow to red to black), flesh color,

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Fig. 8.17. Distribution of sweet cherry (striped area), sour cherry (solid line), and ground cherry (broken line). Source: Faust and Surányi 1997.

and season of ripening. The presence of pollen incompatibility groups makes it essential that this character be understood in laying out plantings. Recent advances have been made by breeding for self-compatibility originally induced by irradiation (Brown et al. 1996). Plum. The plums have the greatest diversity among Prunus species and have been domesticated on three continents (Okie and Weinberger 1996; Faust and Surányi 1999). They are the link between the major subgenera (Watkins 1995). Archeological finds of plums in Europe date to Neolithic times (Faust and Surányi 1999). While there are many species with edible fruit, the principal ones are P. domestica (European plum, 2n=48, hexaploid, native to Europe); P. domestica ssp. P. insititia (damson plum, 2n=48, hexaploid, native to Europe); P. salicina (2n=16, diploid, native to China); and P. americana (American plum, 2n=16, diploid, native to North America). The European plum may be an amphidiploid derived from P. cerasifera, 2n=16, and P. spinosa, 2n=32 (Crane and Lawrence 1956). The present plum industry is based on P. domestica and P. salicina. Neither of these species has wild progenitors and both entered into human use highly developed. The garden plum and the Japanese plum emerged as important fruit crops about 300 BCE. Horticulturally, there are many different types of plums with many different names, shapes, colors, and attributes (e.g., sloe, bullace, damson, green gage, mirabelle). Various plums are converted to juice, liquors,

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brandy (slivovitz), cognac, and cordials, and widely used for baking and confection. Plum trees are especially revered in China for their gnarled branches, profuse early flowering, and fragrance, and the painting of plum trees is a specialized art form. The dried sweet European plums are known as prunes or prune plums. The dried black fruit can be stored almost indefinitely, and is sold partially rehydrated for use as filling for pastries, and for juice. Because of its role in digestive regularity, mention of the word prune has become a source of hilarity in the United States, so much so that the California trade association has changed its name from prune to dried plum. Interspecific hybridization of plums with other Prunus with the same chromosome number are producing a number of new fruits such as plumcot (plum × apricot). C. Chinese and Southeast Asian Fruits 1. Peach. China is the origin of the peach (Prunus persica, Rosaceae), domesticated before 3300–2500 BCE (Faust and Timon 1995). Peach culture dates to 2000 BCE and there are now thousands of cultivars in China (Wang 1985). The peach is mentioned in Chinese literature as early as 1000 BCE and became entwined in Chinese mythology and folklore. A number of forms were selected in different parts of China. In the north, the peach tree is characterized by high chilling requirements, long internodes, single flower buds, upright branching, large flat leaves, and largely cling- or semi-clingstone fruit. In the south, a warm area with mild winters, trees are characterized by more lateral branching and require less chilling; this is the area of peen-tao (a flat fruit now marketed as the donut peach) and the ‘Honey’ type peach, an elongated-pointed (beaked) fruit with a deep suture near the base. In the high mountain area small-fruited peaches are found, including those with smooth seed (P. mira). The peach tree is largely self-pollinated (70 to 85%) so that propagation by seed often provides less variability than in cross-pollinated species. According to Pliny the peach was grown in Greece by 332 BCE. They were probably introduced into Persia from China by seed in the 2nd or 1st century BCE; its name in the West, persica, a misnomer, is based on the incorrect assumption that it originated in Persia. A painting from Herculaneum, near Naples, Italy, destroyed by the eruption of Mount Vesuvius in 79 CE, shows large green free-stone peaches with yellowish flesh (Fig. 8.18); large peaches persisted in Italy as depicted in Renaissance paintings by Campi (1580) and Caravaggio (1590s). The peach was introduced by the Spanish to America soon after the conquests of Cortez and became naturalized in Mexico and the southeastern United States and a number of selections were subsequently made. Until recently, naturalized wild peaches called “Tennessee nat-

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Fig. 8.18.

295

Roman painting of peach from Pompeii, before 79 CE. Source: Jashemski 1979.

urals” were widely used as rootstocks. In addition, soft and whitefleshed English selections were introduced. The peach is unadapted to England and was grown as a novelty or a conservatory plant. In 1850, an introduction from China via England called ‘Chinese Cling’ (‘Shanghai’), adapted from the southern group of Chinese cultivars, was to completely change the peach industry. This introduction proved to be well adapted to warm, moist summer conditions. Open-pollinated progeny (‘Chinese Cling’ was pollen sterile) included ‘Belle of Georgia’ and ‘Elberta’. ‘Elberta’ is large and highly flavored, and firm enough to withstand shipping, in contrast with the naturalized North American seedlings. Descendants of this material now forms the basis of the modern United States and European peach industry (Scorza and Okie 1990). Extensive breeding activity has occurred in the United States, with California breeders concentrating on rubbery-fleshed cultivars for processing and both east and west coast breeders emphasizing a succession of freestone cultivars for fresh market (Scorza and Sherman 1996). As a result, various types have been selected that include a complete range of maturity, round to flat fruit shape, yellow to red fruit color, freestone and clingstone, white, yellow and red flesh, and pubescent and nonpubescent. The “fuzzless” peach, known as nectarine, which resembles a cross between a plum and a peach, is the result of a mutation that

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occurs commonly in peach, probably a deletion, because back-mutation from nectarine to peach is unknown. Extensive efforts for extending the adaptability of peach have been carried out by breeding for low chilling requirement and cold hardiness. One of the problems with peach adaptation to cold climates is that freeze avoidance is due to deep supercooling which limits cold hardiness (Layne 1992). In peach, temperatures between –20° and –25°C kill flowers, and temperatures below –30° damage the wood. Breeding for low chilling requirement has extended the range to subtropical areas. Soil-borne problems have become increasingly important and nematode-resistant rootstocks have been selected. Recently, size-controlling rootstocks have been sought. There are a number of mutations for different growth types from dwarf to weeping that may be useful (Fideghelli 2003). 2. Citrus. Rutaceae consists of six genera of which three, Citrus, Poncirus (trifoliate orange), and Fortunella (kumquat), have commercial value. The genus Citrus includes a diverse group of evergreen Old World fruits originating in southeast Asia (eastern India, Burma, and southern China) and is now grown throughout the tropics and subtropics worldwide (Soost and Roose 1996). The principal horticultural groups include citron (C. medica), lemon (C. limon), lime (C. aurantifolia), pummelo (C. grandis), sour orange (C. aurantium), sweet orange (C. sinensis), mandarin (C. reticulata), and grapefruit (C. paradisi). These “species” designations are a convenient if inaccurate way to consider this group. Domestication of all but grapefruit occurred before 1000 BCE. The taxonomy of citrus is controversial and reflects philosophical differences in defining relationships in an ancient group of cultivated plants, complicated by outcrossing, apomixis, and selection (Roose et al. 1995). Thus, Toshio Tanaka proposed 162 species; Walter T. Swingle recognized 16 species, while R. W. Scora, based on biochemical analysis has concluded three primary species: C. medica (citron), C. reticulata (mandarin), and C. grandis (pummelo). Molecular evidence supports the hybrid origin of many so-called species. The Japanese botanist, Tyozaburo Tanaka, has proposed a line of demarcation (now known as the Tanaka Line) from the northeast border of India to the island of Hainan that separates the development of Citrus species and close relatives. The lemon, lime, citron, and pummelo, sweet and sour orange occur south of this line while most mandarins as well as Poncirus and Fortunella occur north of this line. Most citrus is cross-pollinated (pummelo is cleistogamous) and many are characterized by apomixes (associated with nucellar polyembrony), permitting cloning to be carried out by seed propagation.

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Sexual hybridization between cultivated species has led to many new types of citrus, as indicated by their names. These include tangelos (tangerine × grapefruit), tangor (tangerine × orange), orangelo (orange × grapefruit), citradia (Poncirus × sour orange), and citrangequat (citrange × kumquat). Interspecific hybridization is the mechanism for the origin of the grapefruit (forbidden fruit) that first appeared in Barbados in the 18th century. It is derived from a natural cross of the non-apomictic pummelo (called a Shaddock in Barbados) with the apomictic sweet orange (Gmitter 1995). The grapefruit inherited the apomictic character from the sweet orange and can be proliferated by seed propagation. All grapefruit seem to be a single clone; subsequent changes, such as seedlessness and pink flesh and fruit, have occurred via selection of somatic mutations. The first citrus to reach the West was the citron, whose name, C. medica, suggests Media (Persia now Iran) as the source. According to De Candolle, citron is found in the Himalayas in India and was reported in Iran in 300 BCE. It may have reached the West before this date and Goor and Nurock (1968) suggest it was referred to in Nippur in South Babylon as early as 4000 BCE, but this is not confirmed by Postgate (1987). It is not mentioned in Egyptian writings until the 2nd century CE. Biblical references to the “fruit of the goodly tree” considered the etrog (the Hebrew name of the citron in Leviticus (23:40) would date it to about 1200 BCE. It soon became a sacred tree to Jews and the citron (with the style attached) is still offered as gifts at Succoth (the Feast of Tabernacles). The citron became associated with winter by the Romans, and citrons are found in mosaics celebrating the seasons (D. Parrish, pers. commun.). The baroque painter Bimbi (1648–1729) included various citrons in his paintings of fruit cultivars (Consiglio Naazionale delle Ricerche 1982). The sweet and sour orange reached Europe in the 11th century via the Arabs. The Arabic name haranj, Persian harang, and Spanish Naranja is the source of the name for the color we know as orange. The lemon and pumello were introduced to Europe in the 12th century and the lime in the 13th. The greatest wave of citrus into Europe was in the 15th century from merchants arriving from southeast Asia overland. A second wave occurred in the 16th century by Portuguese sailors who introduced citrus of higher commercial quality. The development of protected culture (orangeries) in the European Renaissance to overwinter citrus trees initiated a vogue for the nobility and the wealthy, who enjoyed the fragrant flower and beautiful fruit, to produce citrus in containers. Mandarins, which originated in Vietnam and China, were important fruits in China and Japan in the 12th century but did not reach Europe until 1805 (Roose et al. 1995).

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Although propagation from polyembronic (nucellar) citrus seed is true-to-type, the long juvenile period, as well as juvenile characters such as thorniness and thick albedo, make propagation by grafting desirable. Various citrus have been used as rootstocks. However, because seedlings are virus-free, nucellar clones have been used to eliminate viruses from citrus. Recently meristem grafting, combined with thermotherapy, has been used to produce virus-free clones. Selection in citrus is associated with changes in size, shape, color of flesh and peel, acidity, ease of peeling (mostly from mandarins), and seedlessness. Seedlessness may be induced by x-rays. In the United States, the popularity of orange juice has created a tremendous industry for concentrate, and so-called “fresh” chilled juice. Much of citrus processing has moved to Brazil where the climate around Sao Paulo is very suitable for citrus. Easy-peel citrus is desirable for consumer use and represents the future of the fresh fruit industry. 3. Banana and Plantain. Bananas and plantains (Musa species, Musaceae) are indigenous to southeast Asia and the Pacific (Simmonds 1995). These are large, herbaceous, perennial plants with a pseudostem consisting of tightly whirled leaf sheaths through which the inflorescence emerges. Bananas are the name for fresh fruit types, while plantains refer to cooking types. They are propagated vegetatively by corms (the true stem) through offshoots. The early evolution and migration of the banana is unknown but is ancient. Banana is referred to by Theophrastus and certainly originated thousands of years earlier. Bananas reached west Africa before European contact, and a few clones from there reached the New World in the late 15th century. They were widespread in the tropics by the end of the 16th century. The wild bananas are seedy and diploid. The cultivated bananas and plantains exhibit a combination of parthenocarpy, sterility, and polyploidy, principally triploidy. Parthenocarpy results from complementary dominant genes found in wild species of M. acuminata. Parthenocarpic triploids arose from unreduced gametes (2n+n) and are seedless. These types must have been immediately selected and multiplied by offshoots. Hybrids between diploid M. acuminata (AA genomes) and M. bulbisiana (BB genomes) produced diploid AB hybrids and AAB and ABB triploids, the plantains and cooking bananas. These are assumed to have originated by migration of the edible diploid, male-fertile AA types into area of M. balbisiana (BB) followed by nonreduction to produce triploidy. Some tetraploid hybrids (AAAB, AABB, and ABBB) also exist. Some AAA triploid types (known as the Cavendish group) include the main bananas of commerce. Selection for somatic mutations has resulted in a number of clones but they are very similar and the banana indus-

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try is threatened by the perils of monoclonal culture. Breeding efforts have begun but are beset by the difficulties of working with a seedless crop. Efforts are being made to produce new AAA triploids from AA × AAAA crosses and breeding efforts are underway for the cooking types taking advantage of unreduced gametes (Ortiz 2003). 4. Mango. Important crops of the Anarcardiaceae include the mango (Mangifera indica) and pistachio (Pistacia vera) native to East Asia, and the cashew (Anacardium accidentale) native to tropical Brazil. The mango, the most popular fruit in India, is now the fourth most important fruit crop in the world. In 2001 over 25.1 million tonnes of mango were produced from about 90 countries, with about 77% produced in Asia (Galán Saúco 2003). The mango originated in the Indo-Burma region and grows wild in the forest of northeast India and is an allopolyploid between M. indica and M. sylvatica. Known for 4 to 6 thousands years, the mango spread through the Indian subcontinent, and reached Malaya in the about the 5th century BCE (Purseglove 1968), East Africa in the 10th century, and the New World in the beginning of the 19th century. It was successfully introduced to Florida in 1861. Mango is consumed fresh, dried, or processed and is widely used in chutneys. To mango enthusiasts, the mango is a fruit that the peach only aspires to become. Nucellar polyembryony is widespread in the mango, allowing vegetative propagation of some types from seed. Two major groups of mango are recognized, the Indian race (monoembryonic) and the Philippine race (polyembryonic). Diversity in mango is very high for fruit size, skin color, and flesh quality that ranges from fibrous to smooth with flavor from turpentine-like to mild. Many generations of selection have produced cultivars free of seed fibers without the turpentine flavor. A number of attractively colored cultivars selected in Florida (Tommy Atkins, Kent, Keitt, Haden, Van Dyke, Sensation Palmer, and Parvin) mainly derived from Indian clones such as ‘Mulgoba’ predominates in the export market. 5. Persimmon. The genus Diospyros (literally “fruit of god”), Ebenaceae (x=15) consists of about 400 species widely distributed in the tropics of Asia, Africa, and the Americas. The temperate species, D. kaki, (2n=90) known as kaki or Japanese persimmon, originated in China. It is a beloved fruit in China, Korea, and Japan and has been known from prehistoric times but there is some production in the United States (California), Italy, Israel, Brazil, Australia and New Zealand (Yonemori et al. 2000). The American species D. virginiana, (2n=60, 90) known as persimmon, a name of the Algonquin Indians of Virginia, is a backyard fruit with a number of local selections, but there is essentially no industry, and it is debatable if the fruit can be considered domesticated.

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Kaki is an attractive fruit with yellow, orange, to deep red skin color, while the intense red color of the autumn leaves makes it an attractive ornamental. The species is considered polygamo-dioecious with three types of sex expression: pistillate, monoecious, and polygamo-monoecious (hermaphroditic, pistillate, and staminate flowers) suggesting selection away from dioecy during the domestication process. There are presently over a 1000 known cultivars with distinct types resulting from mutations involving parthenocarpy and astringency. Parthenocarpy is an important factor controlling productivity because fruit drop in seeded types is a major problem; the higher the parthenocarpy the less the early fruit drop. Parthenocarpy also eliminates the problem of providing pollenizors for pistillate cultivars. Seedless fruits are preferred by the consumer. The developing fruit are extremely astringent due to soluble tannins in the vacuoles. Non-astringent (NA) cultivars lose astringency naturally during fruit development, whereas astringent cultivars (A) retain astringency until maturity. Astringency can be reduced by ethylene application. There is a further classification of cultivars based on the effect of seed on flesh color: pollination variant (PV) and pollination constant (PC). The flesh of PV types becomes dark around the seed, whereas the flesh color of PC types is uninfluenced by seed. The four types of fruit (PCNA, PVNA and PCA and PVA are categorized in Table 8.3. The goal of modern fruit breeding is to obtain parthenocarpic PCNA offspring. Breeding efforts are now facilitated by molecular markers. 6. Kiwifruit. The kiwifruit (Actinidia deliciosa, Actinidiaceae, 2n=174, x=29), a dioecious vine, is an example of an ancient fruit species domesticated in the 20th century (Ferguson and Bollard 1990). It derives from a gathered fruit known as mihautao (monkey peach), which had long been appreciated in China (Fig. 8.19) but was collected rather than cultivated (Ferguson 1990). Introduced to the United States and New Zealand early in the 20th century by the plant explorer E. H. (Chinese) Wilson, it was referred to as Chinese gooseberry in the United States. Although it remained a curiosity in the United States, New Zealand growers and nurserymen succeeded in domesticating the crop by selecting suitable male and female clones, as well as developing techniques for cultivation. One seedling selected by A. Hayward Wright and subsequently named ‘Hayward’, became the mainstay of the world industry. The fruit was exported to the United States where it was promoted by Frieda Caplan, a marketer of new crops. In 1959 the relatively unattractive brown fruit received the new name kiwifruit after the kiwi, an endemic flightless bird often used as a nickname for New Zealanders. Kiwifruit has a pleasant but weak flavor with very high Vitamin C

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Table 8.3. Horticultural classification of persimmon cultivars by astringency and flesh color of fruit. Source: Yonemori et al. 2000. Seed effect

NA (Non-astringent)

A (Astringent)

PC (Pollination constant)

PCNA Non-astringent at maturity whether seeded or not. Flesh color unaffected by seed at maturity. Tannins are not coagulated by ethanol treatment at immature stages when fruit is still astringent. (VIGz)

PCA Astringent at maturity unless treated. Flesh color unaffected by seed at maturity. Tannins are coagulated by ethanol treatment even at immature stages. (VDGy)

PV (Pollination

PVNA Non-astringent at maturity only if seeded. Flesh turns brown at maturity if seeded. Tannins are coagulated by ethanol treatment even at immature stages. (VDG)

PVA Astringent at maturity unless treated. Brown flesh color only around seed at maturity. Tannins are coagulated by ethanol treatment even at immature stages. (VDG)

variant)

z y

VIG; Volatile-independent group. VDG; Volatile-dependent group.

Fig. 8.19. Fruit of mihautao (kiwifruit) taken by W. H. Wilson in western Szechuan, China in 1908. Source: Ferguson and Bollard 1990.

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content, but the nutritious quality of the fruit has not been promoted; rather, it was the beautiful and unique appearance of the sliced flesh, which is used as a garnish on bakery products or as a component of mixed fruit, made this fruit popular worldwide. The long storage life of the fruit made it possible for New Zealand to export the fruit year-round. The popularity of the crop made millionaires of many New Zealand growers, but as kiwifruit began to be grown in such countries as the United States, Italy, and Chile, the boom crashed and New Zealand growers had to struggle to survive. Kiwifruit is consumed out of hand in New Zealand, usually scooped with a spoon, but this technique has not caught on, and further expansion is probably linked to development of a simple method for peeling. A yellow-fleshed kiwifruit marketed as Zespri™ Gold (A. chinesis, 2n=58, a tetraploid) was recently introduced, and the New Zealand growers are attempting to control its distribution. It is too early to know if this will succeed. A small-fruited, hardy and fuzzless species (A. aguta, 2n=96, 114), sometimes called tara fig, is now cultivated in gardens, but has not been commercialized. D. American Fruits 1. Strawberry. Strawberry is the most widely grown of the berry fruits, sometimes called small fruits based on plant size (Darrow 1966). Strawberries are herb-like perennial plants, but the compressed stem may become woody. The plant propagates naturally from runners or from crown divisions. Strawberry has an interesting history. Fragaria vesca, Rosaceae known as the wood strawberry, a diploid species (2n=16) with bisexual flowers, has small, aromatic fruit and is found in Europe, northern Asia, northern Africa, and North America. It was mentioned by Pliny in the 1st century and in the 14th century white- and red-fruited selections and, later, everbearing types (F. silvestris) were cultivated in Europe, both as a fruit crop and as an ornamental, little changed from the wild. It is still being grown in Europe as a gourmet fruit. Later, a tetraploid species, F. moschata (2n=32) known as the hautbois, was briefly grown in England. The modern strawberry is derived from hybrids between two octoploid (2n=56) native American species, both usually dioecious: F. virginiana, indigenous to the East coast of North America but reaching Europe in the 17th century, and F. chiloensis, native from Alaska to Chile. Hybrids between these two species were produced naturally in Brest, France early in the 18th century when a pistillate clone of the large-fruited F. chiloensis, introduced by Amédee François Frézier, a French army officer (and spy) whose family name curiously derives from the French word (fraise) for strawberry, was inter-planted with sta-

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minate plants of F. virginiana. The new hybrids (now known as Fragaria × ananassa or pineapple strawberry, after their shape and aromatic flavor) initiated the modern strawberry industry. Through the years selection has resulted in tremendous changes as the plant evolved from a predominantly dioecious species into a hermaphroditic species, in which flowers contain both stamens and pistils. As a result of extensive breeding in the 20th century (Hancock et al. 1996) fruit size has been greatly increased as well as fruit firmness (too firm for some), and resistance to a number of leaf diseases. Flowering and runnering are both affected by photoperiod but adaptation has been increased with the introgression of the day-neutral character from F. virginiana. Although strawberry is widely adapted, the strawberry industry is now concentrated in California in the United States, in Spain, and in Italy. Some strawberries are now grown in greenhouses. 2. Brambles (Rubus). The genus Rubus, Rosaceae (x=7), is very diverse and many species throughout the world are harvested from the wild. The cultivated Rubus species, known collectively as brambles, includes red raspberry (R. idaeus), black raspberry (R. occidentalis) both diploids, 2n=14, and polyploid blackberries (many species of Rubus) and, now, many interspecific hybrids between raspberry and blackberry, such as loganberry, and tayberry (2n=42), and boysenberry (2n=49) (Jennings 1995; Daubeny 1996). Rubus is divided into sections: Idaeobatus and Eubatus. Idaeobatus (raspberries) is often divided into two subspecies, R. idaeus vulgatus, the red raspberry of northern Europe and Asia, and R. idaeus striosus, the red raspberry of North America. Eubatus (blackberries) is native to the New World. Brambles have delicious flavors but marketing has been a problem because of the soft texture of the fruit. The first records of the European raspberry date to antiquity and Pliny writes of wild raspberries as coming from Mt. Ida. The European raspberry has been cultivated since medieval times but there is little evidence of distinct types. During the 19th century, crosses between European raspberry (subspecies vulgatus) and North American types (subspecies strigosus), made in both continents, gave rise to present-day cultivars, and recent breeding efforts have introduced new genes from a number of Rubus species. The black raspberry (R. occidentalis), indigenous to eastern North America, was first cultivated in the early 19th century, and subsequently purple types were derived from hybrids between black and red raspberry. The blackberry has a complex origin and includes wild species from diploid (2n=14) to dodecaploid (2n=84). Trailing blackberries are selections from R. ursinus. Many other local species are cultivated, including dewberries (R. trivialis) in the southern United States, R. parvifolius in

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China and Japan, and R. phoenicolasius in Japan. Attempts at domestication were first reported in Europe in the late 17th century with R. laciniatus. In eastern North America, selection from natural stands of interspecific crosses contributed to domestication. The main problem was excessive thorniness. A spine-free variant of the octoploid ‘Austin Mayes’ called ‘Austin Thornless’, served as a dominant source of spinelessness. Later recessive sources of spinelessness were obtained. Selection among various Rubus has concentrated on thornlessness, plant habit, fruit quality, fruit size, disease and pest resistance, adaptability to machine harvest, chilling requirements, and postharvest life (Daubeny 1996). 3. Vacciniums. Species of Vaccinium, Ericaceae (x=12), include cultivated blueberries and cranberry, both domesticated in the 20th century. The lingonberry is widely gathered but can hardly be considered domesticated despite recent selections of elite types; cultivation consists almost entirely of managing natural stands. A number of Vaccinium species such as bilberry (V. myrtillus) and bog bilberry (V. uliginosum) are potential domesticates (Galletta and Ballington 1996). Blueberry. There are five major classes of blueberry grown commercially: lowbush, highbush, halfhigh, southern highbush (low chill), and rabbiteye. In lowbush (tetraploid V. angustifolium, diploid V. myrtilloides, diploid V. boreale), improvement has originated from selection of wild clones (Galletta and Ballington 1996). There is still an industry from natural stands of lowbush blueberry in Maine and commercial lowbush production is still more than 95% from unselected wild plants. Highbush blueberries are derived from tetraploid genotypes of V. corymbosum, although some have V. angustifolium in their pedigree. These types were developed by F. V. Coville, USDA botanist from 1888 to 1937, and subsequent breeders. Halfhigh blueberries are derivatives of lowbush-highbush hybrids, usually V. angustifolium × V. corymbosum, but breeding in Finland and Poland has emphasized hybridization between V. corymbosum and tetraploid genotypes of V. uliginosum to improve cold hardiness and tolerance to Fusicoccum canker. Southern highbush blueberries are similar to highbush types but have a lowchilling requirement as a result of crosses with V. darrowi and other species. Finally, rabbiteye blueberries, highly vigorous plants adapted to the southern United States, belong to the highly polymorphic hexaploid V. ashei. Recent progress in breeding has resulted in increased fruit size, productivity, and adaptability to mechanical harvest. Cranberry. Cranberries are usually divided into two species, the circumboreal V. oxycoccus, and the large-fruited V. macrocarpon, native to

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eastern North America. In the 18th and 19th centuries the large-fruited cranberry was gathered from natural bogs, but substantial plantings had been made by 1850 in New Jersey, Massachusetts, and Wisconsin, and the technology of wetland cultivation was developed (Dale et al. 1994). Domestication of V. macrocarpon, which is adapted to lowland acidic soils, has been achieved by selection from elite wild plantings and subsequent hybridization (Galletta and Ballington 1996). Most cranberry cultivars originated as single-plant selections of native bog populations, and four of them still accounted for 90% of the total American production area in 1990. Although cranberry was a traditional holiday fruit consumed at Thanksgiving feasts in the United States, expansion of the industry has occurred with innovative processing involving freezing, juice production—particularly mixtures with other juices—and as a dried fruit. Cranberry has also been considered a health food and the juice is recommended for urinary tract health in women. Lingonberry. Vaccinium vitis-idaea is a circumboreal, woody, dwarf to low-growing, rhizomatous, evergreen shrub. The plant is indigenous to Scandinavia and has been harvested from natural stands since the Bronze Age, based on remnants of lingonberry wine in Danish graves (Hjalmarsson and Ortiz 2001). Icelandic law of the 13th century limited berry picking in other people’s land to that consumed on the spot, indicating its importance as human food. Although elite selections have been made, there is essentially no industry aside from the management of wild stands. Lingonberry has a long history of commerce and 20 million kg of fruit were exported from Sweden, typically to Germany in 1902. Lingonberry preserves, once a traditional delicacy in Scandinavia, are now considered a luxury food. 4. Pineapple. The pineapple (Ananas comosus, Bromeliaceae) was domesticated in pre-Columbian tropical South America, probably north of the Amazon River with a possible secondary center in southeast Brazil (Leal 1995; Leal and Cioppens d’Eeckenbrugge 1996). Selection was based on fruit size, seedlessness and parthenocarpy, long, fibrous smooth leaves and ease of vegetative propagation. In fact, the pineapple can be propagated in a number of ways (stumps, offshoots, slips, and the fruit crown). The pineapple plant is very tough and desiccation resistant and wide distribution occurred in the Americas though human migration and exchange. It was present in all adapted areas of the New World at the time of the European encounter with America (Fig. 8.20). The early description by Pigafetta in 1519 is exuberant: “this fruit resembles a pine cone and is extremely sweet and savoury; in fact it is the most exquisite fruit in existence.”

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Fig. 8.20. The first description of pineapple in 1535 shows a fruit similar to present-day cultivars.

The pineapple has changed little from that era except that genetic variability has been greatly reduced. There are about 6 cultivar groups (Spanish, Cayenne, Queen, Pernambuco, and Maiopure), but only two ‘Cayenne’ and ‘Queen’ were commercially important for most of the 20th century despite the fact that the pineapple became associated with a tremendous processing industry. The ‘Cayenne’ cultivar has been the mainstay of the processing industry because of its robust, large, romboid fruit, which produces many slices, the most valued product. Despite a number of industry-sponsored breeding programs, it has not really been displaced. Continual selection of somatic mutants is important, espe-

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cially for less spiny types (‘Smooth Cayenne’), and for improved fruit shape and quality. ‘Cayenne’ is not highly valued for fresh fruit, but recently a new cultivar, derived from sexual hybridization at the Pineapple Research Institute, now defunct, has become very popular for fresh fruit and is being marketed as ‘Del Monte Gold’. 5. Avocado. Persea is one of 50 mainly tropical genera of the Lauraceae, most of them from the New World. The early history of avocado (P. americana) is unknown but it seems to have originated from southern Mexico to present-day Panama (Bergh and Lahav 1966). There are remnants of seed from 7000 BCE with evidence of selection after 2000 BCE based on seed size (Bergh 1995). The Spanish conquistadores found the avocado (from the Aztec ahucalte) cultivated from northern Mexico to Peru. The avocado are one of the oily fruits, consumed either fresh as part of a salad, or, in Brazil, whipped into a creamy dessert, and also used as a primitive soap. Three horticultural races or subspecies (ecotypes) based on their presumed area of origin are recognized (Mexican, Guatemalan, and West Indian) but it is now clear that the West Indian race is a lowland type originating in the western coast of Central America. In general, the Mexican and Guatemalan types are considered subtropical and native to highlands, while the West Indian are adapted to tropical lowlands. The largest fruit are found in the Guatemalan types, but oil content is highest in Mexican race. The avocado has perfect flowers but cross-pollination is promoted by protogynous dichogamy with synchronous complementarity. Type A types are functionally pistillate in the morning and functionally staminate the following afternoon, while B types are pistillate in the afternoon and staminate the following morning. Thus, both A and B types are required for successful pollination. Most of the current cultivars are based on selection for high oil as well as relatively small seed size, high productivity, and disease resistance. ‘Fuerte’, the most important present-day cultivar, was selected in 1911, from seedlings by the Rodiles family near Atlixco, Mexico. Breeding efforts are hampered by long juvenility, sometimes as long as 15 years. ‘Haas’, originating as a chance seedling in California, is increasing in importance. 6. Papaya. Carica, Euphorbiacia, a genus with about 40 species is indigenous to tropical America (Purseglove 1968). Papaya (Carica papaya), derived from natural hybridization involving C. peltata, is a frost-tender, non-woody plant reaching a height of 8 m. It is widely grown as a dooryard plant throughout the tropics for the melon-sized fruit usually

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consumed for breakfast. The proteolytic enzyme papain has been exploited as a meat tenderizer and has been used for some surgical procedures. The tree is commonly dioecious, although there are types that are considered polygamo-dioecious with both hermaphroditic and pistillate plants. In these types (e.g., ‘Solo’, the popular Hawaiian cultivar with fruit averaging about 600 g) fruit shape of the hermaphroditic types is pyriform (the preferred type) while the fruits from pistillate trees are round. The tree has a short juvenile phase but is short lived. Papaya is one of the few fruits that are propagated exclusively by seed. Fruit size varies from small to very large, up to 9 kg. The fruit, yellow when mature, has flesh color from orange to red. Recently the inbred ‘Solo’ papaya has increased in popularity for export to the United States and Japan and is now widely grown in Brazil, although large-fruited Mexican types are also exported to the United States. The papaya has serious virus susceptibility and cannot be grown in Florida for this reason. Recently, resistance to the tomato mosaic virus has been introduced by transgene technology.

IV. GENETIC CHANGES AND CULTURAL FACTORS IN DOMESTICATION The genetic changes associated with domestication in fruit crops is presented in Table 8.4. Despite some well-known exceptions, fruit crops are characterized by a number of common features. Noteworthy is the obvious appeal of taste—often a combination of sweetness and acidity with many considered delicious because of aromatic constituents. The appealing, sweet taste of many fruit is probably a trait associated with natural selection for seed dispersal mediated by mammals. Most fruit crops are highly cross-pollinated, and tree fruits generally have long juvenility and long-life. Some fruit crops have the ability to propagate vegetatively. Subsequent progress in the improvement of fruit cultivars resulted from continual selection of seedling populations, and from intercrosses among elite clones or with wild or introduced clones, that vastly speeded up the process. This process has been very efficient and in spite of progress in plant breeding, replacing grower-selected clones has not been easy (Table 8.5). A review of the fruit crops discussed above indicates that the origins of fruit growing evolved from an interaction of genetic changes and cultivation technology, often unique for each species. Some idea of how this has occurred can best be inferred from the history of two recent domesticates: cranberry and kiwifruit. What occurred in these crops probably occurred in the past with others, although each crop is unique with its own set of problems and prospects, and each has its own story. Both cranberry and

8. THE ORIGINS OF FRUITS, FRUIT GROWING, AND FRUIT BREEDING Table 8.4.

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Genetic changes associated with domestication in fruit crops.

Breakdown of dioecy carob, fig, grape, papaya, strawberry (unchanged, date palm, kiwifruit) Loss of self-incompatibility cherry Parthenocarpy & seedlessness apple & pear, banana & plantain, citrus, fig, grape, persimmon, pineapple Allopolyploidy banana & plantain, blackberry & raspberry, blueberry, citrus, tart cherry, European plum, strawberry Polyploidy Triploidy: Tetraploid: Hexaploid: Octaploid:

banana and plantain, apple, pear tart cherry, raspberry, blackberry, blueberry, kiwifruit (Actinidia sinensis) European plums, kiwifruit (A. deliciosa) strawberry

Loss of toxic substances “Sweet” seed: almond Nonastringency: apple & pear, persimmon, pomegranate Ease of vegetative propagation Offshoots: date palm Rooting: apple (rootstock) Nucellar embryony: citrus, mango Loss of spines, thorns, or pubescence apple, brambles, citrus, peach, pear, pineapple

kiwifruit were widely appreciated and entered commerce from wild stands long before domestication. The cranberry had been collected in North America since colonial America, but only became cultivated in the 19th century. Successful cultivation involved developing a series of practices to grow a plant adapted to aquatic conditions. Cranberry cultivation Table 8.5.

Effects of organized fruit breeding on the commercial world industry.

Negligible

Slight

Moderate

Major

Banana & plantain Chestnut Date palm Fig Grape (wine) Lingonberry Olive Pomegranate

Cranberry Cherry (tart) Citrus Hazelnut Papaya Persimmon Pear European Pineapple

Almond Apple Apricot Avocado Pear Asian Pecan

Blueberry Brambles (raspberry & blackberry) Cherry (sweet) Currants Grape (table) Strawberry Peach & nectarine Plum

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has recently been adopted in Chile. The kiwifruit, a dioecious vine native to China but never cultivated there, has been appreciated since the 8th century in China and probably much earlier. It was introduced to England and North America in the beginning of the 20th century, but New Zealand claims the honor of domestication. While the plant was introduced to England and the United States, the plant languished there, emphasizing the key role of champions (see IV D). A cultivation system worked out by New Zealand nurserymen and growers involved training and pruning on a trellis, with provision for pollination. The preferred pistillate and staminate clones (‘Hayward’ and ‘Bruno’, respectively) were selected from seed introduced into New Zealand from China. After the germplasm was selected, cultivation techniques established, and markets developed, the technology was quickly transferred and kiwifruit became a world fruit crop in less than 25 years. In both cranberry and kiwifruit, the early elite selections of wild plants were of high quality and could be vegetatively propagated—by cuttings in the case of cranberry and grafting in the case of kiwifruit. Selection, combined with the ability to fix unique combinations by vegetative propagation, was the key breeding technique in these two crops, as in all fruit crops. Breeding work has continued but even after 100 years, the selections made very early still dominate the industry. In both cranberry and kiwifruit, related species are under consideration as potential new crops. In kiwifruit, the related yellow-fleshed Actinidia chinensis has been introduced, and the small-fruited, hardy A. arguta (also known as tara fig) is under consideration as a new domesticate and now widely planted in northern home gardens. In the vacciniums, two related crops—blueberry (especially lowbush types in Maine) and lingonberry—were also widely appreciated and harvested from the wild, but with remarkably different outcomes. Blueberry had more promise as a commercial fruit than did cranberry or lingonberry because the fruit could be consumed fresh as well as processed and there was greater diversity in a number of species. While the domesticates of cranberry and kiwifruit are little changed from their wild forms, the blueberry has undergone remarkable transformation due to interspecific hybridization and ploidy manipulation. The culture of blueberry was dependent on the understanding that the vacciniums are an acid-loving species and required the ammonium form of nitrogen. Intensive selection and breeding with various species of different ploidy levels transformed this crop into a relatively large industry of wide adaptation. Lingonberry, on the other hand, a large Scandinavia export crop from forest-collection, never became domesticated, probably because there was no shortage of collectable fruit. This crop is still based on merely managed wild plantings.

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A. Mutation as an Agent of Domestication Many domesticated fruit crops differ from their wild progenitors by a few characters that have appeared as mutations (Table 8.4). Typically these mutations are not advantageous to the plant in its natural setting as they reduce fitness, but would clearly have been immediately selected by humans. The changes from bitter to sweet seed in almond and seeded to seedless fruits along with parthenocarpy (banana and plantain, citrus, fig, grape, persimmon, and pineapple) would have negative fitness but very positive selective value. Parthenocarpy has two advantages: it eliminates the need for pollination, and is one path to seedlessness that has proved important in grape, banana, and citrus. In dioecious fruit and nut crops, mutations inducing hermaphrodism [carob (Zohary 2002), grape, papaya, and strawberry] are associated with domestication. Other mutations associated with domestication include loss of spininess (brambles, pineapple, pome fruits, and citrus), loss of fruit pubescence (peach), and changes in growth habit mutations (pome and stone fruits). In many fruit crops, fruit color mutations (sports) have become increasingly important, especially in apple, pear, and grapefruit. Many of these mutations are not heritable because they do not occur in the appropriate meristematic layer. B. Interspecific Hybridization and Polyploidization Many of our fruit crops have resulted from interspecific hybridization, polyploidy, or both (Table 8.4). This is particularly obvious in Actinidia, Citrus, Fragaria, Musa, Prunus, Rubus, and Vaccinium. The evolutionary divergence within these genera into different “species” is often associated with polyploidy. These changes represent the divergence of interbreeding populations that became isolated, known as nominalistic species (Spooner et al. 2003). Domestication within these groups and subsequent transfer by human migration would facilitate intercrosses. This development has been well worked out with bread wheat (Triticum vulagare), a hexaploid amphidiploid of the genomic constitution AABBDD. Genomic analysis has identified the three species involved: AA from Triticum urartu, or einkorn, BB from Aegilops speltoides, and DD from Aegilops tauschi or goatgrass. The cross of emmer (AABB) with goatgrass (DD) is presumed to have occurred about 6000 BCE. This process occurred in many fruit crops (cherry, banana and plantains, citrus, brambles) as well as in deliberate chromosome manipulation (blueberry). Recently, “interspecific hybridization” has been used to create new fruits in citrus (tangelos, tangors), Prunus (plumcot), and Rubus (tayberry).

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C. Hybridization and Selection Zohary and Spiegel-Roy (1975) have concluded that spontaneous hybridization between wild races and cultivated clones was critical to the early domestication of fruits. Selection from sexual recombinants is still the dominant force in the domestication process as well as modern fruit breeding. The isolation of elite selections, combined with mass plantings, created a situation where mass selection and recurrent selection could operate naturally. This has recently been confirmed in apple, where elite selections from Kazakhstan are very close to cultivated varieties. In the case of apple, Barrie Juniper (pers. commun.) has made the case that selection for large fruit may have been due to selection by bears over millions of years. Selection from sexual recombination can be clearly followed in North America, now considered a secondary center of origin for the cultivated apple. In colonial America, starting in the 1600s, the apple was imported by immigrants, some as scions but most as seeds from Holland, Germany, France, and the British Isles (Beech 1905). In 1634, Lord Baltimore instructed the first Maryland settlers to carry “kernalls of pears and apples, especially of Permains and Deesons, for making thereafter of Cider and Perry” (Calhoun 1995). Pioneers were encouraged to plant apples and the requirement for settling Ohio (1787–1788) included that the settler must harvest at least 50 apple or pear trees and 20 peach trees (Morgan and Richards 1993). Apples, once introduced, were carried far into the wilderness by Native Americans, traders, and missionaries and became naturalized. In 1806, Jonathan Chapman (the legendary Johnny Appleseed), born in 1744 in Leominster, Massachusetts, distributed apple seeds from cider mills in western Pennsylvania and founded a nursery in West Virginia, distributing seeds along the way. The apple flourished in the new territories with the greatest use for hard cider, the distilled product called apple jack, and vinegar for preservation of fruits and vegetables. Many of the imported apple clones were unadapted, and the selection of natural seedlings from orchards became the glory of 19th century American pomology. In 1905, 698 apple cultivars were described in Beech’s (1905) Apples of New York. Roueché (1975) estimated that 100,000 clones have been selected from literally hundreds of millions of seedlings and evaluated by millions of fruit growers. In the United States the screening of open-pollinated, chance seedlings resulted in thousands of selections, many of which proved to be outstanding cultivars, including Golden Delicious, Delicious, Jonathan, McIntosh, Rome Beauty, York Imperial, Stayman Winesap, Yellow Newtown, Winesap, Rhode Island Greening, Northern Spy, and Gravenstein. ‘Golden Deli-

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cious’ had a profound influence on apple growing in Europe in the 20th century and further proved to be a prepotent parent producing many important new cultivars from breeding efforts (Janick et al. 1996). Among the characters that are influenced by selection is the ease of propagation. The key to domestication of fruit crops is vegetative propagation of elite types, followed by natural intercrosses between elites and wild species. This can clearly be seen in the date palm, in which domesticates, but not wild species, are naturally propagated by offshoots. It also occurs in the apple; dwarfing rootstocks are clonally propagated by layering (stooling) and this character, at least before the advent of micropropagation by tissue culture, is an essential trait. A number of nut trees, black walnut and pecan in particular, have been difficult to propagate vegetatively and are often still propagated from seeds, resulting in a negative effect on productivity. Selection for fruit quality, based on flavor, color, and shelf life, is the goal of modern fruit breeding programs. Selection is nowhere more important than in pineapple where the processing industry was long based on a single cultivar, ‘Cayenne’ and its spineless sport (‘Smooth Cayenne’) that was uniquely adapted to producing canned slices. A sweeter, yellow fleshed seedling produced from hybridization is now being marketed as ‘DelMonte Gold’ and is transforming the world fresh fruit industry because of better fresh fruit quality and appearance. D. Champions The decisive contribution to domestication made by individuals is unknown in most fruit crops and these great horticulturists are largely unremembered and unsung. However, in a few cases of recent domestication, key persons have been identified. These champions are essential to the domestication process. In the case of the kiwifruit, this includes the great plant hunter E. F. Wilson, who introduced the fruit to Britain; a missionary, Katie Frazier, who imported seed to New Zealand, possibly derived from Wilson; and H. R. (Hayward) Wright, who selected the pistillate clone that bears his name. The technology for orchard development was made in New Zealand from grafted plants, principally by F. J. Walker in the town of Wanganui. Domestication of blueberry was initiated by a single researcher, Frederick Vernon Coville (1888–1937), USDA botanist, who recognized the potential of this species, and later had a fortunate collaboration with Elizabeth White, a blueberry enthusiast from Whitesbog, New Jersey. Later influential researchers included George Darrow and Arlen Draper, both USDA researchers. In strawberry, Amédee François Frézier introduced

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F. chiloensis to France, resulting in the fortuitous hybridization with F. virginiana, and the great French botanist, Antoine Nicolas Duchesne, who explained the dioecious nature of Fragaria, interpreted F. ananassa as an interspecific hybrid, and identified hermaphroditic types. The origin of the grapefruit (once known as forbidden fruit), which was discovered from natural intercrosses between orange and pummelo, called shaddock in Barbados, can be traced to the trader Philip Chaddock, who in the mid17th century introduced the pummelo to Barbados. Domestication does not just happen but is carried out by acts of real people and they need to be honored. E. Lost Fruits Although a number of fruits have Cinderella stories, many lose out. Some fruits, once popular and widely grown, fall into disuse. Perhaps this can be considered reverse domestication. Examples include sycomore fig, medlar, quince, and the Asian apple (M. asiatica). A number of fruits languish as regional fruits. Thus, Ribes species (gooseberries, red and black currants) have never been popular in North America, the Asian persimmon (kaki) has never been really popular outside of Asia, and grapefruit, popular in the United States, is only slowly being adopted in Europe and Asia. F. Fruit Breeding Fruit breeding as an organized activity is a 19th century innovation. Its origins trace to mass selection efforts in strawberry (see III D 1) and pear (see III B 1). Thomas Andrew Knight (1759–1838) was the first to improve fruits by selection from genetic recombination derived from inter-pollinations of clones. An early proponent of the development of plant improvement through cross breeding and selection, he literally initiated the field of fruit breeding (Knight 1806). He released a number of improved fruit cultivars (apple and pear, cherry strawberry, redcurrant and strawberry, and cherry, nectarine, and plum). His studies on the effects of pollen in the garden pea on seed characters presaged the work of Gregor Mendel carried out 40 years later. He describes dominance and segregation, although he failed to make the brilliant leap of Mendel in relating phenotypic characters to the factors we now know as genes. Gregor Mendel, the father of genetics, was also involved in apple and pear breeding programs. In the United States, fruit breeding became a part of research at the state and federal experiment stations and a number of important breed-

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ing programs were initiated throughout the United States. Fruit breeding also became an activity of the private sector. Luther Burbank (1849–1926) was the first to consider fruit breeding as a commercial endeavor, and although he distrusted Mendelism, he was a staunch believer in the evolutional theories of Darwin (Crow 2001). At present, private breeders are an important part of Prunus (especially peach and nectarine and plum) and recently strawberry and raspberry. Although fruit breeding has been a major activity since early in the 20th century, the results have been uneven and vary from ineffectual to extraordinarily successful (Table 8.3). Many of the world fruit industries are still based on grower-selected clones. The reason for the lack of progress is two-fold. First, vegetative propagation permits the genetic fixation of naturally occurring variation. Because of the vast populations involved in seedling orchards, the quality of the selected clones over hundreds and even thousands of years of selection is very high. Second, the difficulties and expense inherent in fruit breeding have inhibited long-term breeding programs. Progress from breeding a number of fruit crops, however, has shown significant advances in the second half of the 20th century and selections from controlled crosses are increasingly important in many crops. In apple, although chance seedlings such as ‘Delicious’ and ‘Golden Delicious’ have long dominated the world market, ‘Fuji’, a seedling derived from a Japanese breeding program (‘Ralls Janet’ × ‘Delicious’), is now the leading world cultivar. G. Predicting Future Changes Knowledge of domestication should be used to predict future changes, and to help domesticate new candidates. Thus one might anticipate that in kiwifruit, hermaphroditic mutations would eliminate the need for staminate clones as pollinators, and that fruit skin mutations or breeding could lead to non-pubescent clones, with more attractive and more edible fruit surface. The use of interspecific hybridization should lead to improvement in banana and plantain (now in jeopardy because of lack of genetic diversity), the creation of new stone fruits, and the creation of new seedless, easy-to-peel citrus. A number of tropical fruits are candidates for commercialization, provided postharvest technology can be improved. One of the likely candidates for domestication is pitaya (species of Selenicereus, Hylocereus, and Cereus), an extremely attractive fruit of columnar cactus, but breeding efforts to improve quality are required, since many selections are somewhat insipid (Mizrahi et al. 2002). A number of generalizations can be made concerning the origin and future development of fruit crops. The first is that most fruit crops are

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little removed from wild species, some perhaps by only a few generations, so that continued progress should be possible. The key breeding system has evolved from selection of elite clones followed by fixation by vegetative propagation. The development of fruit culture is based on an interaction between genetic changes and cultural practices. Indeed, in many fruit crops, once desirable clones are discovered, intensive efforts have been made to prop them up by cultural practices. These include artificial pollination, the use of disease-resistant and size controlling rootstocks, extensive methods of disease control, including complex schedules of pesticide application, the control of fruit size and annual bearing by manual and chemical fruit and flower thinning, the control of fruit abscission with growth regulators, and extensive pruning and training systems. Despite some intensive breeding programs, extremely successful in Prunus, strawberry, and blueberry, many of our fruit cultivars are ancient and based on grower-selected seedlings and somatic mutations (Table 8.2). Advances in molecular genetics may overcome some of the limitations to conventional fruit breeding based on sexual recombination by increasing selection efficiency using molecular markers and by transgene technology whereby individual genes from various sources may be inserted without disturbing unique genetic combinations. Progress has already been achieved in papaya (virus resistance) and apple (fireblight resistance) but fears of consumer resistance is a problem. LITERATURE CITED Bahrani, Z. 2002. Peformativity and the image: Narrative, representation, and the Uruk vase. p. 15–22. In: E. Ehrenberg (ed.), Leaving no stones unturned: Essays on the ancient Near East and Egypt in honor of Donald P. Hansen. Eisenbrauns, Winona Lake, IN. Beech, S. A. 1905. The apples of New York. Vol. I. J. B. Lyon Co. Bell, R. L., H. A. Quamme, R. E. C. Layne, and R. Skirvin. 1996. Pears. p. 441–513. In: J. Janick and J. N. Moore (eds.), Fruit breeding. Vol. I. Wiley, NY. Bergh, B. O., and E. Lahav. 1966. Avocados. p. 113–166. In: J. Janick and J. N. Moore (eds.), Fruit breeding. Vol. I. Wiley, NY. Bergh, B. O. 1995. Avocado: Persea Americana: (Lauraceae). p. 240–245. In: J. Smartt and N. W. Simmonds (eds.), Evolution of crop plants. 2nd ed. Longman Scientific & Technical. Essex, England. Blázquez, J. M. 1996. The origin and expansion of olive cultivation. p. 19–20. World Olive Encyclopaedia, Madrid. Brown, S. K., A. F. Iezzoni, and H. W. Fogle. 1996. Cherries. p. 213–255. In: J. Janick and J. N. Moore (eds.), Fruit breeding. Vol. I. Wiley, NY. Calhoun, Jr., C. L. 1995. Old southern apples. The McDonald & Woodward Publishing Co., Blacksburg, VA.

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321 Errata for Fig. 6.1 from William G. Hill and Bünger, L. 2004. Inferences on the Genetics of Quantitative Traits from Long-term Selection in Laboratory and Domestic Animals, in Plant Breeding Reviews, Volume 24, Part 2, edited by Jules Janick. John Wiley & Sons, Hoboken, NJ. 02:17.00

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Subject Index Volume 25 A

D

Almond domestication, 290–291 Anthocyanin pigmentation, 89–114 Apple domestication, 286–289 Apricot domestication, 291–292 Avocado domestication, 307

Date palm domestication, 272–277 Doubled haploid breeding, 57–88 Drought resistance, maize, 173–253

B Banana & plantain domestication, 298–299 Biography, Stanley J. Peloquin, 1–19 Biotechnology politics, 21–55 Blueberry domestication, 304 Bramble domestication, 303 Breeding doubled haploids, 57–88 drought tolerance, maize, 173–253 flower color, 89–114 fruit crops-, 255–320 maize drought tolerance, 173–253 mitochondrial genetics, 115–138 potato, 1–19 sorghum male sterility, 139–172 transgene technology, 105–108

F Fig domestication, 281–285 Flavonoid chemistry, 91–94 Flower color, 89–114 Fruit: breeding, 255–320 domestication, 255–320 origins, 255–320 G Genetics domestication, 255–320 flower color, 89–114 mitochondrial, 115–138 Germplasm rights, 21–55 Grain breeding, maize drought tolerance 173–253 sorghum male sterility, 139–172 Grape domestication, 279–281

C Cherry domestication, 202–293 Citrus domestication, 296–298 Cranberry domestication, 304–305 Cytoplasm, breeding, 115–138 incompatibility, 115–138 male sterility, 115–138, 139–172 sorghum male sterility, 139–172

H Haploidy, doubled, 57–88 K Kiwifruit domestication, 300–301

Plant Breeding Reviews, Volume 25. Edited by Jules Janick. ISBN 0471666939 © 2005 John Wiley & Sons, Inc. 323

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SUBJECT INDEX

L

P

Lingonberry domestication, 305–307

Maize drought tolerance, 173–253 Male sterility: genetics, 115–138, 139–172 sorghum, 139–172 Mango domestication, 299 Mitochondrial genetics, 115–138

Papaya domestication, 307–308 Peach domestication 294–296 Pear domestication, 289–290 Peloquin, Stanley J., (biography), 1–19 Persimmon domestication, 299–300 Pineapple domestication, 305–307 Plant breeders’ rights, 21–55 Plant breeding politics, 21–55 Plum domestication, 293–294 Pomegranate domestication, 285–286

O

S

Olive domestication, 277–279

Sorghum male sterility, 139–172 Strawberry domestication, 302–303

M

Cumulative Subject Index (Volumes 1–25) A Adaptation: blueberry, rabbiteye, 5:351–352 durum wheat, 5:29–31 genetics, 3:21–167 testing, 12:271–297 Aglaonema breeding, 23:267–269 Alexander, Denton, E. (biography), 22:1–7 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 domestication, 25:290–291 transformation, 16:103 Alocasia breeding, 23:269 Alstroemaria, mutation breeding, 6:75 Amaranth: breeding, 19:227–285 cytoplasm, 23:191 genetic resources, 19:227–285 Animals, long term selection 24(2):169–210, 211–234– 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 maize aleurone, 8:91–137 pigmentation, 25:89–114 Anthurium breeding, 23:269–271 Antifungal proteins, 14:39–88 Antimetabolite resistance, cell selection, 4:139–141, 159–160 Apomixis: breeding, 18:13–86 genetics, 18:13–86 reproductive barriers, 11:92–96 rice, 17:114–116 Apple: Domestication, 25:286–289 genetics, 9:333–366 rootstocks, 1:294–394 transformation, 16:101–102 Apricot: domestication, 25:291–292 transformation, 16:102 Arachis, see Peanut in vitro culture, 2:218–224 Artichoke breeding, 12:253–269 Avena sativa, see Oat Avocado domestication, 25:307 Azalea, mutation breeding, 6:75–76

B Bacillus thuringensis, 12:19–45 Bacteria, long-term selection, 24(2):225–265 Bacterial diseases: apple rootstocks, 1:362–365 cell selection, 4:163–164 cowpea, 15:238–239 potato, 19:113–122

Plant Breeding Reviews, Volume 25. Edited by Jules Janick. ISBN 0471666939 © 2005 John Wiley & Sons, Inc. 325

326 Bacterial diseases (cont.) raspberry, 6:281–282 soybean, 1:209–212 sweet potato, 4:333–336 transformation fruit crops, 16:110 Banana: breeding, 2:135–155 domestication, 25:298–299 transformation, 16:105–106 Barley: anther culture, 15:141–186 breeding methods, 5:95–138 diversity, 21:234–235 doubled haploid breeding, 15:141–186 gametoclonal variation, 5:368–370 haploids in breeding, 3:219–252 molelcular markers, 21:181–220 photoperiodic response, 3:74, 89–92, 99 vernalization, 3:109 Bean (Phaseolus): breeding, 1:59–102; 10:199–269; 23:21–72 breeding mixtures, 4:245–272 breeding (tropics), 10:199–269 heat tolerance, 10:149 in vitro culture, 2:234–237 long-term selection, 24(2):69–74 photoperiodic response, 3:71–73, 86–92; 16:102–109 protein, 1:59–102 rhizobia interaction, 23:21–72 Beet (table) breeding, 22:357–388 Beta, see Beet Biochemical markers, 9:37–61 Biography: Alexander, Denton E., 22:1–7 Allard, Robert W., 12:1–17 Bringhurst, Royce S., 9:1–8 Burton, Glenn W., 3:1–19 Coyne, Dermot E., 23:1–19 Downey, Richard K., 18:1–12 Dudley, J.W., 24(1):1–10 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 Peloquin, Stanley J., 25:1–19 Ryder, Edward J., 16:1–14 Sears, Ernest Robert, 10:1–2

CUMULATIVE SUBJECT INDEX Simmonds, Norman W., 20:1–13 Sprague, George F., 2:1–11 Vogel, Orville A., 5:1–10 Vuylsteke, Dirk R., 21:1–25 Weinberger, John H., 11:1–10 Yuan, Longping, 17:1–13 Biotechnology politics, 25:21–55 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 domestication, 25:304 rabbiteye, 5:307–357 Brachiaria, apomixis, 18:36–39, 49–51 Bramble: domestication, 25:303 transformation, 16:105 Brassica, see Cole crops Brassica: napus, see Canola, Rutabaga rapa, see Canola Brassicaceae: incompatibility, 15:23–27 molecular mapping, 14:19–23 Breeding: Aglaonema, 23:267–269 alfalfa via tissue culture, 4:123–152 almond, 8:313–338 Alocasia, 23:269 amaranth, 19:227–285 apomixis, 18:13–86 apple, 9:333–366 apple rootstocks, 1:294–394 banana, 2:135–155 barley, 3:219–252; 5:95–138 bean, 1:59–102; 4:245–272; 23:21–72 beet (table), 22:357–388 biochemical markers, 9:37–61 blackberry, 8:249–312 black walnut, 1:236–266 blueberry, rabbiteye, 5:307–357 bromeliad, 23:275–276 cactus, 20:135–166 Calathea, 23:276 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

CUMULATIVE SUBJECT INDEX 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 cytoplasmic DNA, 12:175–210 diallel analysis, 9:9–36 Dieffenbachia, 271–272 doubled haploids, 15:141–186; 25:57–88 Dracaena, 23:277 drought tolerance, maize, 25:173–253 durum wheat, 5:11–40 Epepremnum, 23: 272–2mn epistasis, 21:27–92 exotic maize, 14:165–187 fern, 23:276 fescue, 3:313–342 Ficus, 23:276 Flower color, 25:89–114 foliage plant, 23:245–290 forest tree, 8:139–188 fruit crops, 25:255–320 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 Hedera, 23:279–280 herbicide-resistant crops, 11:155–198 heritability, 22:9–111 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 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; 25:173–253 mitochondrial genetics, 25:115–238 molecular markers, 9:37–61, 10:184–190; 12:195–226; 13:11–86; 14:13–37, 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181–220, 23:73–174

327 mosaics, 15:43–84 mushroom, 8:189–215 negatively associated traits, 13:141–177 oat, 6:167–207 oil palm, 4:175–201; 22:165–219 onion, 20:67–103 palms, 23:280–281 pasture legumes, 5:237–305 pea, snap, 212:93–138 peanut, 22:297–356 pearl millet, 1:162–182 perennial rye, 13:265–292 persimmon, 19:191–225 Philodendron, 23:273 plantain, 2:150–151; 14:267–320; 21:211–25 potato, 3:274–277; 9:217–332; 16:15–86; 19:59–155, 25:1–19 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; 23:73–174 rose, 17:159–189 rutabaga, 8:217–248 sesame, 16:179–228 snap pea, 21:93–138 somatic hybridization, 20:167–225 sorghum male sterility, 25:139–172 soybean, 1:183–235; 3:289–311; 4:203–243; 21:212–307 soybean hybrids, 21:212–307 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 Syngonium, 23:274 tomato, 4:273–311 transgene technology, 25:105–108 triticale, 5:41–93; 8:43–90 Vigna, 8:19–42

328 Breeding (cont.) 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 white clover, 17:191–223 wild rice, 14:237–265 Bringhurst, Royce S. (biography), 9:1–8 Broadbean, in vitro culture, 2:244–245 Bromeliad breeding, 23:275–276 Burton, Glenn W. (biography), 3:1–19 C Cactus: breeding, 20:135–166 domestication, 20:135–166 Cajanus, in vitro culture, 2:224 Calathea breeding, 23:276 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: breeding, 2:73–134 long-term selection, 24(2):74–79 Castanea, see Chestnut Cell selection, 4:139–145, 153–173 Cereal breeding, see Grain breeding Cereal diversity, 21:221–261 Cherry, see Sweet cherry domestication, 25:202–293 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: domestication, 25:296–298 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

CUMULATIVE SUBJECT INDEX 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 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 Coyne, Dermot E. (biography), 23:1–19 Cranberry domestication, 25:304–305 Crop domestication and selection, 24(2):1–44 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 Cybrids. 3:205–210; 20: 206–209 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

CUMULATIVE SUBJECT INDEX potato, 25:1–19 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: breeding, 23: 175–210; 25:115–138 cybrids, 3:205–210; 20:206–209 incompatibility, 25:115–138 male sterility, 25:115–138,139–172 molecular biology of male sterility, 10:23–51 organelles, 2:283–302; 6:361–393 pearl millet, 1:166 petunia, 1:43–45 sorghum male sterility, 25:139–172 wheat, 2:308–319 D Dahlia, mutation breeding, 6:75 Date palm domestication, 25:272–277 Daucus, see Carrot Diallel cross, 9:9–36 Dieffenbachia breeding, 23:271–272 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

329 virus resistance, 12:47–79 wheat rust, 13:293–343 Diversity in land races, 21:221–261 DNA methylation, 18:87–176 Doubled haploid breeding, 15:141–186; 25:57–88 Downey, Richard K. (biography), 18:1–12 Dracaena breeding, 23:277 Draper, Arlen D. (biography), 13:1–10 Drought resistance: durum wheat, 5:30–31 maize, 25:173–253 soybean breeding, 4:203–243 wheat adaptation, 12:135–146 Dudley, J.W. (biography), 24(1):1–10 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 Endosperm: balance number, 25:6–7 maize, 1:139–161 sweet corn, 1:139–161 Endothia parasitica, 4:355–357 Epepremnum breeding, 23:272–273 Escherichia coli, long-term selection, 24(2):225–224 Epistasis, 21:27–92. Evolution: coffee, 2:157–193 fruit, 25: 255–320 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 Fern breeding, 23:276 Fescue, 3:313–342 Festuca, see Fescue Fig domestication, 25:281–285 Flavonoid chemistry, 25:91–94 Floral biology: almond, 8:314–320 blackberry, 8:267–269

330 Floral biology (cont.) black walnut, 1:238–244 cassava, 2:78–82 chestnut, 4:352–353 coffee, 2:163–164 coleus, 3:348–349 color, 25:89–114 fescue, 3:315–316 garlic: 23:211–244 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 Flower color, 25:89–114 Forage breeding: alfalfa inbreeding, 13:209–233 diversity, 21:221–261 fescue, 3:313–342 perennials, 11:251–274 white clover, 17:191–223 Foliage plant breeding, 23:245–290 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 Fragaria, see Strawberry 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 blueberry, 13:1–10 blueberry, rabbiteye, 5:307–357 breeding, 25:255–320 cactus, 20:135–166 cherry, 9:367–388 citrus, 8:339–374 coffee, 2:157–193 domestication, 25:255–320 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

CUMULATIVE SUBJECT INDEX origins, 25:255–320 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 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 breeding, 6:81, 23:211–244 Genes: action, 15:315–374

CUMULATIVE SUBJECT INDEX 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 Rhizobium, 23:39–47 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 salt resistance, 22:389–425 transformation by particle bombardment, 13:231–260 transgene technology, 25:105–108 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 apomixis, 18:13–86 apple, 9:333–366 Bacillus thuringensis, 12:19–45 bean seed protein, 1:59–102 beet, 22:357–376 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 chimeras, 15:43–84 chrysanthemums, 14:321 clover, white, 17:191–223 coffee, 2:165–170 coleus, 3:3–53 cowpea, 15:215–274 cytoplasm, 23:175–210

331 DNA methylation, 18:87–176 domestication, 25:255–320 durum wheat, 5:11–40 forest trees, 8:139–188 flower color, 25:89–114 fruit crop transformation, 16:87–134 gene action, 15:315–374 history, 24(1):11–40 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 markers to manage germplasm, 13:11–86 maturity, 3:21–167 metabolism and heterosis, 10:53–59 mitochondrial, 25:115–138. 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 pea, 21:110–120 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 quantitative trait loci in animals selection, 24(2):169–210, 211–224 reproductive barriers, 11:11–154

332 Genetics (cont.) rhizobia, 21–72 rice, hybrid, 17:15–156, 23:73–174 rose, 17:171–172 rutabaga, 8:217–248 salt resistance, 22:389–425 selection, 24(1):111–131, 143–151, 269–290 snap pea, 21:110–120 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

CUMULATIVE SUBJECT INDEX 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 peanut, 22:297–356 pearl millet, 1:167–170 plantain, 14:267–320 potato, 9:219–223 preservation, 2:265–282; 23:291–344 rights, 25:21–55 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 diversity, 21:221–261 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; 22:3–4; 24(1): 11–40, 41–59, 61–78; 24(2): 53–64, 109–151; 25:173–253 maize history, 24(2): 31–59, 41–59, 61–78 oat, 6:167–207 pearl millet, 1:162–182 rice, 17:15–156; 24(2):64–67 sorghum, 25:139–172 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; 22:221–297; 24(2):67–69 wild rice, 14:237–265 Grape: domestication, 25:279–281 transformation, 16:103–104 Grapefruit: breeding, 13:345–363 evolution, 13:345–363

CUMULATIVE SUBJECT INDEX 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 doubled, 15:141–186; 25:57–88 maize, 11:199–224 petunia, 1:16–18, 44–45 potato, 3:274–277; 16:15–86 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 Heritability estimation, 22:9–111 Heterosis: gene action, 15:315–374 overdominance, 17:225–257 plant breeding, 12:227–251 plant metabolism, 10:53–90 rice, 17:24–33 soybean, 21:263–320 Honeycomb: breeding, 18:177–249 selection, 13:87–139, 18:177–249 Hordeum, see Barley 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 maize high oil selection, 24(1):153–175 maize long-term selection, 24(2):43–64, 109–151 maize history, 24(1): 31–59, 41–59, 61–78

333 overdominance, 17:225–257 rice, 17:15–156 soybean, 21:263;-320 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 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

334 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 wheat, 22:221–297 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 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: domestication, 25:300–301 transformation, 16:104 L Lactuca sativa, see Lettuce Landraces, diversity, 21:221–263 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

CUMULATIVE SUBJECT INDEX 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 Lingonberry domestication, 25:300–301 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 cytoplasm, 23:189 doubled haploid breeding, 15:141–186 drought tolerance, 25:173–253 exotic germplasm utilization, 14:165–187 high oil, 22:3–4; 24(1):153–175 history of hybrids, 23(1): 11–40, 41–59, 61–78 honeycomb breeding, 18:226–227 hybrid breeding, 17:249–251 insect resistance, 6:209–243 long-term selection 24(2):53–64, 109–151 male sterility, 10:23–51 marker-assisted selection. 24(1):293–309 mobile elements, 4:81–122 mutations, 5:139–180 origins, 20:15–66 origins of hybrids, 24(1):31–50, 41–59, 61–78 overdominance, 17:225–257 physiological changes with selection, 24(1):143–151 protein, 1:103–138 quality protein, 9:181–216

CUMULATIVE SUBJECT INDEX recurrent selection, 9:115–179; 14:139–163 RFLF changes with selection, 24(1):111–131 selection for oil and protein, 24(1):79–110, 153–175 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 genetics, 25:115–138, 139–172 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 sorghum, 139–172 soybean, 21:277–291 wheat, 2:303–319 Malus spp, see Apple Malus ×domestica, see Apple Malvaceae, molecular mapping, 14:25–27 Mango: domestication, 25:277–279 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 Mitochondrial genetics, 6:377–380; 25:115–138 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

335 molecular markers, 9:37–61, 10:184–190; 12:195–226; 13:11–86; 14:13–37; 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181–220, 23:73–174 quantitative trait loci, 15:85–139 salt resistance, 22:389–425 somaclonal variation, 16:229–268 somatic hybridization, 20:167–225 soybean nodulation, 11:275–318 strawberry, 21:139–180 transposable (mobile) elements, 4:81–122; 8:91–137 virus resistance, 12:47–79 wheat improvement, 11:235–250 Molecular markers, 9:37–61, 10:184–190; 12:195–226; 13:11–86; 14:13–37; 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181–220, 23:73–174 alfalfa, 10:184–190 apomixis, 18:40–42 barley, 21:181–220 clover, white, 17:212–215 forest crops, 19:31–68 fruit crops, 12:195–226 maize selection, 24(1):293–309 mapping, 14:13–37 plant genetic resource mangement, 13:11–86 rice, 17:113–114, 23:73–124 rose, 17:179 somaclonal variation, 16:238–243 wheat, 21:181–220 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

336 Mutants and mutation (cont.) gametoclonal variation, 5:359–391 homeotic floral, 9:63–99 induced, 2:13–72 long term selection variation, 24(1):227–247 maize, 1:139–161, 4:81–122; 5:139–180 mobile elements, see Transposable elements mosaics, 15:43–84 petunia, 1:34–40 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 preservation of genetic resources, 23:291–34 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

CUMULATIVE SUBJECT INDEX 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: breeding, 4:175–201, 22:165–219 in vitro culture, 4:175–201 Oilseed breeding: canola, 18:1–20 oil palm, 4:175–201; 22:165–219 sesame, 16:179–228 soybean, 1:183–235; 3:289–311; 4:203–245; 11:275–318; 15:275–313 Olive domestication, 25:277–279 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 Palm (Arecaceae): foliage breeding, 23:280–281 oil palm breeding, 4:175–201; 22:165–219. Panicum maximum, apomixis, 18:34–36, 47–49 Papaya domestication, 25:307–308 transformation, 16:105–106 Parthenium argentatum, see Guayule Paspalum, apomixis, 18:51–52 Paspalum notatum, see Pensacola bahiagrass Passionfruit transformation, 16:105

CUMULATIVE SUBJECT INDEX Pasture legumes, interspecific hybridization, 5:237–305 Pea: breeding, 21:93–138 flowering, 3:81–86, 89–92 in vitro culture, 2:236–237 Peach: cold hardiness breeding, 10:271–308 domestication, 25:294–296 transformation, 16:102 Peanut: breeding, 22:297–356 in vitro culture, 2:218–224 Pear: domestication, 25:289–290 transformation, 16:102 Pearl millet: apomixis, 18:55–56 breeding, 1:162–182 Pecan transformation, 16:103 Peloquin, Stanley, J. (biography), 25:1–19 Pennisetum americanum, see Pearl millet Pensacola bahiagrass, 9:101–113 apomixis, 18:51–52 selection, 9:101–113 Pepino transformation, 16:107 Peppermint, mutation breeding, 6:81–82 Perennial grasses, breeding, 11:251–274 Perennial rye breeding, 13:261–288 Persimmon: breeding, 19:191–225 domestication, 25:299–300 Petunia spp., genetics, 1:1–58 Phaseolin, 1:59–102 Phaseolus vulgaris, see Bean Philodendrum breeding, 23:273 Phytophthora fragariae, 2:195–214 Pigeon pea, in vitro culture, 2:224 Pineapple domestication, 25:305–307 Pistil, reproductive function, 4:9–79 Pisum, see Pea Plant breeders; rights, 25:21–55 Plant breeding politics, 25:21–55 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; 21:1–25 domestication, 25: 298 Plastid genetics, 6:364–376, see also Organelle

337 Plum: domestication, 25:293–294 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; 25:1–19 reproductive barriers, 11:98–105 sweet potato, 4:371 triticale, 5:11–40 Pomegranate domestication, 25:285–286 Population genetics, see Quantitative Genetics Potato: breeding, 9:217–332, 19:69–165 cytoplasm, 23:187–189 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 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

338 Psophocarpus, in vitro culture, 2:237–238 Q Quantitative genetics: epistasis, 21:27–92 forest trees, 8:139–188 gene interaction, 24(1):269–290 genotype x environment interaction, 16:135–178 heritability, 22:9–111 maize RFLP changes with selection, 24(1):111–131 mutation variation, 24(1): 227–247 overdominance, 17:225–257 population size & selection, 24(1):249–268 selection limits, 24(1):177–225 statistics, 17:296–300 trait loci (QTL), 15:85–139; 19:31–68 variance, 22:113–163 Quantitative trait loci (QTL), 15:85–138; 19:31–68 animal selection, 24(2):169–210, 211–224 selection limits: 24(1):177–225 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 foliage plants, 23:255–259 garlic, 23:211–244 Rhizobia, 23:21–72 Rhododendron, mutation breeding, 6:75–76 Rice, see also Wild rice: anther culture, 15:141–186 apomixis, 18:65 cytoplasm, 23:189 doubled haploid breeding, 15:141–186 gametoclonal variation, 5:362–364

CUMULATIVE SUBJECT INDEX heat tolerance, 10:151–152 honeycomb breeding, 18:224–226 hybrid breeding, 17:1–15, 15–156; 23:73–174 long-term selection 24(2): 64–67 molecular markers, 73–174 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 yeast systems, 22:389–425 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 garlic, 23:211–244 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, 23:73–174 soybean, 1:183–235, 3:289–311 synthetic, 7:173–174 variegation, 4:81–86 wheat (hybrid), 2:313–317 Selection, see also Breeding bacteria, 24(2): 225–265 bean, 24(2): 69–74 cell, 4:139–145, 153–173 crops of the developing world, 24(2):45–88 divergent selection for maize ear length, 24(2):153– 168 domestication, 24(2):1–44

CUMULATIVE SUBJECT INDEX Escherichia coli, 24(2): 225–265 gene interaction, 24(1):269–290 genetic models, 24(1):177–225 honeycomb design, 13:87–139; 18:177–249 limits, 24(1):177–225 maize high oil, 24(1):153–175 maize history, 24(1):11–40, 41–59, 61–78 maize long term, 24(1):79–110, 111–131, 133–151; 24(2):53– 64, 109–151 maize oil & protein, 24(1):79–110, 153–175 maize physiological changes, 24(1):133–151 maize RFLP changes, 24(1):111–131 marker assisted, 9:37–61, 10:184–190; 12:195–226; 13:11–86; 14:13–37; 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181–220, 23:73–174, 24(1):293–309 mutation variation, 24(1):227–268 population size, 24(1):249–268 productivity gains in US crops, 24(2):89–106 quantitative trait loci, 24(1):311–335 recurrent restricted phenotypic, 9:101–113 recurrent selection in maize, 9:115–179; 14:139–163 rice, 24(2): 64–67 wheat, 24(2): 67–69 Sesame breeding, 16:179–228 Sesamum indicum, see Sesame Simmonds, N.W. (biography), 21:1–13 Snap pea breeding, 21:93–138 Solanaceae: incompatibility, 15:27–34 molecular mapping, 14:27–28 Solanum tuberosum, see Potato 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:

339 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: male sterility, 25:139–172 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 hybrid breeding, 21:263–307 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: advanced methods, 22:113–163 history, 17:259–316 Strawberry: biotechnology, 21: 139–180 domestication, 25:302–303 red stele resistance breeding, 2:195–214 transformation, 16:104 Stress resistance: cell selection, 4:141–143, 161–163 transformation fruit crops, 16:115 Stylosanthes, in vitro culture, 2:238–240 Sugarcane: mutation breeding, 6:82–84 and Saccharum complex, 16:269–288 Sweet cherry: Domestication, 25:202–293 pollen-incompatibility and selffertility, 9:367–388 transformation, 16:102

340 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 garlic, 23:211–244 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 Toxin resistance, cell selection, 4:163–165 Transformation: alfalfa, 10:190–192 cereals, 13:231–260 fruit crops, 16:87–134 mushroom, 8:206

CUMULATIVE SUBJECT INDEX rice, 17:179–180 somaclonal variation, 16:229–268 white clover, 17:193–211 Transpiration efficiency, 12:81–113 Trilobium, long-term selection, 24(2):211–224 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 Triticosecale, see Triticale Triticum: Aestivum, see Wheat Turgidum, see Durum wheat 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 Variance estimation, 22:113–163 Vegetable breeding: artichoke, 12:253–269 bean, 1:59–102; 4:245–272, 24(2):69–74 bean (tropics), 10:199–269 beet (table), 22:257–388 carrot 19: 157–190 cassava, 2:73–134; 24(2):74–79 cucumber, 6:323–359 cucurbit insect and mite resistance, 10:309–360 lettuce, 1:267–293; 16:1–14; 20:105:133 mushroom, 8:189–215 onion, 20:67–103

CUMULATIVE SUBJECT INDEX

341

pea, 21:93–138 peanut, 22:297–356 potato, 9:217–232; 16:15–86l; 19:69–165 rutabaga, 8:217–248 snap pea, 21: 93–138 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 Vuylsteke, Dirk R. (biography), 21:1–25

chemical hybridization, 3:169–191 cold hardiness adaptation, 12:124–135 cytogenetics, 10:5–15 cytoplasm, 23:189–190 diversity, 21:236–237 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 insect resistance, 22:221–297 in vitro adaptation, 12:115–162 long-term selection, 24(2):67–69 molecular biology, 11:235–250 molecular markers, 21:191–220 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

W

Z

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

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

Y Yeast, salt resistance, 22:389–425 Yuan, Longping (biography), 17:1–13.

Cumulative Contributor Index (Volumes 1–25) Abdalla, O.S., 8:43 Acquaah, G., 9:63 Aldwinckle, H.S., 1:294 Alexander, D.E., 24(1): 53 Anderson, N.O., 10:93; 11:11 Aronson, A.I., 12:19 Ascher, P.D., 10:93 Ashri, A., 16:179 Baggett, J.R. 21:93 Baltensperger, D.D., 19:227 Barker, T., 25:173 Basnizki, J., 12:253 Beck, D.L., 17:191 Beebe, S., .23:21–72 Beineke, W.F., 1:236 Bell, A.E., 24(2): 211 Below, F.E., 24(1):133 Berzonsky, W.A., 22:221 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; 22:389 Bretting, P.K., 13:11 Broertjes, C., 6:55 Brown, A.H.D., 221 Brown, J.W.S., 1:59 Brown, S.K., 9:333,367 Bünger, L., 24(2):169

Burnham, C.R., 4:347 Burton, G.W., 1:162; 9:101 Burton, J.W., 21:263 Byrne, D., 2:73 Campbell, K.G., 15:187 Campos, H., 25: 173 Cantrell, R.G., 5:11 Carputo, C., 25:1 Carvalho, A., 2:157 Casas, A.M., 13:235 Cervantes-Martinez, C.T., 22:9 Chen, J., 23: 245 Chew, P.S., 22:165 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, M, 24(2):109; 25:173 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 Dekkers, J.C.M., 24(1):311 Deroles, S.C., 14:321 Dhillon, B.S., 14:139 Dickmann, D.I., 12:163 Ding, H., 22:221

Plant Breeding Reviews, Volume 25. Edited by Jules Janick. ISBN 0471666939 © 2005 John Wiley & Sons, Inc. 342

Dodds, P.N., 15:19 Dolan, D., 25:175 Donini, P., 21:181 Draper, A.D., 2:195 Dudley, J.W. 24(1): 79 Dumas, C., 4:9 Duncan, D.R., 4:153 Duvick, D.N., 24(2):109 Echt, C.S., 10:169 Edmeades, G., 25:173 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 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, B.P., 25:57 Forster, R.L.S., 17:191 Fowler, C., 25:21 Frusciante, L., 35:1 Frei, U., 23:175 French, D.W., 4:347 Gai, J., 21:263 Galiba, G., 12:115 Galletta, G.J., 2:195

CUMULATIVE CONTRIBUTOR INDEX Gepts, P., 24(2):1 Gmitter, F.G., Jr., 8:339; 13:345 Gold, M.A., 12:163 Goldman, I.L. 19:15; 20:67; 22:357; 24(1):61; 24(2)89 Goodnight, C.J, 24(1):269 Gradziel, T.M., 15:43 Gressel, J., 11:155; 18:251 Gresshof, P.M., 11:275 Griesbach, R.J., 25:89 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 Habben, J., 25:173 Haley, S.D., 22:221 Hall, A.E., 10:129; 12:81; 15:215 Hall, H.K., 8:249 Hallauer, A.R., 9:115; 14:1,165; 24(2):153 Hamblin, J., 4:245 Hancock, J.F., 13:1 Hancock, J.R., 9:1 Hanna, W.W., 13:179 Harlan, J.R., 3:1 Harris, M.O., 22: 221 Hasegawa, P.M. 13:235; 14:39: 22:389 Havey, M.J., 20:67 Henny, R.J., 23:245 Hill, W.G., 24(2):169 Hillel, J., 12:195 Hodgkin, T., 21:221 Hokanson, S.C., 21:139 Holbrook, C.C., 22: 297 Holland, J.B: 21: 27; 22: 9 Hor, T.Y., 22:165 Hunt, L.A., 16:135 Hutchinson, J.R., 5:181 Hymowitz, T., 8:1; 16:289 Janick, J., 1:xi; 23:1, 25:255 Jansky, S., 19:77 Jayaram, Ch., 8:91

Jenderek, M.M., 23:211 Johnson, A.A.T., 16:229; 20:167 Johnson, R., 24(1):293 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 Kasha, K.J., 3:219 Keep, E., 6:245 Kleinhofs, A., 2:13 Keightley, P.D., 24(1):227 Knox, R.B., 4:9 Koebner, R.M.D., 21:181 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 Lamb, R.J., 22:221 Lambert, R.J., 22: 1; 24(1):79, 153 Lamborn, C., 21:93 Lamkey, K.R., 15:1; 24(1):xi; 24(2):xi Lavi, U., 12:195 Layne, R.E.C., 10:271 Lebowitz, R.J., 3:343 Lee, M., 24(2):153 Lehmann, J.W., 19:227 Lenski, R.E., 24(2):225 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 Lower, R.L., 25:21 Lukaszewski, A.J., 5:41 Lyrene, P.M., 5:307 McCoy, T.J., 4:123; 10:169 McCreight, J.D., 1:267; 16:1

343 McDaniel, R.G., 2:283 McKeand, S.E., 19:41 McKenzie, R.I.H., 22: 221 McRae, D.H., 3:169 Mackenzie, S.A., 25:115n Maas, J. L., 21: 139 Maheswaran, G., 5:181 Maizonnier, D., 1:11 Marcotrigiano, M., 15:43 Martin, F.W., 4:313 Matsumoto, T.K. 22:389 Medina-Filho, H.P., 2:157 Mejaya, I.J., 24(1): 53 Mikkilineni, V., 24(1):111 Miles, D., 24(2):211 Miles, J.W., 24(2):45 Miller, R., 14:321 Mondragon Jacobo, C., 20:135 Moose, S.P., 24(1):133 Morrison, R.A., 5:359 Mowder, J.D., 7:57 Mroginski, L.A., 2:215 Muir, W.M., 24(2):211 Mumm, R.H., 24(1):1 Murphy, A.M., 9:217 Mutschler, M.A., 4:1 Myers, J.R., 21:93 Myers, O., Jr., 4:203 Myers, R.L., 19:227. Namkoong, G., 8:139 Navazio, J., 22:357 Neuffer, M.G., 5:139 Newbigin, E., 15:19 Nyquist, W.E., 22:9 Ohm, H.W., 22:221 O’Malley, D.M., 19:41 Ortiz, R., 14:267; 16:15; 21:1; 25:1, 139 Palmer, R.G., 15:275, 21:263 Pandy, S., 14:139; 24(2):45 Pardo, J. M., 22:389 Parliman, B.J., 3:361 Paterson, A.H., 14:13 Patterson, F.L., 22:221 Peairs, F.B., 22:221 Pedersen, J.F., 11:251 Perdue, R.E., Jr., 7:67

344 Peiretti, E.G., 23:175 Peterson, P.A., 4:81; 8:91 Polidorus, A.N., 18:87 Porter, D.A., 22:221 Porter, R.A., 14:237 Powell, W., 21:181 Proudfoot, K.G., 8:217 Rackow, G., 18:1 Raina, S.K., 15:141 Ramage, R.T., 5:95 Ramesh, S., 25:139 Ramming, D.W., 11:1 Ratcliffe, R.H., 22:221 Ray, D.T., 6:93 Reddy, B.V.S., 25:139 Redei, G.P., 10:1; 24(1):11 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 Rochefored.T.R., 24(1):111 Ron Parra, J., 14:165 Roos, E.E., 7:129 Ross, A.J., 24(2):153 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 Schaber, M.A. 24(2):89 Schussler, J., 25:173 Schneerman, M.C. 24(1):133 Schroeck, G., 20:67 Scott, D.H., 2:195 Seabrook, J.E.A., 9:217 Sears, E.R., 11:225 Seebauer, J.R., 24(1):133 Shands, Hazel L., 6:167 Shands, Henry L., 7:1, 5 Shannon, J.C., 1:139

CUMULATIVE CONTRIBUTOR INDEX Shanower, T.G., 22:221 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; 23:211 Singh, B.B., 15:215 Singh, R.J., 16:289 Singh, S.P., 10:199 Singh, Z., 16:87 Slabbert, M.M., 19:227 Sleper, D.A., 3:313 Sleugh, B.B., 19 Smith, J.S.C., 24(2):109 Smith, S.E., 6:361 Snoeck, C., 23:21 Socias i Company, R., 8:313 Sobral, B.W.S., 16:269 Stalker, H.T., 22:297 Soh, A.C., 22:165 Sondahl, M.R., 2:157 Spoor, W., 20: 1 Steadman, J.R., 23:1 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 Sun, H. 21:263 Tai, G.C.C., 9:217 Talbert, L.E., 11:235 Tan, C.C., 22:165 Tarn, T.R., 9:217 Tehrani, G., 9:367 Teshome, A., 21:221 Thomas, W.T.B., 25:57 Thompson, A.E., 6:93 Tiefenthaler, A.E. 24(2):89 Towill, L.E., 7:159, 13:179 Tracy, W.F., 14:189; 24(2):89 Troyer, A.F., 24(1):41 Tsaftaris, A.S., 18:87 Tsai, C.Y., 1:103 Ullrich, S.E., 2:13 Uribelarrea, M., 24(1):133 Vanderleyden, J., .23:21 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 Volk, G.M., 23:291 Vuylsteke, D., 14:267 Wallace, D.H., 3:21; 13:141 Walsh, B. 24(1):177 Wan, Y., 11:199 Waters, C., 23:291 Weber, K., 24(1):249 Weeden, N.F., 6:11 Wehner, T.C., 6:323 Wenzel, G. 23:175 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 Wong, G., 22: 165 Woodfield, D.R., 17:191 Wright, D., 25:173 Wright, G.C., 12:81 Wu, L., 8:189 Wu, R., 19:41 Xin, Y., 17:1,15 Xu, S., 22:113 Xu, Y., 15:85; 23:73 Yamada, M., 19:191 Yan, W., 13:141 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 Zinselmeier, C., 25:173 Zohary, D., 12:253