Genetic Resources, Chromosome Engineering, and Crop Improvement: Oilseed Crops, Volume 4 (Genetic Resources, Chromosome Engineering, and Crop Improvement)

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Volume 4

GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT Oilseed Crops

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GENETIC RESOURCES, CHROMOSOME ENGINEERING, A N D C R O P I M P R OV E M E N T S E R I E S

Series Editor, Ram J. Singh

Genetic Resources, Chromosome Engineering, and Crop Improvement Volume 1: Grain Legumes edited by Ram J. Singh and Prem P. Jauhar Genetic Resources, Chromosome Engineering, and Crop Improvement Volume 2: Cereals edited by Ram J. Singh and Prem P. Jauhar Genetic Resources, Chromosome Engineering, and Crop Improvement Volume 3: Vegetable Crops edited by Ram J. Singh

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GENETIC RESOURCES, CHROMOSOME ENGINEERING, A N D C R O P I M P R OV E M E N T S E R I E S

Series Editor, Ram J. Singh

Volume 4

GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT Oilseed Crops edited by

Ram J. Singh

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

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CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2007 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-3639-2 (Hardcover) International Standard Book Number-13: 978-0-8493-3639-3 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

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Dedication

Gerhard Röbbelen This book is dedicated to Professor Gerhard Röbbelen, whose early and innovative cytogenetic research contributions on Arabidopsis thaliana (L.) Heynh. and Brassica species are being verified by molecular methods. He laid the foundation of the first international Arabidopsis core collection and newsletter. He pioneered in breeding of high-yielding rapeseed cultivars with improved oil and meal quality and was awarded honorary doctorates in agriculture (Dr. Agr. h.c.) from three highly prestigious European universities. During his more than 30 years of professorship, many national and international pre- and postdoctoral fellows were beneficiaries of his expertise.

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Preface What is a weed? A plant whose virtues have not been discovered. — Ralph Waldo Emerson (1803–1882)

Exotic germplasm, often known as weeds, are wild relatives of crops. These wild relatives may contain many undesirable qualities, but they may also harbor a few traits of economic importance that can be transferred to cultigens by conventional chromosome engineering, as well as by molecular methods. Major cereals, grain legumes, oilseeds, vegetables, and forage crops have been an integral part of human civilization since time immemorial. They were selected and domesticated from their wild progenitors. These crops are the primary food source for the world human and animal populations. The majority of the oilseed crops originated in the Old World. Groundnut and sunflower originated in the New World. Today, groundnut and sunflower are important oil crops and are produced throughout the world. Vegetable oils are an excellent source of highly nutritional cooking oil for humans, and oil cake and meal products provide protein-rich meal for animals. Pharmaceutical industries use vegetable oils for producing cosmetics, medicines, and numerous oil-based products. Oil processing industries refine heart healthy oil as well as by-products. Various methods of oil extraction and purification and development of foods for human and animal use have been discovered, invented, and improved upon since humans identified oilseed crops and learned their use. The indigenous Indian method of oil extraction, known as ghani, is still practiced in the villages of India. Modern oil processing industries have constructed refineries worldwide. Processing industries extract oil from the meal and remove antinutritional elements during processing. Oilseed crops are one of the wonders of nature because they are used for human food (salad oil, margarine, Vanaspati, shortening, cooking, and bakery) and animal feed, as well as for other commercial applications (pharmaceutical products, soap, paints and resins, coatings, linoleum, cosmetics, lubrication, chemicals, plastic coatings, and ethanol). However, information on oil extraction, refinement, and production of meals for human, animal, and technical use is beyond the scope of this book. Because of their healthful fatty acid composition, vegetable oils provide us heart-healthy polyunsaturated fatty acids that are free of hydrogenated trans fatty acids. However, vegetable oils turn unhealthy when reheated at high temperatures, as they release a toxin called 4-hydroxy-trans2-nonenal (HNE). Numerous studies link HNE consumption to increased risks for cardiovascular disease, stroke, Parkinson’s, Alzheimer’s, and Huntington’s diseases, liver ailments, and even cancer (). Sunflower oil is not commonly used for industrial purposes because of its generally higher value, compared to other oilseeds. However, it is used to some extent in some paints, varnishes, and plastics. It imparts desirable semidrying properties, without the yellowing problems associated with oils high in linolenic acid. Sunflower oil is also used in the manufacture of soaps and detergents. Vegetable oils have potential value for the production of adhesives, agrichemicals, surfactants, plastics and plastic additives, fabric softeners, synthetic lubricants, and coatings. Actual use depends, to a large extent, on its price, relative to that of petroleum and petro-based chemicals. The prices of oilseeds and their products depend on the supply of oilseeds, and the market is supply oriented. Some oilseed crops (soybean, groundnut, sunflower, sesame) may be consumed raw or cooked as food. Non-oilseed or confectionery sunflower seeds usually have very large, black with whitestriped achenes and are used as a confection or snack food, roasted and salted. Sunflower kernels are also used in the baking industry, as a condiment for salads, and other foods. Non-dehulled or partially dehulled sunflower meal can be substituted successfully for soybean meal of equal protein percentage in feeding ruminant animals. Achenes are also used for feeding birds and in small animal feed. Sesame has been primarily an oil crop, but the seeds are also used in baked goods, for condiments, and in making sweets such as halva and candy bars, and in fast foods.

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Soybean accounts for 53% of all the oilseed crops (excluding perennial tree crops) production listed by the Food and Agriculture Organization (FAO) (). Thus, soybean plays a dominant role in the international trade, foreign exchange earnings, and oil processing because it is rich in protein (40%) and oil (20%). Rapeseed contains 36 to 50% oil (on a dry matter basis), while the oil-free meal contains 33 to 48% protein. Soybean, although originating in China, is produced mainly by the U.S., Brazil, and Argentina. Soybean is considered a miracle crop of the Orient because in addition to oil and protein, the root nodules of soybean contain rhizobial bacteria, which play a positive role in crop rotations with cereals and help replenish soil’s nitrogen supply. It has been used for generations in India, especially in the hills, as both animal and human feed. The intensive varietal improvement of oilseed crops for high yield and improved nutritional quality (elimination of antinutritional quality) are the primary breeding objectives of various national (public institutions and private industries) and international programs. The groundnut varietal improvement program is among those mandated by the International Crops Research Institute for the Semiarid Tropics (ICRISAT), Patancheru, India. The Asian Vegetable Research and Development Center (AVRDC), Shanhua, Taiwan, breeds edible vegetable soybean known as edamame. The International Soybean Program (INTSOY), established in 1973, at the University of Illinois at Urbana-Champaign, promotes soybean products worldwide and is an international soybean resource. However, it does not have a soybean varietal improvement program. Most genetic improvement of oilseed crops has been accomplished by conventional breeding assisted by germplasm resources, cytogenetics, plant pathology, entomology, agronomy, cell and tissue cultures, and molecular biology. A major technological breakthrough by genetic engineering was the creation of Roundup Ready® soybean and rapeseed (widely known as canola). Roundup Ready soybean is widely grown in the U.S. and rapeseed in Canada. Production of both crops is expanding in other countries of the world. Three books on oilseed crops are in print: Salunkhe and Desai (1986) published a book on postharvest biotechnology of oilseeds (CRC Press); Röbbelen, Downey, and Ashri (1989) edited Oil Crops of the World (McGraw-Hill); and Weiss (2000) revised (second edition) his book on oilseed crops. With the exception of soybean and rapeseed, however, there are no consolidated accounts of germplasm resources, cytogenetic manipulations, biotechnological approaches, and breeding of the remaining oilseed crops. Volume 4 in the Genetic Resources, Chromosome Engineering, and Crop Improvement series provides a comprehensive, consolidated resource for seven oilseed crops. Worldrenowned scientists contributed chapters on the vegetable oilseed crops of their expertise. Chapter 1 summarizes the landmark research done in the seven oilseed crops discussed in this book. Each chapter provides a comprehensive account of the origin of the crop, its genetic resources in various gene pools, basic and molecular cytogenetics, conventional breeding, and the modern tools of molecular genetics and biotechnology. Appropriate germplasm collections can be an excellent source for genetic enhancement of various traits in oilseed crops and for broadening their genetic base. The genetic base of oilseed crops is extremely narrow. In view of this, three gene pools have now been identified by scientists: primary (GP-1), secondary (GP-2), and tertiary (GP-3) for each crop. The recommendation is to use GP-2 and GP-3 resources in producing widely adapted varieties. Utilization of these resources in producing high-yielding cultivars, resistant to abiotic and biotic stresses, and with improved nutritional qualities, is discussed in this book. Seven major oilseed crops, such as soybean (Chapter 2), groundnut (Chapter 3), cottonseed (Chapter 4), sunflower (Chapter 5), safflower (Chapter 6), Brassica oilseeds (Chapter 7), and sesame (Chapter 8), are included in this book. Linseed has been excluded from this volume (per Allan Green, personal communication). Perennial tree crops like coconut, babassu nut, oil palms, and jojoba are also not included. Each chapter has been written by one or more experts in the field. I am extremely grateful to all the authors for their outstanding contributions, and to the reviewers of all the chapters. I have been fortunate to know them both professionally and personally, and our communication has been very cordial and friendly. I am particularly indebted to Thomas G. Isleib, Govindjee,

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and Joseph Nicholas for their comments and suggestions on some of the chapters. Finally, I thank Steven G. Pueppke, former associate dean and research director at the University of Illinois, Urbana, for his support and encouragement. This book is intended for scientists, professionals, and graduate students whose interests center upon genetic improvement of crops in general and major oilseed crops in particular. This book is intended as a reference for plant breeders, taxonomists, cytogeneticists, germplasm explorers, pathologists, entomologists, physiologists, agronomists, molecular biologists, food technologists, and biotechnologists. Graduate students in these disciplines, with an adequate background in genetics, as well as other researchers interested in biology and agriculture will also find this volume a worthwhile source of reference. I sincerely hope that the information assembled will help in the much needed genetic amelioration of oilseed crops to feed the ever-expanding global population. I anticipate that this book will enhance awareness regarding nutritive values of oilseeds, preventing malnutrition worldwide. Ram J. Singh Urbana-Champaign, Illinois E-mail: [email protected]

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The Editor Ram J. Singh, M.Sc., Ph.D., is an agronomist–plant cytogeneticist in the Department of Crop Sciences, University of Illinois at Urbana-Champaign. He received his Ph.D. degree in plant cytogenetics under the guidance of the late Professor Takumi Tsuchiya from Colorado State University, Fort Collins, CO. He benefited greatly from the cytogenetic expertise of Drs. T. Tsuchiya, G. Röbbelen, and G. S. Khush. Dr. Singh conceived, planned, and conducted pioneering research related to cytogenetic problems in barley, rice, rye, wheat, oat, and soybean. He isolated monotelotrisomics and acrotrisomics in barley, identified them by Giemsa C- and N-banding techniques, and determined chromosome arm–linkage group relationships. In rice, he established a complete set of primary trisomics and determined a chromosome–linkage group relationship. In soybean (Glycine max), he established genomic relationships among species of the genus Glycine, and each species was assigned a genome symbol based on cytogenetics and molecular methods. Singh constructed, for the first time, a soybean chromosome map based on pachytene chromosome analysis that laid the foundation for creating a global soybean map. By using fluorescent genomic in situ hybridization, he confirmed the tetraploid origin of the soybean. Dr. Singh has published 69 research papers in reputable international journals, including American Journal of Botany, Chromosoma, Crop Science, Genetics, Genome, Journal of Heredity, Plant Breeding, and Theoretical and Applied Genetics. In addition, he has summarized his research results by writing 13 book chapters. His book Plant Cytogenetics (first edition, 1993; second edition, 2003) is widely used for teaching graduate students. Dr. Singh has presented research findings as an invited speaker at national and international meetings. In 2000, he received the Academic Professional Award for Excellence: Innovative and Creativity from the University of Illinois at Urbana-Champaign. He was invited as visiting professor (October 12, 2004 to January 12, 2005) by Professor Kiichi Fukui, Osaka University, Osaka, Japan. He is the editor of the Genetic Resources, Chromosome Engineering, and Crop Improvement series and has published Grain Legumes, Volume 1, and Cereals, Volume 2, and Vegetable Crops, Volume 3.

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Contributors Amram Ashri R.H. Smith Institute of Plant Sciences and Genetics in Agriculture Hebrew University of Jerusalem Rehovot, Israel Gyuhwa Chung Department of Biotechnology Yosu National University Yosu, Chonnam, South Korea Wolfgang Friedt Department of Plant Breeding Justus Liebig University of Giessen Giessen, Germany Corley Holbrook USDA-ARS Tifton, Georgia Chao-Chien Jan USDA-ARS Northern Crop Science Laboratory Fargo, North Dakota R.J. Kohel Crop Germplasm Research Unit College Station, Texas Boshou Liao Oil Crops Research Institute of Chinese Academy of Agricultural Sciences Wuhan, Hubei, China Wilfried Lühs Department of Plant Breeding Justus Liebig University of Giessen Giessen, Germany

Randall L. Nelson USDA-ARS Department of Crop Sciences National Soybean Research Laboratory University of Illinois Urbana, Illinois N. Nimbkar Nimbkar Agricultural Research Institute (NARI) Tambmal, Phaltan, Maharashtra, India Gerald J. Seiler USDA-ARS Northern Crop Science Laboratory Fargo, North Dakota Ram J. Singh Department of Crop Sciences National Soybean Research Laboratory University of Illinois Urbana, Illinois Vrijendra Singh Nimbkar Agricultural Research Institute (NARI) Tambmal, Phaltan, Maharashtra, India Rod Snowdon Department of Plant Breeding Justus Liebig University of Giessen Giessen, Germany J.Z. Yu Crop Germplasm Research Unit College Station, Texas

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Contents Chapter 1 Landmark Research in Oilseed Crops ...............................................................................................1 Ram J. Singh Chapter 2 Soybean (Glycine max (L.) Merr.)...................................................................................................13 Ram J. Singh, Randall L. Nelson, and Gyuhwa Chung Chapter 3 Groundnut.........................................................................................................................................51 Boshou Liao and Corley Holbrook Chapter 4 Cottonseed ........................................................................................................................................89 R.J. Kohel and J.Z. Yu Chapter 5 Sunflower........................................................................................................................................103 Chao-Chien Jan and Gerald J. Seiler Chapter 6 Safflower (Carthamus tinctorius L.)..............................................................................................167 Vrijendra Singh and N. Nimbkar Chapter 7 Brassica Oilseeds ...........................................................................................................................195 Rod Snowdon, Wilfried Lühs, and Wolfgang Friedt Chapter 8 Sesame (Sesamum indicum L.)......................................................................................................231 Amram Ashri Index...............................................................................................................................................291

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CHAPTER 1 Landmark Research in Oilseed Crops Ram J. Singh

CONTENTS 1.1 1.2 1.3 1.4 1.5

Introduction...............................................................................................................................1 Importance of Oilseed Crops ...................................................................................................3 Establishment of International and National Programs...........................................................5 Development of Oilseed Crops Processing Industries ............................................................6 Gene Pools of Oilseed Crops...................................................................................................6 1.5.1 Primary Gene Pool .......................................................................................................6 1.5.2 Secondary Gene Pool ...................................................................................................7 1.5.3 Tertiary Gene Pool .......................................................................................................7 1.6 Germplasm Resources for Oilseed Crops................................................................................7 1.7 Germplasm Enhancement for Oilseed Crops ..........................................................................8 1.7.1 Breeding for High Yield ...............................................................................................9 1.7.2 Breeding Oilseeds for Antinutritional Elements..........................................................9 1.7.3 Breeding for High Oil and Protein ............................................................................10 1.7.4 Breeding for High-Quality Fatty Acids .....................................................................10 1.7.5 Development of Breeding Methods ...........................................................................11 1.8 Conclusions.............................................................................................................................11 References ........................................................................................................................................11 Acknowledgment..............................................................................................................................12

1.1 INTRODUCTION Oilseed crops and human civilization have coevolved in a symbiotic way since ancient time. Soybean, a staple food in China, was domesticated there more than 5000 years ago (Chapter 2). Sesame seed was also known in ancient times, and rapeseed was mentioned in the Indian Sanskrit writings from 2000 B.C. (Hatje, 1989). Charred sesame seeds, about 5000 years old, were found in archaeological excavations in Harapa (Pakistan). Sesame was also known in antiquity in Anatolia (Turkey) and Mesopotamia (now Iraq). Its oil was used in food preparation, lighting, and personal grooming. Sesame is not mentioned in the Bible, but it was well known in the Hellenic and Roman eras in the Middle East (Chapter 8). The domesticated groundnut has been cultivated and utilized in South America for over 3500 years. The earliest archaeological evidence of groundnut in Peru may date back to 1500 B.C. (Chapter 3). 1

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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT

Sunflower 8%

Cottonseed 17%

Groundnut 9% Linseed 1%

Soybean 52% Sesame 1%

Figure 1.1

Rapeseed & Mustard Safflower 12% 0%

Pie diagram showing the world production of eight major oilseed crops in 2005. (From www.faostat.fao.org.)

Million Hectares

World Area Oilseed Harvested in 2005 100 90 80 70 60 50 40 30 20 10 0 Soybean

Figure 1.2

Cottonseed Rapeseed & Groundnut Mustard

Sunflower

Sesame

Linseed

Safflower

Total world area of eight major oilseed crops in 2005. (From www.faostat.fao.org.)

Oilseed crops are diverse in the plant kingdom and belong to several families. Soybean and groundnut are members of the legume family Fabaceae (Leguminoseae) and have been grown in rotation, with other crops benefiting from the nitrogen fixation feature of soybean and groundnut. Rhizobium and related genera in the root nodules of legumes fix atmospheric nitrogen. Much of the nitrogen remains in the soil and is available for subsequent crops. Before 1945, soybean in the U.S. was used as much for forage as for grain. During World War II, the emphasis of soybean production and utilization shifted to a source of oil (Chapter 2). Soybean and groundnut are sometimes considered pulse crops. However, they are now considered oilseeds because they contain more than 20% oil and are used extensively for oil and meal (K. Siddique, personal communication, May 18, 2004). Cottonseed belongs to the family Malvaceae, sunflower and safflower to Asteraceae, Brassica oilseeds to Brassicaceae, sesame to Pedaliacea, and linseed to Linaceae. Seven primary annual oilseed crops are included in Oilseed Crops, Volume 4, of the series Genetic Resources, Chromosome Engineering, and Crop Improvement. They are soybean (Chapter 2), groundnut (peanut) (Chapter 3), cottonseed (Chapter 4), sunflower (Chapter 5), safflower (Chapter 6), Brassica oilseeds (Chapter 7), and sesame (Chapter 8). Soybean production accounted in the year 2005 for 52% of the mainly edible oil production of the seven major annual oilseed crops listed above (www.FAOSTAT.org). It was followed by cottonseed (17%), rapeseed and mustard (12%), groundnut (9%), and sunflower (8%). Linseed (mainly industrial oil) and sesame’s contribution is 1%, and safflower’s was the lowest (Figure 1.1). Soybean is grown on 91,386,621 ha worldwide, while safflower is cultivated on 812,687 ha (Figure 1.2). Other sources of oil, e.g., perennial tree crops

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3

Table 1.1 Classification of the Oils Based on Their Utilization (Hatje, 1989) Edible Oil Uses

Industrial Oil Uses

Salad oil Margarine Vanaspati Shortenings Cooking oils Fats for the bakery confectionary industry and mayonnaise manufacturers Oils for the fish and canning industry

Pharmaceutical products Soap Paints and resins, coatings Linoleum Cosmetics Lubrication Chemicals, candies Technical products Plastic coatings

Feed fats

like coconut, babassu nut, oil palms, olive, castor, and jojoba, are not included in this volume. Oil is also produced from maize, commonly known as corn oil, but is a major cereal crop, described in the Volume 2 (Cereals) of this series. Cottonseed is grown mainly for the fibers, but oil is extracted from the seed (Chapter 4). Brassica species are grown as a vegetable crop (Vegetable Crops, Volume 3 of this series) and for oil and meal (Chapter 7). The main goal of this chapter is to summarize recent knowledge and achievements related to genetic resources, their taxonomy, diversity, collection, conservation, evaluation, and utilization in breeding for seven major vegetable oilseeds of economic importance.

1.2 IMPORTANCE OF OILSEED CROPS Some oilseed crops are used as food for humans and feed for animals. They have been characterized based on their edible and industrial uses (Table 1.1). Vegetable proteins are the most economical source of protein for a large proportion of the population in Asia, Africa, and other impoverished countries. Protein energy malnutrition (PEM) affects every fourth child worldwide. Geographically, more than 70% of PEM children live in Asia, 26% in Africa, and 4% in Latin America and the Caribbean (http://www.worldhunger.org). Oilseed meals and by-products (soy meal, soy milk, and tofu) are a healthy, rich source of protein. Consumption of vegetable protein can reduce PEM for vegetarian populations worldwide. Vegetable oils are also used by industries for producing pharmaceutical products: soap, varnishes, paints, putty, printing inks, erasers, coating, plastics, and greases. Linseed oil is used in protective coatings. Thus, oilseeds, oils/fats, and oil cake/meal play an important role in foreign exchange earnings. Price is determined by supply and demand (Weiss, 2000). Soybean is a miracle crop of many uses. It is rich in high-quality protein with essential amino acids, vitamins (vitamin B, including folate), fiber, essential fatty acids (omega-3 fatty acid, alpha linolenic acid), phytochemicals, and lecithins. Lecithin is an emulsifier used in pharmaceuticals, cosmetics, paints, plastics, and food (in the manufacture of margarine, bakery goods, and chocolate products), and also in compounds for animal feed (Hatje, 1989). Consumption of soybean oil, meal, and products aids in the prevention of heart disease, cancer, kidney disease, osteoporosis, diabetes, and obesity (www.soyfoodilliois.uiuc.edu). Soy milk and tofu do not contain lactose, saturated fat, and cholesterol. Soy milk is an excellent substitute for animal milk and milk products that contain lactose, saturated fat, cholesterol, and antibiotics. De-oiled soy flour, rich in protein, mixed with cereal flour in a ratio of 1:3 is a healthy dietary supplement for vegetarian populations. Food manufacturers add a small amount of soy flour to their baked goods because soy flour extends shelf-life (www.nsrl.uiuc.edu). In the U.S., the sunflower industry has developed a mid-oleic (550 to 700 g/kg) sunflower oil called NuSun® (National Sunflower Association, Bismarck, ND), which possesses a significant advantage over several other popular oils, such as soybean and canola, because it does not have to be

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Table 1.2 Fatty Acid Compositions (%) of Major Oilseed Crops (Vles and Gottenbos, 1989) Fat Type and Source Saturated Butter Beef Lard Coconut Palm Monounsaturated Olive Groundnut Rapeseed Polyunsaturated Maize Soybean Sunflower Safflower

Polyunsaturated Total Linoleic

Saturated

Monounsaturated

63 55 43 90 50

33 41 47 8 39

4 4 10 2 11

1 2 9 2 10

17 18 6

73 52 67

10 30 27

10 30 17

15 16 12 10

30 24 21 15

55 60 67 75

55 53 67 75

hydrogenated prior to its use as a frying oil, and therefore has negligible trans fatty acids. This oil offers desirable frying and flavor characteristics, increases the life span of the heated oil, and confers a healthful fatty acid composition with adequate levels of heart-healthy polyunsaturated fatty acids, yet is free of hydrogenated trans fatty acids. In addition, the increased oleic acid content has the added benefit of slightly lowering saturated palmitic and stearic fatty acid concentrations (Chapter 5). Sesame seeds are used either decorticated or whole in sweets such as sesame seed bars and halva and in baked goods or milled to obtain high-grade edible oil or tahini, an oily paste (Chapter 8). Sesame oil contains natural antioxidants known as lignans (see Section 8.6.6), which prevents rancidity and gives it a long shelf-life. Mixing small amounts of sesame oil with other oil-containing products (e.g., peanut butter) prolongs their shelf-life. Traditionally, sesame oil has been used in home cosmetic preparations and has been reported to ease joint pains when used as an ointment. It is used in the preparation of cosmetic creams and as a carrier for medicines. Sesame oil has been used in some areas for lighting and as a synergist for pyrethrum insecticides. Protein-rich flour can be made from sesame meal. However, in traditional growing areas, sesame meal has been used to feed livestock and as a manure. Sesame stover, remaining after threshing, is sometimes used as fuel. Its use as roughage for farm animals is discouraged because of its woody nature, although goats do feed on it (Chapter 8). Consumers prefer products prepared from vegetable oils such as soybean, cottonseed, sunflower, safflower, rapeseed (also known as canola oil), and corn (maize). These oils are heart-friendly because they contain unsaturated fatty acids. Supermarkets are full of products such as cooking oil, cookies, breads, salad dressing, margarines, shortenings, processed foods, cosmetics, and medicines. Due to consumer demand, fast food chains have now switched to vegetable oils, replacing animal fat (lard), palm kernel, and coconut oil, for making french fries. Saturated fatty acids are major components of lipids that are solid at room temperature. Coconut oil and palm kernel oil that remain solid at room temperature are known as fats, while oils remain liquid at room temperature. Most vegetable oils are liquid at room temperature and have unsaturated fatty acids as their major components. The fatty acid compositions of major oil-producing crops and animals are listed in Table 1.2. Soybean oil is used as industrial lubricants and printing ink. Soybean oil-based paint, cosmetics, and biodegradable crayons are biofriendly by-products of soybean. Soy diesel and by-products are environmentally friendly replacements for their petroleum-based counterparts. Safflower oil may be used in urethane resins, caulks, putties, and linoleum. Sunflower oil is not commonly used for industrial purposes because of its generally higher value, compared to other oils. However, it is

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used to some extent in some paints, varnishes, and plastics because of its good semidrying properties, without the yellowing problems associated with oils high in linolenic acid. Sunflower oil is also used in the manufacture of soaps and detergents. Along with other vegetable oils, it has potential value for the production of adhesives, agrichemicals, surfactants, additional plastics and plastic additives, fabric softeners, synthetic lubricants, and coatings. Actual use will depend to a large extent on the price, relative to that of petroleum and petro-based chemicals (Chapter 5). In India, safflower has been used since time immemorial to extract an orange-red dye from its brilliant florets for food and fabrics and also for the red circle (sindur) on the forehead. Safflower oil is nonallergenic and considered ideal for cosmetics, paints, alkyd resins and coatings, and herbal health tea. Flowers or florets of safflower have medicinal properties, useful to treat hypertension, cardiovascular diseases, arthritis, spondylosis, and sterility in both men and women. SemBioss — a Calgary-based (Canada) company — has transformed safflower that facilitates the genetic attachment of target proteins of interest to oleosin, the primary protein coating the oil-containing vesicles (oil bodies) of the seed. Such attachment permits the target protein to be purified along with the oil body fraction, which, upon centrifugation, floats to the surface of a slurry of ground seeds and water slurry (Stratosome™ system) (Chapter 6). Rapeseed or canola, with its high erucic acid content (22:1n-9), has considerable advantage in specific applications, such as stability at high temperatures, durability, and the ability to remain fluid at low temperatures. Today, methyl esters derived from rapeseed oil are widely used as a diesel substitute (biodiesel). In Europe, low erucic acid rapeseed is a primary feedstock (Chapter 7). Oilseed crops contain several antinutritional elements, aflatoxin, and allergens. Soybean contains a trypsin inhibitor that can be removed by heating soybean seed prior to oil extraction. Aflatoxin is generally produced by molds and is common, particularly in groundnut, and may cause death if contaminated products are consumed. This chemical has also been found in sunflower and soybeans. Erucic acid and glucosinolate found in rapeseed have harmful physiopathological effects on human health. Cottonseeds, devoid of pigment glands (gossypol), are highly desirable for their high nutritional value and for their unique protein properties (Chapter 4). Consumer concern about food quality of groundnut has increased. Groundnut is susceptible to Aspergillus infection, which could result in aflatoxin production during production, processing, storage, and transportation. Generally, contamination by aflatoxin is more serious in the warm tropical and subtropical regions and in systems with poor curing and storage and management. Groundnut and groundnut products can produce an allergic reaction in about 0.6% of the population. Trace amounts of groundnut protein could lead to fatal anaphylactic reactions in individuals allergic to groundnut. Groundnut is dry roasted, which apparently increases the allergic properties of the proteins. Refined groundnut oil does not contain protein, and thus the oil is normally allergen-free. It should be cautioned that vegetable oils heated at high temperature (200°C) may produce carcinogenic chemicals (Salunkhe and Desai, 1986).

1.3 ESTABLISHMENT OF INTERNATIONAL AND NATIONAL PROGRAMS The following international and national centers have been established for oilseed crops research: 1. International Crops Research Institute for the Semiarid Tropics (ICRISAT), Patancheru, India (www.icrisat.org): Groundnut is one of mandated crops of the institute. ICRISAT has the largest collection of domesticated groundnut, consisting of 15,000 accessions from 92 countries. The U.S. Department of Agriculture (USDA) germplasm collection of groundnut contains more than 8000 accessions (Chapter 3). Groundnut is grown in nearly 100 countries. Developing countries account for 96% of the global groundnut production area and 92% of the global production. ICRISAT collects, maintains, and breeds improved groundnut lines and distributes them upon request.

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2. Asian Vegetable Research and Development Center (AVRDC), Shanhua, Taiwan (www.avrdc.org): Of the several vegetable crops, AVRDC breeds edible vegetable soybean known as edamame (fresh green soybeans). 3. The International Soybean Program (INTSOY), established in 1973 at the University of Illinois at Urbana-Champaign, promotes soybean products worldwide and is an international soybean resource. However, INTSOY does not have a soybean varietal improvement program. 4. International Plant Genetics Resources Institute (IPGRI), Rome, Italy (www.ipgri.cgiar.org): This institute is involved in the conservation of germplasm of several oilseed crops. 5. National programs: National (public) and private industries worldwide have oilseed crops improvement programs. For example, USDA-ARS at Urbana, IL, maintains soybean germplasm collection (www.ars-grin.gov), and the National Soybean Research Laboratory at the University of Illinois, Urbana (www.nsrl.uiuc.edu), concentrates on soybean research and product promotion. Monsanto (www.monsanto.com) and Pioneer (www.pioneer.com), private industries in the U.S., are improving soybean and rapeseed (canola) through conventional and biotechnology methods. Sesaco (www.sesaco.net), established in 1978, works on all aspects of sesame, including varietal improvement, exporting, importing, processing, product development, and bulk sales. Public sectors conduct basic research on oilseed crops, develop unique and improved germplasm lines, and release germplasm lines for further improvement by sophisticated private sectors. Private companies release varieties and market produce to consumers.

1.4 DEVELOPMENT OF OILSEED CROPS PROCESSING INDUSTRIES Oilseeds have been crushed to extract oil from the seeds since prehistoric times. The oldest record of oil extraction is from about 2000 B.C. (Weiss, 2000). New and efficient oil extraction, purification, processing, and storage methods developed as human civilization progressed. Oilseeds are cleaned of impurities to produce high-quality oil. They are crushed, flaked, and hulled before they are pressed by an expeller process or treated with solvent to extract oil. Weiss (2000) described the history of the development of oil processing methods used worldwide. Information on major oilseed crops processing is published annually (www.soyatech.com). Archer Daniels Midland (ADM) Company, Decatur, IL, is one of the world’s largest agricultural processors of soybean, maize, wheat, and cocoa. ADM turns these crops into soy meal and oil, maize (corn) sweeteners, flour, cocoa, and chocolate. ADM also produces ethanol, biodiesel, and other value-added ingredients for animal nutrition and industrial products (www.admworld.com).

1.5 GENE POOLS OF OILSEED CROPS Based on reviewing literature on hybridization, Harlan and de Wet (1971) proposed a three-gene pool concept, primary (GP-1), secondary (GP-2), and tertiary (GP-3), for utilization of germplasm resources for crop improvement. Genetic resources are developed with integrated, multidisciplinary approaches through plant exploration, taxonomy, genetics, cytogenetics, plant breeding, microbiology, plant pathology, entomology, agronomy, physiology, distant hybridization, and molecular biology, including cell and tissue culture, DNA analyses, and genetic transformation. These efforts have produced superior oilseed cultivars with resistant to abiotic and biotic stresses and improved oil quality and quantity. The concept of primary, secondary, and tertiary gene pools and genetic transformation has played a key role in improving oilseed crops (Chapters 2 to 8), as in other crops. 1.5.1

Primary Gene Pool

The primary gene pool (GP-1), consisting of landraces and biological species, has been identified for oilseed crops described in this volume. Wild progenitors of cultivated oilseed crops are identified,

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postulated, and proposed based on geographical distribution, classical taxonomy, cytogenetics, and molecular methods (Chapters 2 to 8). Each chapter describes the primary gene pool in detail. For example, the GP-1 for soybean (2n = 40) is only its wild annual progenitor Glycine soja (2n = 40), and for rapeseed, Brassica carinata (Ethiopian mustard; 2n = 34), Brassica juncea (Indian mustard, brown mustard; 2n = 36), Brassica napus ssp. napus (oilseed rape, fodder rape; 2n = 38), B. napus ssp. napobrassica (Swede; 2n = 38), and B. napus ssp. napus var. pabularia (leaf rape, kale; 2n = 38) (Table 7.1). 1.5.2

Secondary Gene Pool

The secondary gene pool (GP-2) includes all species that can be hybridized with GP-1 with at least some fertility in F1s resulting in gene transfer (Harlan and de Wet, 1971). Glycine max does not have a GP-2 (Chapter 2). The GP-2 for Brassica oilseeds includes B. nigra, B. oleracea (includes crop varieties, B. alboglabra, B. bourgeaui, B. cretica, B. hilarionis, B. incana, B. insularis, B. macrocarpa, B. montana, B. rupestris, and B. villosa), and B. rapa (includes wild and cultivated varieties) (Table 7.1). 1.5.3

Tertiary Gene Pool

The tertiary gene pool (GP-3) is the extreme outer limit of potential genetic resources. Prezygotic barriers can cause partial or complete hybridization failure, inhibiting introgression between GP-1 and GP-3 (Singh, 2003). GP-3 is available for oilseed crops (Chapters 2 to 8). The tertiary gene pool in soybean includes 26 wild perennial species of the subgenus Glycine. All species are diverse morphologically and genomically and grow in wide agro-geo climatic environments. GP-3 may harbor traits of economic importance. These traits can be transferred to soybean through wide hybridization, and such technology is being developed (Chapter 2). Rapeseed has a large number of species, and the chromosome number ranges from 2n = 14 to 90 (Chapter 7). Several newly described wild species of cottonseed from Australia belong to GP-3. Technology is being developed to transfer useful genes to cultivated crops from GP-3. Exotic species for sesame (Chapter 8) still need to be studied.

1.6 GERMPLASM RESOURCES FOR OILSEED CROPS National and international institutes and private industries for the seven oilseed crops presented in this volume collect, maintain, disseminate, and develop breeding lines and varieties. Cultivars with traits such as resistance to abiotic and biotic stresses, high yield, and improved nutritional quality and quantities, particularly protein, oil, and fatty acids, have been developed and maintained. Plant exploration of landraces and wild relatives of the seven oilseed crops (Chapters 2 to 8) described in this book is extensive. Collected species have been characterized based on classical taxonomy, cytogenetics, and molecular methods for soybean, groundnut, cotton, sunflower, safflower, and Brassica oilseeds. A clear understanding of cytogenetics and molecular genetics is lacking for sesame. Of the seven oilseed crops described in this volume, groundnut and sunflower originated in the New World. Furthermore, tetraploid cotton (Gossypium barbadanse and G. hirsutum; 2n = 52; AD genome), 13 diploid (2n = 26; D genome) species originated in the New World, and diploid cultivated (2n = 26; A genome) and wild species originated in the Old World. Several new diploid wild species of Gossypium have been collected and identified in Australia (Chapter 4). The center of origin of soybean is China, and its wild perennial relatives are widespread in Australia (Chapter 2). Sesame originated in India (Chapter 8). Safflower, a minor oilseed crop (Figure 1.1 and Figure 1.2), originated in the Middle East (Chapter 6). Linseed or flax has been grown for oil and fiber since 6000 B.C. and was grown primarily for its fiber in the Near East. In Abyssinia

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(Ethiopia), flax is used as a cereal (Vavilov, 1992). Basic chromosome numbers in the genus Linum are x = 8, 9, 10, 12, 14, 15, and 16, and cultivated linseed contains 2n = 30 (Lay and Dybing, 1989). Due to the large number of closely related cruciferous crop species, an enormous variety of germplasm resources are available in international genebank collections for evaluation and introgression of traits of agronomical interest into oilseed Brassica breeding materials (Chapter 7). Use of cytogenetics and molecular genetics has helped establish the genomic relationships among species and discovered the ancestral species of soybean, groundnut, cotton, Brassica oilseeds, safflower, and sunflower. Somatic chromosomes of oilseed crops are small and morphologically indistinguishable (Chapters 2 to 8). Pachytene chromosomes have been used to construct karyotypes of soybean (Chapter 2) and Brassica oilseed (Chapter 7). A cytogenetic study of oilseed crops has not parallels that of to cereals. Researchers have begun to develop molecular maps for oilseed crops without establishing chromosome–linkage group relationships. The only exception is Brassica oilseed (Chapter 7). The Multinational Brassica Genome Project (MBGP) has been established by international Brassica researchers to coordinate Brassica genomics activities and to achieve common goals, i.e., to develop freely available genetic resources for Brassica genome analysis. These include mapping populations, markers, genomic libraries, expressed sequence tags (ESTs), and genomic sequences. Using a computational tool developed by the Plant Biotechnology Center of Latrobe University in Australia, the end sequences of the B. rapa bacterial artificial chromosomes (BACs) are being comparatively mapped onto the Arabidopsis genome. Scientists are producing comprehensive data for navigation between homoeologous sequences in the genomes of B. rapa, B. oleracea, and A. thaliana (Chapter 7). Molecular linkage groups in soybean have been developed by several laboratories using various molecular markers, but linkage groups—cytologically identifiable chromosomes—are unknown. Twenty possible primary trisomic and 15 tetrasomic stocks have been produced in soybean. Eleven molecular linkage groups have been associated with chromosomes by primary trisomics, but these stocks are not available to reconfirm primary trisomic results and to continue this study (Chapter 2). In 2005 the U.S. National Science Foundation granted $4.5 million for sequencing the soybean genome to a team of researchers headed by Dr. Scott Jackson (http://news.uns.purdue.edu/html4ever/2005/ 051018.Jackson.soygenome.html). High-yielding oilseed cultivars produced by either conventional methods or genetic transformation are a threat to the natural habitats of the allied species and genera. It is very important, therefore, that the invaluable germplasm of allied species and genera be collected before they become extinct. International and national institutions are collecting, characterizing, and preserving indigenous cultivars, landraces, and wild relatives in medium- and long-term storage genebanks (Chapters 2 to 8).

1.7 GERMPLASM ENHANCEMENT FOR OILSEED CROPS Varietal improvement programs develop elite breeding lines. Development of these lines is dependent upon the breeding objectives, the type of oilseed crops (cross-and self-pollinated, longand short-term reproductive cycle), end use products (meals, oils, and by-products), breeding methods (conventional vs. molecular-aided breeding), and loss vs. benefit of raising a particular crop (supply-and-demand market value). Other important qualities include inheritance patterns and heritability of the selected characters, such as antinutritional factors (gossypol in cotton, aflatoxin in groundnut, erucic acid and glucosinolate in rapeseed). Other trait objectives are improved quantity and proportions and quality of fatty acids, lignan antioxidant, and immunoglobulin E (IgE)-mediated food allergens in sesame, yield heterosis, plant architecture modification, earliness, stress resistance, resistance to shattering, and lodging. These breeding lines may be improved from the available germplasm, mutation breeding, and genetic transformation (Chapters 2 to 8).

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The core collection concept has been utilized in germplasm collection, maintenance, and breeding programs for most of the oilseed crops. The core collection approach to germplasm evaluation is a two-stage approach. The first stage involves examining all accessions in the core collection for a desired trait. This information is then used to determine which clusters of accessions in the entire germplasm collection should be examined during the second stage of screening. Theoretically, the probability of finding additional accessions with a desired characteristic should be highest in these clusters (Chapter 3). For example, the core collection for ICRISAT 14,310 groundnut accessions was reduced to 1704 accessions. Further evaluation reduced the core collection to 184 accessions, known as the mini-core (core of core). The mini-core of 184 represents the entire 14,310 ICRISAT collection. It has been suggested that the mini-core collection may be used to improve the efficiency of identifying desirable traits in the core collection and in the entire collection (Chapter 3). There are major cotton collections in France, the People’s Republic of China, Russia, the U.S., and Uzbekistan. The U.S. cotton germplasm bank has 9000 accessions (Chapter 4). The National Plant Germplasm System (NPGS) maintains 3860 sunflower accessions from 59 countries and is one of the largest and most genetically diverse ex situ collections in the world. The core subset of sunflower consists of 112 accessions (Chapter 5). India maintains 7316 safflower accessions at the Germplasm Management Unit (GMU) of the Directorate of Oilseeds Research, Hyderabad. The safflower germplasm collection in the U.S. contains 2288 accessions (Chapter 6). Passport data for over 19,000 Brassica oilseed accessions are available in the ECP/GR Central Crop Database (www.cgn.wur.nl/pgr/collections/brasedb/). From a total of 3787 entries of B. napus, a preliminary core collection of 200 accessions was selected covering all the systematic groups. This core collection was further reduced to around 150 accessions from 1100 accessions that cover the broad variations of agriculturally important traits (Chapter 7). The National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India, maintains 6658 sesame accessions. Other countries, such as China, the U.S., the Republic of Korea, and Venezuela, maintain other sesame collections. Core collections of sesame are being developed to eliminate duplicate and triplicate collections in India and China (Chapter 8). 1.7.1

Breeding for High Yield

The present genetic base of oilseed crops is extremely narrow because breeders have largely confined their varietal improvement programs to GP-1. Soybean breeders have not exploited G. soja, a wild annual progenitor of soybean, and wild perennial species to broaden the genetic base of modern soybean cultivars. Soybean yield in India has been under 1 MT/ha. Mutation breeding has produced soybean cultivars with high yield in China, Japan, and Korea (Chapter 2). Groundnut breeders in China used only two local varieties — Fahuasheng and Shitouqui — in varietal improvement. Their pedigree could be traced to more than 60% of the groundnut cultivars released. Most of the groundnut varieties released are from conventional methods (pedigree, backcross, mass selection, and single seed descent). The most prevalent species of cultivated cotton, G. hirsutum, accounts for 90% of the world production (Chapter 4). In cotton, earliness is a desired trait that could help minimize production inputs, avoid hazardous weather, or facilitate double-cropping systems (Chapter 4). The use of the cytoplasmic male sterility (CMS) system has been suggested to produce hybrid cotton, groundnut, safflower, and rapeseed. Mutation breeding technology has been discussed for varietal improvement of soybean, groundnut, cottonseed, sunflower, safflower, rapeseed, and sesame. 1.7.2

Breeding Oilseeds for Antinutritional Elements

The nutritional value and industrial suitability of oilseed crops are determined by fatty acid compositions. Oilseed crops may also contain antinutritional elements. These antinutritional

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elements may be removed by plant breeding and seed processing. Soybean seed protein contains an undesirable Kunitz trypsin inhibitor, which may be removed by treating the moist seed with heat (Chapter 2). Groundnut cultivars resistant to Aspergillus are being developed to eliminate aflatoxin contamination (Chapter 3). Cottonseed and aerial portions of the plant contain pigment glands, known as gossypol and gossypol precursors, the greatest quality detriment in cottonseeds. The gossypol trait is a complex developmental trait. Biotechnological tools are being used to produce gossypol-free cotton. A breeding program in California resulted in the release of a cultivar with decreased seed gossypol and increased seed-oil content. However, gossypol-free cottonseed has not been produced and remains an unrealized potential (Chapter 4). Sunflower protein contains chlorogenic acid that gives an undesirable greenish color to sunflower meal (Chapter 5). Safflower varieties are spiny. It is desirable to produce spineless varieties. In China, the entire safflower production uses spineless cultivars. In India, earlier developed spineless cultivars produced lower yields than spiny cultivars. However, breeders have produced spineless cultivars with yields equal to those of spiny cultivars (Chapter 6). Brassica oilseeds contain erucic acid and glucosinolate, both of which are considered antinutritional elements. Varietal improvement has produced double-zero canola cultivars with very low erucic acid and low glucosinolates. Canola, low in saturated fatty acids, meets all the requirements of prime edible oil. Improvement of the C18 fatty acid composition in rapeseed has been achieved by selecting altered linoleate/linolenate genotypes after chemical mutagenesis (Chapter 7). Sesame seeds contain immunoglobulin E (IgE)-mediated food allergens, and sesame seed allergy is becoming more prevalent due to the wider and expanding use of sesame seeds in baked goods and fast foods. At least 10 allergenic proteins in sesame seeds have been identified. Sesame seeds taste bitter and sweet. Sweet sesame seeds are preferred by both consumers and processors. The bitter taste in the seeds is caused by an oxalic acid compound in the seed coat that can be reduced markedly by decortication (Chapter 8). 1.7.3

Breeding for High Oil and Protein

The ultimate aim of oilseed breeders is to produce high oil and protein contents. However, variation in oil and protein contents depends on genotype and environmental factors (Chapter 5). Soybean seed oil is the major vegetable oil (53%) among oilseed crops produced in the world. Oil and protein contents are negatively correlated. Breeding efforts to increase soybean oil above 20% and protein above 40% have been unsuccessful because oil and protein contents are negatively correlated to seed yield (Chapter 2). Oil content in groundnut ranges from 35 to 60%, with an average of about 50%. Several groundnut varieties with high oil content and yield have been developed in China (Chapter 3). Selection for high protein usually results in lower oil content because of the negative correlation between the two traits (Chapter 5). Indian safflower varieties contain 28 to 32% oil. However, safflower lines with oil content as high as 55% have been reported (Chapter 6). The oil content in Brassica oil crop species ranges from 36 to 50%, while oil-free meal contains 33 to 48% protein (Chapter 7). Sesame seed that contains about 50% oil is close to its biological limit. Higher oil content may lead to lower yields. Black sesame seed contains significantly less oil (46.8%) than white (55.0%) or brown (54.2%) seeds. Black-seeded sesame is not used for oil extraction because it colors the oil (Chapter 8). 1.7.4

Breeding for High-Quality Fatty Acids

Quality of oil is determined by the fatty acid composition of the oil. Vegetable oils rich in polyor monounsaturated fatty acids are considered desirable because they aid in reducing blood cholesterol (Table 1.2). Mutagenesis has played a major role in modifying fatty acids of several oilseed crops (Chapters 2, 5, and 7). Genetic transformation may improve desired fatty acid content if such a system is developed as has been for soybean (Chapter 2).

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Development of Breeding Methods

Breeding methodologies have been developed depending upon breeding systems. Oilseed crops may range from highly self-pollinated (soybean, groundnut) to often cross-pollinated (cotton, sunflower, safflower, Brassica oilseeds, sesame) plants. Hybrid sunflower, safflower, and rapeseed are produced using cytoplasmic male sterility (Chapters 5 to 7). Conventional breeding methods (selection, pedigree, bulk, backcross, single-seed descent) have produced oilseed crops with high yield and containing resistance to biotic stresses and tolerance to abiotic stresses. The use of the haploidy technique (double-haploid (DH)), somatic hybridization, and genetic transformation have improved rapeseed cultivars (Chapter 7). Stable glyphosate-tolerant soybean, known as Roundup Ready® soybean, canola, and insecticide-tolerant cotton (Bt-cotton) have been produced by genetic transformation (Chapters 2 and 7).

1.8 CONCLUSIONS 1. Oilseed crops coevolved with human civilization. They are used as food for humans and feed for animals, and they are characterized on the basis of their edible and industrial uses. Crop rotation of soybean or groundnut with cereals enriches the soil because these legumes fix nitrogen in symbiotic association with Rhizobium species. 2. Of the seven major annual oilseed crops discussed in this volume, 52% of the oil is derived from soybean production, followed by cottonseed (17%), rapeseed and mustard (12%), groundnut (9%), and sunflower (8%). Sesame contribution is 1%, and safflower is the lowest. 3. Oilseed crops are heart-friendly because they contain unsaturated fatty acids. Supermarkets contain various kinds of cooking oil produced from soybean, canola, corn, groundnut, and sunflower, and these oils are major ingredients to produce dressings, margarines, shortenings, processed foods, cosmetics, and medicines. Cottonseed oil is also used in baking items. 4. Vegetable oils are also used by industries for producing pharmaceutical products, soaps, varnishes, paints, putties, printing inks, erasers, coatings, plastics, biodiesel, and greases. Oilseeds, oils/fats, and oil cake/meal play an economic role in foreign economies. 5. Oilseed crop breeders have confined their efforts to the primary gene pool (GP-1). Exploitation of the secondary (GP-2) and tertiary (GP-3) gene pools is hampered because of pre- and postzygotic barriers. Researchers have conducted extensive plant explorations to collect primitive cultivars, landraces, and wild relatives before the spread of high-yielding varieties and environmental factors make them extinct. These invaluable genetic materials are deposited in genebanks for mediumand long-term storage. 6. Oilseed crop industries have developed equipment and processing methods to remove antinutritional factors from oil and meal. 7. Breeders have achieved substantial yield gain by conventional and genetic transformation methods. 8. Molecular biology has added to the scope of plant breeding in oilseed crops, providing an option to manipulate plant genes of economic importance. The process has barely begun in some oilseed crops. Researchers will have to strive to combine the best conventional and modern molecular approaches to improve oilseed crops to keep them economically viable global crops.

REFERENCES Harlan, R.R. and J.M.J. de Wet. 1971. Toward a rational classification of cultivated plants. Taxon 20: 509–517. Hatje, G. 1989. World importance of oil crops and their products. In Oil Crops of the World. G. Röbbelen, R.K. Downey, and A. Ashri, Eds. McGraw-Hill, New York, pp. 1–21. Lay, C.L. and C.D. Dybing. 1989. Linseed. In Oil Crops of the World. G. Röbbelen, R.K. Downey, and A. Ashri, Eds. McGraw-Hill, New York, pp. 416–430. Salunkhe, D.K. and B.B. Desai. 1986. Postharvest Biotechnology of Oilseeds. CRC Press, Boca Raton, FL. Singh, R.J. 2003. Plant Cytogenetics, 2nd ed. CRC Press, Boca Raton, FL.

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Vavilov, N.I. 1992. Origin and Geography of Cultivated Plants. D. Löve, Trans. Cambridge University Press, Cambridge, U.K. Vles, R.O. and J.J. Gottenbos. 1989. Nutritional characteristics and food uses of vegetable oils. In Oil Crops of the World. G. Röbbelen, R.K. Downey, and A. Ashri, Eds. McGraw-Hill, New York, pp. 63–86. Weiss, E.A. 2000. Oilseed Crops, 2nd ed. Blackwell Science Ltd., London.

ACKNOWLEDGMENT The author thanks Drs. Govindjee, G. Seiler, A. Ashri, R. Snowdon, and C.C. Jan for their critical review of this chapter.

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CHAPTER 2 Soybean (Glycine max (L.) Merr.) Ram J. Singh, Randall L. Nelson, and Gyuhwa Chung

CONTENTS 2.1 2.2 2.3

2.4

2.5

Introduction.............................................................................................................................14 Domestication and Dissemination of Soybean ......................................................................15 Botany.....................................................................................................................................17 2.3.1 Taxonomy ...................................................................................................................17 2.3.2 Morphology ................................................................................................................17 Gene Pools of Soybean ..........................................................................................................19 2.4.1 Soybean GP-1.............................................................................................................19 2.4.2 Soybean GP-2.............................................................................................................20 2.4.3 Soybean GP-3.............................................................................................................21 Cytogenetics ...........................................................................................................................21 2.5.1 Evolution of the Glycine Genome .............................................................................25 2.5.2 Genomic Relationships among Diploid Species........................................................27 2.5.2.1 Genome Designation...................................................................................27 2.5.2.2 Classical Taxonomy ....................................................................................27 2.5.2.3 Crossing Affinity .........................................................................................28 2.5.2.4 Chromosome Pairing...................................................................................28 2.5.2.5 Molecular Methods .....................................................................................29 2.5.3 Polyploid Complexes of G. tabacina and G. tomentella ..........................................31 2.5.3.1 Glycine tabacina (2n = 80).........................................................................31 2.5.3.2 Glycine tomentella (2n = 78, 80) ...............................................................32 2.5.4 Chromosomal Aberrations: Structural Changes.........................................................32 2.5.5 Chromosomal Aberrations: Numerical Changes .......................................................32 2.5.5.1 Autopolyploidy............................................................................................32 2.5.5.2 Aneuploidy ..................................................................................................33 2.5.5.2.1 Primary Trisomics.....................................................................34 2.5.5.2.2 Monosomics ..............................................................................33 2.5.5.2.3 Tetrasomics ...............................................................................34 2.5.6 Linkage Mapping........................................................................................................34 2.5.6.1 Chromosome Map.......................................................................................34 2.5.6.2 Classical Genetic Linkage Map..................................................................35 2.5.6.3 Molecular Linkage Map..............................................................................35 13

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2.6

Soybean Germplasm Enhancement........................................................................................36 2.6.1 Conventional Breeding ...............................................................................................36 2.6.2 Interspecific Hybridization .........................................................................................37 2.6.3 Intersubgeneric Hybridization ....................................................................................37 2.6.4 Mutation Breeding......................................................................................................38 2.6.5 Biotechnology.............................................................................................................40 2.6.5.1 Plant Regeneration from Callus and Cell Suspension Cultures ................41 2.6.5.2 Protoplasts Culture ......................................................................................41 2.6.5.3 Genetic Transformation...............................................................................41 2.6.6 Potential to Produce Hybrid Soybeans ......................................................................42 2.7 Summary.................................................................................................................................43 References ........................................................................................................................................43

2.1 INTRODUCTION The soybean (Glycine max (L.) Merr.; 2n = 40) is an economically important leguminous seed crop for feed and food products that are rich in seed protein (40%) and oil (20%). Soybean is ranked number one in world production in the international trade markets among the major oil crops, such as cottonseed, groundnut (peanut), sunflower seed, rapeseed, linseed, sesame seed, and safflower (see Chapter 1). Soybean is widely grown in the U.S., Brazil, Argentina, China, and India. In the past 45 years (1961 to 2004), the U.S. has been the leader in soybean production, and currently produces more than half (53%) of the world production. Soybean yield per hectare has increased over 60% in China and Brazil, over 30% in Argentina and the U.S., but has remained unchanged in India (Figure 2.1; FAO STAT, 2004). The enormous economic value of the soybean was realized in the first two decades of the 20th century. Osborne and Mendel (1917) demonstrated experimentally that heated soybean meal promoted growth in rat at a normal rate, which was in contrast with raw soybean meal. This study resulted in the establishment of soybean processing industries in the U.S. Mr. A.E. Staley Sr. laid the foundation for operational soybean processing facilities in Decatur, IL, in 1922 (Windish, 1981). Soybean is processed worldwide for oil and meal and has great influence on other oilseeds in the international trade. Soybean Yield (Mt) per Hectare 3.00

Yield (Mt/Ha)

2.50 2.00 1.50 1.00

2003

2001

1999

1997

1995

1993

1991

1989

1987

1985

1983

1981

1979

1977

1975

1973

1971

1969

1967

1965

1963

0.00

1961

0.50

Year Argentina

Figure 2.1

Brazil

China

India

USA

A graphic representation of soybean yield (Mt/ha) in the U.S., Argentina, Brazil, China, and India from 1961 to 2004. (From www.FAOSTAT.org.)

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Soybean is not considered a model plant for cytogenetic studies because of the large number of chromosomes (2n = 40), the small and similar chromosome sizes (1.42 to 2.84 μm), and the lack of morphological distinguishing landmarks (Singh, 2003). Using DNA markers, a linkage map with 20 linkage groups has been developed, but not all linkage groups have been associated with the respective chromosomes. A pachytene karyogram (Singh and Hymowitz, 1988) and a set of the possible 20 primary trisomics (Xu et al., 2002c) have been established, but currently the primary trisomics seeds are not available. (Hymowitz, personal communication). Cytogenetics of the soybean is far behind that of maize, barley, rice, wheat, tomato, and others (Singh, 2003). The objective of this chapter is to present information on the origin and domestication of the soybean, the maintenance and use of Glycine genetic resources, and the available genetic and cytogenetic tools for exploiting available germplasm for soybean improvement.

2.2 DOMESTICATION AND DISSEMINATION OF SOYBEAN Soybean was domesticated from the wild soybean, G. soja Sieb. & Zucc. (formerly G. formosana Hosokawa; G. ussuriensis Regal & Maack (Fukuda, 1933; Hermann, 1962)), which is an annual weedy-form climber whose pods contain black seeds that shatter at maturity. G. soja grows wild in China, far eastern Russia, the Korean peninsula, Taiwan, and Japan (Hymowitz, 1970; Singh and Hymowitz, 1999). The wild soybean seed has a wide range of protein concentration (31 to 52%), similar to that of soybean, but is generally much lower in oil (9 to 12%) (Hymowitz et al., 1972). The cultivated soybean and its progenitor G. soja belong to the subgenus Soja (Moench.) F.J. Herm., and both are cross compatible, contain 2n = 40 chromosomes, and produce vigorous fertile intermediate F1 hybrids (Fukuda, 1933; Palmer et al., 1987; Singh and Hymowitz, 1988, 1989). Based on linguistic, geographical, and historical literature, the soybean was likely domesticated during or prior to the Shang dynasty, which ruled China between 1700 to 1100 B.C. (Gai, 1997; Gai and Guo, 2001; Hymowitz and Newell, 1981; Qiu et al., 1999). There is no definitive research that establishes the location of the domestication of soybean. Different authors have indicated that the soybean may have been domesticated in northeastern China (Fukuda, 1933), the middle and lower Yellow River Valley of central China (Hymowitz and Newell, 1981; Xu, 1986; Zhou et al., 1998), southern China (Wang et al., 1973; Gai et al., 1999, 2000; Shimamoto et al., 1998), in a corridor from southwest to northeast China, which included Sichuan, Shaanxi, Shanxi, Hebei, and Shandong provinces (Zhou et al., 1999), or simultaneously at multiple centers (Lu, 1977; Dong et al., 2001). Soybean is reported to have come to the Korean peninsula around the fourth or fifth century B.C.E. (Kim, 1993). Kihara (1969) reported that rice (Oryza sativa L.) was introduced to Japan on Kyushu Island around the third century B.C.E. and that soybean arrived in Japan about the same time as rice. It is possible that soybean in Japan could have come from either Korea or China. Using cluster analyses of DNA marker data from primitive germplasm from China, Japan, and the Republic of Korea, Li and Nelson (2001) showed that accessions from Japan and the Republic of Korea were clearly distinct from Chinese accessions, but not from each other, and were less diverse than the accessions from China. Because soybean was introduced into Japan and Korea centuries earlier than to any other country, these data provide justification that they be considered the secondary center of diversity. It is difficult to find any published history about the spread of soybean to other Asian countries, but soybean may have been cultivated in Vietnam, Indonesia, India, and Nepal longer than other areas of southeastern and central Asia. These, plus other Asian countries, may be considered the tertiary center of diversity. Missionaries and sailors brought the soybean to Europe from China and Japan. Soybean was grown in 1740 in botanical gardens in France and in 1790 in the Royal Botanical Garden, Kew, England. Soybean was cultivated in several European countries, but the acreage was very limited (Morse, 1927). Soybean was introduced to North America from China by Samuel Bowen in 1765

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210,000,000 200,000,000 190,000,000 180,000,000 170,000,000 160,000,000 150,000,000 140,000,000 130,000,000 120,000,000 110,000,000 100,000,000 90,000,000 80,000,000 70,000,000 60,000,000 50,000,000 40,000,000 30,000,000 20,000,000 10,000,000 0

Hectares

Metric Tons

1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

Hectares & Metric Tons

World Soy Production & Area 1961 - 2004

Years

Figure 2.2

A graphic representation of world soybean production and area under cultivation from 1961 to 2004. (From www.FAOSTAT.org.)

and was planted in Greenwich, located at Thunderbolt, a few miles east of Savannah, GA (Hymowitz and Harlan, 1983). Since 1765, soybean has been introduced into the U.S. several times by seed dealers, merchants, plant explorations, and various individuals, but the oldest extant cultivar in the U.S. was not introduced until ca. 1880 (Bernard et al., 1987). Seed and Plant Introduction was established in 1898 within the U.S. Department of Agriculture (USDA) and initiated the introduction of a large number of soybean accessions from Asian countries (Morse, 1927). This facilitated centralized plant introduction activities and preserved records of imported accessions. William J. Morse and P.H. Dorsett conducted plant exploration trips to China, Japan, India, and Korea to enhance the U.S. germplasm resources. They collected (1924–1932) 5,420 accessions of the 7,867 soybean introductions documented between 1898 and 1944 (Bernard et al., 1987). Currently, USDA/ARS at Urbana maintains 18,837 accessions of soybean (G. max) and 1,258 accessions of wild annual soybean (G. soja). Accessions of 16 wild perennial species of the subgenus Glycine are also preserved (http://www.ars-grin.gov/cgi-bin/npgs/html/site_holding.pl?SOY). When the USDA Soybean Germplasm Collection was established in 1949, only 1677 accessions had been preserved in the collections of individual soybean scientists. Before 1945, soybean in the U.S. was used as much for forage as for grain. During World War II, the emphasis of soybean production and utilization shifted to a source of oil. Argentina, Brazil, China, India, and the U.S. are currently the five largest producers of soybeans in the world. According to Food and Agriculture Organization (FAO) statistics (http://faostat.fao.org/), these five countries had approximately 90% of the harvested area and produced approximately 93% of the total crop in each year between 1961 and 2004 (Figure 2.2 and Figure 2.3). The consistency of these figures belies the dramatic changes that have occurred in total area harvested, total production, and the distribution of that production among the five leading countries. In 1961, China had 42% and the U.S. 46% of nearly 24 million ha harvested. The other three countries had 1% or less. The U.S. produced 69% and China 23% of the 23.8 million metric tons. Over the next 20 years, soybean production continued to expand in the U.S. and declined slightly in China. In 1973, 60% of the area harvested and 71% of the production was in the U.S., and only 20% of the area and 14% of the production was in China. Brazil increased from 1 to 10% of the area harvested between 1961 and 1973. Total area harvested in 1973 was 33.8 million ha, and that produced 55.9 million metric

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Average Annual Soy Production and Area per Decade (1960 - 2004)

200,000,000

Hectares & Metric Tons

180,000,000

Area

160,000,000

Production

140,000,000 120,000,000 100,000,000 80,000,000 60,000,000 40,000,000 20,000,000 0 1960s

1970s

1980s

1990s

2000s

Decades Figure 2.3

A graphic representation of average annual soybean production and area per decade (1960 to 2004). (From www.FAOSTAT.org.)

tons of soybeans. In 2004, the area harvested and total soybean production both reached new highs, with 91.4 million ha and 189.3 million metric tons (Figure 2.3). The distribution of the harvested area has also changed significantly (Figure 2.2 and Figure 2.3). Only 33% of the harvested area is in the U.S. Brazil and Argentina have 24 and 16%, respectively. With 11% of current world total, China has nearly the same amount of harvested area as it did in 1961. India, like South America, has greatly increased its production area to 8% of the world total. 2.3 BOTANY 2.3.1

Taxonomy

The taxonomy of the soybean is as follows: Order Family Subfamily Tribe Subtribe Genus Subgenus Species

2.3.2

Fabales Fabaceae (Leguminosae) Papilionoideae Phaseoleae Glycininae Glycine Willd. Soja (Moench) F.J. Herm. max (L.) Merr.

Morphology

Soybean is an annual plant. It exhibits taproot growth initially, followed later by a large number of secondary roots. Roots establish a symbiotic relationship with the bacterium (Bradyrhizobium

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wings

androecium

standard

keel

gynoecium B

sepals A

anthers

stigma style

filament ovary C

Figure 2.4

D

(See color insert following page 144.) Reproductive organs (identified) of soybean: (A) complete mature flower, (B) mature androecium and gynoecium, (C) a mature gynoecium with stigma, style, and ovary, and (D) mature anthers with five anthers on longer filament (outer whorl), four anthers on shorter filament (inner whorl), and one free anther always below the stigma.

japonicum) through formation of root nodules. Soybean has four different types of leaves: the seed (first pair of simple cotyledons leaves; epigeal germination), simple primary leaves, pinnately trifoliolate leaves, and prophylls (a pair of 1-mm-long simple leaves at the base of each lateral branch) (For a detailed description of vegetable morphology, see Lersten and Carlson, 2004.). There are two loci that are known to control stem termination (Dt1 and Dt2) (Woodworth, 1933; Bernard, 1972). With the determinate stem type (dt1) there is usually little growth in stem length after flowering with blunt stem termination and a terminal raceme, whereas with the indeterminate stem type (Dt1) stem elongation and node production continue after flowering, producing a longer, more tapered main stem and branches. There is considerable variation in stem growth within each of these two types, with time of flowering and time of maturity having major effects on stem morphology. An intermediate phenotype is conditioned by the Dt2 genotype and is called semideterminate (Bernard, 1972). Thompson et al. (1997) identified a third allele (dt1-t) at the Dt1 locus. It produces a phenotype for plant height that is similar to Dt2 but with fewer main stem nodes and larger terminal leaflets. Soybean plants enter into the reproductive stage following vegetative growth. Axilary buds develop into clusters of 2 to 35 flowers. From 20 to 80% of the flowers abscise. The earliest and latest flowers produced generally abort most often. Soybean has a typical papilionaceous flower with a tubular calyx of five unequal sepals, and a five-parted corolla. The corolla consists of a standard (posterior banner) petal, two lateral wings, and two anterior keel petals contacting each other but not fused (Figure 2.4A). (See color insert following page 144). Stamens are clustered around the stigma, ensuring self-pollination (Figure 2.4B). The gynoecium constitutes an ovary, style, and stigma (Figure 2.4C). As many as four ovules appear in the ovary. Nine stamens are arranged in two whorls; the outer and inner whorls contain five and four stamens, respectively. The two whorls of nine stamens align themselves into a single whorl on a staminal tube. The larger and older stamens alternate with the smaller and younger stamens in sequence around the developing gynoecium. The single (10th stamen) free stamen is the last to appear (Figure 2.4D). Soybean is

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highly self-pollinated with natural crossing usually less than 1% because stamens are elevated so that the anthers form a ring around the stigma; thus, pollen is shed directly on the stigma, ensuring self-pollination (Carlson and Lersten, 2004). Pollen is shed on the stigma. Pollen tubes travel through style and enter into the filiform apparatus. The pollen tube tip bursts and releases two sperm nuclei. One sperm nucleus fuses with the egg and forms a zygote, while the second sperm unites with the secondary nucleus, forming an endosperm. Mature seeds develop from 50 to 80 days after fertilization. They are devoid of endosperm and contain two large fleshy cotyledons, a plumule with two well-developed primary leaves enclosing one trifoliolate leaf primordium, a hypocotyl-radicle axis, a micropyle, a hilum with central fissure, and a raphe (see Carlson and Lersten, 2004). The inflorescence of each node of soybean plant may develop into 1 to more than 20 pods. A plant may have up to 400 pods. The soybean pod is similar to that of other legumes. A pod usually contains 1 to 3 seeds and rarely 4 seeds (Figure 2.5A, B, and C), except for plants that have the have to na allele that produces narrow leaflets and a much higher proportion of 4-seeded pods. Since soybean was introduced into the U.S. from several geographical regions of East Asia, the response to the adopted country was extremely variable. Morse (1927) realized the problem and developed the concept of relative maturity groups based on critical day length. He identified five soybean maturity groups (MGs) (1 = southern through 5 = northern) in the soybean-growing regions of the U.S. Morse et al. (1949) reclassified the maturity groups of soybean and divided varieties into nine maturity groups (0 through VIII). Maturity group 0 and I cultivars were those adapted to the northern part of the country, reverse of the previous classification of Morse (1927). Succeeding maturity groups contained cultivars adapted to areas farther south, with those of group VIII suited for the Gulf Coast region. Now, 13 (000 to X) maturity groups have been established for the appropriate latitude at which maximum commercial yield is produced (Figure 2.6).

2.4 GENE POOLS OF SOYBEAN Harlan and de Wet (1971) developed the concept of three — primary (GP-1), secondary (GP-2), and tertiary (GP-3) — gene pools based on the success rate of hybridization among species. The clear understanding of taxonomic and evolutionary relationships between cultigen and its wild relatives is a prerequisite for the exploitation of primary, secondary, and tertiary gene pools. 2.4.1

Soybean GP-1

Soybean GP-1 consists of biological species that can be crossed to produce vigorous hybrids that exhibit normal meiotic chromosome pairing and possess total seed fertility. Gene segregation is normal and gene exchange is generally easy. Based on this definition, all soybean (G. max) germplasm and the wild soybean, G. soja Sieb. & Zucc., are included in GP-1. Both species have 2n = 40 chromosomes, hybridize easily, and produce normal fertile F1 hybrids; meiotic chromosome pairing is normal, but may differ by a reciprocal translocation (Palmer et al., 1987; Singh and Hymowitz, 1988) or by an inversion (Ahmad et al., 1977). Skvortzow (1927) characterized a distinct species Glycine gracilis Skvortzow; however, subsequent cytogenetic research demonstrated that this species is a hybrid derivative of G. max and G. soja (Fukuda, 1933; Karasawa, 1952; Singh and Hymowitz, 1989), and chloroplast DNA variation suggested that subgenus Soja precludes G. gracilis as an independent species (Shoemaker et al., 1986). However, Chen and Nelson (2004) found that former G. gracilis accessions in the USDA Soybean Germplasm Collection are distinct from both G. max and G. soja based on either morphogical data or SSR allelic diversity. The largest collection of soybean germplasm is the National Crop Gene Bank in Beijing, China, with 25,034 accessions of G. max and 6172 accessions of wild soybean (http://icgr.caas.net.cn/ cgris_english.html). The USDA Soybean Germplasm Collection at Urbana, IL, is the second largest

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Figure 2.5

Mature pods of soybean. A) One, two, three, and four seeded pods; often soybean plants produce one to three seeded pods. B) A mature open pod containing three seeds. C) A mature open pod containing four seeds.

collection, with 18,567 accessions of G. max and 1117 accessions of G. soja (http://www.ars-grin.gov/ cgi-bin/npgs/html/site_holding.pl?SOY). More than 70 countries maintain soybean germplasm collections, and the total world collection exceeds 170,000 accessions, but the number of duplicates is unknown (Carter et al., 2004; IPGRI, 2005). 2.4.2

Soybean GP-2

GP-2 species can hybridize with GP-1 easily, and F1 plants exhibit at least some seed fertility (Harlan and de Wet, 1971). G. max is without GP-2 because no known species has such a relationship

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Figure 2.6

21

Maturity groups (MGs) of soybean cultivars relative to the area in which they would be full season cultivars with a late spring planting date. (From Fehr, W.R., in Oil Crop of the World, Röbbelen, G. et al., Eds., McGraw-Hill, New York, 1989, pp. 283–300. With permission.)

with soybean (Figure 2.7). It is possible that species in the soybean GP-2 exist in Southeast Asia, where the Glycine genus may have originated. It is merely a speculation, and extensive plant exploration in this part of the world is required to validate this assumption. 2.4.3

Soybean GP-3

GP-3 is the extreme outer limit of potential genetic resources. Hybrids between GP-1 and GP-3 are anomalous, lethal, or completely sterile, and gene transfer is not possible or requires radical techniques (Harlan and de Wet, 1971). Based on this definition, GP-3 includes the 26 wild perennial species of the subgenus Glycine. These species are indigenous to Australia and are geographically isolated from G. max and G. soja (Figure 2.8). Table 2.1 shows species, 2n chromosome number, nuclear and plastome genomes, and geographical distribution of the Glycine species. Only three species (G. argyrea, G. canescens, and G. tomentella) have been hybridized with soybean. The USDA Soybean Germplasm Collection maintains 919 accessions of the 16 wild perennial species (http://www.ars-grin.gov/cgi-bin/npgs/html/site_holding.pl?SOY).

2.5 CYTOGENETICS Cytogenetics of soybean has lagged behind that of the other economically important crops, such as maize, wheat, rice, barley, tomato, and faba bean. Soybean is not considered a model crop for cytogenetic studies because it contains a high chromosome number (2n = 40; Karpechenko, 1925; verified by Fukuda, 1933; Veatch, 1934) and small and symmetrical chromosome size (1.42 to 2.84 μm; Sen and Vidyabhusan, 1960), lacks morphological landmarks by Giemsa C-banding technique (Ladizinsky et al., 1979), and only one pair of nucleolus organizer chromosomes is

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Tertiary gene pool GP-3 26 wild perennial species Secondary gene pool GP-2 unknown

Glycine soja Primary gene pool GP-1 soybean cultivars land races

Secondary gene pool GP-2 unknown Tertiary gene pool GP-3 26 wild perennial species

Figure 2.7

Gene pools of the soybean based on the classification of Harlan and de Wet, (1971). Primary gene pool (GP-1) contains land races and its wild annual progenitor Glycine soja. Secondary gene pool (GP-2) is not identified. Tertiary gene pool (GP-3) constitutes 26 perennial wild species.

Soybean and wild annual Glycine soja common ancestor of the genus Glycine with n=x=10 is unknown

Twenty six perennial wild Glycine species

Figure 2.8

A geographical map showing the home of Glycine; the common progenitor (2n = 2x = 20) of G. soja and soybean (annual) and 26 wild species (perennial) is unknown. It may be extinct or not yet identified. Soybean is domesticated in China from G. soja, and 26 wild perennial Glycine species were not domesticated in Australia.

G. curvata Tindale G. cyrotoloba Tindale G. falcata Benth. G. gracei B.E.Pfeil and Craven** G. hirticaulis Tindale and Craven

G. lactovirens Tindale and Craven G. latifolia (Benth.) Newell and Hymowitz G. latrobeana (Meissn.) Benth. G. microphylla (Benth.) Tindale G. montis-douglas B.E.Pfeil and Craven** G. peratosa B. E. Pfeil and Tindale G. pescadrensis Hayata G. pindanica Tindale and Craven G. pullenii B. Pfeil, Tindale and Craven G. rubiginosa Tindale and B. E. Pfeil G. stenophita B. Pfeil and Tindale

7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. G. syndetika B.E.Pfeil and Craven** 24. G. dolichocarpa Tateishi and Ohashi** 25. G. tabacina (Labill.) Benth.

G. albicans Tindale and Craven G. aphyonota B. Pfeil G. arenaria Tindale G. argyrea Tindale G. canescens F. J. Hermann G. clandestina Wendl.

1. 2. 3. 4. 5. 6.

Species

Table 2.1 Taxonomy of the Genus Glycine Willd

I1 B1 A3 B ? A5** AB1** H2 H3** A 4* B3**

A6 D1A** B2 BB1;BB2;B1B2

40 80** 40 80

C1 C F ? H1, (??)

I I*3 H A2 A A1

Cb PI-Number

?** B* B*

A B A B ? A* A A A* A* B

C C A ? A, (A)*

A A* A A A A

373990 373992

441000

440954 378705

440996 595818

IL1246 IL943 IL1247 378709 483196 440956

505166 440962 505179

505204 505151 440932 440958

Subgenus Glycine

40 40 40 40 40 80 40 40 40 40 40 40 80 40 40 40 40

40 40 40 40 40 40

2n

Genome Symbol Na

1317 1314

1300

2916 1433 2951 2599 1874 2600

1849 1184 1155 3124 2876 1956 2720 1697 1385 1867

2049 2589 1305 1420 1853 1126

G Number

Aust. : Q Taiwan** Aust. :Q, NSW Aust. :Q, NSW, V, SA; West Central and South Pacific Islands

Aust. : Q Aust. : Q, NSW Aust. : Q, NT, WA Aust. : NT Aust. : NT Aust. : NT Aust. : WA Aust. : Q, NSW Aust. : V, SA, T Aust. : Q, NSW, V, SA, T Aust. : NT Aust. : WA Aust. : Q, NSW; Taiwan, Japan Aust. : WA Aust. : WA Aust. : NSW, SA, WA Aust. : Q, NSW; (Japan ??)

Aust. : WA Aust. : WA Aust. : WA Aust. : Q Aust. : Q, NSW, V, SA, NT, WA Aust. : Q, NSW, V, SA, T

Distribution

continued

Provenance of 378705 is probably not Japan**. Pfeil et al. 2006

Pfeil et al. 2006

Pfeil et al. 2006

Atypical western slopes ssp.**

Comment

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SOYBEAN (GLYCINE MAX (L.) MERR.) 23

A A* A* A* A* A* A* A*

H2* D2 D 3E AE E H2** D A6** D D2** D H2** 505294 505203 441001 509501 505286 441005 483219 330961

440998 505222

PI-Number

G1

G G

G 51762

1943 1303 1133 1487 1945 1188 1927 1348

1858 1749

G Number

Subgenus Soja (Moench) F. J. Hermann

A A*

E D

Cb

China, Japan, Russia, Korea, Taiwan Cultigen; worldwide

Aust. : WA Aust. : WA, NT Aust. : Q, NSW; PNG Aust. : NSW Aust. : WA Aust. : Q; Taiwan Aust. : Q, NT, WA ; PNG; Timor Aust. : Q, NT, WA; Philippines, Taiwan

Aust. : Q Aust. : Q, WA, PNG

Distribution

I picked a counted accession that USDA and we both have**

Comment

*J.J. Doyle, Personal communication **A.H.D. Brown, Personal communication Molecular group (Isozyme groups) Abbreviation:PI, Plant introduction; G, CSIRO number; Aust, Australia; Q, Queensland; NSW, New South Wales; NT, Northern Territory; SA, South Australia; V, Victoria; WA, Western Australia; T, Tasmania; PNG, Papua New Guinea

N , Nuclear; C , Chloroplast

b

40

a

2. G. max (L.) Merr.

40 40 78 78 78 80 80 80

D5B D5A T1 T5 T6 T2 T3 T4

40

38 40

D1, D2 D3

2n

Genome Symbol Na

24

1. G. soja Sieb.&Zucc.

26. G. tomentella Hayata

Species

Mol. Group

Table 2.1 (continued) Taxonomy of the Genus Glycine Willd

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Figure 2.9

25

A mitotic metaphase cell of soybean with 2n = 40 chromosomes. One pair of chromosomes containing a nucleolus organizer region (NOR) can be distinguished, while 38 chromosomes are almost similar.

occasionally visible (Figure 2.9). Yanagisawa et al. (1991) separated 40 soybean mitotic metaphase chromosomes into five groups (A, B, C, D, E) by using a chromosome image analyzing system (CHIAS). Group A included a pair of nucleolus organizer (satellite) chromosomes, group B included two submedian chromosomes with a gap at the center of the long-arm contraction, and groups C, D, and E consisted 10, 14, and 12 chromosomes, respectively. However, pachytene chromosomes exhibit defined euchromatin and heterochromatin differentiation (Singh and Hymowitz, 1988). Heterochromatin is distributed proximal to and on either side of the centromeres on the long and short arms, and 6 of the 20 short arms are very heterochromatic (Figure 2.10 and Figure 2.11). It is interesting to note that 36% of the soybean genome is heterochromatic, which is higher than that observed (29%) for tomato (Barton, 1950). This latter feature makes soybean pachytene chromosomes unique (Singh, 2003). 2.5.1

Evolution of the Glycine Genome

The basic chromosome number x = 10 has been proposed for G. max (Darlington and Wylie, 1955). Based on this proposal, Singh (2003) hypothesized a putative ancestor with 2n = 20 chromosomes for the genus Glycine and carrying at least one pair of nucleolus organizer regions (NORs). Although such a progenitor is currently unknown, it would be most likely found in Southeast Asia (Cambodia, Laos, and Vietnam). Whether tetraploization (2n = 4x = 40) involved auto(spontaneous chromosomes doubling) or allo- (interspecific hybridization followed by chromosome doubling) polyploidy of the progenitor species, and whether it occurred prior to dissemination or after, cannot be substantiated experimentally, because we do not know where the progenitor of the genus Glycine originated. The progenitor of the wild perennial species of the subgenus Glycine radiated out into several morphotypes depending on the growing conditions in the Australian subcontinent. These species have never been domesticated and remained as wild perennials. In contrast, the pathway of migration from a common progenitor to China is assumed: wild perennial (2n = 4x = 40; unknown extinct) wild annual (2n = 4x = 40; G. soja) soybean (2n = 4x = 40; G. max) (Figure 2.8). All species of the genus Glycine exhibit diploid-like meiosis and are inbreeders (Singh and Hymowitz, 1985a). Allopolyploidization probably played a key role in the speciation of the genus Glycine. This implies that the 40-chromosome Glycine species and 80-chromosome G. tabacina and G. tomentella are allotetraploid and allooctoploid, respectively (Singh and Hymowitz, 1985b). Meiotic pairing in haploid (2n = 20; range = 0II to 5II) soybean (Crane et al., 1982) and in interspecific hybrids (Singh and Hymowitz, 1985b, 1985c; Singh et al., 1988; Grant et al., 1984), fluorescent in situ hybridization

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Pachytene Chromosomes of Soybean

K

7

D2

9

17

16 A2

6

8

5

18

11 C1

N

4

G

15

A1

14

3

L

19

2 D1a

1

10

12

F

H

13

20

Figure 2.10 The pachytene chromosome complement of G. max × G. soja F1 hybrid. Each figure shows a different chromosome, 1 to 20. Arrows indicate centromere location. The letter above the number represents the molecular linkage group. (From Singh, R.J. and Hymowitz, T., Theor. Appl. Genet., 76, 705–711, 1988. With kind permission of Springer Science and Business Media.)

Kinetochore

Figure 2.11 Proposed idiogram, based on Figure 2.10, of the pachytene chromosomes of the soybean. Dotted arrows show totally heterochromatic short arm. Chromosome 13 is a nucleolus organizer chromosome. (From Singh, R.J. and Hymowitz, T., Theor. Appl. Genet., 76, 705–711, 1988. With kind permission of Springer Science and Business Media.)

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A

B

C

Figure 2.12 (See color insert following page 144.) Morphology of three Glycine species grown in the greenhouse: (A) G. tomentella (2n = 78); (B) G. cyrtoloba (2n = 40), showing the characteristic curved pod (arrow); and (C) G. falcata (2n = 40), showing the characteristic falcate trait of pods.

(Singh et al., 2001; Pagel et al., 2004), and molecular studies (Shoemaker et al., 1996) elucidate that soybean is of tetraploid origin. 2.5.2

Genomic Relationships among Diploid Species

Understanding the genomic relationships among species is important to systematists, evolutionary biologists, cytogeneticists, molecular biologists, and plant breeders. The taxonomic nomenclature of species and their evolutionary relationships can be refined by cytogenetic evidence such as chromosome morphology, crossability, hybrid viability, meiotic chromosome pairing, and molecular (isozymes and nuclear, chloroplast, and mitochondrial DNA markers) approaches. Thus, phylogenetic relationships among species can be understood more precisely by a multidisciplinary approach rather than through reliance on a single technique (Singh, 2003). Broué et al. (1977) established, for the first time, phylogenetic relationships among four species of the subgenus Glycine by starch gel electrophoresis. Extensive plant exploration, cytogenetic, and molecular studies currently have identified 26 species in the subgenus Glycine, while only 6 species were recognized by Broué et al. (1977). 2.5.2.1 Genome Designation The genomes of diploid Glycine species are assigned capital-letter symbols according to the degree of chromosome homology between species in F1 hybrids (Kihara and Lilienfeld, 1932). Similar letter symbols are designated for species with interspecific F1 hybrids that show normal chromosome pairing. Placing a subscript after the letter indicates minor chromosome differentiation, such as inversions or translocations. Highly differentiated species are designated by different letter symbols, because their hybrids exhibit highly irregular chromosome pairing and hybrids are completely sterile. Singh and Hymowitz (1985b, 1985c) conceived assigning genome symbols to Glycine species based on cytogenetic results. Molecular methods helped to assign genome symbols to those species where cytogenetic information was not obtained (Singh et al., 1992a, 1998b; Kollipara et al., 1993, 1995, 1997; Doyle et al., 2002; Brown et al., 2002; Table 2.1). 2.5.2.2 Classical Taxonomy Classical taxonomy has played a major role in the identification and nomenclature of new species in the subgenus genus Glycine (Table 2.1). G. clandestina (2n = 40) has been observed to be a morphologically highly variable species (Hermann, 1962). Stems of wild perennial species are twining, climbing (Figure 2.12A), (See color insert following page 144) or procumbent and exhibit

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A

B Figure 2.13 Meiosis in intragenomic F1 hybrid of G. latifolia (B1B1; 2n = 40) × G. microphylla (BB; 2n = 40). (A) Metaphase I, showing 20 bivalents. (From Singh, R.J., unpublished result.) (B) Anaphase I, showing a chromatin bridge and an acentric fragment (paracentric inversion) in an interspecific hybrid of G. clandestina (A1A1; 2n = 40) × G. canescens (AA; 2n = 40). (From Singh, R.J. et al., Genome, 30, 166–176, 1988. With permission.)

morphologically distinct traits. G. cyrtoloba and G. curvata contain curved pods (Figure 2.12B), G. microphylla, G. latifolia, and G. tabacina carry adventitious roots, and falcate pods (Figure 2.12C) are a unique trait for G. falcata. Table 2.1 contains 26 wild perennial species of the subgenus Glycine and 2 species of the subgenus Soja. 2.5.2.3 Crossing Affinity Crossability rate is an excellent indirect measure for estimating the degree of genomic relationship between parental species. Interspecific crosses involving parental species with similar genomes usually set normal pods and seeds, while in crosses between genomically dissimilar species, seed abortion is common and hybrids are sterile (Singh, 2003). 2.5.2.4 Chromosome Pairing The degree of chromosome pairing in interspecific hybrids provides an important cytogenetic context for infering phylogenetic relationships among diploid species, enhances our understanding of evolution of the genus, and provides information about the ancestral species. Generally, species with similar genomes exhibit complete or almost complete chromosome pairing (intragenomic chromosome pairing) in their hybrid (Figure 2.13A). Sometimes, species differ by chromosomal interchanges or by paracentric inversion (Figure 2.13B). Based on classical taxonomy, G. soja and G. max are different species (Hermann, 1962). However, both species carry 2n = 40 chromosomes, hybridize readily, produce viable, vigorous, and fertile hybrids, and sometimes differ by a reciprocal translocation (Karasawa, 1936; Palmer et al., 1987; Singh and Hymowitz, 1988) or by a paracentric

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Figure 2.14 Meiosis in intergenomic F1 hybrid of G. latifolia (B1B1; 2n = 40) × G. canescens (AA; 2n = 40); metaphase I, showing 20 univalents + 10 bivalents. (From Singh, R.J. and Hymowitz, T., Theor. Appl. Genet., 71, 221–230, 1985. With kind permission of Springer Science and Business Media.)

inversion (Ahmad et al., 1977). Therefore, G. soja and G. max are now assigned genome symbols G and G1, respectively. In the genus Glycine, all F1 plants from crosses among A- (G. canescens, G. argyrea, and G. clandestina) and B- (G. microphylla, G. latifolia, and G. tabacina) genome species display 20 bivalents in the majority of sporocytes. The extent of chromosome association in the hybrids of genomically dissimilar species elucidates structural homology in the parental chromosomes, and hence furnishes evidence regarding the progenitor species (Singh, 2003). Usually the F1 generated from genomically unlike parents (different biological species) are germinated through in vitro techniques. In general, hybrids are weak, slow in vegetative and reproductive growth, and sterile. In the subgenus Glycine, A and B genome species hybrids show an average chromosome association of 19.7I + 10.2II (A3 × B1) and 20.9I + 9.5II (A × B1) (Figure 2.14). This suggests strongly that one genome is common in A and B genome species. Furthermore, the common genome may be the progenitor species with 2n = 20 chromosomes. Hybrid seed inviability, seedling lethality, and vegetative lethality are common occurrences in intergenomic crosses (Singh et al., 1988). G. cyrtoloba (C genome) and G. curvata (C1 genome) contain only a curved pod (Figure 2.12B), a distinct morphological trait that distinguishes these species from other species (Tindale, 1984). These species also express two pairs of nucleolus organizer chromosomes at mitotic metaphase by Feulgen staining and by fluorescent in situ hybridization (FISH) (Singh et al., 2001), while other species of the genus Glycine express one pair; it is feasible that the second pair is either silent or has lost its NOR activity. Variable (semihomologous-homoeologous) and minimal chromosome pairing are common in intergenomic F1 hybrids. A wrong conclusion can be drawn if genome designation of species is based on classical taxonomy. For example, aneudiploid (2n = 38) G. tomentella is morphologically similar to 40-, 78-, and 80-chromosome tomentellas. By contrast, limited chromosome pairing was observed between 40-chromosome G. tomentella and G. canescens (A genome) (Figure 2.15A). G. falcata is morphologically distinct among 23 wild perennial species of the genus Glycine and 2 species of subgenus G. soja. Chromosome pairing results (B1 × F, 37.8I + 1.1II; A × F, 38.7I + 0.6II) support the uniqueness of genome (F) of G. falcata because it showed the minimum chromosome synapsis with A and B genomes (Figure 2.15B). Similar observation was recorded by Doyle et al. (1996). 2.5.2.5 Molecular Methods During the past decade, literature on genomic relationships (plant phylogenetic relationships) has been dominated by molecular data, including nuclear sequence variation in the internal transcribed spacer (ITS) region of rDNA, extranuclear (chloroplast and mitochondrial) DNA variation, and

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A

B

Figure 2.15 Meiosis in interspecific Glycine hybrids. (A) Metaphase I, showing 31 univalents + 4 bivalents in G. tomentella (2n = 38; EE) × G. canescens (2n = 40; AA). (B) Metaphase I, showing 40 univalents in G. clandestina (A1A1; 2n = 40) × G. canescens (AA; 2n = 40) × G. falcata (FF; 2n = 40). (From Singh, R.J. et al., Genome, 30, 166–176, 1988. With permission.)

genomic in situ hybridization (GISH) by multicolor FISH. This latter approach is extremely powerful, where production of interspecific or intergeneric hybrids is not feasible by conventional methods (Singh, 2003). Molecular tools verified cytogenetic results that G. max and G. soja are genomically similar (Doyle, 1988; Zhu et al., 1995; Kollipara et al., 1993, 1995, 1997). The sequence divergence between G. soja and G. max was 0.2% (Kollipara et al., 1997). Kollipara et al. (1997) determined phylogenetic relationships among 18 species of the genus Glycine and two species of the subgenus Soja from nucleotide sequence variation in the ITS region of nuclear ribosomal DNA. This study helped to assign a genome symbol to five species: H to Glycine arenaria, H1 to G. hirticaulis, H2 to G. pindanica, I to G. albicans, and I1 to G. lactovirens. The cytogenetic relationship of these five species is unavailable, as they are difficult to grow in the greenhouse at Urbana, IL, and verified by histone, H3-D gene sequences and genomes were assigned to G. aphyonota (I3), G. peratosa (A5), G. pullenii (H3), and G. stenophita (B3) (Brown et al., 2002; Doyle et al., 2002; A.H.D. Brown and J.J. Doyle, personal communication, 2004). The ITS region (nrDNA) is a multigene family. However, in the soybean, the nrDNA is mapped to a single locus on the short arm of chromosome 13 based on the location of the nucleolus organizer region by pachytene chromosome analysis (Singh and Hymowitz, 1988) and also by FISH using ITS as a probe (Singh et al., 2001). The wild perennial Glycine species also contain one pair of NOR chromosome, like those in the soybean, except for G. curvata and G. cyrtoloba, which have two NOR chromosomes (Singh et al., 2001). Of the 26 wild perennial Glycine species, G. tomentella is unique because it constitutes four cytotypes (2n = 38, 40, 78, 80). Aneudiploid (2n = 38) G. tomentella is distributed in a restricted region of Queensland. The diploid (2n = 40) cytotype is distributed in Queensland, Northern Territory, Western Australia, and Papua New Guinea. The isozyme banding pattern grouped aneudiploid into two isozyme groups (D1 and D2) and the diploid form into six isozyme (D3A, D3B, D3C, D4, D5, and D6) groups (Doyle and Brown, 1985). Cytogenetics revealed that D1 and D2 isozyme groups carry a similar genome, and Singh et al. (1988) assigned the E genome symbol.

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The D4 isozyme group G. tomentella contains PI441000 (D3 genome) and has close affinity cytogenetically with A genome species (Singh et al., 1988; Grant et al., 1984). Although the D4 isozyme group is morphologically distinct from the A genome species, it does have long and narrow leaves and a longer pod length, which is a characteristic feature of A genome species that distinguishes it from other diploid G. tomentella accessions (Kollipara et al., 1998). Based on DNA sequence variation at the single-copy nuclear locus histone H3-D, Brown et al. (2002) also grouped D4 isozyme accessions with A genome species; D4 isozyme group G. tomentella should be designated the A6 genome symbol. PI441000 has been designated a new species, Glycine syndetika, status (Table 2.1) Pfeil et al., 2006. They also separated the D5 isozyme group into D5A and D5B. 2.5.3

Polyploid Complexes of G. tabacina and G. tomentella

Of the 26 species of the subgenus Glycine, G. hirticaulis, G. tabacina, and G. tomentella contain 2n = 40 and 2n = 80 chromosomes. Furthermore, G. tomentella consists of aneudiploid (2n = 38) and aneutetraploid (2n = 78) accessions. Tetraploid G. hirticaulis, described by Tindale and Craven (1988), has restricted geographical distribution. Tateishi and Ohashi (1992) described a morphological variant of G. tomentella, found in Taiwan as Glycine dolichocarpa Tateishi and Ohashi. It contains 2n = 80 chromosomes. On the other hand, G. tomentella (2n = 78) is found in Australia and Papua New Guinea and G. tomentella (2n = 80) is distributed in Australia, Papua New Guinea, the Philippines, Timor Island of Indonesia, and Taiwan. Tetraploid tabacinas and tomentellas are allopolyploid complexes of multiple origins (Singh and Hymowitz, 1985c; Singh et al., 1989; Kollipara et al., 1994; Hsing et al., 2001; Doyle et al., 2002; Rauscher et al., 2004). Classification of the polyploid G. tabacina and G. tomentella accessions into discretely defined, reproductively isolated groups using various methods (morphological, cytogenetic, biochemical, and molecular) helps us to better understand the origin of the species complex (Kollipara et al., 1994). 2.5.3.1 Glycine tabacina (2n = 80) Diploid G. tabacina is indigenous to Australia, while tetraploid (2n = 80) cytotype is found sympatrically with diploids in Australia and in the islands of the south Pacific (New Caledonia, Vanuatu, Fiji, Tonga, Niue) and west-central Pacific (Taiwan, Ryuku, Marianas) (Singh et al., 1992b). Morphological observations (Costanza and Hymowitz, 1987), cytogenetic investigation (Singh et al., 1987, 1992b), and molecular studies (Doyle et al., 1990a, 1990b, 1999a) have shown two distinct groups in the 80-chromosome G. tabacina. It is an allopolyploid complex and is of multiple origins. The one group contains adventitious roots [with adventitious roots (WAR)], while the other group lacks adventitious roots [no adventitious roots (NAR)]. All the intraspecific F1 hybrids within each group showed normal meiosis and complete fertility. However, F1 hybrids between the groups were sterile owing to disturbed meiosis. At metaphase I, a model chromosome association of 40I + 20II was recorded (Singh et al., 1987). This indicates that both groups have one genome in common and differ for the second genome. Singh et al. (1992b) proposed, based on cytogenetics, that the 80-chromosome G. tabacina (NAR) is a complex, probably synthesized from the A genome (G. canescens, G. clandestina, G. argyrea, D4 isozyme group G. tomentella), and G. tabacina (WAR) evolved through segmental allopolyploidy from the B genome (G. latifolia, G. mictophylla, G. tabacina). Doyle et al. (1999b) suggested, based on sequencing of histone H3-D locus, the multiple origins with gene exchange among lineage increases the genetic base of a polyploid and helps in better colonization of polyploid G. tabacina relative to its diploid progenitors. Hybridization is unlikely in a highly self-inbreeder in nature; however, F1 hybrids among B genome species are completely fertile (Putievsky and Broué, 1979; Newell and Hymowitz, 1983; Grant et al., 1984; Singh and Hymowitz, 1985c). Since B genome species are sympatric (Doyle et al., 1999a), adventitious root trait is controlled by a recessive gene (Singh et al., 1987) and Bowman–Birk inhibitor (BBI) is present in A genome species, including

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80-chromosome G. tabacina (NAR), but absent in B genome species and 80-chromosome G. tabacina (WAR). This suggests segmental allotetraploid origin of 80-chromosome G. tabacina (WAR) and true allotetraploid origin of 80-chromosome G. tabacina (NAR) (Singh et al., 1992b). 2.5.3.2 Glycine tomentella (2n = 78, 80) Diploid-like meiosis, isozyme banding patterns among the accessions and meiotic pairing in intraspecific and interspecific F1 hybrids, wide geographical distribution, and aggressive and vigorous growth habits suggest that 78- and 80-chromosome tomentellas are of allopolyploid origin and are polyploid complex (Putievsky and Broué, 1979; Newell and Hymowitz, 1983; Grant et al., 1984; Singh and Hymowitz, 1985a, 1985b, 1985c; Singh et al., 1987, 1989; Kollipara et al., 1994; Doyle et al., 1986). Morphologically, four cytotypes (2n = 38, 40, 78, 80) are indistinguishable. This suggests that one or both ancestors of 78- and 80- chromosome tomentellas may be 38- and 40- chromosome G. tomentella. The isozyme banding pattern revealed three groups (T1, T5, T6) in aneutetraploid and three groups (T2, T3, T4) in tetraploid G. tomentella (Doyle and Brown, 1985, 1989; Doyle et al., 1986). Cytogenetics, biochemical, and molecular methods verified isozyme results and clearly demonstrated the distinct reproductively isolated genomic groups in aneutetraploid and tetraploid tomentellas (Kollipara et al., 1994). This strongly supports three distinct isozyme and genomic groups for aneutetraploid T1 (D3E genome), T5 (AE genome), and T6 (D1E genome) and tetraploid T2 (D1A genome), T3 (D1D2 genome), and T4 (D1D3 genome) G. tomentella. Various groups within 78- and 80-chromosome tomentellas originated in Australia by allopolyploidization, most likely through multiple independent events (Kollipara et al., 1994; Doyle et al., 1999b). 2.5.4

Chromosomal Aberrations: Structural Changes

Chromosomal structural changes such as deficiencies, duplications, interchanges, and inversions have not been systematically produced, identified, and used in physical genetic mapping in soybean. An interchange of spontaneous origin in soybean (Sadanaga and Grindeland, 1984) has been used to locate the w1 (white flower) locus on the satellite chromosome (chromosome 13) in soybean. Palmer et al. (1987) surveyed 56 G. soja accessions from China and the Soviet Union, which also included PI81762 studied by Singh and Hymowitz (1988). They concluded that these accessions have a single similar or identical interchange. Singh and Hymowitz (1988) examined an interspecific F1 hybrid of soybean × PI81762 and observed that one quadrivalent was always associated with the nucleolus. Inversions (paracentric and pericentric) are neither produced nor used in physical mapping of soybean genome. Study has been limited to identifying a paracentric inversion in the soybean × G. soja hybrid (Ahmad et al., 1977; Palmer et al., 2000). Wild perennial Glycine species with similar genomes are differentiated by a paracentric inversion (Singh, 2003), as the majority of sporocytes show normal pairing at metaphase I, but at anaphase I a chromatin bridge and an acentric fragment are observed. 2.5.5

Chromosomal Aberrations: Numerical Changes

2.5.5.1 Autopolyploidy Haploid (Crane et al., 1982), triploid (Chen and Palmer, 1985), and tetraploid soybean plants have been reported. Haploid and triploid are completely sterile, and tetraploid soybean produces few one or two large seeded pods. Tetraploid soybean has no commercial value. A tetraploid × diploid cross has failed to produce an autotriploid, an excellent source for producing primary trisomics (Singh, 2003). However, Chen and Palmer (1985) identified autotriploid from the progenies of male-sterile lines, but the derived autotriploid was not used to produce primary trisomics. Xu et al. (2000a) found a hypertriploid (2n = 3x + 1 = 61) plant from a cross T31 (a homozygous recessive glabrous

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A

B

Figure 2.16 Meiotic chromosome configurations at metaphase I in Triplo 20 of the soybean. (A) A cell with 1III (arrow) + 19II. (B) A cell with 20II + 1I. (From Xu, S.J. et al., Crop Sci., 40, 1543–1551, 2000c. With permission.)

(pp)) × T190-47-3 (an unidentified primary trisomic). The hypertriploid plant produced 98 selfed seeds, and the chromosome number ranged from 2n = 50 to 69. The chromosome number in hypertriploid × diploid seeds ranged from 2n = 44 to 56. These lines were not used to produce primary trisomics. 2.5.5.2 Aneuploidy 2.5.5.2.1 Primary Trisomics Primary trisomics in soybean (an individual with normal chromosome complements plus an extra complete chromosome; 2n = 2x + 1 = 41) have been isolated from the progenies of asynaptic and desynaptic mutants (Palmer, 1976; Gwyn et al., 1985; Xu et al., 2000c). Four primary (2n = 41) trisomics (Tri A, B, C, and D) examined by Gwyn et al. (1985) were similar to the diploid (2n = 40), and this was attributed to the polyploid nature of soybean (Palmer, 1976). Skorupska et al. (1989) identified Tri S that contained three satellite chromosomes. Singh and Hymowitz (1988) developed a chromosome map for soybean by pachytene chromosome analysis. Singh and Hymowitz (1991) identified, by using a pachytene chromosome, Tri A as Triplo 5, Tri C as Triplo 1, Tri D as Triplo 4, and Tri S as Triplo 13. Xu et al. (2000c) verified the identification of these four primary trisomics. They isolated and tentatively identified 16 additional primaries from 37 aneuploid lines (2n = 41, 42, 43). These aneuploid lines originated from the progenies of asynaptic and desynaptic mutants that were supplied by Reid Palmer. Triplo 1 contains the longest chromosome and Triplo 20 the smallest chromosome. At metaphase I of meiosis, a majority of microsporocytes in primary trisomics exhibit either 1III + 19II (Figure 2.16A) or 20II + 1I (Figure 2.16B). The average female transmission of 20 soybean primary trisomics was 42%, with a range of 27 (triplo 20) to 59% (triplo 9). The female transmission rate has been estimated from the hybrid population (Xu et al., 2000c). This may be the reason for the high female transmission rate of the extra chromosome in primary trisomics of soybean; heterozygosity often favors the higher female transmission rate (Singh, 2003). Three marker genes, Eu1 (seed urease), Lx1 (lipoxygenase 1), and P2 (puberulent), were located on chromosomes 5, 13, and 20, respectively. Zou et al. (2003b) associated yellow leaf mutant y10 to chromosome 3 by the primary trisomics method.

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Figure 2.17 A mitotic metaphase cell of soybean tetrasomic for chromosome 13 showing four SAT chromosomes (arrows). (From Singh, R.J., Plant Cytogenetics, 2nd ed., CRC Press, Boca Raton, FL, 2003. With permission.)

2.5.5.2.2 Monosomics In soybean, monosomics (2n – 1 = 39; an individual lacking one chromosome is called monosomic) have been isolated in the progenies of Triplo 3 and Triplo 6 and were designated as mono-3 and mono-6 (Xu et al., 2000b). Morphologically, mono-3 was smaller with reduced vigor while mono-6 was similar to the disomics. Female transmission in mono-3 was 6.5%, while mono-6 was not transmitted among 105 S1 plants. Skorupska and Palmer (1987) reported two monosomic plants among 94 S1 KS-6 progeny. The monosomics were not identified for a particular chromosome. The transmission rate in monosomics is sporadic in soybean. It was concluded that monosomics in soybean are viable and fertile and can be produced; however, no systematic effort is being made to isolate monosomics in this economically important crop. 2.5.5.2.3 Tetrasomics In soybean, tetrasomics (2n + 2 = 42), an individual carrying two extra chromosomes in addition to its normal somatic chromosome complement (Figure 2.17), are identified in low frequencies from the selfed progenies of primaries (2n = 41) (R.J. Singh, unpublished results). Soybean tetrasomics are viable and, compared to their counterpart, primary trisomics, are slow in vegetative and reproductive growth and partially to completely fertile. Primary trisomics are morphologically indistinguishable except triplo 1, 13, and 17, but the extra two chromosomes cause morphological characteristic modification in tetrasomics. Gwyn and Palmer (1989) observed, based on morphological measurement, that tetrasomics and double trisomics (2n + 1 + 1) could be distinguished accurately from their disomics sibs. Tetrasomics mostly breed true, and occasionally related trisomics (2n = 41) and diploids (2n = 40) are identified. Most primary trisomics plants are produced from tetrasomics × disomics crosses. Tetrasomics in the soybean are unique cytogenetic stocks, as they are unviable in diploid crops such as maize, barley, rice, and tomato. The occurrence of tetrasomics suggests that soybean is an ancient tetraploid but behaves like a true diploid. 2.5.6

Linkage Mapping

Chromosome, genetic, and cytogenetic maps in economically important crops like rice, maize, barley, wheat, and tomato were developed first, and molecular maps were followed. By contrast, it has been the reverse in the case of soybeans; several molecular maps have been developed first and these maps are now being associated with the chromosomes by primary trisomics and molecular cytogenetics.

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2.5.6.1 Chromosome Map Although the precise chromosome number of soybean was determined in 1925 (Karpechenko, 1925), the chromosome map was not developed based on somatic metaphase chromosomes because chromosomes are symmetrical and only a pair of nucleolus organizer chromosomes is identified in one of the best chromosome spreads. Singh and Hymowitz (1988) constructed a chromosome map of soybean by using pachytene chromosomes (Figure 2.10). This pioneering research has set the stage to produce all possible primary trisomics in the soybean (Xu et al., 2000c). 2.5.6.2 Classical Genetic Linkage Map A genetic linkage map with 20 linkage groups, designated the Classical Genetic Linkage Map (CGLM) of soybean, has been proposed (Palmer et al., 2004). The CGLM groups 2, 3, 12, 13, 15, 16, 18, 20, and (21?); each has two qualitative trait loci. Thus, the genetic linkage map of soybean is not saturated with classical markers compared to other economical important crops. 2.5.6.3 Molecular Linkage Map In soybean, restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and simple sequence repeats (SSR) have been used to develop molecular linkage groups (MLGs) (Cregan et al., 1999; Shoemaker et al., 2004). Cregan et al. (1999) and Song et al. (2004) integrated several molecular linkage maps into one map with 20 molecular linkage groups. Song et al. (2004) used the JoinMap software program to integrate the data from five soybean mapping populations (Minsoy × Noir 1, Minsoy × Archer, Archer × Noir 1, Clark × Harosoy, A81-3560022 × PI468916). The integrated genetic map spans 2523.6 cM of Kosambi map distance across 20 linkage groups and consists of 1849 markers, including 1015 SSRs, 709 RFLPs, 73 RAPDs, 24 classical traits, 6 AFLPs, 10 isozymes, and 12 other markers. However, MLGs are not all associated with the individual soybean chromosomes. By using SSR markers from 20 MLGs and primary trisomics, Zou et al. (2003a) found the following relationships between chromosomes and MLGs: MLG

CGLM

Chromosome

A1 A2 B1 B2 C1 C2 D1a + Q D1b + W D2 E F G H I J K L M N O

— 07, 09 — 17 21 01 03 11 20 14 08, 13 18 20 04 19 02, 12 05 — 10 15

05 08 — — 14 — 01 — 17 — 13 18 — 20 — 9 19 — 03 —

Note: MLG = molecular linkage group; CLGM = classical genetic linkage map.

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Nine MLGs have not been associated with the chromosomes. Segregation distortion is common using primary trisomics and SSR markers. This may be due to the preferential selection of gametes containing certain genotypes (genomic divergence) (Zou et al., 2006). Soybean scientists are focusing on sequencing the soybean genome, but no major effort is under way to develop a universal cytogenetic map for the soybean, as has been accomplished in other crops.

2.6 SOYBEAN GERMPLASM ENHANCEMENT 2.6.1

Conventional Breeding

Only a small fraction of the genetic diversity available is currently used in soybean breeding worldwide. The two oldest and largest national breeding programs in the world are in China and the U.S. The genetic base of soybean in the U.S. and Canada is narrow (Gizlice et al., 1996; Burton, 1997; Singh and Hymowitz, 1999). Based on pedigrees of North American public soybean cultivars released from 1947 through 1988, 6 ancestral lines supply 60% of the genetic base for U.S. soybean production, and an additional 16 progenitors provide another 30% (Gizlice et al., 1996). Analyses of DNA markers indicate that these ancestral lines are quite diverse (Thompson and Nelson, 1998) and contain as much diversity as a relatively large set of exotic introductions that have been used in recent population development and selection for yield improvement (Brown-Guedira et al., 2000; Kisha et al., 1998; Li et al., 2001). Efforts to expand the genetic base of soybean production in the U.S. are not new (Hartwig, 1973), but recent results have been more promising. Improved germplasm has been released that contained 50% exotic parentage by pedigree and exceeded the yield of the best cultivars in regional testing, and another released line that is derived from 100% exotic parentage equaled the best cultivar in regional testing (Nelson and Johnson, 2006). The genetic base of soybean breeding in China is much larger than in the U.S. The pedigrees of 651 Chinese soybean cultivars released from 1923 to 1995 contain 339 ancestors, and as many as 190 ancestral lines contributed 80% of the genetic base of production (Cui et al., 2000). Based on both pedigrees (Cui et al., 2000) and DNA markers (Li et al., 2001), the genetic bases of the three major soybean-growing regions of China — northeastern, central, and southern — are distinct and could be considered independent gene pools. The initiation or expansion of many breeding programs in southern China and the intentional efforts to broaden the genetic base of soybean cultivars in northern China have resulted in the incorporation of much new germplasm in the past 25 years (Carter et al., 2004). Despite the apparent genetic limitation of a narrow genetic base for world soybean production, soybean breeding has continued to make significant progress. Analyzing data collected from 60 years of cooperative regional tests in the production area of the U.S. and Canada, Wilcox (2001) concluded that annual rates of yield improvement in kg ha–1 were 21.6 (MG 00), 25.8 (MG 0), 30.4 (MG I), 29.3 (MG II), 30.6 (MG III), and 29.5 (MG IV). Rates of yield improvement in recent years were equal to or greater than those in earlier years. In 1961 the world average yield was 1.13 Mt/ha. In 2004, that had been raised to 2.23 Mt/ha (Figure 2.2). There are a variety of breeding procedures that have been and are currently being used (Orf et al., 2004), but the use of single seed descent and winter nurseries has had a major impact by greatly reducing the time from hybridization to yield testing. Increased mechanization and computerization have also increased the efficiencies of most breeding programs. There is still a large discrepancy among mean yields of the top producing countries. The average yield in Argentina, Brazil, and the U.S. from 2000 to 2005 was nearly 2.6 Mt/ha. During the same period, the average yield in China was 1.7 Mt/ha and in India 0.9 Mt/ha (Figure 2.1). These values represent not only the genetic potential of the cultivars within each country, but also differences in environmental conditions. Lack of inputs, marginal soils, and complex cropping systems can reduce yield. Soybean contains several antinutritional factors. Kunitz trypsin inhibitor protein is one of the major antinutritional elements present in raw mature soybeans. Kunitz (1945) isolated, identified,

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and crystallized the protein that inhibited the proteolytic action of trypsin, which is commonly known as Kunitz trypsin inhibitor (SBTI-A2). Treating the moist seed with heat destroys antinutritional factors. A mutant without SBTI-A2 has been identified (Singh et al., 1969), and Bernard et al. (1991) registered L81-4590 as a Kunitz soybean (registration 271, PI542044) cultivar for commercial production. 2.6.2

Interspecific Hybridization

Soybean breeders have not fully exploited the wealth of genetic diversity from exotic germplasm, including soybean’s progenitor G. soja (Singh and Hymowitz, 1999; Carter et al., 2004). G. soja may be an excellent source of genetic variability, although it harbors several undesirable genetic traits, for example, vining, lodging susceptibility, lack of complete leaf abscission, seed shattering, and small black seed coat (Carpenter and Fehr, 1986; Carter et al., 2004). However, G. soja has been shown to be more genetically diverse than G. max (Choi et al., 1999; Li and Nelson, 2001), and the undesirable traits can be separated from the desirable traits during the course of selection in successive backcross generations and perhaps through marker-assisted selection. Attempts to broaden the genetic base of soybeans by utilizing G. soja were reported by Hartwig (1973), Ertle and Fehr (1985), Carpenter and Fehr (1986), and Carter et al. (2004). Hartwig (1973) reported highly productive and high protein lines derived from soybean and G. soja hybrids. Ertle and Fehr (1985) concluded that introgression of G. soja germplasm into the two soybean cultivars was not an effective method for increasing their yield potential. To obtain a relatively high frequency of useful segregates for cultivar development, three backcrosses to the soybean were preferred. Two small-seeded (seed of <100 mg) cultivars, such as ‘SS201’ and ‘SS202’, have been developed where G. soja was used as a nonrecurrent parent with two backcrosses to a G. max cultivar. (Fehr et al., 1990a, 1990b). The small-seeded cultivars are used for sprouts and the fermented Japanese product natto. Qian et al. (1996) have recorded the accessions of G. soja that are potential sources of additional genes that restrict nodulation of soybean with specific strains of Bradyrhizobium. They concluded that introgression of such genes could result in soybean cultivars that exclude some of the indigenous strains and become nodulated with commercial strains that are more efficient in fixing nitrogen. Since 1970, soybean production increased rapidly in Brazil and Argentina, and the initial varietal improvement program was from the germplasm introduced from the U.S. and other countries. 2.6.3

Intersubgeneric Hybridization

The 26 wild perennial species of the subgenus Glycine have not been exploited in soybean breeding programs. These species are extremely diverse morphologically, cytologically, and genomically, grow in very diverse climatic and soil conditions, and have a wide geographical distribution (Singh and Hymowitz, 1999). Wild perennial Glycine species have great potential for soybean improvement. They are a rich source of agronomically useful genes, such as resistance to soybean rust (Phakopsora pachyrhizi Sydow), soybean brown spot (Septoria glycines Hemmi.), powdery mildew (Microsphaera diffusa Cke. & Pk.), phytophthora root rot (Phytophthora sojae H.J. Kaufmann & J.W. Gerdemann), white mold (Sclerotinia sclerotiorum (Lib. De Bary)), sudden death syndrome (Fusarium solani (Mart.) Sacc.), tobacco ring spot, yellow mosaic virus, alfalfa mosaic virus, and soybean cyst nematode (SCN) (Heterodera glycines Ichinohe), and tolerance to certain herbicides and salt (Singh and Hymowitz, 1999). Soybean rust is one of the major soybean diseases in China, Thailand, India, Australia, and Taiwan. A significant reduction (80%) in yield may be caused by the pathogen (Hartman, 1996). Soybean rust has been reported in Puerto Rico and Brazil (Bonde and Peterson, 1996). Killgore (1996) reported soybean rust on vegetable soybeans grown on the islands of Kauai, Oahu, and Hawaii. Significant yield loss (>10%) is predicted in nearly all soybean-growing areas. However,

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the greatest loss (up to 50%) could occur in the Mississippi delta and the southeastern coastal areas (Yang, 1996). On November 10, 2004, the U.S. Department of Agriculture announced the presence of soybean rust on soybean leaf samples taken from two plots associated with a Louisiana State University research farm on November 6. This was the first instance of soybean rust to be found in the continental U.S. (http://www.asa-cssa-sssa.org/soybean_rust.html). Several researchers have attempted to hybridize wild perennial Glycine species with the soybean, but only a few sterile intersubgeneric F1 hybrid combinations have been reported (Newell et al., 1987; Singh et al., 1999). Thus far, only Singh et al. (1990, 1993) have successfully produced backcross-derived fertile progenies from the soybean and a wild perennial, Glycine tomentella (2n = 78). Monosomic alien addition lines (MAALs) and modified diploid (2n = 40) lines are being isolated and identified (Singh et al., 1998a). The modified diploid lines could be screened for pests and pathogens. Riggs et al. (1998) reported the introgression of SCN resistance from G. tomentella into modified derived diploid soybean lines. This study sets the stage for the exploitation of perennial germplasm to broaden the genetic base of the cultivated soybean. 2.6.4

Mutation Breeding

Mutation breeding in soybeans has lagged behind that of other economically important crops. Micke et al. (1985) compiled information on cultivars produced using induced mutations. They listed 17 soybean cultivars developed by various mutagens: 10 cultivars from China (Hei Noun 4, 5, 7, 8, 16, and 26; Mu Shi 6; and Tai Nung 1(R) and 2(R); and Tie Feng 18); 3 cultivars from Japan (Nanbushirome, Raiden, and Raiko); and one cultivar from each of Bulgaria (Boriana), Algeria (Cerag Nr. 1), Korea (KEX-2), and the former U.S.S.R. (Universal I). All cultivars from Japan were high yielding, because they were resistant to nematodes. Cultivar KEX-2 from Korea was earlier maturing (11 days) with a higher yield (ca. 16%) and larger seed. Karmakar and Bhatnagar (1996) listed 43 soybean cultivars released in India from 1969 to 1993. Three cultivars (Birsa Soy1, VL Soy1, and NRC2) were developed by mutagenesis, five cultivars (Bragg, Lee, Improved Pelican, Hardee, and Monetta) were direct introductions from the U.S., and the remaining cultivars were selected from introductions and single crosses (two parents). Buss (1983) isolated a recessive genetic male-sterile (gms) line from a M3 generation of ‘Essex’ soybean that had been irradiated with neutrons. Allelism tests revealed that the gms line inherited independently from ms1, ms2, ms3, and ms4. Thus, the newly identified ‘Essex’ gms gene was assigned the symbol ms5. By using chemical mutagenesis (ethyl methanesulfonate (EMS), N-nitroso-N-methylurea (NMU), or ethyl nitrosourea (ENU)), Sebastian and Chaleff (1987) and Sebastian et al. (1989) isolated soybean lines with increased tolerance for sulfonylurea herbicides. Sebastian and Chaleff (1987) identified four single recessive genes. Allele tests revealed that each mutation resided at one of three loci (hs1, hs2, or hs3). They observed, in biochemical studies, that the mutants did not contain an altered form of acetolactate synthase (the site of action of sulfonylurea herbicide). In subsequent studies, Sebastian et al. (1989) identified a monogenic semidominant mutant that was nonallelic to the hs1, hs2, and hs3 genes. They assigned the gene symbol Als1 to the line that was resistant to the action of sulfonylurea herbicide. Carroll et al. (1985) mutagenized soybean seeds of cv. Bragg with EMS. They isolated 15 independent nitrate-tolerant symbiotic (nts) mutants from 2500 M2 families. Mutant nts382 was studied extensively. In the presence of KNO3, nts382 produced six times more nodules than those observed in control ‘Bragg’ grown under identical culture conditions. Song et al. (1995) evaluated yield, N2 fixation, and the effects on cereal crops grown subsequent to the harvest of intermediate supernodulating (two times), extreme supernodulating (six times), and nonnodulating mutants of ‘Bragg’, genotypes derived from the mutants, and commercial cultivars. The experiment was conducted for 6 years at two locations. The results were as follows: (1) the supernodulators and ‘Manark’ were similar, with values 13 to 21% above those for ‘Centaur’; (2) in the plots fertilized

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Table 2.2 Changes in Fatty Acid Content in Soybean Produced through Mutagensis Fatty Acids Palmitic

Content Level High

Low Stearic

High

Oleic

High

Linolenic

Low High Low

Mutagen

Reference

Ethyl methanesulfonate

Wilcox and Cavins, 1990; Fehr et al., 1991 Fehr et al., 1991 Wilcox and Cavins, 1990 Fehr et al., 1991 Hammond and Fehr, 1983b Rahman et al., 1997 Brossman and Wilcox, 1984 Rahman et al., 1996 Hammond and Fehr, 1983b Brossman and Wilcox, 1984 Takagi et al., 1989 Hammond and Fehr, 1983a; Brossman and Wilcox, 1984

N-nitroso-N-methyl-urea Ethyl methanesulfonate N-nitroso-N-methyl-urea Sodium azide X-rays Ethyl methanesulfonate X-rays Sodium azide Ethyl methanesulfonate X-ray Ethyl methanesulfonate

Table 2.3 Fatty Acid Composition of Mutants A5 and A6 and Their parents, FA9525 and FA8077 (Hammond and Fehr, 1983a, 1983b)

Mutagen EMS Sodium azide

Genotype A5 FA9525 A6 FA8077

Palmitic 16:0 9.3 9.3 8.0 8.4

Fatty Acid (%) Stearic Oleic Linoleic 18:0 18:2 18:2 3.9 3.1 28.1 4.4

39.8 39.1 19.8 42.8

42.9 42.9 35.5 36.7

Linolenic 18:3 4.1 6.3 6.6 7.6

Arachidic

2.0 <1.0

with nitrogen, the supernodulators exhibited higher activity than the commercial cultivars, including ‘Manark’; (3) grain yield of the supernodulators was either the same or up to 25% less than those of ‘Bragg’ and ‘Centaur’; and (4) oats and barley sown immediately after soybean harvest produced significantly greater yields than after commercial soybean cultivars. Soybean seed oil is the major vegetable oil (52%) among oilseed crops produced in the world (Figure 1.1). Genetic studies have elucidated that oil synthesis in soybean is determined largely by the genotype of the maternal plants, because the oil content of F1 plants was not significantly different from those of selfed seeds of the female parent (Singh and Hadley, 1968). Similarly, fatty acid composition in soybean seed is determined by the maternal parent (Hammond et al., 1972). Breeding efforts to increase soybean oil above approximately 20% have been unsuccessful, because oil content and seed yield have a negative relationship (Burton, 1985). Soybean breeders initiated programs to improve soybean oil quality (Table 2.2). The principal fatty acids in soybean oil are palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3). A common soybean cultivar contains 11% palmitic, 3% stearic, 22% oleic, 56% linoleic, and 8% linolenic acid (Wilcox, 1985). The high linolenic acid content (7 to 9%) is associated with poor flavor (fishy, painty, grassy, melony) and stability in soybean oil (Dutton et al., 1951). Mounts et al. (1988) analyzed the fatty acid composition of more than 5000 soybean samples from both northern and southern soybean germplasm collections and identified one line, PI361099B, with low linolenic (4.2%) and normal oleic acid content. The low linolenic acid content of PI361088B remained stable regardless of environmental conditions, which suggests that soybean germplasm lacks a strain with a linolenic acid content of 3% or less (Hammond and Fehr, 1975). Mutagenesis has been an excellent tool for creating variability for fatty acid content in soybeans. From the M4 generation, following the use of EMS, Hammond and Fehr (1983a) selected a line, designated A5, that contained an average of 4.1% linolenic acid, while the parent (FA9525) contained 6.3%; the content of other fatty acids remained unchanged (Table 2.3). They also isolated a line with

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an elevated stearic acid (28.1%) content and a marked reduction in oleic acid (19.8%), designated A6, from an M2 population of sodium azide-treated seeds of FA8077 (Hammond and Fehr, 1983b; Table 3.3). Wilcox et al. (1984) identified a genetically stable low linolenic acid (3.4%) mutant from ca. 15,000 M2 plants, where seeds of soybean cv. Century had been treated with EMS. The linolenic acid content of seeds in the M2 populations ranged from 3.4 to 11.1%, and for ‘Century’, it ranged from 6.6 to 9.4%. In contrast, Takagi et al. (1989) developed a line with high linolenic acid content (18.4%) by treating seeds of cv. Bay with x-ray irradiation. ‘Bay’ contains 9.4% linolenic acid. Linolenic acid is essential in the mammalian diet. In some mammals, lack of linolenic acid causes skin lesions, lowered learning ability, stunted growth, and mental retardation (Coscina et al., 1986). Wilcox and Cavins (1987) assigned gene symbols for linolenic acid content: Fan Fan for high levels (= 7.2 ± 0.11%), Fan fan for intermediate levels (= 5.2 ± 0.07%), and fan fan for low levels (= 3.2 ± 0.13%). They observed that linolenic acid content was controlled by the genotype of the embryo rather than by the genotype of the maternal parent. Rahman et al. (1994) observed no maternal and cytoplasmic effects for linolenic acid content. It has also been demonstrated that low linolenic acid content is a quantitative trait (Fehr et al., 1992). Palmitic (16:0) and stearic (18:0) acids are the two main saturated fatty acids in the soybean. Fehr et al. (1991) produced a mutant containing reduced palmitic acid content by treating soybean cv. A1937 with NMU. Low palmitic acid content was controlled by two different alleles at two different loci. They assigned the genotypes fap1 fap1 and fapx fapx. This line contains 44 g kg–1 palmitic acid, the lowest content known in soybean. Wilcox and Cavins (1990) isolated two mutants, C1726 (registration GP-116; PI532833) and C1727 (registration GP-117; PI532834) from cv. Century by EMS treatment. Mutant C1726 contained 8.5% palmitic acid, and mutant C1727 contained 17.2% palmitic acid, while ‘Century’ had 11.2%. Both mutants bred true for low and high palmitic acid content. Genetic studies revealed that alleles from two independent loci segregated for palmitic acid percentage and that the gene action was additive. The gene symbol fap1 was assigned to an allele in C1726 that acts to lower the palmitic acid level in soybean oil, and fap2 was assigned to an allele in C1727 that acts to increase the palmitic acid level (Erickson et al., 1988). A reduction in palmitic acid content improves the quality of oil. An elevated palmitic acid content enhances its use in the production of food products, such as shortening and margarine (Schnebly et al., 1994). Rahman et al. (1996) examined the genetics of mutants with high oleic acid content (M11 and M23) produced by x-ray irradiation. Low oleic acid content in cv. Bay was partially dominant to the high oleic acid content in mutant M23, but completely dominant to the high oleic acid content in mutant M11. An inverse relationship between oleic and linolenic acid content in both mutants was recorded. Oil with high levels of oleic acid is less susceptible to oxidative changes during refining, storage, and frying (Miller et al., 1987). Mutagenesis can sometimes be used to break the linkage between two closely linked genes. The grassy-beany flavor in soybeans and soybean products is caused by lipoxygenases. Three soybean lipoxygenases (L1, L2, and L3) have been characterized. L1 and L2 are linked. Hajika et al. (1995) isolated a line without L1, L2, and L3 liposygenases by γ-ray irradiation. Soybean plants lacking lipoxygenases showed normal plant growth and yield. The production of soybeans without lipoxygenases is cost-effective, because heat treatment to inactivate these enzymes will not be required in the processing of soybean food products. Thus, mutation breeding provides an alternative method to wide hybridization and biotechnology. 2.6.5

Biotechnology

Conventional plant breeding has failed to revolutionize gains in soybean yield. Biotechnology is considered an innovative science with which to broaden the genetic base of crops by overcoming the genetic barriers in extremely distant crosses. Biotechnological methods include somaclonal variation, cybrids, and recombinant DNA technology. Thus, genetic engineering is one of the

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alternatives for developing high-yielding soybeans with high protein and oil content, resistance to pests and pathogens, and tolerance to herbicides. 2.6.5.1 Plant Regeneration from Callus and Cell Suspension Cultures Soybeans have received attention from tissue culture scientists of both public and proprietary institutes in order to generate normal soybean plants with increased genetic variability. However, soybean has been one of the most recalcitrant plant species as far as plant regeneration from tissue cultures is concerned (Lippmann and Lippmann, 1984). Ranch et al. (1985) first achieved a controlled somatic embryogenesis system for initiation, proliferation, and fertile plant regeneration using Murashige and Skoog (1962; MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D). Barwale et al. (1986) also regenerated plants via embryogenesis and shoot organogenesis by changing medium composition; embryogenesis resulted when explants were planted on MS medium containing naphthalene acetic acid (NAA), while the addition of benzylaminopurine (BAP) with a high concentration of MS minor salts resulted in organogenesis. However, these regeneration systems are relatively inefficient, complicated, and genotype dependent. A simple, efficient, and more rapid regeneration procedure through somatic embryogenesis (Komatsuda et al., 1992; Samoylov et al., 1998) and shoot organogenesis (Shetty et al., 1992; Reichert et al., 2003) has been established. The regeneration efficiency from soybean cell cultures is highly dependent on genotype regardless of the protocol routes (Komatsuda and Ohyama, 1988; Hofmann et al., 2004), but genotype-independent regeneration protocols have also been reported (Bailey et al., 1993; Tomlin et al., 2002; Reichert et al., 2003; Sairam et al., 2003). It is concluded that the early maturity genotypes, immature embryos, and MS-based medium are ideal factors for plant regeneration in soybean tissue culture. Morphological variants in soybean have been obtained through cell and tissue culture (Graybosch et al., 1987; Bailey et al., 1993), and although these research efforts failed to deliver high-yielding soybeans, methodologies were developed to regenerate complete soybean plants, a prerequisite for genetic transformation. Soybean has been regenerated by suspension cultures. Christianson et al. (1983) regenerated through embryogenesis from immature embryo-derived cell cultures at very low frequency. Soon after, Li et al. (1985) reported a regeneration system by single cells derived from the frozen immature embryos. Suspension cultures are potentially useful for the application of modern biotechnologies to soybean improvement, particularly for the selection of mutant cell lines, if cultures were totipotent. Haploid induction through anther culture is a useful tool for the production of homogeneous plants; microspore-derived whole plant production has not yet been reported in soybean, except callus development from anthers cultured on several media modifications (de Moraes, 2004). 2.6.5.2 Protoplasts Culture Protoplasts provide techniques for genetic manipulation and plant improvement programs at the cellular level, in particular the induction of somaclonal variation, somatic hybridization, and transformation. Because of economic significance of soybean, researchers have long sought to improve and optimize the protoplast culture system. Plant regeneration from soybean protoplasts has been difficult. There are only few reports where success has been achieved in plant regeneration from soybean protoplasts. Wei and Xu (1988) first established a routine plant regeneration system from immature cotyledon protoplasts. 2.6.5.3 Genetic Transformation Foreign genes of economic importance can be delivered into soybeans by Agrobacterium (Hinchee et al., 1988; Olhoft et al., 2001) and particle bombardment (Sato et al., 1993; Kinney, 1996; reviewed by Finer et al., 1995; Christou, 1997; Furutani and Hidaka, 2004). Padgette et al.

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(1995) reported a stable glyphosate-tolerant soybean line (known as Roundup Ready®) that had been developed using the Agrobacterium-mediated gene transfer method. Kinney (1996) produced a high oleic acid content (84%) soybean through particle bombardmentmediated transformation. The high oleic acid-containing transgenic soybean lines were stable over a number of different environments during a single growing season and were competitive in terms of yield with the parental commercial soybean line. High lysine (up to 12%) soybeans lines have been produced by transformation. Normal soybeans contain about 6% lysine. The high lysine trait was stable in R2 and R3 seeds. Soybean transformants with a lysine content higher than 15% carried wrinkled seed coat and exhibited poor germination (Falco et al., 1995). Soybean transformation methods are not routinely reproducible (Christou, 1997). Soybean transformations are often sterile, and sterility is attributed mostly to chromosomal aberrations (Singh et al., 1998c). Frequently, unexpected segregations and low expression or disappearance of foreign genes have been observed. Genes may be physically present but may be poorly expressed or totally lost in subsequent generations. This may be explained by the poorly understood phenomenon of cosuppression or gene silencing (Stam et al., 1997). 2.6.6

Potential to Produce Hybrid Soybeans

Attempts to produce commercial hybrid soybean cultivars have not succeeded because (1) a good system of producing male-sterile plants is generally not available; (2) soybean pollen must be carried by insect vectors and soybean flowers are generally unattractive to these insects, so even on malesterile plants seed set is often low; and (3) the difficulty in producing hybrids greatly limits the parental combinations that can be tested in order to find commercially acceptable heterosis. Patent 4,545,146 (October 8, 1985) has been granted for hybrid soybean production (Davis, 1985). The methodology remains on the books, but its application in hybrid soybean production has not been realized. Sun et al. (1997) isolated a stable, cytoplasmic-nuclear male-sterile soybean line (cms; A line) and its maintainer (B line) from an interspecific hybrid between G. max and G. soja. Average pollen sterility in all BC4 plants was about 98%, and the parallel crosses showed that the female was normal. This system has been used to develop experimental hybrid cultivars. Several genic (nuclear) male-sterile (gms) soybean lines (ms1 through ms9) are available, and this literature was summarized by Palmer et al. (2004). Jin et al. (1997) identified a gms mutant not allelic to any previously described soybean gms lines. Male-sterile lines can be used to produce hybrid seeds. More than 99% of the seed set on monogenic ms1 ms1 male-sterile plants is the result of natural crossing (Brim, 1973). Distinguishing morphological markers that are visible in seedlings and tightly linked with gms would facilitate early identification of gms plants (Skorupska and Palmer, 1989). Skorupska and Palmer (1989) recorded close linkage between the w1 locus (white flower and green hypocotyls) and the ms6 locus. By utilizing w1 ms6 genetic stock, Lewers et al. (1996) suggested a cosegregation method for hybrid soybean production: purple hypocotyl seedlings are removed shortly after germination, leaving only male-sterile plants, and any remaining recombinants with purple flower are removed at flowering. They used the terms traditional and dilution to describe the other methods for hybrid soybean seed production. The cosegregation method produced higher seed yield, better efficiency, and equal or better seed quality than the traditional and dilution methods. The cosegregation method may be used for male-sterile-facilitated selection, and the cyclic mating system and marker-assisted recurrent selection (Lewers and Palmer, 1997) for cultivar development. The degree of heterosis is an important issue in hybrid soybean production. Nelson and Bernard (1984) examined 27 hybrid combinations. Five hybrids yielded 13 to 19% more than their better parent in at least one season. Manjarrez-Sandoval et al. (1997) recorded heterosis of yield as high as 11% across locations. This may justify resources for breeding hybrid soybean cultivars, but the continual improvement of inbred cultivars will make this a difficult goal to achieve.

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Pfeil, B.E. et al. 2006. Three new species of northern Australian Glycine (Fabaceae, Phaseolae), including G. gracei, G. montis-douglas, and G. syndetika. Aust. Syst. Bot. 19: 245–258. Putievsky, E. and P. Broué. 1979. Cytogenetics of hybrids among perennial species of Glycine subgenus Glycine. Aust. J. Bot. 27: 713–723. Qian, D. et al. 1996. Plant genetic study of restricted nodulation in soybean. Crop Sci. 36: 243–249. Qiu, L. et al. 1999. The history and use of primitive varieties in Chinese soybean breeding. In Proceedings of the World Soybean Research Conference VI, Chicago, August 4–7, 1999. H.E. Kauffman, Ed. Superior Print, Champaign, IL, pp. 165–172. Rahman, S.M., Y. Takagi, and T. Kinoshita. 1996. Genetic control of high oleic acid content in the seed oil of two soybean mutants. Crop Sci. 36: 1125–1128. Rahman, S.M., Y. Takagi, and S. Towata. 1994. Inheritance of high linolenic acid content in the soybean mutant line B739. Breed. Sci. 44: 267–270. Ranch, J.P., L. Oglesby, and A.C. Zielinski. 1985. Plant regeneration from embryo-derived tissue cultures of soybean. In Vitro Cell. Dev. Biol. 21: 653–658. Rauscher, J.T., J.J. Doyle, and A.H.D. Brown. 2004. Multiple origins and nrDNA internal transcribed spacer homeologue evolution in the Glycine tomentella (Leguminosae) allopolyploid complex. Genetics 166: 987–998. Reichert, N.A., M.M.Young, and A. Woods. 2003. Adventitious organogenic regeneration from soybean genotypes representing nine maturity groups. Plant Cell Tiss. Organ Cult. 75: 273–277. Riggs, R.D. et al. 1998. Possible transfer of resistance to Heterodera glycines from Glycine tomentalla to Glycine max. J. Nematol. 30: 547–552. Sadanaga, K. and R.L. Grindeland. 1984. Locating the w1 locus on the satellite chromosome in soybean. Crop Sci. 24: 147–151. Sairam, R.V. et al. 2003. A study on the effect of genotypes, plant growth regulators and sugars in promoting plant regeneration via organogenesis from soybean cotyledonary nodal callus. Plant Cell Tiss. Organ Cult. 75: 79–85. Samoylov, V.M. et al. 1998. A liquid-medium based protocol for rapid regeneration from embryogenic soybean cultures. Plant Cell Rep. 18: 49–54. Sato, S. et al. 1993. Stable transformation via particle bombardment in two different soybean regeneration systems. Plant Cell Rep. 12: 408–413. Schnebly, S.R. et al. 1994. Inheritance of reduced and elevated palmitate in mutant lines of soybean. Crop Sci. 34: 829–833. Sebastian, S.A. and R.S. Chaleff. 1987. Soybean mutants with increased tolerance for sulfonylurea herbicides. Crop Sci. 27: 948–952. Sebastian, S.A. et al. 1989. Semidominant soybean mutation for resistance to sulfonylurea herbicides. Crop Sci. 29: 1403–1408. Sen, N.K. and R.V. Vidyabhusan. 1960. Tetraploid soybeans. Euphytica 9: 317–322. Shetty, K., Y. Asano, and K. Oosawa. 1992. Stimulation of in vitro shoot organogenesis in Glycine max (Merrill) by allantoin and amides. Plant Sci. 81: 245–251. Shimamoto, Y. et al. 1998. RFLPs of chloroplast and mitochondrial DNA in wild soybean, Glycine soja, growing in China. Genet. Resour. Crop Evol. 45: 433–439. Shoemaker, R.C., P.B. Cregan, and L.O. Vodkin. 2004. Soybean genomics. In Soybeans: Improvement, Production, and Uses, 3rd ed., Agronomy Monograph 16. H.R. Boerma and J.E. Specht, Eds. American Society of Agronomy/Crop Science Society of America/Soil Science Society of America, pp. 235–263. Shoemaker, R.C. et al. 1986. Chloroplast DNA variation in the genus Glycine subgenus Soja. J. Hered. 77: 26–30. Shormaker, R.C. et al. 1996. Genome duplication in soybean (Glycine subgenus soja). Genetics 144: 329–338. Singh, B.B. and H.H. Hadley. 1968. Material control of oil synthesis in soybeans, Glycine max (L.) Merr. Crop Sci. 8: 622–625. Singh, R.J. 2003. Plant Cytogenetics, 2nd ed. CRC Press, Boca Raton, FL. Singh, R.J. and T. Hymowitz. 1985a. Diploid-like meiotic behavior in synthesized amphidiploids of the genus Glycine Willd. subgenus Glycine. Genome 27: 655–660. Singh, R.J. and T. Hymowitz. 1985b. The genomic relationships among six wild perennial species of the genus Glycine subgenus Glycine Willd. Theor. Appl. Genet. 71: 221–230. Singh, R.J. and T. Hymowitz. 1985c. Intra- and interspecific hybridization in the genus Glycine, subgenus Glycine Willd.: chromosome pairing and genome relationships. Z. Pflanzenzüchtg. 95: 289–310.

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Singh, R.J. and T. Hymowitz. 1988. The genomic relationship between Glycine max (L.) Merr. and G. soja Sieb. and Zucc. as revealed by pachytene chromosome analysis. Theor. Appl. Genet. 76: 705–711. Singh, R.J. and T. Hymowitz. 1989. The genomic relationships among Glycine soja Sieb. and Zucc., G. max (L.) Merr. and ‘G. gracilis’ Skvortz. Plant Breed. 103: 171–173. Singh, R.J. and T. Hymowitz. 1991. Identification of four primary trisomics of soybean by pachytene chromosome analysis. J. Hered. 82: 75–77. Singh, R.J. and T. Hymowitz. 1999. Soybean genetic resources and crop improvement. Genome 42: 605–616. Singh, R.J., H.H. Kim, and T. Hymowitz. 2001. Distribution of rDNA loci in the genus Glycine Willd. Theor. Appl. Genet. 103: 212–218. Singh, R.J., K.P. Kollipara, and T. Hymowitz. 1987. Polyploid complexes of Glycine tabacina (Labill.) Benth. and G. tomentella Hayata revealed by cytogenetic analysis. Genome 29: 490–497. Singh, R.J., K.P. Kollipara, and T. Hymowitz. 1988. Further data on the genomic relationships among wild perennial species (2n = 40) of the genus Glycine Willd. Genome 30: 166–176. Singh, R.J., K.P. Kollipara, and T. Hymowitz. 1989. Ancestors of 80- and 78-chromosome Glycine tomentella Hayata (Leguminosae). Genome 32: 796–801. Singh, R.J., K.P. Kollipara, and T. Hymowitz. 1990. Backcross derived progeny from soybean and Glycine tomentella Hayata intersubgeneric hybrids. Crop Sci. 30: 871–874. Singh, R.J., K.P. Kollipara, and T. Hymowitz. 1992a. Genomic relationships among diploid wild perennial species of the genus Glycine Willd. subgenus Glycine revealed by crossability, meiotic chromosome pairing and seed protein electrophoresis. Theor. Appl. Genet. 85: 276–282. Singh, R.J., K.P. Kollipara, and T. Hymowitz. 1993. Backcross (BC2-BC4)-derived fertile plants from Glycine max and G. tomentella intersubgeneric hybrids. Crop Sci. 33: 1002–1007. Singh, R.J., K.P. Kollipara, and T. Hymowitz. 1998a. Monosomic alien addition lines derived from Glycine max (L.) Merr., and G. tomentella Hayata: production, characterization, and breeding behavior. Crop Sci. 38: 1483–1489. Singh, R.J., K.P. Kollipara, and T. Hymowitz. 1998b. The genomes of Glycine canescens F. J. Herm., and G. tomentella Hayata of Western Australia and their phylogenetic relationships in the genus Glycine Willd. Genome 41: 669–679. Singh, R.J. et al. 1992b. Putative ancestors of 80-chromosome Glycine tabacina. Genome 35: 140–146. Singh, R.J. et al. 1998c. Cytological characterization of transgenic soybean. Theor. Appl. Genet. 96: 319–324. Singh, L., C.M. Wilson, and H.H. Hadley. 1969. Genetic differences in soybean trypsin inhibitors separated by disc electrophoresis. Crop Sci. 9: 489–491. Skorupska, H. and R.G. Palmer. 1987. Monosomics from synaptic Ks mutant. Soybean Genet. Newsl. 14: 174–178. Skorupska, H. and R.G. Palmer. 1989. Genetics and cytology of ms6 male-sterile soybean. J. Hered. 80: 304–310. Skorupska, H. et al. 1989. Detection of ribosomal RNA genes in soybean, Glycine max (L.) Merr., by in situ hybridization. Genome 32: 1091–1095. Skvortzow, B.W. 1927. The Soy Bean: Wild and Cultivated in Eastern Asia, Series A, No. 22. Manchuria Research Society, Natural History Section, Harbin, China (in Russian and English). Song, L. et al. 1995. Field assessment of supernodulating genotypes of soybean for yield, N2 fixation and benefit to subsequent crops. Soil Biol. Biochem. 27: 563–569. Song, Q.J. et al. 2004. A new integrated genetic linkage map of the soybean. Theor. Appl. Genet. 109: 122–128. Stam, M., J.N.M. Mol, and J.M. Kooter. 1997. The silence of genes in transgenic plants. Ann. Bot. (London) 79: 3–12. Sun, H., L. Zhao, and M. Huang. 1997. Cytoplasmic-nuclear male sterile soybean line from interspecific crosses between G. max and G. soja. In Proceedings of the 5th World Soybean Research Conference, Chiang Mai, Thailand, February 21–27, 1994. B. Napompeth, Ed. Kasetsart University Press, Bangkok, Thailand, pp. 99–102. Takagi, Y. et al. 1989. High linolenic acid mutant in soybean induced by x-ray irradiation. Jpn. J. Breed. 39: 403–409. Tateishi, Y. and H. Ohashi. 1992. Taxonomic studies on Glycine of Taiwan. J. Jpn. Bot. 67: 127–147. Thompson, J.A., R.L. Bernard, and R.L. Nelson. 1997. A third allele at soybean dt locus. Crop Sci. 37: 757–762. Thompson, J.A. and R.L. Nelson. 1998. Utilization of diverse germplasm for soybean yield improvement. Crop Sci. 38: 1362–1368. Tindale, M.D. 1984. Two new eastern Australian species of Glycine Willd. (Fabaceae). Brunonia 7: 207–213.

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Tindale, M.D. and L.A. Craven. 1988. Three new species of Glycine (Fabaceae, Phaseolae) from North-Western Australia, with notes on amphicarpy in the genus. Aust. Syst. Bot. 1: 399–410. Tomlin, E.S. et al. 2002. Screening of soybean, Glycine max (L.) Merrill, lines for somatic embryo induction and maturation capability from immature cotyledons. In Vitro Cell. Dev. Biol. 38: 543–548. Veatch, C. 1934. Chromosomes of the soy bean. Bot. Gaz. 96: 189. Wang, J.L. et al. 1973. Analysis of the photoperiod ecotypes of soybeans from northern and southern China. J. Agric. 7: 169–180 (in Chinese). Wei, J. and Z. Xu. 1988. Plant regeneration from protoplasts of soybean (Glycine max L.). Plant Cell Rep. 7: 348–351. Wilcox, J.R. 1985. Breeding soybeans for improved oil quantity and quality. In Proceedings of the 3rd World Soybean Research Conference, Ames, IA, August 12–17, 1984. R. Shibles, Ed. Westview Press, Boulder, CO, pp. 380–386. Wilcox, J.R. 2001. Sixty years of improvement in publicly developed elite soybean lines. Crop Sci. 49: 1711–1716. Wilcox, J.R. and J.F. Cavins. 1990. Registration of C1726 and C1727 soybean germplasm with altered levels of palmitic acid. Crop Sci. 30: 240. Wilcox, J.R. and J.F. Cavins. 1987. Gene symbol assigned for linolenic acid mutant in the soybean. J. Hered. 410. Wilcox, J.R., J.F. Cavins, and N.C. Nielsen. 1984. Genetic alteration of soybean oil composition by a chemical mutagen. J. Am. Oil Chem. Soc. 61: 97–100. Windish, L.G. 1981. The Soybean Pioneers. M&D Printing, Henry, IL. Woodworth, C.M. 1933. Genetics of the soybean. J. Am. Soc. Agron. 25: 36–51. Xu, B. 1986. New evidence about the geographic origin of soybean. Soybean Sci. 5: 123–130 (in Chinese). Xu, S.J., R.J. Singh, and T. Hymowitz. 2000b. Monosomics in soybean: origin, identification, cytology, and breeding behavior. Crop Sci. 40: 985–989. Xu, S.J. et al. 2000a. Hypertriploid in soybean: origin, identification, cytology, and breeding behavior. Crop Sci. 40: 72–77. Xu, S.J. et al. 2000c. Primary trisomics in soybean: origin, identification, breeding behavior, and use in linkage mapping. Crop Sci. 40: 1543–1551. Yanagisawa, T. et al. 1991. Marker chromosomes commonly observed in the genus Glycine. Theor. Appl. Genet. 81: 606–612. Yang, X.B. 1996. Assessment and management of the risk of soybean rust. In Proceedings of the Soybean Rust Workshop, Urbana, IL, August 9–11, 1995, National Soybean Research Laboratory Publication 1. J.B. Sinclair and G.L. Hartman, Eds. College of Agricultural, Consumer, and Environmental Sciences, Urbana, IL, pp. 52–63. Zhou, X.A. et al. 1998. The genetic diversity and center of origin of Chinese cultivated soybean. Sci. Agric. Sin. 31: 37–43 (in Chinese). Zhou, X. et al. 1999. Study on the center of genetic diversity and origin of cultivated soybean. In Proceedings of the World Soybean Research Conference VI, Chicago, August 4–7, 1999. H.E. Kauffman, Ed. Superior Print, Champaign, IL, p. 510. Zhu, T. et al. 1995. A single nuclear locus phylogeny of soybean based on DNA sequence. Theor. Appl. Genet. 90: 991–999. Zou, J.J. et al. 2006. SSR markers exhibit trisomic segregation distortion in soybean. Crop Sci. 46: 1456–1461. Zou, J.J., R.J. Singh, and T. Hymowitz. 2003b. Association of the yellow leaf (y10) mutant to soybean chromosome 3. J. Hered. 94: 352–354. Zou, J.J. et al. 2003a. Assignment of molecular linkage groups to soybean chromosomes by primary trisomics. Theor. Appl. Genet. 107: 745–750.

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CHAPTER 3 Groundnut Boshou Liao and Corley Holbrook

CONTENTS 3.1 3.2

Introduction.............................................................................................................................51 Genetic Resources ..................................................................................................................54 3.2.1 Origin and Distribution of Species of the Genus Arachis.........................................54 3.2.2 Dissemination of Groundnut ......................................................................................54 3.2.3 Taxonomy of Arachis Species....................................................................................55 3.2.4 Collection and Conservation of Groundnut Germplasm and Wild Relatives ...........57 3.2.5 Evaluation and Core Collections................................................................................59 3.3 Cytogenetics and Genomes ....................................................................................................62 3.4 Crop Improvement..................................................................................................................63 3.4.1 Traditional Breeding Methods....................................................................................63 3.4.2 Interspecific Hybridization .........................................................................................64 3.4.3 Mutation Breeding......................................................................................................66 3.4.4 Marker-Assisted Selection..........................................................................................66 3.4.5 Genetic Transformation ..............................................................................................68 3.4.6 Breeding Progress for Important Traits .....................................................................69 3.4.6.1 Resistance to Foliar Diseases .....................................................................69 3.4.6.2 Resistance to Soilborne Fungi Diseases.....................................................71 3.4.6.3 Resistance to Bacterial Wilt........................................................................72 3.4.6.4 Resistance to Nematodes ............................................................................73 3.4.6.5 Resistance to Aflatoxin Contamination ......................................................73 3.4.6.6 Improved Drought Tolerance ......................................................................75 3.4.6.7 High Oil Content and Improved Oil Quality .............................................75 3.5 Looking Ahead .......................................................................................................................76 References ........................................................................................................................................77

3.1 INTRODUCTION Cultivated groundnut (Arachis hypogaea L.), also known as peanut, is an important source of plant oil and protein worldwide. It is also an important source of farmers’ cash income in countries

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Figure 3.1

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Groundnut growing under mulch conditions in central China.

where it is grown. This leguminous crop has been extensively cultivated in more than 100 countries between latitudes 40°N and 40°S since the late 19th century. During 2000 to 2003, the global average annual sowing area under groundnut was 24.67 million ha, with a total production of 35.01 million tonnes and a yield of 1421 kg ha–1 (FAO, 2000–2003). Asia and Africa have been the largest producers, with about 92% of the global groundnut crop grown there. The remaining 8% comes from North America, the Caribbean, Europe, and Oceania. Groundnut production in a few countries, including the U.S. and Australia, is completely mechanized, but in many other regions the production is mostly operated by hand. Nearly 94% of groundnut was produced in the developing world, mostly under rainfed conditions (Dwivedi et al., 2003). Currently, the major groundnut-producing countries are China, India, Indonesia, Myanmar, and Vietnam in Asia; Nigeria, Sudan, Democratic Republic of Congo, Chad, Mozambique, Zimbabwe, Burkina Faso, Uganda, and Mali in Africa; the U.S.; and Argentina, Brazil, and Mexico in Central and South America. India has had the largest groundnut sowing area for many years, but it has ranked below China in total production since 1993, as both sowing area and yield in China have considerably increased. China has contributed the most to the increase in global groundnut production during the past two decades. The key factors contributing to the increased yields in China include application of improved cultivars and cultural practices such as polyethylene film mulching (Figure 3.1) and balanced fertilization (Wan, 2003). In contrast, groundnut yields in Africa are relatively low, with many countries reporting yields as low as 500 to 800 kg ha–1 due to various reasons. Although the Central and South American regions contribute only about 3.4% of the world groundnut production, relatively high yields of 2200 kg ha–1 in Argentina have been reported. Global production of groundnut is projected to increase in the future, especially in the developing countries.

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Approximately 53% of the total global production of groundnut is crushed for edible oil, 32% is used for confectionery consumption, and the remaining 15% is used for animal feed and seed for production (Dwivedi et al., 2003). Thus, groundnut is one of the major oilseed crops worldwide. The current ratio of groundnut utilization is not expected to change much in the near future, and groundnuts will continue to be cultivated as a major edible oil source in many developing countries, especially in the largest producing countries, such as India and China, where edible oil supply is likely still in shortage (Liao, 2001). Most groundnut will be used as direct food consumption in the developed countries. Groundnut cake after oil extraction can be used in human food or incorporated into animal feeds (Savage and Keenan, 1994). Groundnut haulms constitute approximately 45% of the total plant biomass and provide excellent forage for cattle and swine in many regions. Haulms are rich in protein and more palatable than many other fodders (Cook and Crosthwaite, 1994). Thus, groundnut plays an important role in sustainable agricultural systems in addition to the diversified utilization of the kernels. Genetic improvement is crucial for groundnut industry development. Enhancement of productivity and quality are high priorities in most groundnut research programs worldwide. There are many biotic and abiotic constraints to groundnut production in various ecoagricultural systems. The important widespread biotic constraints are foliar diseases, including late leaf spot (Phaeoisariopsis personata (Berk. & Curtis) Deighton), early leaf spot (Cercospora arachidicola Hori), rust (Puccinia arachidis Sperg.), and web blotch (Didymella arachidicola (Chock.) Taber, Pettit & Philley); and stem or root rot caused by soilborne fungi, such as Sclerotium rolfsii Sacc., bacterial wilt (Ralstonia solanacearum (E.F. Smith)), groundnut rosette virus, peanut clump virus, peanut bud necrosis, peanut stunt virus, peanut strip virus, tomato spotted wilt virus, nematodes, leaf miner, and Spodoptera spp. The abiotic constraints are drought, acid soil, low soil fertility, and low temperature. Based on the estimate made by International Crops Research Institute for the Semiarid Tropics (ICRISAT), late leaf spot, drought, and rust are the most important constraints in terms of economic losses globally (Dwivedi et al., 2003). Much of the global plant breeding effort is being redirected from only developing cultivars with high yields to ones that are also incorporating genes conferring resistance or tolerance to the important constraints to genotypes with adaptability to certain locations. A consumer concern about food quality of groundnut has become increasingly important. Groundnuts are susceptible to Aspergillus infection, which can result in aflatoxin contamination during production, processing, storage, and transportation. Generally, aflatoxin contamination is more serious in the warm tropical and subtropical regions and in those systems with poor management. Another concern for groundnuts and groundnut products is allergens, as groundnuts have proteins that result in allergic reactions in about 0.6% of the population (Li et al., 2000). Trace amounts of groundnut protein could lead to fatal anaphylactic reactions in individuals allergic to groundnuts. Dry roasting of peanut has been reported to increase the allergenic properties of the proteins (Maleki et al., 2000). Refined groundnut oil does not contain protein, and thus the oil is normally allergen-free. However, when the seed is cold pressed, as is done in many parts of the world, protein remains in the oil used for cooking, and allergic reactions could occur. With the increase of production inputs such as pesticides and plant regulators, chemical residue in groundnut is attracting much more concern in many regions. Genetic enhancement is one of the key approaches addressing the food quality and food safety of groundnut. The history of groundnut germplasm and breeding research can be traced back to the early 20th century, and improved cultivars have predominated production for more than 50 years in most groundnut-producing regions in the world. More recent progress has been made in broadening the genetic diversity of groundnut cultivars by combining traditional and advanced technologies, including introgression of genes from wild Arachis species, marker-assisted selection, and transformation of alien genes. The purpose of this chapter is to review the global progress on genetic resources and varietal enhancement of groundnut.

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3.2 GENETIC RESOURCES 3.2.1

Origin and Distribution of Species of the Genus Arachis

The plant species of genus Arachis originated and evolved in South America. The genus consists of many diploid and several tetraploid species. The unique characteristic of the species in genus Arachis is that they flower aboveground but produce fruits below the soil surface, which is different from almost all other plant species. Several species of Arachis have been cultivated for their edible seeds or for forage, whereas only the cultivated groundnut has been domesticated and widely distributed. The cultivated groundnut is a tetraploid species and a member of the Fabaceae, tribe Aeschynomeneae, subtribe Stylosanthinae in genus Arachis. It is believed to have evolved from a natural hybridization combination of two diploid species. The cultivated groundnut was described by Linnaeus in 1753 as Arachis (from the Greek arachos, meaning “weed”) and hypogaea (meaning “underground chamber”) (Hammons, 1982). Geographically, the cultivated groundnut is believed to have originated in the southern Bolivia to northern Argentina region of South America. In this region, many genotypes have been found with primitive characteristics in plant, pod, and seed. Similarly, wild species of Arachis are native to a large region of South America, extending from the foothills of the Andes to the Atlantic and from the northern shores of Brazil to about 34˚S in Uruguay, and the greatest amounts of variation are found in Brazil (Gregory et al., 1980) or the Mato Grosso region of Brazil to eastern Bolivia (Stalker et al., 1994). However, when specifically comparing A. hypogaea to other species, the greatest probability of finding unique genes is in the north-central, northeast, south, and southeast regions of Brazil. These are the areas where species distantly related to the domesticated groundnut are found, as well as ones that are cross-incompatible with A. hypogaea. Recent evidence indicates that northwest Peru may be another possible site for the origin of the cultivated peanut. Valls et al. (1985) reported that species distributions are nearly continuous, and there is an extensive amount of distributional overlap among taxa in different sections of the genus. In total, 68 wild species have been described (Krapovickas and Gregory, 1994) among the 1300 accessions collected from South America. For A. hypogaea, six centers of diversity have been identified in South America, including the geographic regions of (1) Guarani (Paraguay-Paraná), (2) upper Amazon and west coast of Peru, (3) Goiás and Minas Gerais of Brazil, (4) Rondonia and northwest Mato Grosso of Brazil, (5) southwest Amazon in Bolivia, and (6) northeastern Brazil. 3.2.2

Dissemination of Groundnut

The domesticated groundnut has been cultivated and utilized in South America for at least 3500 years, as the earliest archaeological evidence of groundnut in Peru dates back to 1500 B.C. (Hammons, 1982). It is generally believed that the groundnut was transported from South America to other continents after the New World was discovered. Dubard (1906) first tried to explain the movement of groundnut from South America to other countries. He thought that the two groups of groundnut, a two-seeded Brazilian and a three-seeded Peruvian, were basically disseminated by eastward and westward routes, respectively, and then widely distributed in the world. Dubard believed the three-seeded Peruvian group (var. hirsuta) was transported by the westward route from Peru to the western Pacific, China, Indonesia, and Madagascar. There is a concurrence in morphology and configuration of pod samples from China, Indonesia, and Madagascar and between those and the groundnuts found in the tombs at Ancon in Peru. Krapovickas (1968) also concluded that the groundnut moved up the west coast from Peru to Mexico, and across the Pacific islands and then to the Philippines, Malaysia, China, Indonesia, and Madagascar. The other route of the groundnut dissemination was the eastward path to Europe and then Africa. The groundnut was believed to be first introduced into North America from Brazil through Africa by the way of the slave trade (Stalker and Simpson, 1995).

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Asia has been the largest continent for groundnut production since the late 19th century, even though it is most remote from the originating regions of the crop. Krapovickas (1968) believed the groundnut that was first introduced in Asia was most likely the Peruvian type (var. hirsuta), and the Peruvian type groundnut reached Asia (Philippines) through Spanish ships. The estimated time of initial introduction of groundnut to China was the middle of the 16th century, while Goverich thought it was in 1608 (Sun, 1998). Hammons (1982) indicated that the earliest Chinese references about groundnut were later than the time of discovery of the Americas. The sources of information of Hammon’s description were from some reference about China written in English by foreign missionaries during their stay in the country in the early 20th century. However, the access and understanding of Chinese ancient literature might have been limited to the missionaries for several reasons, including the language barrier; thus, the references might be incomplete, as a description for groundnut could be seen in some ancient agricultural literature dated before 1500 (Sun, 1998). There is some evidence to support the possibility that groundnut journeyed from the western coast of South America to China well before the “rediscovery” of America (Person and Moriarity, 1980). According to the limited description about groundnut in China before the 19th century, all the varieties cultivated belonged to the Chinese dragon type (var. hirsuta), based on their runner plant growth habit, long growth period, three or four seeds per pod, strong pod beak, and deep pod reticulation (Sun, 1998). Up to now, about 250 landraces of dragon type germplasm lines with diversified botanical and agronomic characters have been collected from various provinces. One interesting characteristic in the dragon type germplasm is resistance to bacterial wilt caused by R. solanacearum. More than 50 lines are highly resistant to the disease, and all the resistant landraces are from the southern regions of China, where bacterial wilt has been prevalent, while no landrace of this type collected from northern regions with less bacterial wilt severity has been identified as resistant. It is also interesting to note that very few germplasm lines of var. hirsuta or dragon type have been collected outside China. If the dragon groundnut was transported by Europeans in their travel to and from South America, it should be popular among the germplasm lines in Africa and other places. It is well recognized that even among germplasm from South America, germplasm of var. hirsuta is rare. In the agronomic views, the characteristics of var. hirsuta are undesirable, including long growth period, low yield, difficulty in harvest, and long seed dormancy compared to other types, which could be reasons why this variety was less cultivated even in South America, because the local people could find other types to grow. In China, the dragon groundnuts have been replaced by other types since the 1950s, and except for some remote mountain areas, this variety is rarely grown in production. There is no doubt that the discovery of the Americas by European explorers speeded the dissemination of groundnut to many places, including China, and there must have been many instances of introduction. The large-podded Virginia type was introduced from the U.S. into Shandong Province of China in the late 1870s by missionaries, while the Spanish and Valencia types are thought to have been introduced from Southeast Asian countries like the Philippines or Indonesia in the 19th century (Sun, 1998). 3.2.3

Taxonomy of Arachis Species

The genus Arachis has been divided into nine sections (Krapovickas and Gregory, 1994), which consists of diploid (2n = 2x = 20 or 2n = 2x = 18), tetraploid (2n = 4x = 40), and aneuploid species (2n = 2x = 18). Studies on wild species of Arachis trace back to the 1840s, while studies on taxonomy of the species were initiated in the 1930s. Several specialists, including Krapovickas, Gregory, Simpson, Moss, Stalker, and Valls, have contributed greatly to the classification of the wild species. Diversity apparently occurred in the genus as the species became separated into major South American watersheds, and species of sections Erectoides and Extranervosae and the diploids of section Rhizomatosae are believed to be the most ancient (Holbrook and Stalker, 2003). The brief description of each of the nine sections as described by Krapovickas and Gregory (1994) is as follows:

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Section Arachis: Leaves tetrafoliolate, plants erect or decumbent, pegs near 45° angle of soil penetration. Plants are annual or perennial. 2n = 2x = 20, 2n = 4x = 40. Type species: A. hypogaea L. Section Caulorrhizae: Leaves tetrafoliolate, stems with roots/root primordial at the nodes. Plants are perennial. 2n = 2x = 20. Type species: A. repens Handro. Section Erectoides: Leaves tetrafoliolate, plants erect or decumbent, with flowers and fruits grouped generally at the plant base. Roots with enlarged laterals are common in most species. Plants are perennial. Some pegs up to 1 m or longer. 2n = 2x = 20. Type species: A. benthamii Handro. Section Extranervosae: Leaves tetrafoliolate and roots are various sizes and shapes (but basically cylindrical). Standard petal contains red lines on the back side and all flowers are normal with an expanded corolla. Plants are perennial with 2n = 2x = 20. Type species: A. prostrata Benth. Section Heteranthae: Leaves tetrafoliolate, taproot root system, but fibrous and without enlargements. Standard petal contains red lines on front only or on both sides. Flowers are dimorphic: normal and open or very small and closed, small with corolla not exceeding the calyx. Plants are annual or biannual with 2n = 2x = 20. Type species: A. dardani Krapov. & W.C. Gregory (= Ambinervosae Krap. et Greg. nom. und.). Section Procumbentes: Leaves tetrafoliolate, stems with roots occurring in internodes, pegs thickened, horizontal and, in many cases, long. Plants are perennial with 2n = 2x = 20. Type species: A. rigonii Krapov. & W.C. Gregory. Section Rhizomatosae: Leaves tetrafoliolate, plants with rhizomes. Plants are perennial with 2n = 2x = 20 (Ser. Prorhizomatosae), 2n = 4x = 40 (Ser. Rhizomatosae). Type species: A. glabrata Benth. Section Trierectoides: Leaves trifoliolate, hypocotyl tuberiform, plants erect, flowers and fruits primarily at the base of the main stem. Pegs very long, growing horizontal and superficial. Plants are perennial with 2n = 2x = 20. Type species: A. guaranitica Chodat & Hassl. Section Triseminatae: Leaves tetrafoliolate, fruits with two or four segments, lateral branches decumbent with flowers and fruits along with their length. Standard petal contains red lines on both sides. Cotyledons contain ribs on the upper surface (after plant emergence). Plants are perennial with 2n = 2x = 20. Type species: A. triseminata Krapov. & W.C. Gregory.

The cultivated groundnut is divided into two subspecies that are primarily distinguished by branching pattern and distribution of vegetative and reproductive nodes along the main stem and lateral branches. Subspecies hypogaea has two botanical varieties (hypogaea and hirsuta Köhler), and subspecies fastigiata Waldron has four botanical varieties (fastigiata, vulgaris Harz, peruviana Krapov. & W.C. Gregory, and aequatoriana Krapov. & W.C. Gregory) (Krapovickas and Gregory, 1994). Isleib and Wynne (1983) grouped lines using principal component analysis and found that most morphological differences are observed between subspecies. Based on Amplified Fragment Length Polymorphism (AFLP) markers, He and Prakash (1997) thought that the botanical varieties aequatoriana and peruviana were closer to subspecies hypogaea than subspecies fastigiata, and the wild A. monticola was not distinct from the cultivated A. hypogaea. Although A. hypogaea is believed to have originated east of the Andes Mountains, the oldest archaeological findings are in Peru (Banks, 1987; Banks et al., 1993), where groundnut predates the remains of maize (Zea mays L.) in the region of the Casma Valley. This Peruvian site may be the oldest simply because of good preservation conditions of pods in the dry climate, or there could have been a secondary domestication event, although the available molecular data indicate a single origin of A. hypogaea (Kochert et al., 1996). By using simple sequence repeat (SSR) and a sequence-tagged microsatellite site (STMS), Ferguson et al. (2004) identified 89 alleles in groundnuts, varying from 2 to 17 per locus, with an average of 7.4 alleles per locus. Greater differentiation was observed between the botanical varieties. Ferguson also concluded that Rogers’ modified distance among botanical varieties revealed the similarity of subspecies fastigiata Waldron, vulgaris C. Harz, and aequatoriana Krapov. & W.C. Gregory but did not support the inclusion of var. peruviana Krapov. & W.C. Gregory in this grouping. Moreover, the results suggest that subsp. hypogaea var. hypogaea and var. hirsuta Köhler are not closely related, and therefore should not hold the same subspecific ranking.

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Figure 3.2

3.2.4

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(See color insert following page 144.) Diversity for testa color in groundnut cultivars.

Collection and Conservation of Groundnut Germplasm and Wild Relatives

Germplasm resources are the base of crop improvement. Domesticated groundnut had been disseminated to different locations in the world from South America for hundreds of years and has been cultivated in the originating region for thousands of years. Systematic collection and conservation of groundnut germplasm was initiated in the middle of the 19th century. Since 1959, there have been more than 40 collection trips in South America for A. hypogaea and wild species (Stalker and Simpson, 1995). In addition, a large number of A. hypogaea accessions were introduced into the U.S. from Africa during the 1960s (Holbrook and Stalker, 2003). Globally, more than 40,000 accessions of cultivated groundnut have been assembled, and these accessions are stored in collections in the U.S., the International Crops Research Institute for the Semiarid Tropics (ICRISAT) in India, Argentina, Brazil, China, and Indonesia. The largest collection of domesticated groundnut consisting of about 15,000 accessions from 92 countries is at ICRISAT (Upadhyaya et al., 2001; Dwivedi et al., 2003). The USDA germplasm collection of groundnut contains over 8000 accessions (Holbrook, 2001). The groundnut collection in China contains over 6300 accessions (Liao, 2003). The groundnut germplasm collection in each country normally includes landraces, materials introduced from foreign countries, and improved materials or cultivars developed through breeding. For example, among 6390 accessions of A. hypogaea in China, around 2400 are landraces, 2173 are introduced lines, and the remaining were generated from breeding programs at various institutions. Testa color diversity is common in cultivated groundnut (Figure 3.2) (See color insert following page 144). However, the overlap of groundnut germplasm among countries or institutions is not clear. More than 1300 accessions of wild Arachis species have been collected (Stalker and Simpson, 1995), and importantly, many of these are cross-compatible with the domesticated species.

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A. villosulicarpa Hoehen, which grows in the northeastern region of Mato Grosso, Brazil, and A. stenosperma Krapov. & W.C. Gregory, which grows in central and southeast Brazil, have also been cultivated for their seeds (Gregory et al., 1973; Simpson et al., 1993b). In the case of A. stenosperma, seeds were apparently carried by either the native people or missionaries from central Brazil to the southeast because plants of the species are found at abandoned missionary sites. Several wild species have been used for forages, including A. glabrata, which has several cultivars under cultivation (Prine et al., 1986, 1990). Unfortunately, this species produces few seeds, and propagation is entirely through rhizomes. A. pintoi Krapov. & W.C. Gregory is cultivated as a forage crop in South America and Australia (Asakawa and Ramirez, 1989; Cameron et al., 1989). It produces large numbers of seeds and is relatively easy to establish under field conditions. Numerous other species have been used as ornamentals, including A. repens Handro, which is used extensively as a roadside and landscape plant in Central and South America (Stalker and Simpson, 1995). Large breeding efforts have been undertaken since the mid-1950s to characterize species for agronomically useful traits (Lynch and Mack, 1995; Stalker and Simpson, 1995), and many accessions have been identified with high levels of disease and insect resistance. Large wild Arachis species collections are also maintained at Texas A&M, North Carolina State University, the International Crops Research Institute for the Semiarid Tropics (ICRISAT) in India, the National Center of Genetic Resources (CENARGEN) in Brazil, and the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences (CAAS) in China. Preservation of accessions in the A. hypogaea collection is generally straightforward, and regeneration decisions are based on the total number and age of seeds available in storage and the number of requests made by the user community. Groundnut seed can be stored for several years if temperature and humidity can be regulated. Sanders et al. (1982) indicated that the sum of temperature (F) plus relative humidity should be less than 100 to have optimal seed storage. Under ideal storage conditions, groundnuts remain viable for 15 or more years, and seeds could be stored for 20 years with good germination percentages under the storage conditions of 10°C and 45% relative humidity. Lu (2001) found that the grounndut seeds had a germination ratio over 98% after 10 to 12 years’ storage in the gene bank at the Chinese Academy of Agricultural Sciences. Wild Arachis accessions are more difficult to maintain than ones of the domesticated groundnut. Species of Arachis occupy a wide range of habitats in South America, and accessions can be lost before adequate growing conditions under cultivation can be discovered, especially for species that produce few seeds. Both annual and perennial species exist in nature, and the annuals generally produce greater numbers of seeds than perennials. Of the more than 1300 Arachis wild species accessions that have been collected, about 800 remain in germplasm nurseries (Stalker and Simpson, 1995). A. marginata Gardner is an example of a species that is very difficult to maintain in the U.S. because plants grow very slowly and are weak. The Triseminales species (A. tuberosa Benth. and A. guaranitica Chodat & Hass1) enter permanent dormancy when seeds are dried, but seeds have been kept viable for several years in moist sphagnum moss. Under long day conditions, many species flower profusely (Figure 3.3) but do not produce pegs, whereas under short day conditions, many species have a very high reproductive efficiency but do not produce many flowers (Stalker and Wynne, 1983). More than 25% of the species accessions currently in germplasm nurseries (especially the Rhizomatosae species) produce very few seeds and are maintained as vegetative materials in greenhouses. Many perennial species will not produce seeds when grown under greenhouse conditions, and only a few seeds when propagated in the field. Because groundnuts produce pegs, seed mixture can be caused by either placing pots close to each other in the greenhouse or failing to isolate plots in the field. Several curators only propagate the wild species collection under greenhouse conditions, but this results in few seeds being produced and restricts evaluations for agronomically useful traits. Germplasm exchange is an important issue for continued crop genetic enhancement. However, germplasm exchange has become more difficult during recent years, as many countries have

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Figure 3.3

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Plants and flowers of wild species Arachis glabrata.

imposed more strict quarantine and exchange policies. Diseases such as rosette virus are found only in Africa, and other production areas have attempted to avoid introducing this virus. A 1- to 2-year observation period under greenhouse conditions is generally imposed to restrict introductions of unwanted diseases. The USDA Plant Introduction Station at Griffin, GA, has routinely screened introductions for peanut stripe virus (PStV) using bioassays since the mid-1980s. Intellectual property rights issues have not greatly affected the movement of groundnut germplasm inside most countries, but since the Convention on Biological Diversity in 1993, international seed exchange has become significantly more tedious and restricted (Williams and Williams, 2001). Germplasm obtained prior to 1993 at the Consultative Group on International Agricultural Research (CGIAR) centers, including ICRISAT, which has a mandate to preserve Arachis genetic resources, is freely available, but germplasm obtained since then is subject to the terms of the Convention on Biological Diversity. A memorandum of understanding has been signed by the USDA and ICRISAT to facilitate exchange of germplasm (Shands and Bertram, 2000). 3.2.5

Evaluation and Core Collections

Descriptor lists have been published for groundnut by the International Board for Plant Genetic Resources (IBPGR) and ICRISAT (1992) and the USDA (Pittman, 1995), which has greatly enhanced the evaluation of groundnut germplasm. A large number of accessions in the ICRISAT collection have been evaluated for morphological traits, water use efficiency, and reactions to many disease and insect pests of groundnut (Upadhyaya et al., 2001). Upadhyaya et al. (2002a) described the geographical patterns of diversity for morphological and agronomic traits in the 13,342 groundnut accessions germplasm contained in the ICRISAT gene bank. The germplasm accessions were

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characterized for 16 morphological descriptors, 10 agronomic traits in two seasons, and reaction to early leaf spot and groundnut rosette virus disease. Phenotypic variation was found for most traits in all the regions. South America exhibited 100% of the range of variation for 12 of the 16 morphological descriptors and on average showed the most variation. The Shannon–Weaver diversity index was variable in different regions for different traits. Three of the six botanical varieties, aequatoriana, hirsuta, and peruviana, were poorly represented, indicating the need for additional collection. Principal component analysis (PCA) using 38 traits and clustering on the first seven PC scores delineated three regional clusters, consisting of North America, Middle East, and East Asia in the first cluster, South America in the second cluster, and West Africa, Europe, Central Africa, South Asia, Oceania, South Africa, East Africa, Southeast Asia, Central Asia, and Caribbean in the third cluster. Extensive evaluation for groundnut germplasm has also been conducted in China (Liao, 2003). A core collection was selected to represent the U.S. groundnut germplasm collection and has been used to stimulate germplasm evaluation. Holbrook et al. (1993) used data on groundnut from the Germplasm Resources Information Network (GRIN) to select this core collection. The U.S. germplasm collection was first stratified by country of origin and then divided into nine sets based on the amount of additional information available for accessions and on the number of accessions per country of origin. Multivariate analysis and random selection resulted in the selection of 831 accessions from the U.S. germplasm collection. Accessions included in the core collection are noted in the GRIN, and the relationship between the individual accessions and the clustering procedure used to develop the core collection is available in two table formats on diskette. The core collection approach to germplasm evaluation has two stages. The first stage involves examining all accessions in the core collection for a desired characteristic. This information is then used to determine which clusters of accessions in the entire germplasm collection should be examined during the second stage of screening. Theoretically, the probability of finding additional accessions with a desired characteristic would be highest in these clusters (Holbrook et al., 1993). Data for resistance to late leaf spot (Cercosporidium personatum (Berk. & M.A. Curtis)) (Holbrook and Anderson, 1995) and data for resistance to the peanut root-knot nematode (Meloidogyne arenaria (Neal) Chitwook race 1) (Holbrook et al., 2000c) were used to evaluate the effectiveness of a two-stage core screening approach in identifying resistance in the entire collection. Both studies clearly demonstrated that the core collection approach can be used to improve the efficiency of germplasm evaluations. A major benefit of having a core collection for the U.S. germplasm collection has been a significant increase in groundnut germplasm evaluation work (Holbrook, 1999). Accessions in the core collection have been evaluated for resistance to tomato spotted wilt virus (TSWV) (Anderson et al., 1996a), Cylindrocladium black rot (CBR; C. parasiticum Crous, Wingefield & Alfenas) and early leaf spot (Cercospora arachidicola Hori) (Isleib et al., 1995), the peanut rootknot nematode (Holbrook et al., 2000b), preharvest aflatoxin contamination (PAC) (Holbrook et al., 1998b), Rhizoctonia limb rot (Rhizoctonia solani Kuhn AG-4) (Franke et al., 1999), and Sclerotinia blight and pepper spot (Leptosphaerulina crassiasca) (Damicone et al., 2003). The accessions in the core collection also have been used to identify genetic variation for oil content (Holbrook et al., 1998a) and fatty acid composition (Hammond et al., 1997). Holbrook and Anderson (1993) evaluated accessions in the core collection for eight aboveground and nine belowground plant descriptors. Using these data, it was possible to make inferences about the adequacy of the entire germplasm collection (Holbrook, 1997). It was concluded that additional peanut accessions should be collected from Columbia, Venezuela, Uruguay, and possibly Bolivia. Data generated from research with the U.S. peanut core collection have been used to examine the geographical distribution of resistance to five diseases to obtain a better understanding of the genetic variability available in the entire U.S. peanut germplasm collection (Holbrook and Isleib, 2001). Countries of origin that are valuable sources of resistance to these important diseases of

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peanut were identified. These observations should enable peanut breeders to more efficiently utilize the genes for disease resistance that are available in the U.S. germplasm collection. A core collection has also been developed to represent the A. hypogaea germplasm maintained by ICRISAT (Upadhyaya et al., 2003). This core collection was developed from a total of 14,310 accessions using an approach slightly different from that used by Holbrook et al. (1993). The ICRISAT groundnut collection was first stratified by botanical variety within subspecies, and then stratified by country of origin. Accessions of the same botanical variety, but from small and adjacent countries with similar agroclimates, were grouped together. This resulted in 75 groups, and accessions within each group were then clustered using multivariate statistical analysis. Approximately 10% of the accessions from each cluster were randomly sampled, resulting in a core collection consisting of 1704 entries. Mean comparisons using t test and distribution using chi-square test and Wilcoxon’s rank-sum nonparametric test on different descriptors indicated that the genetic variation available for these traits in the entire collection has been preserved in the core collection. The Shannon–Weaver diversity index for different traits was also similar in the entire collection and core collection. The important phenotypic correlations between different traits, which may be under the control of coadapted gene complexes, were preserved in the core collection. Upadhyaya et al. (2005) reported screening of the groundnut core collection for Asia, consisting of 504 accessions for 22 agronomic traits, to select diverse superior germplasm accessions for use as parents in improvement programs. On the basis of performance compared to control cultivars in different environments, 15 var. fastigiata, 20 var. vulgaris, and 25 var. hypogaea accessions from 14 countries were selected. The selected accessions and control cultivars were grouped using scores of the first 15 principal components (PCs) in fastigiata, 20 PCs in vulgaris, and 21 PCs in hypogaea. The clustering by Ward’s method indicated that the selected accessions were diverse from the control cultivars. A core collection was selected to represent the Chinese germplasm collection (Jiang et al., 2004). All accessions in the germplasm collection were first sorted into five large groups based on the peanut germplasm classification system used in China. Multivariate analysis of all morphological data was then used to form clusters of accessions within these groups. Random sampling was used to select a core collection of 582 accessions. Upadhyaya and Ortiz (2001) suggested a strategy for sampling the entire and core collections for developing a mini-core subset that contains about 1% of total accessions in the entire collection but captures most of the useful variation of the crop. A core of core collections (or mini-cores) has been selected to represent the ICRISAT and U.S. groundnut germplasm collections. For the ICRISAT collection, Upadhyaya et al. (2002b) developed a groundnut mini-core collection consisting of 184 accessions based on morphological, agronomic, and quality traits. Ward’s method of clustering was used to separate core collection accessions into groups of similar accessions. Newman Keuls’ test for means, Levene’s test for variances, and a chi-square test for frequency distribution analysis for different traits indicated that the variation available in the core collection has been preserved in the mini-core subset. Holbrook and Dong (2005) selected a core of the core collection for the U.S. germplasm collection using data for eight aboveground and eight belowground morphological characteristics for all accessions from the core collection. Cluster analysis was used on these data to partition the core accessions into groups, and random sampling was used to select a 10% sample from each group. The result was a core of the core collection (mini-core) consisting of 112 accessions. Holbrook and Dong (2005) also used data on resistances to four diseases to evaluate the mini-core concept. Results indicated that the mini-core collection can be used to improve the efficiency of identifying desirable traits in the core collection and in the entire collection. The core of the core approach should be particularly useful for traits that are difficult or expensive to measure.

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3.3 CYTOGENETICS AND GENOMES There have been many reports of investigation on cytological or cytogenetical aspects of the domesticated groundnut and its wild relative species in the genus. For several decades since the 1930s, both diploid (2n = 2x = 20) and tetraploid (2n = 4x = 40) species in the genus Arachis have been well documented based on extensive characterization. However, a few aneuploid species with 18 chromosomes, including A. decora Krapov., W.C. Gregory & Valls, A. palustris Krapov., W.C. Gregory & Valls, and A. praecox Krapov., W.C. Gregory & Valls (Lavia, 1996, 1998; Krapovickas and Lavia, 2000), have also been identified by more recent investigations (Holbrook and Stalker, 2003). The chromosomes of Arachis species are small, ranging from 1.4 to 3.9 μm in length. Several species have been karyotyped (Stalker and Dalmacio, 1981; Singh and Moss, 1982; Stalker, 1985, 1991; Lavia, 1998) among the materials collected. Considerable cytogenetic and genomic differentiation has been observed among different sections. Tetraploid species are found in section Arachis, including A. hypogaea and A. monticola Krapov. & Rigoni, and in sections Extranervosae and Rhizomatosae. Polyploidy is believed to have originated and evolved independently in different sections (Smartt and Stalker, 1982), and diploids are likely to be more ancient than tetraploids. It has been observed that species in section Arachis generally have metacentric chromosomes, with the exception of A. glandulifera Stalker, which is highly asymmetrical (Stalker, 1991). Karyotypically, six chromosomes appear to be similar, whereas the nucleolus organizer region has been found in different positions on the seventh chromosome, and there are many structural changes in the other three chromosomes (Murty et al., 1985; Bharathi et al., 1983; Kirti et al., 1983; Jahnavi and Murty, 1985). It it is believed that species in sections Erectoides, Extranervosae, and Triseminatae are more ancient than those in sections Arachis and Rhizomatosae. Section Arachis consists of 27 described species, including A. hypogaea. This section is important because introgression from closely related species into A. hypogaea should be easier than from more distantly related species in other sections. Three genomes have been defined in section Arachis, including the A genome found in most species; the B genome species as represented by A. batizocoi Krapov. & W.C. Gregory, A. ipaensis Krapov. & W.C. Gregory, A. cruziana Krapov., W.C. Gregory & C.E. Simpson (Burow et al., 1996), and likely A. williamsii Krapov. & W.C. Gregory (Lavia, 1996; Tallury et al., 2001), A. hoehnei Krapov. & W.C. Gregory, and A. magna Krapov., W.C. Gregory & C.E. Simpson; and the D genome represented by A. glandulifera (Stalker, 1991). It has been found that translocations could be common in A. batizocoi (Stalker et al., 1991), and the nucleolar organizer chromosome is karyotypically different in botanical varieties of A. hypogaea (Stalker and Dalmacio, 1986). Other genomes have been proposed in sections Ambinervosae (Am), Erectoides (E), Caulorhizae (C), Extranervosae (Ex), and Triseminalae (T) (Smartt and Stalker, 1982). Tetraploids in section Rhizomatosae appear to have similarities to the genomes of sections Erectoides and Arachis (Stalker, 1981). Smartt and Gregory (1967) concluded that A. hypogaea has A and B genomes. Kochert et al. (1991) supported this by restriction fragment length polymorphism (RFLP) analysis. In their results, most gel lanes had two bands in A. hypogaea and only one band in diploids. Molecular data indicate that the domesticated groundnut had a single-event origin and that introgression from related species into A. hypogaea has been extremely limited (Kochert et al., 1996). A. monticola is the only additional tetraploid species in the section Arachis, which Stalker and Simpson (1995) concluded as a weedy derivative of A. hypogaea rather than its progenitor. Within A. hypogaea, cytological differentiation has been reported in meiotic and somatic cells among different subspecies (Stalker, 1985). Secondary constrictions have been observed on five different chromosomes in cultivars of var. hypogaea, fastigiata, and vulgaris (Stalker and Dalmacio, 1986), from which translocation is speculated to be relatively common in A. hypogaea. Molecular evidence indicates that A. monticola is nearly indistinguishable from A. hypogaea. Raina and Mukai (1999) used genomic in situ hybridization for investigating genome organization and evolution of cultivated groundnut in relation with wild species

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and confirmed the allopolyploid nature of A. hypogaea and A. monticola. Their results also supported the view that wild A. monticola and cultivated A. hypogaea are very closely related, and A. villosa and A. ipaensis are the diploid wild progenitors of the tetraploid species. Generally, less work has been done for chromosome engineering in the cultivated groundnut. Efforts have been made on introgression of genes from wild relative species into the cultivated groundnut, and chromosome engineering was involved. This aspect is described in the next section.

3.4 CROP IMPROVEMENT 3.4.1

Traditional Breeding Methods

The domesticated groundnut is widely cultivated and utilized. Globally, most groundnuts are crushed for oil, especially in certain developing countries such as India and China. In the U.S. and several other countries, most groundnuts are consumed as foods, including peanut butter, snack, and roasted pods or seeds, and only those deemed unsuitable for human consumption are used for oil stocks. While the overall portion for oil extraction will be stable, the edible groundnuts seem to be more diversified in the future due to market development and breeding progress. Several authors have estimated the role of crop breeding in the groundnut industry. Isleib et al. (2001) estimated that resistant cultivars have had an economic impact of more than $200 million annually for U.S. groundnut producers. The largest positive impact on groundnut production has come through development of cultivars with resistance to Sclerotinia blight, root-knot nematode, and TSWV. Wan (2003) estimated that application of new cultivars has contributed at least 30% of the groundnut yield increase in China. It is common that fewer groundnut cultivars have been used in the developed countries than in the developing countries. During the 1970s and 1980s, three cultivars, including Florunner, Florigiant, and Star, prevailed in production in the U.S., but more than 40 cultivars were grown in China and India. The number of released cultivars may not necessarily reflect the breeding effort or its effectiveness, but the diversified production conditions and lack of effective seed system for quick seed extension in China and India have been the reasons for more groundnut cultivars used in production. Standard breeding methods for self-pollinated crops have been widely used to develop groundnut cultivars (Isleib and Wynne, 1992; Isleib et al., 1994; Knauft and Ozias-Akins, 1995; Knauft and Wynne, 1995; Sun, 1998; Wan, 2003). Cultivars that have been widely distributed are commonly used as parents in hybridization programs, and thus the genetic base of groundnut cultivars has historically been quite narrow. In China, two local groundnut varieties, Fuhuasheng and Shitouqi, have been extensively used in breeding, and their pedigree could be traced in more than 60% of the groundnut cultivars released (Sun, 1998; Xue and Isleib, 2002). However, since the late 1980s, a large number of diverse cultivars have been released in major groundnut-growing countries, and consequently, the genetic base of the commercially produced germplasm is much broader at present. Parental selection is an important consideration in plant breeding, and with uniformity requirements imposed by the groundnut processing industries, the genetic base of groundnut will continue to be relatively narrow in the future. Pedigree breeding is commonly used in groundnut as in many other self-pollinated crops. The backcross method is becoming more frequent as useful qualitatively inherited traits are identified. Mass selection is used infrequently in groundnut because of negative correlations between disease resistance and yield (Knauft and Wynne, 1995). Use of the single-seed descent method has greatly increased in recent years. Advantages of single-seed descent include savings in space and resources (Isleib et al., 1994). Recurrent selection has received little attention in groundnut breeding because of the efforts needed to make a large number of crosses, but Halward et al. (1991) concluded that genetic gain for multiple traits could be made in a population derived from an interspecific cross between A. hypogaea and A. cardenasii Krapov. & W.C. Gregory.

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Production of F1 hybrids on a commencial scale is not a viable option in groundnut because of the difficulties encountered in making crosses. There does not appear to be a large advantage to early generation testing in groundnut, in large part because most breeding efforts with the crop are for quantitatively inherited traits. It is clear that most improved groundnut cultivars have been developed through conventional hybridization breeding. Besides selection for productivity, more emphasis is being given to resistance to diseases, pests, and abiotic stresses and quality traits. Facilities for evaluating resistance and quality traits are crucial for breeding progress. 3.4.2

Interspecific Hybridization

Transferring genes conferring important resistance or quality traits from wild relative species of the genus Arachis has been a key strategy in groundnut varietal improvement. High levels of resistance to the many diseases and insect pests have generated much interest in creating interspecific hybrids. Although transformation technologies are alternatives for utilizing resistant species that will not hybridize with A. hypogaea, genes or gene complexes that confer high levels of disease or insect resistance have not been isolated in groundnut. Until agronomically useful genes are isolated and shown to be stably expressed by transformation technologies, interspecific hybridization is the most promising method to introgress genes from related Arachis species that are not present in the cultivated groundnut. Gregory and Gregory (1979) conducted the most comprehensive crossing study in groundnut by a diallel crossing program with 100 accessions of Arachis species. Their studies indicated that crosses between species of different sections resulted in very few hybrids, and all progenies of such crosses were sterile. Hybrids within sectional groups were easier to produce, and fertility levels were generally higher; however, a considerable amount of sterility can still occur (Stalker et al., 1991). Bridge crosses have also been attempted with species in sections Rhizomatosae and Erectoides, but Stalker (1985) concluded that the germplasm outside section Arachis is inaccessible to the domesticated groundnut through conventional sexual hybridization. However, Mallikarjuna and Sastri (2002) reported production of hybrids between A. hypogaea cv. MK 374 and A. glabrata through interspecific pollinations and embryo culture. The hybrids produced had morphological characters of both parents plus floral abnormalities not seen in either parent. Cytological research showed variable chromosome association and also homoeology between the genomes of A. hypogaea and A. glabrata. The hybrids inherited resistance to rust, late leaf spot, peanut bud necrosis, and peanut stripe diseases from the pollen parent A. glabrata. Shen et al. (1995) also reported fertile hybrids between A. hypogaea and A. glabrata Benth. Restricted fertilization is not believed to be the cause of hybridization failures in most interspecific crosses (Sastri and Moss, 1982). However, application of gibberellic acid or mentor pollen to the stigma at the time of pollination may increase the frequency of pegging (Sastri and Moss, 1982; Stalker et al., 1987). Using annual species as female parents vs. perennial species usually results in a higher success rate, which may be related to perennials having smaller stigmas and being surrounded by a protective ring of hairs (Lu et al., 1990). Because of the apparent immunity of species in section Rhizomatosae to leaf spots and many viruses, research programs in the U.S. prior to 1980 (as well as much of the work at ICRISAT since that time) have concentrated on introgressing genes from A. glabrata into A. hypogaea. However, only species in section Arachis are readily available for gene introgression. Hybrids of A. hypogaea × A. monticola are relatively easy to produce. Because most of the diseases and insects that are problematic in A. hypogaea are also a problem with A. monticola, and considering that A. monticola has fragile pegs and one-seeded pods, it has generally not been considered a good parent for breeding. Diploid species of the section Arachis have greater potential for cultivar improvement than A. monticola because many accessions have very high levels of resistance to many economically important insect pests (Stalker and Campbell, 1983; Lynch and

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Mack, 1995) and diseases (Stalker and Moss, 1987; Stalker and Simpson, 1995). Several methods have been attempted to introgress genes into A. hypogaea from diploid species, with each having advantages and disadvantages, but none of them lead to quick introgression of desired genes because of sterility in progenies and limited genetic recombination. As many species contain traits not found at high levels in A. hypogaea, the efforts for producing interspecific hybrids would still be a key approach in crop improvement. Introgressing useful genes from wild species into A. hypogaea is normally influenced by sterility barriers due to different ploidy levels, genomic incompatibilities, and cryptic genetic differences. Constraints to obtaining hybrids may occur at the time of fertilization, during early cell division of the embryo, or during later embryo development. Even when hybrids are obtained, genetic recombination is often restricted, and desired genes may not be incorporated into the A. hypogaea genome properly. Thus, simply obtaining fertile and stable 40-chromosome progenies from interspecific hybridization does not guarantee gene incorporation into the desired genome. Introgression of useful traits in groundnut is a two-step process where a trait is first incorporated into the A. hypogaea genome (which also results in progenies with many unfavorable traits), and then a plant breeding program is initiated to enhance yield and quality traits while at the same time selecting for the desired trait. Hybrids of A. hypogaea with wild species of section Arachis with both A and B genomes have been obtained in many institutions. Krapovickas and Rigoini (1951) produced the first interspecific hybrid with A. villosa var. correntina, and since then, many other interspecific combinations have been successful (Stalker and Moss, 1987; Stalker and Simpson, 1995). Several interspecific hybrids are more difficult to obtain (Stalker et al., 1991), although most of the diploid species could hybridize relatively easily with the domesticated groundnut. Normally, hybridization could be more successful when the domesticated species is used as the female parent. Direct hybridization between A. hypogaea and diploids results in sterile triploid hybrids. Fertility can be restored at the hexaploid level with colchicine treatment on the vegetative cuttings (Singh et al., 1991b), or sometimes by simply propagating plants under field conditions for a prolonged growth period (Singh and Moss, 1984). Hexaploids are expected to have 30 bivalents, but many plants are cytologically unstable (Company et al., 1982) and thus produce very few seeds. Hexaploid × diploid (and reciprocal) crosses normally abort, so a one-step program to lower the chromosome number to the tetraploid level is less possible (Halward and Stalker, 1987). Reducing the chromosome number to 2n = 40 has been accomplished by several methods, including self-pollination and subsequent chromosome loss (Stalker, 1992) and by backcrossing with A. hypogaea. When hexaploids are backcrossed with the tetraploid A. hypogaea, the pentaploid hybrids are mostly sterile and produce few flowers, and continued backcrossing is not practical. Unexpectedly, some pentaploids have 25 bivalents (Company et al., 1982), which indicates that there is more genomic similarity between A. hypogaea and related diploid species than the commonly designated A and B genomic designations would imply. When pentaploid plants are allowed to self-pollinate, a few will produce viable aneuploid progenies and a second generation of selfing will usually result in tetraploid progenies from which fertile lines can be selected. Garcia (1995) conducted studies on introgressing genes from diploid species to A. hypogaea by crossing the cultivated groundnut with several diploid species, restoring fertility after colchicine treatment of F1s to produce fertile hexaploids, and then selfing hexaploids and backcrossing with a recurrent parent at each respective generation after selfing. Garcia found that increasing numbers of molecular markers were lost during each selfing generation, which likely resulted from meiotic irregularities and subsequent random loss of genes. Garcia et al. (1995) also analyzed a tetraploid population derived by selfing hexaploids and reported introgression of A. cardenasii (A genome species) genes into A. hypogaea in 10 of the 11 linkage groups on a molecular map. The authors concluded that this was evidence that the genomes of A. hypogaea are similar and that the species is not a true allopolyploid. Some progenies selected from the highly diverse A. hypogaea × A. cardenasii hybrid population were highly resistant to early leaf spot, late leaf spot, root-knot nematode, southern corn rootworm (Diabrotica undecimpunctata howardi Barber), and potato leafhopper (Empoasca

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fabae (Harris)). Several germplasm lines have been released from these progenies (Stalker and Beute, 1993; Moss et al., 1997; Stalker et al., 2002a, 2002b; Stalker and Lynch, 2002). Another method to produce tetraploid interspecific hybrids is to produce autotetraploids or amphiploids of Arachis species prior to crossing with A. hypogaea. Polyploids are relatively easy to produce in groundnut by treating germinating seeds with colchicine, but cytological identification of polyploid branches is necessary to confirm chromosome numbers of reproductive tissues. Autotetraploids have been produced with at least eight diploid species of section Arachis (Singh, 1986a), but they are generally weak plants with poor survival for more than one growing season. Thus, crossing programs between autotetraploids and A. hypogaea need to be conducted as soon as plants are cytologically identified. Germplasm lines have not been released from this cytological pathway. Furthermore, amphidiploids can also be produced between two or more diploid species before crossing with A. hypogaea. Gardner and Stalker (1983) created amphidiploids of hybrids between A genome diploids and observed high bivalent association in the F1 hybrids with A. hypogaea. When amphidiploids are created by crossing A. batizocoi (B genome) with A genome species, there are usually greater numbers of bivalents in the polyploids. Although advantages exist for using A. batizocoi as a parent in crosses to enhance fertility restoration, this species is susceptible to late leaf spot and other diseases, which may result in unfavorable traits being introduced into breeding lines. Rust-resistant hybrids have been selected from progenies of A. hypogaea × amphiploid (A. batizocoi × A. duranensis Krapov. & W.C. Gregory) and (A. correntina × A. batizocoi) (Singh, 1986b). Simpson et al. (1993a) released TxAG6, a germplasm line derived from a 4x (A. batizocoi × (A. cardenasii × A. diogoi)) cross, as well as its backcross with A. hypogaea cv. Florunner. Both germplasm lines were highly resistant to the root-knot nematode, M. arenaria. The cultivar COAN with resistance to M. arenaria and M. javanica was derived from a backcross program using this amphiploid and ‘Florunner’ as the recurrent parent. In China, extensive efforts have been made for introgressing useful genes from wild Arachis species to the cultivated groundnut. A bacterial wilt-resistant cultivar, Yuanza 9102, a selected progeny of A. hypogaea cv. Baisha 1016 with A. diogoi, has been released in Henan Province (Liao, 2003). Several germplasm lines derived from wide crossing with high oil content have been obtained (Sun, 1998). 3.4.3

Mutation Breeding

In the U.S., the mutation breeding methodology was used extensively in the late 1950s to early 1970s, but the materials produced were not widely utilized in groundnut improvement or production. In China, mutation breeding of groundnut has been conducted in several institutions since the 1960s, and several cultivars have been developed with mutants as breeding parents (Qiu, 1992), among which Yueyou 551 and 8130 have been extensively cultivated. Groundnut mutants with variation in content of protein, oil, amino acid, and fatty acid components have been obtained by treating a Spainish type cultivar, Baisha 1016 (Qiu and Feng, 1998). Mutants with improved resistance or tolerance to salt, leaf spot, and drought stress have been reported (Qiu and Feng, 1998). The effectiveness of mutation breeding can be enhanced with other approaches such as hybridization and tissue culture. At least nine groundnut cultivars have been developed by mutation breeding and released in India. Globally, mutation breeding has been less utilized in groundnut improvement in recent years. Wang (2002) reported production of extra large and small pod mutants by chemical mutagenesis. 3.4.4

Marker-Assisted Selection

Isozyme analysis of A. hypogaea has shown little variation (Grieshammer and Wynne, 1990), but polymorphisms are somewhat more frequent among interspecific hybrids (Lacks and Stalker,

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1993). Isozyme studies of species in section Erectoides showed a large amount of variation, and species appeared to associate with members of other sections. Analysis of seed storage proteins has shown that variation exists among species of section Arachis (Singh et al., 1991a; Bianchi-Hall et al., 1993, 1994), but proteins are less useful for discrimination at the species level in the genus. Restriction fragment length polymorphism (RFLP) is the first DNA marker system with a sufficiently large number of polymorphisms that could be used to create linkage maps and to implement indirect selection strategies in crops. In the cultivated groundnut, little molecular variation has been detected by using RFLP technologies (Kochert et al., 1991). However, significant amounts of variation for RFLP (Kochert et al., 1991; Paik-Ro et al., 1992; Halward et al., 1992, 1993) have been observed among various Arachis species. Accessions in section Arachis, representing taxa that will hybridize with A. hypogaea, have been analyzed using RFLPs, and then multivariate analysis has been used to group accessions into clusters (Kochert et al., 1991) that correspond closely with morphological traits (Stalker, 1990). Tetraploids were clearly separated from diploids in both investigations. Stalker et al. (1995a) utilized RFLPs to examine genetic diversity among 18 accessions of A. duranensis Krapov. & W.C. Gregory, and they found more variation between than within accessions, and individual accessions could be identified. Kochert et al. (1996) concluded that the cultivated groundnut originated from a cross between A. duranensis and A. ipaensis; even later, A. villosa and A. ipaensi were reported as progenitors by Raina and Mukai (1999). Chloroplast analysis indicated that A. duranensis was the female progenitor of the cross (Kochert et al., 1996). The first RFLP-based genetic linkage map of groundnut, with a total map distance of 1063 cM, consisting of 117 markers in 11 linkage groups, was developed using an F2 population from an interspecific cross between two diploid species, A. stenosperma and A. cardenasii, in section Arachis (Halward et al., 1993). Fifteen unassociated markers were also reported (Halward et al., 1993). Burow et al. (2001) reported the AFLP-based tetraploid genetic linkage map of groundnut derived from a BC1 population of TxAG6 with Florunner. TxAG6 was an amphidipliod created by doubling the chromosomes of a hybrid of A. batizocoi × (A. cardenasii × A. diogoi). Three hundred and seventy RFLP loci were mapped to 23 linkage groups, with a total map distance of 2210 cM. Garcia et al. (1996) used Random Amplified Polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) technologies to map two dominant genes conferring resistance to the groundnut root-knot nematode M. arenaria race 1. A marker (Z3/265) was closely linked with M. arenaria resistance and subsequently mapped to a linkage group on a backcross map in an area known to contain A. cardenasii introgression. This fragment was cloned to make SCAR and RFLP probes, and linkages were confirmed (Garcia et al., 1996). Burow et al. (1996) also linked RFLP markers to genes conditioning M. arenaria resistance in the tetraploid cross of cultivar Florunner × TxAG6, but it is not known whether the genes identified in the two crosses are the same. In an investigation to link molecular markers with resistance to C. arachidicola, Stalker and Mozingo (2001) reported association of RAPDs with a gene conferring resistance to sporulation, lesion diameter, defoliation, and overall rating in an interspecific hybrid with A. cardenasii in the pedigree. In addition, they associated markers with Cylindrocladium black rot resistance and sporulation to C. arachidicola in a cross between NC 7 and PI109839. He and Prakash (1997) used 28 primer pairs to generate 111 AFLP markers in A. hypogaea. They reported a greater amount of variation using this technology than any other molecular marker technique. However, other studies conducted with cultivated groundnut have shown less variation than reported by He and Prakash (1997). Simple sequence repeat (SSR) markers are highly variable, codominant, easily detected from relatively little amounts of DNA after polymerase chain reaction (PCR) amplification, and reportedly more variable than other marker systems. Hopkins et al. (1999) reported six polymorphic SSRs in A. hypogaea, with the number of fragments amplified per SSR ranging from 2 to 14. Herselman (2003) reported the use of AFLP markers in identifying genetic variation among Southern African groundnut germplasm. MluI/MseI primer combinations were successfully used to detect polymorphisms between closely related cultivated

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groundnut genotypes. Herselman et al. (2004) used a total of 308 AFLP primer combinations to identify markers associated with aphid resistance in groundnut, and 20 putative markers were found, of which 12 mapped to five linkage groups covering a map distance of 139.4 cM. Raina et al. (2001) used 21 random and 29 SSR primers to assess genetic variation and interrelationships among subspecies and botanical varieties of A. hypogaea, and phylogenetic relationships among cultivated groundnut and wild species. In contrast with the previous generalization that peanut accessions lack genetic variation, both random and SSR primers revealed 42.7 and 54.4% polymorphism, respectively, among 220 and 124 genetic loci amplified from 13 accessions. The dendrograms based on RAPD, ISSR (Inter Simple Sequence Repeat), and RAPD + ISSR data precisely organized the five botanical varieties of the two subspecies into five clusters. The results also support the view that A. monticola and A. hypogaea are very closely related and indicate that A. villosa and A. ipaensis are the diploid wild progenitors of these tetraploid species. 3.4.5

Genetic Transformation

Application of transformation techniques is the feasible alternative for accessing genes in the tertiary gene pool (or ones outside the genus Arachis). A reliable tissue culture system to regenerate plants is crucial for utilizing this technology in practice. Research has been conducted for several decades by many investigators to develop plant regeneration systems in groundnut, and considerable progress has been achieved. With the progress of tissue culture technology, many tissue types of groundnut can now be regenerated in vitro, including hypocotyls, immature leaflets, leaf sections, cotyledons, and epicotyls. Although regeneration is highly influenced by genotype, media, light, temperature, and growth regulators, shoots have been obtained from different explants and dedifferentiated callus cultures of groundnut (Ozias-Akins and Gill, 2001). Several investigators found that cells surrounding the central vein of a leaflet of groundnut will produce callus that is more competent for plant regeneration (Cheng et al., 1992; Utomo et al., 1996; Akasaka et al., 2000). Genotypic variation in reaction to plant regeneration has been found in groundnut. Cheng et al. (1994, 1996) reported that only New Mexico Valencia A has been shown to be capable of regeneration after Agrobacterium infection, even though cells of many genotypes are susceptible to Agrobacterium infection. Li et al. (1995) reported that protoplasts could only been regenerated from cells derived from immature cotyledons. In general, groundnut has proven to be a crop that is relatively recalcitrant for cell culture and plant regeneration. To date, Agrobacterium and microprojectile bombardment-mediated transformation have been used in several laboratories in the world. Because actively dividing cells are required to integrate foreign DNA into tissues, embryogenic cultures that divide rapidly are highly desirable, and cotyledons or immature embryos are good sources of callus for transformation in groundnut (Ozias-Akins et al., 1992, 1993; Baker and Wetzstein, 1995). In order to develop cultivars adapted to specific geographic regions, many researchers have used different genotypes in transformation experiments. Transformation techniques can be applied to primary cultures if shoot primordia or somatic embryos are formed directly from an explant, dedifferentiated callus cultures, or repetitive tissue cultures. As in many legume species, suspension cultures are more difficult for groundnut than cultures on semisolid medium because recovery of fertile plants is difficult and genotype dependent (Ozias-Akins and Gill, 2001). Eapen and Gorge (1993) first reported regenerated groundnut plants following Agrobacterium transformation. Since then, several reports have indicated successful transformation of groundnut by Agrobacterium (Cheng et al., 1996; Li et al., 1997; Sharma and Anjaiah, 2000). Yang et al. (2003) reported transformation of groundnut using a modified bacterial mercuric ion reductase gene driven by an actin promoter from Arabidopsis thaliana. Transformed plants have also been obtained by directly inoculating injured embryo axes with Agrobacterium tumefaciens and planting infected seeds in sterilized soil (McKently et al., 1995; Rohini and Rao, 2000). However, microprojectile bombardment is considered to be the most consistent and successful transformation technique for

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groundnut (Holbrook and Stalker, 2003). Several cases of microprojectile bombardment-mediated groundnut transformation have been reported (Ozias-Akins et al., 1993; Singsit et al., 1997; Livingstone and Birch, 1998; Xu, personal communication). Techniques of detecting transient expression can be used to monitor gene integration, but it does not always correlate with successful gene transfer (Altpeter et al., 1996). Thus, selectable markers are usually used to identify transformed cells or tissues. Hygromycin phosphotransferase and neomycin phosphotransferase II are the most frequently used for antibiotic selection in groundnut. For obtaining gene expression in groundnut, gene promoters are the most critical element. The CaMV35S promoter is the most commonly used promoter in groundnut and the only one used to control the hph gene (Ozias-Akins and Gill, 2001). Genes have been inserted into A. hypogaea for resistance to virus (Brar et al., 1994; Li et al., 1997; Yang et al., 1998; Magbanua et al., 2000; Sharma and Anjaiah, 2000) and lesser cornstalk borer (Singsit et al., 1997). Durable resistance to tomato spotted wilt viruses is yet to be achieved (Li et al., 1997; Magbanua et al., 2000), but introduction of the crystalline proteins from Bacillus thuringensis appears to be stable (Ozias-Akins and Gill, 2001). The latter transformation system was developed more to reduce aflatoxin contamination in groundnut through inhibition of feeding the lesser corn stalk borer (Elasmopalpus lignosellus (Zeller)). At present, no transgenic groundnut cultivars have been released for commercial production. Transformation is a strategy for long-term improvement of the crop. Many important traits could be incorporated into the cultivated genome, and transformation technology will become increasingly important for groundnut breeding as more genes with agronomic potential are isolated. 3.4.6

Breeding Progress for Important Traits

3.4.6.1 Resistance to Foliar Diseases Early leaf spot (C. arachidicola), late leaf spot (C. personatum), and rust (P. arachidis) are the most damaging diseases of groundnut worldwide (Dwivedi et al., 2003). Sources of resistance to these three foliar diseases have been identified in A. hypogaea and wild species (Chiteka et al., 1988a, 1988b; Anderson et al., 1993; Singh et al., 1997; Liao, 2003). In general, resistance to these foliar diseases in A. hypogaea is more frequent among the germplasm lines mostly from the region of Tarapoto, Peru, belonging to the Valencia type (Singh et al., 1997). For early leaf spot, most resistant sources originated from secondary centers of diversity in South America, and resistant lines have been identified in var. hypogaea, var. fastigiata, and var. peruviana, but none in var vulgaris (Spanish type). More than 30 resistant lines have been reported (Singh et al., 1997), but most of the lines showed differential disease reactions at different locations, indicating the possible existence of variation of the early leaf spot pathogen. Some of the lines identified as resistant in the U.S. were susceptible in India (Singh et al., 1997). However, some lines showed more stable resistance across locations. An interspecific derivative, ICG 13917, is stably resistant to early leaf spot in Malawi and three locations in Asia. The geographic distribution of early leaf spot is limited, so breeding for resistance and release of resistant cultivars has also been limited. Late leaf spot is generally more widespread and more destructive than early leaf spot. At ICRISAT, India, over 13,000 accessions have been screened and 69 lines identified as resistant to late leaf spot, with disease scores ranging between 3 and 5 based on a 1 to 9 scale (Singh et al., 1997). Among the 69 resistant lines, 49 are landraces belonging to var. fastigiata or var. peruviana mainly collected from Peru (Singh et al., 1997). In China, 53 genotypes have been identified as resistant to late leaf spot from 5700 accessions, and nearly 60% of these resistant materials belong to var. fastigiata introduced from foreign countries (Liao, 2003). Holbrook and Isleib (2001) observed that resistance to late leaf spot was more frequent than expected in A. hypogaea accessions that originated in Bolivia, and this country was also a valuable source of origin for resistance to

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early leaf spot. Some of the identified resistant sources have been extensively used in breeding programs, and groundnut cultivars with both high-level leaf spot resistance and desirable agronomic traits have recently been released (Branch, 2002). Undesirable genetic linkage between the resistance and low yield, late maturity, low shelling outturn, and heavy pod reticulation is believed to be the reason that resistant germplasm has not been more widely used in breeding. In China, a number of interspecific populations have shown high levels of resistance to late leaf spot and better breeding efficiency, and promising breeding lines have been selected from them. Agronomically, pod traits such as shelling percentage and pod reticulation in the progenies generated from the resistant interspecific populations are more acceptable than those from the resistant ssp. fastigiata lines. Five interspecific germplasm lines with resistance to leaf spot have recently been released in the U.S. (Stalker et al., 2002b). Rust in groundnut has been reported in most tropical and subtropical countries, while it is more serious in tropical regions. More than 13,000 accessions have been screened for rust resistance at ICRISAT, from which 169 lines with disease scores of 5 or less on a 1 to 9 scale (Subrahmanyam et al., 1995) have been reported as resistant. These resistance sources include 135 landraces, of which 80% belong to subsp. fastigiata var. peruviana that originated predominantly from Peru (Singh et al., 1997). Rao (1987) indicated that the rust pathogen on groundnut might have evolved with the host species in South America. In China, 92 accessions have been identified as resistant to rust from 5700 accessions screened (Liao, 2003). Similar to the resistance sources to early and late leaf spot, most of the rust-resistant landraces belonging to var. fastigiata and var. peruviana have poor agronomic traits, including low shelling outturns, thick pod shells, heavy pod reticulation, and unacceptable seed coat colors. Undesirable genetic linkage between rust resistance and the poor pod characters has been observed in most breeding programs and impeded the progress of breeding. For example, among 49 resistant lines used as rust resistance donors in breeding at ICRISAT, only ICG 1697 and ICG 4747 have led to the release of rust-resistant cultivars such as ICGV 86590, FDRS 4, and FDRS 10 in India (Singh et al., 1997), but still with poor pod traits. Several interspecific derivatives with rust resistance transferred from A. batizocoi and A. duranensis might be more valuable, and their use may lead to the release of cultivars with more acceptable agronomic characteristics. Resistance to rust and late leaf spot is reported to be correlated with correlation coefficients ranging from 0.48 to 0.60 (Anderson et al., 1990). Forty-two genotypes resistant to late leaf spot are also resistant to rust (Singh et al., 1997). ICG 13917, an interspecific derivative, has shown high levels of resistance to late leaf spot, early leaf spot, and rust (Singh et al., 1997). Recently, Dwivedi et al. (2002) reported that the remaining green leaf area is an important criterion in selecting resistance for late leaf spot and rust. Several interspecific derivatives, ICGV 99005, 99003, 99012, and 99015 for rust and ICGV 99006, 99013, 99004, 99003, and 99001 for late leaf spot, would be better parents for use in resistance breeding programs (Dwivedi et al., 2002). Resistance to leaf spots has been reported as a complex trait in terms of inheritance (Kornegay et al., 1980; Anderson et al., 1986, 1991; Green and Wynne, 1987; Iroume and Knauft, 1987a, 1987b; Jogloy et al., 1987), and several components contribute to resistance, including initial infection, lesion size, sporulation, and defoliation (Green and Wynne, 1986; Chiteka et al., 1988a, 1988b; Anderson et al., 1993; Waliyar et al., 1993, 1995). Resistance to leaf spot in groundnut has generally been associated with late maturity (Norden et al., 1982; Miller et al., 1990) and vigorous vegetative growth (Liao, 2003). However, Branch and Culbreath (1995) documented a breeding line that had early maturity and tolerance to leaf spot. Aquino et al. (1995) found that latent period and maximum percentage of lesions that sporulated were the components of resistance most highly correlated with late leaf spot disease development. They suggested that using either of these two components to evaluate breeding populations might facilitate more rapid selection of lines with improved levels of resistance. Resistance to rust in the cultivated groundnut is recessive and appears to be controlled by only a few genes, while that in interspecific derivative is governed by partial dominant genes (Singh et al., 1997).

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Until the release of ‘Southern Runner’ in 1984, no commercial cultivar was available with meaningful resistance to late leaf spot in the U.S. The level of resistance in Southern Runner is moderate, and fungicide applications are still needed to obtain optimum yields. Moderate to high levels of resistance are also available in the cultivars Florida MDR 98 (Gorbet and Shokes, 2002b), C-99R (Gorbet and Shokes, 2002a), and ‘Georgia-01R’ (Branch, 2002). In China, Zhonghua 4 and Shanyou 27 with moderate rust resistance have been used in central and southern regions where rust is a problem (Liao, 2003). 3.4.6.2 Resistance to Soilborne Fungi Diseases The most important soilborne fungi diseases of groundnut are stem and pod rots (Sclerotium rolfsii Sacc.), Sclerotinia blight (Sclerotinia minor Jagger), and Cylindrocladium black rot (CBR) (C. parasiticum). Stem and pod rots, also known as white mold, are found throughout the major groundnut-growing areas of the U.S. and cause the greatest yield losses of all soilborne diseases (Backman and Brenneman, 1997). Genetic variation for resistance to white mold exists in A. hypogaea, and sources of resistance have been identified (Branch and Csinos, 1987; Smith et al., 1989; Grichar and Smith, 1992). For stem rot, field screening was more consistent than greenhouse tests for evaluating genotype responses (Shokes et al., 1996). The nonuniform spatial distribution of natural inoculum can be a problem for field evaluations (Shew et al., 1984), and sterilized oat seed inoculated with S. rolfsii has been used to increase pathogen population and improve the uniformity of fungal distribution (Shew et al., 1987; Brenneman et al., 1990; Shokes et al., 1996). However, even with uniform inoculum distribution, individual plants may escape the disease. Shokes et al. (1996) developed an inoculation method, termed the agar disk technique, which can be used to inoculate individual plants to prevent disease escape. This technique has been used to identify resistant breeding lines (Shokes et al., 1998). Several groundnut lines, including NC 2, NCAc18016, and NCAc18416, have been identified as resistant to white mold in the U.S., and several interspecific derivatives with white mold resistance have also been indentified in ICRISAT (Singh et al., 1997). Southern Runner (Gorbet et al., 1987), initially released as a cultivar with partial resistance to late leaf spot, was found to have moderate resistance to stem rot (Brenneman et al., 1990; Grichar and Smith, 1992; Branch and Brenneman, 1993). Gorbet et al. (1997) also observed some resistance to stem rot in Toalson (Simpson et al., 1979), Pronto (Banks and Kirby, 1983), Georgia Browne (Branch, 1994), and Sunbelt Runner (Mixon, 1982). Tamrun 96 (Smith et al., 1998) has shown superior resistance to stem rot compared to other commonly grown cultivars (Besler et al., 1997, 2001). Genetic variation for resistance to CBR has been observed in A. hypogaea. In general, Spanish cultivars are more resistant while Valencia cultivars are most susceptible, and Virginia cultivars are moderately susceptible (Phipps and Beute, 1997). However, CBR-resistant lines of each type have been described, and the partially resistant Virginia type cultivars, NC 8C (Wynne and Beute, 1983), NC 10C (Wynne et al., 1991), and NC 12C (Isleib et al., 1997), and Perry (Isleib et al., 2003) have been released. The earlier cultivars had pod and seed characteristics that were only marginally acceptable to manufacturers, and the newer releases have more acceptable seed traits and higher yields. The inheritance of resistance is complex (Green et al., 1983), and the resistance appears to delay the onset of epidemics rather than the rate of disease progress (Culbreath et al., 1991). Sclerotinia blight is the most important soilborne disease of groundnut in Virginia and Oklahoma in the U.S. (Porter and Melouk, 1997). Sources of resistance to S. minor have been identified (Coffelt and Porter, 1982; Melouk et al., 1989; Akem et al., 1992; Porter et al., 1992), and several moderately resistant cultivars and germplasm lines have been released (Coffelt et al., 1982, 1994; Smith et al., 1991; Kirby et al., 1998). Groundnut cultivars with Spanish ancestry appear to be more resistant to Sclerotinia blight than cultivars or breeding lines from Valencia or Virginia backgrounds (Akem et al., 1992). Wildman et al. (1992) reported that at least two loci are involved in the inheritance of resistance. Screening techniques for identifying resistance have been developed

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(Brenneman et al., 1988; Melouk et al., 1992) that rely on rate of lesion growth and development. The age or developmental stage of the lateral limbs of plants has a marked effect on lesion development (Brenneman et al., 1988), and the younger and more succulent tissues are more susceptible to the disease. Melouk et al. (1992) developed a detached shoot technique that can be used to assess resistance to Sclerotinia blight in groundnut genotypes. Goldman et al. (1995) reported that significant progress had been made in developing runner type groundnut breeding lines with resistance to Sclerotinia blight using field and greenhouse screening tools. 3.4.6.3 Resistance to Bacterial Wilt Bacterial wilt (BW) caused by Ralstonia solanacearum has been the only bacterial disease with significant economical importance in groundnut. Extensive screening of groundnut germplasm, largely based on field evaluation at hot spots in China and Indonesia, has been conducted during the past century (Singh et al., 1997). A high survival ratio under heavy disease pressure has been the most important criteria for resistance identification. Latent infection by the bacteria in groundnut plants has been reported and regarded as an important issue for further resistance enhancement, as latent colonization of R. solanacearum in groundnut without obvious symptoms could influence yield and other traits (Liao et al., 1998). Resistant germplasm lines have also been identified in Vietnam (Hong et al., 1999). Worldwide, more than 120 resistant accessions have been identified, including landraces and improved breeding lines. Among the 5700 germplasm accessions screened in China, 112 accessions are resistant to the disease with a survival ratio of more than 85% (Liao, 2003). Resistant genotypes have been found in four botanical types of the cultivated groundnut — Spanish, Valencia, Virginia, and hirsuta (or Chinese dragon) (Duan et al., 1993) — while 61% of the above 112 resistant lines belong to the dragon type (Liao, 2003). Bacterial wilt resistance has also been identified in some accessions in wild species of Arachis (Liao et al., 1998; Tang and Zhou, 2000). It is interesting to note that many dragon type lines collected from south China where bacterial wilt has been prevalent have been identified as highly resistant, but no resistant landrace collected from north regions in this botanical type has been identified as resistant, indicating that the resistance might have evolved with natural selection pressure. The resistance in groundnut has been proven to be most stable among the host plant species of R. solanacearum. For example, the resistant genotype Schwarz 21 developed more than 80 years ago is still resistant across different regions. New evidence in transferring of bacterial wilt resistance from diploid wild species into the tetraploid cultivated groundnut without obvious undesirable genetic linkage verified that the resistance to bacterial wilt in some wild species is controlled by major genes. Seven new groundnut cultivars with high-level resistance to bacterial wilt and improved agronomic traits have been developed and released to production in China, including Zhonghua 4, Zhonghua 6, Tianfu 11, Yuanza 9102, Yueyou 202-35, Yueyou 79, and Yuhua 14 (Liao, 2003). Zhonghua 6 was also found to have reduced aflatoxin contamination under artificial inoculation compared to other cultivars (Lei et al., 2004). Even though many sources of resistance have been identified, only a few have been sucssessfully used in breeding. In China, most released BWresistant groundnut cultivars had Xiekangqing and Taishan Sanlirou as resistant parents. In Indonesia, only Schwarz 21 and its derivatives have been used as resistant parents. All released BW-resistant cultivars are derived from resistant parents belonging to A. hypogaea subspecies fastigiata, even though many resistant lines in subspecies hypogaea have been used in breeding programs. Due to undesirable genetic linkage, the agronomic traits derived from the subspecies hypogaea are not acceptable in central and southern China, where bacterial wilt is generally serious, even though the resistance in the progenies can be high. This has resulted in a narrow genetic background for the available wilt-resistant cultivars. Further broadening of the genetic base for resistance and adaptation to the environments of diseased areas is highly necessary.

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3.4.6.4 Resistance to Nematodes The peanut root-knot nematode (Meloidogyne arenaria (Neal) Chitwood race 1) is an important pathogen in many groundnut production areas of the world. Most research to develop resistance has been conducted in the U.S. Only moderate levels of resistance have been observed in the naturally occurring germplasm of A. hypogaea (Holbrook and Noe, 1992; Holbrook et al., 1996, 2000b, 2000c). However, very high levels of resistance to M. arenaria exist in related Arachis spp. (Baltensperger et al., 1986; Nelson et al., 1989; Holbrook and Noe, 1990). This resistance has been introgressed into A. hypogaea. Stalker et al. (1995b) introgressed nematode resistance into A. hypogaea (2n = 4x = 40) from A. cardenassii Krapov. and W.C. Gregory (2n = 2x = 20). Restoring fertility of triploid F1 hybrids was accomplished by creating hexaploids and then selfing progenies until 40-chromosome progenies were identified. Garcia et al. (1996) reported that this resistance was conditioned by two dominant genes, where one gene (Mag) inhibited root galling and another gene (Mae) inhibited egg production by M. arenaria. Further development of this germplasm resulted in the release of two highly resistant germplasm lines, GP-NC WS5 and GP-NC WS 6 (Stalker et al., 2002a). Resistance to M. arenaria has also been introgressed into A. hypogaea by means of a complex interspecific hybrid from the three nematode-resistant species, A. batizocoi Krapov. and W.C. Gregory, A. cardenasii, and A. diogoi Hoehne (Simpson, 1991). This work resulted in the release of two highly resistant germplasm lines, T×AG-6 and T×AG-7 (Simpson et al., 1993a). A backcrossing breeding program was then used to introgress the root-knot nematode resistance from T×AG-7 into peanut breeding populations (Starr et al., 1995). This work resulted in the release of ‘COAN’, the first peanut cultivar with resistance to M. arenaria (Simpson and Starr, 2001). The yield potential of COAN was found to be less than that of its recurrent parent, ‘Florunner’ (Starr et al., 1998), but Church et al. (2005) observed significantly higher yield potential in nematode-resistant breeding lines that resulted from two additional backcross generations. Neither of these nematode-resistant cultivars can be grown in some parts of the U.S. because of their extreme susceptibility to tomato spotted wilt virus (TSWV). More recently, breeding lines have been developed that combine resistance to both of these pathogens (Holbrook et al., 2003; Anderson et al., 2006). Garcia et al. (1996) and Burow et al. (1996) independently mapped genes for resistance to the peanut root-knot nematode, which led to the development of molecular markers and the use of marker-assisted selection (MAS) for nematode resistance in groundnut (Choi et al., 1999; Church et al., 2000; Anderson et al., 2004). Building on this early work, Chu et al. (2005) has developed and tested an improved marker that dramatically improved the efficiency of MAS and greatly reduced the cost. This should allow for high-throughput marker-assisted selection. To date, most breeding efforts have focused on the use of a single dominant gene for nematode resistance. The durability of this resistance would probably be enhanced through the discovery and use of additional genes for resistance. Church et al. (2005) discovered a recessive gene for resistance to Meloidogyne arenaria in an interspecific population. Anderson et al. (2004) evaluated a diverse array of interspecific lines and identified material that contained genes for nematode resistance that were introgressed from other wild species, such as A. diogoi and A. batizocoi. Holbrook et al. (2003) and Chu et al. (2006) have also reported on observations that indicate the existence of additional unique genes for nematode resistance in interspecific groundnut material. Some of the genes may also be useful in developing resistance to other Meloidogyne spp. in groundnut. 3.4.6.5 Resistance to Aflatoxin Contamination Many research efforts on improving resistance to aflatoxin contamination in groundnut have been made worldwide. Three types of resistance have been reported, including resistance to

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Aspergillus invasion, resistance to aflatoxin production or formation, and resistance to preharvest aflatoxin contamination (PAC). Resistance to seed infection by Aspergillus ssp. and resistance to aflatoxin formation are assessed by in vitro inoculation and incubation under controlled conditions (Mehan et al., 1991; Xiao et al., 1999; Cole et al., 1995). Correlations between preharvest aflatoxin contamination or field resistance and in vitro resistance have been observed in Africa (Zambettakis et al., 1981; Waliyar et al., 1994) and India (Mehan et al., 1986, 1987). However, inconsistency between in vitro resistance screening and field resistance testing has been reported in several cases (Anderson et al., 1995; Blankenship et al., 1985). Kisyombe et al. (1985) observed significant field resistance in only 1 of the 14 in vitro resistant selections. In vitro resistance to aflatoxin formation in groundnut seeds having been stored for several years with poor emergence capacity has also been observed (Wang et al., 2003). This indicates that the resistance in these genotypes might be related to certain storage proteins. Techniques for resistance assessment are crucial, as the resistance to aflatoxin contamination in groundnut may be influenced by several factors. The effect of temperature and moisture stress on colonization of kernels and aflatoxin contamination of groundnuts has been extensively investigated (Hill et al., 1983; Sanders et al., 1985). Technological advances have also provided great improvement in detection and measuring techniques for assessing aflatoxin contamination (Wilson, 1989), which can more accurately measure resistance in a field or greenhouse environment (Holbrook et al., 1994, 1998b; Anderson et al., 1996b). Holbrook et al. (1994) developed a large-scale field screening technique to directly measure field resistance to PAC by using subsurface irrigation in a desert environment to allow an extended period of drought stress in the pod zone while keeping the plant alive. Sanders et al. (1993) also observed high levels of aflatoxin contamination when groundnuts in the pod zone were artificially stressed with heat and drought while keeping plants nonstressed by providing root zone irrigation. Anderson et al. (1996b) developed a screening technique that can be used in the greenhouse where high amounts of preharvest aflatoxin accumulation were produced by completely isolating the pod zone and restricting moisture to the root zone. Because aflatoxin concentrations are higher when the plant is stressed, development of cultivars with reduced aflatoxin contamination when grown under heat- or drought-stressed conditions is a valuable tool. Twenty-one groundnut genotypes have been identified as resistant to seed invasion of Aspergillus flavus, with a seed infection frequency of less than 2% in a sick plot under imposed drought stress at ICRISAT (Singh et al., 1997). Although significant genotype × environment interactions for seed infection have been reported, some accessions, including ICGs 1326, 3263, 3700, 4749, and 7633, have shown consistent resistance to seed infection in India and Senegal, and most of them also possess resistance to seed colonization under artificial inoculation conditions in the laboratory (Mehan et al., 1991). It is interesting to note that most groundnut genotypes resistant to seed infection and colonization belong to A. hypogaea subsp. fastigiata var. vulgaris with small seeds. Considerable variation in ability to support aflatoxin production has been observed among groundnut genotypes. U 4-7-5 and VRR 245 were identified as resistant to aflatoxin production even though they were susceptible to seed colonization by A. flavus (Singh et al., 1997). Xiao et al. (1999) reported two lines, N1211 and N1322, with the lowest aflatoxin production under artificial inoculation conditions among 1517 groundnut accessions screened. They also found that the resistance in these two genotypes was more stable across seasons (Xiao et al., 1999). Lei et al. (2004) reported that Taishan Zhengzhu and Zhonghua 6 with resistance to bacterial wilt were relatively resistant to aflatoxin formation under artificial inoculation conditions. An important breeding goal in Asia is to develop groundnut cultivars with multiple resistances to bacterial wilt and aflatoxin contamination. Fifteen groundnut accessions with field resistance to PAC have been identified from the U.S. core collection, and these accessions exhibited a 70 to 90% reduction in aflatoxin contamination in comparison to susceptible accessions (Holbrook et al., 1998b). Obviously, direct selection for resistance to aflatoxin contamination is generally expensive, and thus indirect selection methods are valuable. Drought tolerance might be an indirect selection tool for resistance to PAC. In the U.S., Holbrook et al. (2000a) evaluated the resistance to PAC in

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genotypes with varying levels of drought tolerance (Rucker et al., 1995) and concluded that tolerant genotypes had reduced aflatoxin contamination. Aflatoxin contamination was found to be highly related with leaf temperature and visual drought stress ratings. A significant negative correlation was also observed between aflatoxin contamination and yield under drought-stressed conditions. Leaf temperature, visual stress ratings, and yield are less variable and relatively inexpensive to measure compared to the amount of aflatoxin in seed samples. A similar relationship between drought tolerance and reduced aflatoxin contamination was observed in Streeton, a drought-tolerant cultivar in Australia (Cruickshank et al., 2000). This cultivar has up to 40% lower aflatoxin levels during years of high aflatoxin incidence than other cultivars. Physiological studies have shown that the lower aflatoxin incidence is associated with better root water uptake, resulting in better maintenance of crop water status during severe end-of-season drought (Nageswara Rao et al., 2000b). 3.4.6.6 Improved Drought Tolerance Improvement of drought tolerance of groundnut could help in sustaining yield and reducing aflatoxin contamination under drought stress in late growth stage. Drought tolerance in groundnut may be enhanced through improving capability of extracting water from soil (Wright and Nageswara Rao, 1994) or improving water use efficiency of the plant, or both (Hebbar et al., 1994). Drought tolerance has been associated with traits of root systems, such as root weight, length, and size. Increased root volume could be an important criterion for drought tolerance selection (Sun, 1998). Dwarfed plants with more nodes, thick leaflets, and more hair on the leaf surface are also important morphological traits associated with drought tolerance (Sun, 1998). Several physiological traits have been identified as important components for drought tolerance of groundnut, including stomatal conductance, surface wax, water potential, membrane stability, accumulation of cytokinins, and evapotranspiration. Selection of drought tolerance in groundnut could be based on performance of morphological and physiological traits. In conventional breeding, biomass and pod yield under drought stress have been extensively used for selecting drought tolerance. In terms of physiological approaches, some are more expensive than others. Specific leaf area can be easily and inexpensively measured, and it is used in a large-scale screening program for improved drought resistance in Australia (Nageswara Rao et al., 2000a). This research group has demonstrated progress from using physiological traits to indirectly select for high pod yield of groundnut under water-limited conditions. After two cycles of selection, they selected progenies that yielded 30% more than their parents under drought-stressed conditions (Nageswara Rao et al., 2000b). Nageswara Rao et al. (2001) demonstrated the potential utility of using a handheld portable SPAD 502 chlorophyll meter for rapid assessment of specific leaf area (SLA) and specific leaf nitrogen (SLN), which are surrogate measures of transpiration efficiency (TE) in groundnut. 3.4.6.7 High Oil Content and Improved Oil Quality Groundnut cultivars with high oil content are crucial for the oil processing industry, especially in developing countries where most groundnuts are crushed for cooking oil. In China, around 55% of the production is used for oil, and the annual consumption of peanut oil is over 220 million tonnes (Liao, 2003). However, there is still a shortage in the edible oil supply, and importation of additional plant oils is required. To fill this need, additional groundnut production is anticipated. However, groundnut oil is relatively less competitive with other plant oils, such as rapeseed and soybean oil, because of its higher market price. It is estimated that each 1% increase of oil content would increase the processor’s benefit by 7% (Liao, 2003). Extensive germplasm collection and evaluation have been conducted for various traits, including oil content. Among 5700 peanut germplasm accessions tested, the oil content ranged from 35 to 60%, with an average of 50.7% (Liao, 2003). Most accessions with relatively high oil content (over 55%) belong to Spanish type with early maturity. The average

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oil content of the most popular 30 peanut cultivars currently grown in China is 51.4% (Liao, 2003). In recent years, several peanut cultivars with high oil content and high yield have been released, including Zhonghua 5, Zhonghua 8, Yuanza 9102, and Jihua 4 (Liao, 2003; Liao et al., 2003). In breeding, it is relatively easy to select materials with high oil content, early maturity, and drought tolerance. The yield level of cultivars for oil extraction could be enhanced through crossing high oil content parents with large-pod and high-yielding ones. However, it has been proven that it is difficult to integrate high oil content with resistance to certain foliar diseases, such as rust and late leaf spot. All the high oil content cultivars are also found to be more supportive to seed infection of Aspergillus flavus and aflatoxin formation (Wang, personal communication). Quality of groundnut oil is determined by the fatty acid composition. In groundnut, oleic (O) and linoleic (L) acids comprise over 80% of the oil content. Linoleic acid is less saturated and less stable than oleic acid, and the oxidative stability and shelf-life of groundnut and groundnut products can be enhanced by increasing the O/L ratio. Among 5700 germplasm accessions tested in China (Liao, 2003), oleic acid contents ranged from 26.2 to 72.8% and linoleic acid contents ranged from 12.6 to 52.9%. There are 22 genotypes with oleic acid content over 67%. In terms of the average of botanic types, the dragon type (var. hirsuta) has the highest oleic acid content at 51.1% and the Valencia type has the lowest one at 32.2% (Liao, 2003). Most breeding efforts for increasing oleic acid content have been made for the cultivars for export trade during the past decade in China, and the progress in this aspect is limited (Wan, 2003). Norden et al. (1987) examined the fatty acid composition of 494 genotypes and identified two breeding lines with 80% oleic acid and 2% linoleic acid. This was a major deviation from previously known levels of fatty acid composition in groundnut. The high oleate trait in groundnut is controlled by two duplicate genes, ol1 and ol2 (Moore and Knauft, 1989), one of which is common in U.S. runner and Virginia type cultivars (Knauft et al., 1993; Isleib et al., 1996). Early inheritance studies indicated that the genes controlling the higholeate trait exhibited complete recessivity (Moore and Knauft, 1989; Knauft et al., 1993; Isleib et al., 1996). López et al. (2001, 2002) examined the inheritance of high oleic acid in Spanish market type material and observed different patterns of inheritance indicative of three-gene control with dominant and recessive epistasis. Jung et al. (2000b) reported isolation of two genes encoding microsomal oleoyl–PC desaturases, ahFAD2A and ahFAD2B, from the developing groundnut seed with a normal oleate phenotype. Characterization of the genes indicates that ahFAD2A is expressed in both normal and high oleate peanut seeds, but the steady-state level of the ahFAD2B transcript is severely reduced in the high oleate peanut varieties, suggesting that the reduction in ahFAD2B transcript level in the developing seeds is correlated with the high oleate trait (Jung et al., 2000a). Jung et al. (2000a) also examined gene expression by reverse transcriptase (RT)-PCR/restriction digestion in a cross that shows a one-gene segregation pattern for the high oleate trait. The severely reduced level of ahFAD2B transcript correlates absolutely with the high oleate phenotype in this cross, suggesting that the single gene difference is correlated with the ahFAD2B transcript level. When they tested the enzyme activity of the proteins encoded by ahFAD2A and ahFAD2B by expression of the cloned sequences in yeast, only the ahFAD2B gene product showed significant oleoyl-PC desaturase activity. The results suggest that a mutation in ahFAD2A and a significant reduction in levels of the ahFAD2B transcript together cause the high oleate phenotype in peanut varieties, and that one expressed gene encoding a functional enzyme appears to be sufficient for the normal oleate phenotype.

3.5 LOOKING AHEAD Groundnut is becoming more important in several regions in the world, and production of this crop is expected to increase. Genetic enhancement of groundnut will attract more research efforts. Generally speaking, research emphasis will be given to enhancing or stabilizing groundnut productivity

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in various production systems, especially in the low-yielding areas, improving quality for diversified utilization, and ensuring food safety. Progress has been made in collecting and preserving the genetic resources in both A. hypogaea and related Arachis species. Although additional collecting is needed in South and Central America, the available germplasm collection, consisting of 20,000 to 30,000 accessions, is quite large and could represent a major part of the variation in the genus. Evaluation of the collected germplasm has resulted in the identification of much useful material for breeding, but further evaluation is still highly needed. Creating core collections has greatly enhanced evaluation research, and more intensive evaluation of the groundnut germplasm collections in the world could be done by core collection-based strategies. Worldwide, the breeding objectives of groundnut are diverse among locations with different agroecological characteristics and economic situations. In the developed countries, groundnut breeding efforts have shifted emphasis during recent years from mostly selecting for increased yields to selecting high-yielding cultivars with greater resistance to biotic stresses and enhanced quality traits, especially for improving oil profiles and flavor characteristics. Cultivars with multiple sources of high levels of pest resistance will be needed in the future. In the developing countries, most efforts are still being made for enhancing productivity. High yield potential and resistance to major diseases (foliar diseases) and certain viral diseases are the most important breeding objectives. In recent years, high oil content groundnut has attracted more breeding efforts in regions where most groundnuts are crushed for edible oil. However, breeding for resistance or tolerance to drought stress and aflatoxin contamination is a high priority in both developed and developing nations. Further breeding objectives in more countries will also include improving the food quality, such as flavor, and reducing or eliminating allergens. Utilization of wild Arachis species has greatly contributed to enhancing resistance of the cultivated groundnut, including resistance to foliar diseases, nematodes, and viruses. Hopefully, interspecific hybridization will contribute more to improving the quality of the groundnut in the future. Molecular technologies are being developed for groundnut even though little molecular variation has been detected in A. hypogaea. More efforts will be made in identifying useful DNA markers for important traits. A large amount of molecular variation is present in species of Arachis, and marker-assisted selection has great potential for enhancing introgression of useful traits from related species to cultivars. Transgenic technologies have been developed to insert foreign genes into the cultivated groundnut, and the critical component needed for cultivar development is identification of agronomically useful genes. Even cultivars developed with transgenic technologies have not been released for production, but the concerned research would serve as a long-term strategy for traits that are difficult to improve through conventional approaches.

REFERENCES Akasaka, Y., H. Daimin, and M. Mii. 2000. Improved plant regeneration from cultured leaf segments in peanut (Arachis hypogaea L.) by limited exposure to thidiazuron. Plant Sci. 156: 169–175. Akem, C.N., H.A. Melouk, and O.D. Smith. 1992. Field evaluation of peanut genotypes for resistance to Sclerotinia blight. Crop Protect. 11: 345–348. Altpeter, F. et al. 1996. Accelerated production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell Rep. 16: 12–17. Anderson, W.F. et al. 1990. Statistical procedure for assessment of resistance in a multiple foliar disease complex of peanut. Phytopathology 80: 1451–1459. Anderson, W.F., C.C. Holbrook, and T.B. Brenneman. 1993. Resistance to Cercosporidium personatum within peanut germplasm. Peanut Sci. 20: 53–57. Anderson, W.F., C.C. Holbrook, and A.K. Culbreath. 1996a. Screening the core collection for resistance to tomato spotted wilt virus. Peanut Sci. 23: 57–61. Anderson, W.F., C.C. Holbrook, and P. Timper. 2006. Registration of root-knot nematode resistant peanut germplasm lines NR 0812 and NR 0817. Crop Sci. 46: 481–482.

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Li, Z., R.L. Jarret, and J.W. Demski. 1997. Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene. Transgenet. Res. 6: 297–305. Liao, B.S. 2001. Economic aspects of groundnut in food security and agricultural development in China. Cereals Oils 10: 28–29. Liao, B.S. 2003. The Groundnut. Hubei Press for Science and Technology, Wuhan, China (in Chinese). Liao, B.S. et al. 1998. Germplasm screening and breeding for resistance to bacterial wilt in China. In Groundnut Bacterial Wilt: Proceedings of the Fourth Working Group Meeting, May 11–13, 1998, Vietnam Agricultural Science Institute, Hanoi, Vietnam. S. Pande, B. Liao, N.X. Hong, C. Johansen, and C.L.L.Gowda, Eds. International Crops Research Institute for the Semiarid Tropics, India, pp. 75–81. Liao, B.S. et al. 2003. Breeding and utilization of a new peanut cultivar Zhonghua 5 with high oil content. Chin. J. Oil Crops Sci. 25: 86–88. Livingstone, D.M. and R.G. Birch. 1998. Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds. Mol. Breed. 5: 43–51. López, Y. et al. 2001. Genetic factors influencing high oleic acid content in Spanish market-type peanut cultivars. Crop Sci. 41: 51–56. López, Y. et al. 2002. Inheritance of the high-oleic trait in peanut: unsolved puzzle. Proc. Am. Peanut Res. Educ. Soc. 34: 68–69. Lu, J., A. Mayer, and B. Pickersgill. 1990. Stigma morphology and pollination in Arachis L. (Leguminosae). Ann. Bot. 66: 73–82. Lu, X.X. 2001. Vigor test of seeds of crops stored in the national genebank. Plant Genet. Resour. 2: 1–5. Lynch, R.E. and T.P. Mack. 1995. Biological and biotechnical advances for insect management in peanut. In Advances in Peanut Science. H.E. Pattee and H.T. Stalker, Eds. Am. Peanut Res. Educ. Soc., Stillwater, OK, pp. 95–159. Magbanua, Z.V. et al. 2000. Field resistance to tomato spotted wilt virus in transgenic peanut (Arachis hypogaea L.) expressing an antisense nucleocapsid gene sequence. Mol. Breed. 6: 227–236. Maleki, S.J. et al. 2000. The effects of roasting on the allergenic properties of peanut proteins. J. Allergy Clin. Immunol. 106: 763–768. Mallikarjuna, N. and D.C. Sastri. 2002. Morphological, cytological and disease resistance studies of the intersectional hybrid between Arachis hypogaea L. and A. glabrata. Euphytica 126: 161–167. McKently, A.H. et al. 1995. Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants. Plant Cells Rep. 14: 699–703. Mehan, V.K. et al. 1991. The Groundnut Aflatoxin Problem: Review and Literature Database. International Crops Research Institute for the Semiarid Tropics, Patancheru, India. Mehan, V.K., D. McDonald, and K. Rajagopalan. 1987. Resistance of peanut genotypes to seed infection by Aspergillus flavus in field trials in India. Peanut Sci. 14: 17–21. Mehan, V.K. et al. 1986. Effect of genotype and date of harvest on infection of peanut seed by Aspergillus flavus and subsequent contamination with aflatoxin. Peanut Sci. 13: 46–50. Melouk, H.A., C.N. Akem, and C. Bowen. 1992. A detached shoot technique to evaluate the reaction of peanut genotypes to Sclerotinia minor. Peanut Sci. 19: 58–62. Melouk, H.A., C.N. Akem, and O.D. Smith. 1989. Reaction of peanut genotypes to Sclerotinia blight in field plots, 1986 and 1987. Biol. Cult. Tests Control Plant Dis. 4: 39. Miller, I.L. et al. 1990. Influence of maturity and fruit yield on susceptibility of peanut to Cercosporidium personatum (late leaf spot pathogen). Peanut Sci. 17: 52–58. Mixon, A.C. 1982. Registration of Sunbelt Runner peanut (reg. no. 26). Crop Sci. 22: 1086. Moore, K.M. and D.A. Knauft. 1989. The inheritance of high oleic acid in peanut. J. Hered. 80: 252–253. Moss, J.P. et al. 1997. Registration of ICGV 87165 peanut germplasm line with multiple resistance. Crop Sci. 37: 1028. Murty, U.R. et al. 1985. Chromosome morphology and Gregory’s sectional delimitation in the genus Arachis L. In Proceedings of the International Workshop on Cytogenetics of Arachis, Hyderabad, India, October 31–November 2, 1983. J.P. Moss, Ed. International Crops Research Institute for the Semiarid Tropics, Patancheru, India, pp. 81–83. Nageswara Rao, R.C., H.S. Talwar, and G.C. Wright. 2001. Rapid assessment of specific leaf area and leaf nitrogen in peanut (Arachis hypogaea L.) using a chlorophyll meter. J. Agron. Crop Sci. 186: 175–182.

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CHAPTER 4 Cottonseed R.J. Kohel and J.Z. Yu

CONTENTS 4.1 4.2 4.3 4.4

Introduction.............................................................................................................................89 Distribution and Importance...................................................................................................90 Origin and Genetic Resources ...............................................................................................90 Genetic Improvement .............................................................................................................92 4.4.1 Mode of Reproduction ...............................................................................................92 4.4.2 History ........................................................................................................................95 4.4.3 Methods ......................................................................................................................96 4.4.4 Objectives ...................................................................................................................96 4.4.5 Pests ............................................................................................................................97 4.4.6 Cottonseed Quality and Gossypol..............................................................................97 4.5 Utilization of Products ...........................................................................................................99 4.6 Conclusions...........................................................................................................................100 References ......................................................................................................................................100

4.1 INTRODUCTION Cottonseed is the second most important oilseed produced worldwide. Such a position is unique because cottonseed is a secondary product in the production of cotton fibers. The producer grows cotton plants for the production of the seed fibers, and the product is sold as seed cotton, seeds with the fibers still attached; as lint, ginned fibers, for which the cottonseed is used to defray the cost of ginning; or as lint and cottonseed as separate products. The trend is toward the latter, but all variations still exist throughout the world. Cotton breeding is focused on the improving of fiber yield and quality. Breeding programs do not normally consider cottonseed composition in their evaluations, and seed quality is considered primarily as it pertains to planting seed. Seed quality takes into consideration such factors as seed coat integrity and seed size as it influences the seed processing through the ginning process and the processing of fiber in the textile mills. Normal breeding and testing procedures provide for the selection of seedling vigor and stand establishment. However, measurements of compositional quality of the seed are not normally determined in the breeding programs. As a consequence, or in spite of this, cottonseed composition has remained relatively stable. Since seed germination and 89

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seedling growth are dependent on the stored reserves of the cotyledons, which are the fatty acids and amino acids, there is an indirect selection for an optimal level of these components to maintain good seed viability and vigor. The cottonseed processing industry originated in the 19th century as an industry utilizing a by-product (Altschul et al., 1958). Overshadowed by the economic value of lint, the cottonseed industry has exerted little influence on the genetic research, breeding, and production of cotton. The large disparity in the value of lint compared to cottonseed will dictate that resources will be directed toward the improvement of lint. However, the sheer volume of cottonseed produced makes it an important factor in the economics of cotton production. As with most agricultural production, there is the need to maximize the value of the products produced, so ways to increase the farm value of both the lint and cottonseed are important. 4.2 DISTRIBUTION AND IMPORTANCE Cotton originates from tropical to subtropical latitudes, but approximately 50% of the production areas are in temperate zones (Waddle, 1984). The People’s Republic of China, the U.S., India, Pakistan, and countries of the former Soviet Union account for about 80% of the area of production. As the political landscape of the world changes, so do the areas of world cotton production. Greater changes have taken place as to which countries are exporters or importers of cotton lint. The U.S. has become a major exporter of cotton lint and importer of textiles, but in all cases, cottonseed remains a product to be processed and consumed primarily in the domestic economy. A listing of major cotton-producing countries and their production is provided in Table 4.1. The general trend is for an increase in both the amount of cotton grown and the yield per hectare. The value of cottonseed is about one fifth to one sixth that of lint, which keeps the role of cottonseed as a byproduct of lint production, but it is still significant in the economy of cotton production. 4.3 ORIGIN AND GENETIC RESOURCES There are about 50 species of the genus Gossypium (Percival et al., 1999) (see Table 4.2). The origin and systematics of Gossypium have been thoroughly reviewed in books by Kohel and Lewis (1984), and updated by Smith and Cothren (1999). The origin and domestication is detailed by Brubaker et al. (1999) and Wendel et al. (1992). Percival et al. (1999) present the taxonomy and germplasm resources of Gossypium. Here we will present a brief overview of these topics. The cultivated cottons consist of two diploid A genome species (2n = 26), G. aboreum L. and G. herbaceum L., and two tetraploid AD genome species (2n = 4x = 52), G. barbadense L. and G. hirsutum L. The diploid species are placed in genomic groupings and designated A through G plus K. An Asiatic diploid species, A genome, in combination with a diploid of the New World, D genome, are the presumed progenitors of the tetraploids, AD genomes (Figure 4.1). The cultivated diploid species have their center of origin in Africa–Asia, and they are referred to as the Asiatic species because it was in this area that domestication and weaving became established. The early cotton textile industry in Europe was based on the Asiatic species. The tetraploid species have their center of origin in Mexico, Central America, and South America. G. barbadense domestication is thought to be in South America, and G. hirsutum domestication is thought to be in Mexico/Guatemala. The discovery of cottons in the New World by European explorers further fueled the cotton textile industry and the accompanying industrial revolution. Cotton production expanded in the New World, and the New World tetraploids were dispersed by traders to other countries and began to displace the less productive diploids. Upland cotton, G. hirsutum, became the predominant species grown. Today it accounts for about 90% of the world cotton production, G. barbadense accounts for about 10%, and the Asiatic cotton production remains only in marginal growing areas of India and Pakistan (Lee, 1984).

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Table 4.1 Cotton Area, Yield, and Production* World and Selected Countries and Regions

Area (1,000 Ha) 2001-02 2002-03 2003-04

Yield (KG/Ha) Production (1,000 Bales) 2001-02 2002-03 2003-04 2001-02 2002-03 2003-04

Western Hemisphere United States Brazil Paraguay Argentina Mexico Peru Colombia Others

5,596 750 150 165 82 75 46 80

5,205 720 250 160 40 75 51 80

4,880 980 275 250 70 75 55 80

790 1,021 319 396 1,147 534 587 340

727 1,089 348 408 1,034 534 576 340

813 1,155 416 348 933 534 614 340

20,303 3,518 220 300 432 184 124 125

17,375 3,600 400 300 190 184 135 125

18,224 5,200 525 400 300 184 155 125

TOTAL

6,944

6,581

6,665

790

738

820

25,206

22,309

25,113

EU Greece Spain Others

501 410 90 18

466 380 85 18

436 350 85 18

1,123 1,111 1,188 399

1,000 974 1,127 399

1,014 1,014 1,025 399

2,585 2,093 491 33

2,141 1,700 440 33

2,031 1,630 400 33

TOTAL

519

484

454

1,098

978

990

2,618

2,174

2,064

Franc-Zone Africa Mali Burkina Benin Cote d''Ivoire Cameroon Togo Chad Egypt Zimbabwe Sudan Nigeria Tanzania Zambia Mozambique South Africa Uganda Others

2,442 520 350 390 300 200 150 425 315 400 150 375 450 200 155 41 250 311

2,352 450 350 350 300 200 160 425 325 400 180 325 450 200 155 45 250 311

2,457 550 350 375 260 220 160 425 218 450 240 375 387 250 155 75 250 311

401 461 451 447 508 490 399 166 996 191 399 261 113 185 155 441 78 238

361 411 435 389 435 435 408 154 837 299 454 234 138 185 155 435 78 238

418 455 591 450 440 495 476 166 874 254 454 261 132 209 155 508 109 238

4,495 1,100 725 800 700 450 275 325 1,441 350 275 450 234 170 110 83 90 340

3,895 850 700 625 600 400 300 300 1,250 550 375 350 285 170 110 90 90 340

4,715 1,150 950 775 525 500 350 325 875 525 500 450 235 240 110 175 125 340

TOTAL

5,089

4,993

5,168

344

327

349

8,038

7,505

8,290

4,820 8,730 3,130 2,493 1,430 245 515 184

4,200 7,800 2,700 2,444 1,420 265 500 165

5,100 8,400 3,000 2,445 1,400 285 480 185

1,102 307 577 642 746 591 359 769

1,115 313 629 608 721 596 305 627

939 329 552 594 653 554 408 647

24,400 12,300 8,300 7,350 4,900 665 850 650

21,500 11,200 7,800 6,830 4,700 725 700 475

Europe

Africa

Asia and Oceania China; Peoples Re India Pakistan FSU Uzbekistan Tajikistan Turkmenistan Kazakhstan

22,000 12,700 7,600 6,665 4,200 725 900 550 continued

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Table 4.1 (continued) Cotton Area, Yield, and Production* World and Selected Countries and Regions Azerbaijan Kyrgyzstan Turkey Australia Syria Iran Burma Afghanistan Israel Others TOTAL WORLD TOTAL

Area (1,000 Ha) 2001-02 2002-03 2003-04

Yield (KG/Ha) Production (1,000 Bales) 2001-02 2002-03 2003-04 2001-02 2002-03 2003-04

82 37 693 420 260 200 320 50 15 229

65 29 710 225 180 160 320 50 13 205

60 35 710 175 200 130 320 50 13 205

385 824 1,249 1,659 1,298 626 184 370 1,451 367

435 751 1,257 1,451 1,512 646 184 370 1,340 357

599 778 1,257 1,493 1,415 636 184 370 1,340 370

145 140 3,975 3,200 1,550 575 270 85 100 386

130 100 4,100 1,500 1,250 475 270 85 80 336

165 125 4,100 1,200 1,300 380 270 85 80 348

21,360

19,007

20,748

637

635

595

62,491

55,426

56,728

33,912

31,065

33,035

631

613

608

98,353

87,414

92,195

Source: Data abstracted from World Agricultural Production, Circular Series, USDA, FAS, 2004.

As the archaeological records continue to push the domestication of cottons to earlier times for both the New World and Asiatic cottons, the case for independent domestication becomes stronger. With the development of DNA marker technology, these DNA molecular tools have been used to investigate the evolutionary questions of Gossypium and to shed light on the domestication of the cultivated cotton species (Brubaker and Wendel, 1994; Brubaker et al., 1993; Wendel, 1989; Wendel and Albert, 1992). However, while the results of these investigations have added some insight to the process, they have been no more definitive than existing presumptions. Of particular interest in a discussion of cotton as an oilseed crop is that it has been proposed that Asiatic cottons were domesticated initially for their seeds as an animal feed in the Nubian culture, and that subsequently their seed fibers were incorporated into an existing linen textile industry (Chowdhury and Burth, 1971). There are several major Gossypium germplasm collections in the world, such as in France, the People’s Republic of China, Russia, the U.S., and Uzbekistan. The U.S. collection is comprehensive and contains about 9000 accessions; the other major collections are generally quite similar. The tetraploid members of the U.S. collection were evaluated for their compositional quality (Kohel, 1978; Kohel et al., 1985). As one would expect, there was wide variation in the germplasm for seed composition. The greater variability was found in the unimproved cottons. However, these cottons varied for other seed properties, such as seed size and hull thickness, which had a large impact on compositional content.

4.4 GENETIC IMPROVEMENT 4.4.1

Mode of Reproduction

Cotton plants are perennials that have indeterminate growth habits, and most of the wild forms are short-day photoperiodic. The cultivated forms are predominantly day-neutral in flowering response, and they are cultivated as annuals. Growth is characterized by a vegetative monopodial main stem on which the axial buds can produce either monopodial or sympodial, fruiting, branches. Each node of a sympodial branch arises from an axillary bud at the terminus flower. Therefore, continued vegetative plant growth is required to sustain flowering (Mauney, 1984), and the earlier the initiation of sympodial growth, the earlier is the crop production.

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Table 4.2 Name, Genomic Symbols, and Origins of Species of Gossypium Species Name

Genomic Symbol

Origin

Diploids (2n = 26) A. Subgenus Sturtia (R. Brown) Todaro (all the indigenous Australian species) 1. Section: Sturtia (species do not deposit terpenoid aldehydes in the seeds (gossypol)) i. G. sturtianum J.H. Willis ii. G. robinsonii F. Mueller 2. Section: Grandicalyx Fryxell (unusual perennial, thick underground root-stock, fat bodies on seeds) i. G. costulatum Todaro ii. G. cunninghamii Todaro iii. G. exiguum Fryxell, Craven & Stewart iv. G. rotundifolium Fryxell, Craven & Stewart v. G. enthyle Fryxell, Craven & Stewart vi. G. nobile Fryxell, Craven & Stewart vii. G. pilosum Fryxell viii. G. pulchellum (C.A. Gardner) Fryxell ix. G. londonderriense Fryxell, Craven & Stewart x. G. marchantii Fryxell, Craven & Stewart xi. G. populifolium (Bentham) F. Mueller ex. Todaro xii. G. anapoides 3. Section: Hibiscoidea Todaro (species do not deposit terpenoid aldehydes in the seeds) i. G. australe F. Mueller ii. G. nelsonii Fryxell iii. G. bickii Prokhanov B. Subgenus Houzingenia (Fryxell) Fryxell (new world, primarily Mexico; large shrubs or small trees) 1. Section: Houzingenia a. Subsection: Houzingenia i. G. thurberi Todaro ii. G. trilobium (DC) Skovsted b. Subsection: Integrifolia (Todaro) Todaro i. G. davidsonii Kellog ii. G. klotzschianum Anderson b. Subsection: Caducibracteolata Mauer i. G. armourianum Kearney ii. G. harknessi Brandegee iii. G. turneri Fryxell 2. Section: Erioxylum (Rose & Standley) Prokhanov a. Subsection: Erioxylum i. G. aridum (Rose & Standley ex. Rose) Skovsted ii. G. lobatum H. Gentry iii. G. laxum Phillips iv. G. schwendimanii Fryxell & S. Koch b. Subsection: Selera (Ulbrich)Standley i. G. gossypioides (Ulbrich) Fryxell c. Subsection: Austroamericana Fryxell i. G. raimondii Ulbrich C. Subgenus Gossypium L. 1. Section: Gossypium a. Subsection: Gossypium i. G. herbaceum L. ii. G. arboreum L. b. Subsection: Anomala Todaro i. G. anomalum Wawra & Peyritsch

C1 C2

Australia Australia

K K K K K K K K K K K K

Australia Australia Australia Australia Australia Australia Australia Australia Australia Australia Australia Australia

G G G1

Australia Australia Australia

D1 D8

Mexico, Arizona (U.S.) Mexico

D3-d D3-k

California (U.S.) California (U.S.)

D2-1 D2-2 D10

Mexico Mexico Mexico

D4 D7 D9 D11

Mexico Mexico Mexico Mexico

D6

Mexico

D5

Peru

A1 A2

Africa, Asia Asia

B1

Africa

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Table 4.2 (continued) Name, Genomic Symbols, and Origins of Species of Gossypium Species Name

Genomic Symbol

Origin

ii. G. triphyllum (Harvey & Sonder) Hochreutiner iii. G. capitis-viridis Mauer c. Subsection: Longiloba Fryxell i. G. longicalyx J.B. Hutchinson & Lee d. Subsection: Pseudopambak (Prokhanov) Fryxell i. G. benadirense Mattei ii. G. bricchettii (Ulbrich) Vollesen iii. G. vollesenii Fryxell iv. G. stocksii Masters ex. Hooker v. G. somalense (Gürke) J.B. Hutchinson vi. G. areysianum Deflers vii. G. incanum (Schwartz) Hillcoat D. Subgenus Karpas Rafinesque (the allotetraploid cottons)

B2 B3

Africa Cape Verde Islands

F1

Africa

E E E E1 E2 E3 E4

African-Arabian African-Arabian African-Arabian African-Arabian African-Arabian African-Arabian African-Arabian

Allotetraploids (2n = 4x = 52)

AD

i. ii. iii. iv. v.

G. hirsutum L. G. barbadens L. G. tomentosum Nuttall ex. Seemann G. mustelinum Miers ex. Watt G. darwinii Watt

AD1 AD2 AD3 AD4 AD5

Central America South America Hawaiian Islands NE Brazil Galapagos Islands

The cotton plant has perfect flowers that open in the morning, shed pollen, and wither by nightfall. In cultivated cottons, pollen sheds concurrently with flower opening. The anthers surround the stigma, so self-pollination is readily accomplished. The stigmas remain receptive until mid-day or shortly thereafter, depending on weather conditions. The pollen grains are large and sticky, so no wind pollination occurs, but insects, primarily bees, are capable of effecting cross-pollination. Because insects can cause cross-pollination, the cotton plant is often classed as cross-pollinated. However, cotton is completely self-compatible, and any cross-pollination is a function of the numbers of pollinating insects present. In production fields, the amount of cross-pollination is largely a function of the amount and kinds of insecticides used to control pests, and cross-pollination can range from 0 to >30%. Modern cotton cultivars begin to flower about 60 days after planting, flowering continues for about 45 days, and individual bolls mature in about 55 days. Cotton bolls are capsules with 3 to 5 locules, each with 7 to 10 ovules. A boll normally matures 20 to 30 seeds. Variation in the time for each developmental period depends on the genotype of the plant and climatic conditions. Cotton plants are very responsive to stress, and flowers and young bolls are readily shed when plants are subjected to stress. As crop development progresses and bolls are set, the rate of vegetative growth and flowering diminishes and the amount of flower shedding increases. Once a crop of bolls matures sufficiently, cotton plants initiate new growth and flowering activity to produce a second crop of bolls. In most production areas, growth and fruiting are limited by temperature and moisture, and plants produce only a single crop of bolls. If the growing season is long enough, the second crop can increase production. The bulk of storage reserves of the cottonseed are produced during later stages of seed development (Benedict et al., 1976). In areas of temperate climate, low temperatures at the later stages of seed development have a marked influence on seed oil. As temperatures decrease, the rate of boll development decreases, as does the seed-oil content. Relative amounts of individual fatty acids change in response to temperature, but the pattern of response in cottonseed is not clear (Kohel and Cherry, 1984). This relationship is made even less clear by the differences in the developmental age of bolls on a plant at any given time.

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Australian C-genome 2 species

Australian G-genome 3 species African B-genome 3 species

Australian K-genome 12 species

African-Arabian E-genome 7 species

African F-genome 1 species

African-Asian A-genome G. herbaceum G. arboreum

New World AD-genome G. darwinii G. barbadense G. mustelinum G. tomentosum G. hirsutum

Figure 4.1

New World D-genome G. schwendimanli G. lobatum G. aridum G. laxum G. klotzschianum G. davidsonii G. turneri G. harknessii G. armourianum G. trilobum G. thurberi G. gossypioides G. raimondii

The evolutionary relations of species of Gossypium.

The response of seed development and oil content to moisture varies. Chronic moisture stress has not been shown to produce any negative changes on seed-oil content (Cherry et al., 1981). An acute moisture stress at a late stage of boll development may adversely influence seed-oil content because seeds may be arrested in their development. Cotton plants stressed at early stages of boll development or under chronic stress generally compensate by boll shedding so that seed-oil content is affected to a limited extent (Kohel and Benedict, 1984). 4.4.2

History

The most prevalent species of cultivated cotton, G. hirsutum, accounts for 90% of the world production. The center of origin of this species is in Mexico–Central America, and the natives

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practiced a home spinning–weaving industry and cotton commerce when the European explorers arrived. The Europeans were effective in disseminating seed and establishing production throughout their colonial empires. The large growing area that developed in the southern U.S. resulted in the selection of a day-neutral, early-maturing, and prolific type, and repeated introductions from the center of origin contributed to the U.S. germ pool. The U.S. selections provided a germplasm base for further world expansion, especially in temperate areas. Evidence of early direct dissemination from the center of origin is recognizable in cottons of the more tropical regions, because when grown in temperate regions, many cultivars display residual photoperiodic and late-maturity traits. The second most widely grown cultivated species, G. barbadense, accounts for about 10% of world production. Its center of origin is in South and Central America. It includes the Sea Island type of G. barbadense that was grown in the coastal islands of colonial U.S. Sea Island cottons were introduced into Egypt, and following hybridization and selection, G. barbadense became established as the cotton of Egypt. Later, G. barbadense was reintroduced into the Southwest desert of the U.S. Derivatives of this cotton, known as Egyptian, Egyptian-American, and Pima, are known for their long and strong fiber. Marketing of these unique G. barbadense cottons established them as extra-long staple (ELS) cottons, whose classification is defined by specific fiber properties. These ELS fiber characteristics are not known in the native G. barbadense cottons, and some regions of the world grow G. barbadense that does not have fiber with these quality characteristics. 4.4.3

Methods

Cotton breeding is directed toward enhancing the yield of quality fibers, and because the yield of fiber is directly related to the number of seeds produced, both seed and fiber yield are increased. The lack of specific attention to seed quality has not seemed to result in a change of seed composition. Breeding efforts directed to more efficient production systems and the preservation of fiber quality have a similar positive effect on the production and preservation of seed quality. Some of the new mechanization production systems, which include the use of cotton modules, can affect seed quality negatively. Damage results from excessive moisture in the seed cotton when placed in the module, and it is commonly more serious to planting seed quality than to compositional quality (Cherry et al., 1984). Breeding methodologies used in cotton are those normally utilized with self-pollinating crops. Niles and Feaster (1984) provided a review of cotton breeding. The discovery of cytoplasmic malesterile (CMS) and restorer factors (Meyer, 1973, 1975) had stimulated interest in producing hybrid planting seeds. However, hybrid cotton planting seeds are in limited use in areas of the world where abundant, low-cost hand labor is available for hand cross-pollination, primarily in India and the People’s Republic of China. There is no reason to assume that changes in cotton breeding methods would result in a change of emphasis on seed quality. 4.4.4

Objectives

As with most crops, the major aim of cotton breeding is to increase production efficiency and total yield. Since the original cottons were slow-maturing, short-day photoperiodic perennials, and since cultivated cotton is generally produced as an annual crop, the history of cotton breeding has been directed toward increased earliness (Niles et al., 1984). Originally, earliness was needed to maximize yield of a perennial plant within an annual cropping system and to avoid the predation of the boll weevil. Today, earliness is desired to minimize production inputs, avoid hazardous weather, or facilitate double-cropping systems. Plant breeders have been able to select for increased earliness and restricted plant growth to the point where yield potential becomes limiting. The indeterminate growth habit of the cotton plant requires vegetative plant growth to produce flowering

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sites. To overcome these limitations, efforts are directed toward increasing the rate of flowering, and this has met with some success. In addition, selection has been practiced to decrease the period of boll development. Success of such selection could influence seed quality if the rates of the developmental processes in the boll are not increased to compensate for the shortened period of boll development. There are scattered reports of breeding attempts to improve the compositional quality of cottonseed. The reports are characteristically positive, but do not appear to represent any continuous effort to breed for improved seed quality (Cherry et al., 1981). 4.4.5

Pests

Areas of the world suited to cotton production are generally conducive to insect pests that prey on cotton. Favorable climatic conditions and a normally long growing period for cotton plants combine to make insect pests a major problem in cotton production. Sizable breeding efforts are directed toward breeding for pest resistance. Thus far, little success has been realized other than breeding for avoidance through increased earliness. Breeding for long trichomes was successful against the jassid, but resistance to other insects is less dramatic and does not persist under high insect pressures (Ridgway, 1984). Breeding for disease resistance was successful against bacterial blight. Resistance factors with single gene inheritance were identified and included individually or in combination in cultivars in the problem areas. Other diseases are not so readily controlled because the genetics of resistance is either complex or not known (Bell, 1984). A disease of specific concern to cottonseed quality is the infection of bolls by Aspergillus flavus, and the subsequent production of aflatoxin. The occurrence of aflotoxin is limited to a few geographical areas, and its occurrence is highly variable from season to season. The complex interaction of insect vectors, environmental variation, and variable production of aflatoxin has prevented the establishment of any definitive control strategies. 4.4.6

Cottonseed Quality and Gossypol

The discovery of the genes gl2 and gl3, which remove the pigment glands from seed and all aerial portions of the plant, has had the greatest impact on seed quality (McMichael, 1960). These glands contain pigments, gossypol, and gossypol precursors that are the greatest quality detriment in cottonseeds. With the discovery of glandless cotton, much breeding effort was directed toward breeding glandless cultivars. The discovery that chewing insects prefer glandless cotton caused a recession of these breeding efforts, and breeding was redirected toward increasing the gland content of the cotton plant (Jenkins et al., 1966; Lukefahr et al., 1969). Breeding to produce glandless cotton continued for a period, but has essentially ceased in the U.S. The glandless genotype used was from plants that were discovered in the progeny of a cross between Hopi cotton, domesticated by the Hopi Indians, and an Acala type. The original material was very poor agronomically, and breeding efforts were directed toward transferring the glandless genes into well-adapted agronomic backgrounds. Because of the very poor agronomic origin of glandless cotton, there was initial concern about possible genetic linkages or pleiotropic associations between the glandless genes and poor agronomic performance. A glandless mutant was discovered in Egyptian cotton, G. barbadense. This glandless expression was controlled by an incompletely dominant allele (Gl2e) at the Gl2 locus (Kohel and Lee, 1984). The use of a single, incomplete dominant gene that is identifiable as a heterozygote should be easier than using the two recessive genes gl2 and gl3 in the development of glandless cultivars. The limited breeding efforts for glandless cottons throughout the world now utilize the Gl2e gene as the source. Researchers continue to strive to produce a gossypol-free seed. The ideal goal remains to produce a cotton plant with gossypol glands in the vegetative tissues, but not in the seed. The idea of a glanded plant and glandless cottonseed originated with the observation that the wild diploids

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of Australia possess such a characteristic. Efforts have prevailed for over two decades by numerous researchers to introgress the trait into the cultivated tetraploids (Muramato, 1969; Dilday, 1986; Altman et al., 1987; Mergeai et al., 1997; Vroh et al., 1999). The efforts to transfer the trait to G. hirsutum were based on the premise that the trait was homoeologous to either gl2 or gl3. The efforts have not been successful, and most researchers seem to have reached the conclusion that the trait is a complex developmental trait, which may not merit further pursuit. However, the goal of a gossypol-free seed and glanded plant is still pursued. Research efforts have followed three paths to this goal through the use of biotechnological tools. The isolation of delta cadinene synthase genes (Chen et al., 1995, 1996) led researchers to pursue their use to disrupt the pathway of gossypol production in seeds. Transgenic plants with the delta cadinene synthase gene in an antisense construct with a seed-specific promoter were produced. Unfortunately, gossypol was reduced but not eliminated in the seed (Martin et al., 2003). A subsequent study with a similar antisense construct reported that the blockage of the pathway reduced the plant’s defense to disease (Belinda et al., 2005). A second approach to produce gossypol-free cottonseed is to break down the gossypol in the seed glands. This is the research path followed in Ow’s lab (Koshinsky et al., 1994). The third approach is to eliminate the gossypol glands in the seeds, and therefore the deposition of gossypol in the seed glands. The authors are following this route with cotton genomic resources produced in the lab. Their research efforts are directed toward the isolation of the Gl2e gene and combining it with a seed-specific promoter in antisense construction; this is an ongoing longterm effort (Yu et al., 2000a, 2000b; Decanini et al., 2001; Kohel et al., 2001). Despite the limited acceptance of glandless cottons, they did serve to focus increased attention on seed quality. A breeding program in California resulted in the release of a cultivar with decreased seed-gossypol and increased seed-oil content, compared to earlier releases. The U.S. National Cotton Variety Testing program has included seed quality among its evaluations. The author maintains a genetics program for cottonseed quality improvement. There is ongoing research into the pathways of lipid synthesis and the development of transgenic plants to modify the fatty acid profile of cottonseed oil (Chapman et al., 2001; Pirtle et al., 2001). Successful research efforts in producing such transformants have not been adapted in commercial cultivars. Studies of seed-oil content have revealed that it is controlled by predominantly additive gene variation with sizable environmental variation, which leads to lowered heritability (Kohel, 1980). Genetic studies of seed-oil content in Egyptian cultivars have shown larger amounts of nonadditive genetic variance, but these materials had a more restrictive genetic base. Research and breeding efforts to improve seedling vigor and protection against seedling diseases seem to impart better preservation of seed quality. There is no evidence that seed constituents are changed, but rather seed coat changes provide better protection of the embryo. One of the reasons that integration of seed quality into breeding programs will be slow is the lack of means to make seed quality evaluations. Routine fiber quality evaluations are available through relatively inexpensive service laboratories, and certain large breeding programs have their own laboratories. The evaluation of seed quality, on the other hand, for oil, protein, and gossypol is expensive, and test procedures require large quantities of seed for separate analyses of oil and protein and gossypol that are not available in genetic studies or in early stages of plant breeding. Few breeding programs have the resources available for such determinations. Given the relative lower value of seed, compared to fiber, there exists little incentive to devote limited resources to the evaluation and breeding of cottonseed quality. Limited efforts have been used to develop methods for evaluating cottonseed quality. The survey of oil content of the germplasm utilized the broadband nuclear magnetic resonance (NMR) (Kohel, 1978). Kohel has continued his work with methods to measure cottonseed quality, and he has published the results of near-infrared (NIR) equations to determine seed-oil content of cottonseed (Kohel, 1998); he is also analyzing data for NIR equations for additional cottonseed constituents (Kohel, personal communication).

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4.5 UTILIZATION OF PRODUCTS Cottonseed is the second-ranking oilseed in the world in production, from which is produced oil (16%), meal (45%), linters (9%), hulls (26%), and the remainder (4%) as waste and processing losses. Refined cottonseed oil is used in the food industry, and in some parts of the world it is the preferred vegetable oil. The cottonseed meal, remaining after oil extraction, is used as a proteinrich feed for ruminant animals in most areas of the world. Often the hulls are added to the meal as roughage. The contamination of meal by contents of the pigment glands limits the use of cottonseed meal in nonruminant feeds since gossypol in the pigment glands is toxic to nonruminants, such as swine and poultry. Free gossypol is commonly bound by heat treatments, which also binds part of the available lysine. During the 1970s and 1980s there was a large interest in cottonseed quality. The USDA Southern Regional Research Center at New Orleans, LA, devoted major efforts toward cottonseed utilization (Cherry et al., 1984). Results have identified the chemical and physical properties of the proteins, described functional properties, and developed potential products. The presence of pigment glands and their undesirable contamination of cottonseed products remain deterrents to full utilization of cottonseed. In addition to genetic removal of the pigment glands, methods were developed to physically remove them in processing. One such method was the liquid cyclone process. This process offered the potential to produce food-grade products from glanded cottonseed (Gardner et al., 1976). Neither genetic removal or physical removal became successful. The liquid cyclone process never reached full production, and it failed to find financial support to continue its development. The glandless genes were thought to predispose greater susceptibility of the cotton plant to chewing insects. The risk of greater insect susceptibility never allowed widespread adaptation of glandless cultivars that were developed; because of this and other problems associated with product development and financing, further development ceased. From a processing and utilization point of view, gossypol-free cottonseed is highly desirable because it allows the production of traditional products with higher quality and reduced cost and allows expanded uses (Anonymous, 1977). The incentives to improve cottonseed and its products originate not only from the cotton industry, which would benefit directly, but also from a worldwide perspective that would benefit food and feed resources. Cotton will continue to be produced for its fiber because cotton fibers are a renewable resource that is processed into products with highly desirable textile properties. Given that cottonseeds will be produced in the continued production of cotton fibers, a major goal is to efficiently and effectively utilize the seed. Cottonseeds devoid of pigment glands are highly desirable for their high nutritional value and for their unique protein properties. The physical and functional properties of cottonseed proteins are such that they will not only complement existing vegetable proteins, but also offer unique properties not found in other vegetable proteins (Cherry et al., 1984). In the seed-crushing industry, gossypol-free cottonseeds offer advantages directly related to the absence of pigment glands, and all of these result in more efficient processing and higherquality products. The reduction of gossypol in the system eliminates the need of high-energy inputs for heat binding of free gossypol; the oil would require less refining due to the absence of pigments; the meal produced would have a higher lysine value and could be safely fed to both ruminants and nonruminants. In the food industry, gossypol-free cottonseed products can range from the full-fat kernel to defatted flours to protein concentrates. Traditional vegetable protein products could be produced and new product potentials have been identified, capitalizing on the properties of cottonseed proteins. Given the worldwide distribution of cotton production, advances in gossypol-free cottonseed would provide for a new protein source in areas where protein availability is limited. All of these describe the potential for cottonseed utilization, but the elusive gossypol-free seed has not been produced and remains an unrealized potential.

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In recent years, a large market has been established for the feeding of whole cottonseeds to primarily the dairy industry. Research supported by Cotton, Inc. (CI) developed a process in which cottonseeds are coated with a material to facilitate their handling. Normal fuzzy cottonseeds clump or bunch, making them difficult to handle in the traditional feeding operations. The coated cottonseeds, called EZIFLO, allow them to be handled in a manner similar to that of feed grains, which facilitates feeding and formulating feeding rations.

4.6 CONCLUSIONS There are major issues in the implementation of a unique cottonseed improvement. The first is of course one of sufficient economic value to warrant its introduction. Associated with this is the need to produce and deliver a product in sufficient quantity to develop a market. This would require the production, ginning, and delivery separated from other cottonseeds. These are primarily mechanical and logistical considerations. In the production of cotton for lint production, there is no need to be concerned about contamination by cross-pollination because the fiber is a maternal tissue. However, the cottonseed is embryonic tissue and cross-pollination would be a source of contamination. So a unique cottonseed product would require both biological and physical isolation for its production. Cottonseed improvement will never have a major role in commercial cultivar development unless the economic incentives are sufficient to warrant the diversion of resources. It is unlikely that commercial breeding programs are willing or able to conduct research into the improvement of cottonseed quality. The public sector will have to take the lead in cottonseed quality research. Such research will have to identify and develop improvements of sufficient importance and value that they will warrant inclusion in commercial breeding programs.

REFERENCES Altman, D.W., D.M. Stelly, and R.J. Kohel. 1987. Introgression of the glanded-plant and glandless-seed trait from Gossypium sturtianum Willis into cultivated Upland cotton using ovule culture. Crop. Sci. 27: 880–884. Altschul, A.M., C.M. Lyman, and F.H. Thurber. 1958. Cottonseed meal. In Processed Plant Protein Foodstuffs, Altschul, A.M., Ed. Academic Press, New York, pp. 469–534. Anonymous. 1977. Glandless cotton: its significance, status, and prospects. In Conference Proceedings, Dallas, TX, 184 pp. Belinda, J. et al. 2005. Antisense suppression of a (+)-cadinene synthase gene in cotton prevents the induction of this defense response gene during bacterial blight infection but not its constitutive expression. Plant Physiol. 138: 516–528. Bell, A.A. 1984. Cotton protection practices in the USA and world: diseases. In Cotton, Agronomy Monograph 24, Kohel, R.J. and Lewis, C.F., Eds. Am. Soc. Agron., Madison, WI, pp. 288–309. Benedict, C.R., R.J. Kohel, and A.M. Schubert. 1976. Transport of C-assimilates to cottonseed: integrity of funiculus during seed filling stage. Crop Sci. 16: 23–27. Brubaker, C.L., F.M. Bourland, and J.F. Wendel. 1999. In Cotton: Origin, History, Technology, and Production, Smith, C.W. and Cothren, J.T., Eds. John Wiley & Sons, New York, pp. 3–31. Brubaker, C.L., J.A. Koontz, and J.F. Wendel. 1993. Bidirectional cytoplasmic and nuclear introgression in the New World cottons. Am. J. Bot. 80: 1203–1208. Brubaker, C.L. and J.F. Wendel. 1994. Reevaluating the origin of domesticated cotton (Gossypium hirsutum: Malvaceae) using nuclear restriction fragment length polymorphisms (RFLPs). Am. J. Bot. 81: 1309–1326. Chapman, K.D. et al. 2001. Transgenic cotton plants with increased seed oleic acid content. J. Am. Oil Chem. Soc. 78: 941–947.

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Chen, X.Y. et al. 1995. Cloning, expression, and characterization of (+)-cadinene synthase: a catalyst for cotton phytoalexin biosynthesis. Arch. Biochem. Biophys. 324: 255–266. Chen, X.Y. et al. 1996. Cloning and heterologous expression of a second (+)-cadinene synthase from Gossypium arboreum. J. Nat. Prod. 59: 944–951. Cherry, J.P. and H.R. Leffler. 1984. Seed. In Cotton, Agronomy Monograph 24, Kohel, R.J. and Lewis, C.F., Eds. Am. Soc. Agron., Madison, WI, pp. 522–567. Cherry, J.P. et al. 1981. Cottonseed quality: factors affecting feed and food uses. In Proceedings of the Beltwide Cotton Prod. Res. Conf. National Cotton Council America, Memphis, TN, pp. 266–283. Chowdhury, K.A. and G.M. Burth. 1971. Cotton seeds from the Neolithic Egyptian Nubia and the origin of Old World cotton. Linn. Soc. London. Biol. J. 3: 303–312. Decanini, L.I., R.J. Kohel, and J. Yu. 2001. Fine-mapping of the glandless gene in cotton. In Proceedings of the Plant and Animal Genome VIII, San Antonio, TX, p. 631. Dilday, R.H. 1986. Development of cotton plant with glandless seeds and glanded foliage and fruiting forms. Crop Sci. 26: 639–641. Gardner, H.K., R.J. Hron, and H.L.E. Vix. 1976. Removal of pigment glands (gossypol) from cottonseed. Cereal Chem. 53: 549–560. Jenkins, J.N., F.G. Maxwell, and H.N. Lafever. 1966. The comparative preference of insects for glanded and glandless cottons. J. Econ. Entomol. 59: 352–356. Kohel, R.J. 1978. Survey of Gossypium hirsutum L. Germplasm Collections for Seed-Oil Percentage and Seed Characteristics, ARS-S-187. USDA, 38 pp. Kohel, R.J. 1980. Genetic studies of seed oil in cotton. Crop Sci. 20: 784–787. Kohel, R.J. 1998. Evaluation of near infrared reflectance for oil content of cottonseed. J. Cotton Sci. 2: 23–26. Kohel, R.J. and C.R. Benedict. 1984. Year effects in partitioning of dry matter into cotton boll components. Crop Sci. 24: 268–270. Kohel, R.J. and J.P. Cherry. 1984. Variation of cottonseed quality with stratified harvests. Crop Sci. 23: 1119–1124. Kohel, R.J., J. Glueck, and L.W. Rooney. 1985. Comparison of cotton germplasm collections for seed-protein content. Crop Sci. 25: 961–963. Kohel, R.J. and J.A. Lee. 1984. Genetic analysis of Egyptian glandless cotton. Crop Sci. 24: 1119–1121. Kohel, R.J. and C.F. Lewis, Eds. 1984. Cotton, Agronomy Monograph 24, Kohel, R.J. and Lewis, C.F., Eds. Am. Soc. Agron., Madison, WI, 605 pp. Kohel, R.J. et al. 2001. Cottonseed quality and the creation of glanded plants with glandless seeds. In Proceedings of the Genetic Improvement of Cotton Fiber and Seed Quality Workshop, Beltwide Cotton Production–Research Conferences, pp. 271–277. Koshinsky, H.A., H.K. Liao, and D.W. Ow. 1994. Progress in screening microorganisms for gossypol-degrading ability. In Biochemistry of Cotton, Proceedings of the Biochemistry of Cotton Workshop. Cotton, Inc., Galveston, TX, pp. 19–22. Lee, J.A. 1984. Cotton as a world crop. In Cotton, Agronomy Monograph 24, Kohel, R.J. and Lewis, C.F., Eds. Am. Soc. Agron., Madison, WI, pp. 1–15. Lukefahr, M.J., T.N. Shaver, and W.L. Parrot. 1969. Sources and nature of resistance in Gossypium hirsutum to bollworm and tobacco budworms. In Proceedings of the Beltwide Cotton Prod. Res. Conf. National Cotton Council America, Memphis, TN, pp. 81–82. Martin, G.S. et al. 2003. Reduced levels of cadinane sesquiterpenoids in cotton plants expressing antisense (+)-delta-cadinene synthase. Phytochemistry 62: 31–38. Mauney, J.R. 1984. Anatomy and morphology of cultivated cottons. In Cotton, Agronomy Monograph 24, Kohel, R.J. and Lewis, C.F., Eds. Am. Soc. Agron., Madison, WI, pp. 59–80. McMichael, S.C. 1960. Combined effects of the glandless genes gl2 and gl3 on pigment glands in the cotton plant. Agron. J. 46: 385–386. Mergeai, G., J.P. Baudoin, and B.I.Vroh. 1997. Exploitation of trispecific hybrids to introgress the glandless seed and glanded plant trait of Gossypium sturtianum Willis into G. hirsutum L. Biotech. Agron. Soc. Environ. 1: 272–277. Meyer, V.G. 1973. Fertility restorer genes for cytoplasmic male sterility from Gossypium harknessii. In Proceedings of the Beltwide Cotton Prod. Res. Conf. National Cotton Council America, Memphis, TN, p. 65. Meyer, V.G. 1975. Male sterility from Gossypium harknessii. J. Hered. 66: 23–27.

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Muramato, H. 1969. Hexaploid cotton: some plant and fiber properties. Crop Sci. 9: 27–29. Niles, G.A. and C.V. Feaster. 1984. Breeding In Cotton, Agronomy Monograph 24, Kohel, R.J. and Lewis, C.F., Eds. Am. Soc. Agron., Madison, WI, pp. 202–231. Percival, A.E., J.F. Wendel, and J.M. Stewart. 1999. Taxonomy and germplasm resources. In Cotton: Origin, History, Technology, and Production, Smith, C.W. and Cothren, J.T., Eds. John Wiley & Sons, New York, pp. 33–63. Pirtle, I. et al. 2001. Molecular cloning and functional expression of the gene for a cotton delta-12 desaturase (FAD2). Biochim. Biophys. Acta 1522: 122–129. Ridgway, R.L. 1984. Cotton protection practices in the USA and world: insects. In Cotton, Agronomy Monograph 24, Kohel, R.J. and Lewis, C.F., Eds. Am. Soc. Agron., Madison, WI, pp. 265–287. Smith, C.W. and J.T. Cothren, Eds. 1999. Cotton: Origin, History, Technology, and Production. John Wiley & Sons, New York, 850 pp. Vroh, B.I. et al. 1999. Development of high-gossypol cotton plants with low-gossypol seeds using trispecies bridge crosses and in vitro culture of seed embryos. Euphytica 106: 243–251. Waddle, B.A. 1984. Crop growing practices. In Cotton, Agronomy Monograph 24, Kohel, R.J. and Lewis, C.F., Eds. Am. Soc. Agron., Madison, WI, pp. 233–263. Wendel, J.F. 1989. New world tetraploid cottons contain old world cytoplasm. Proc. Natl. Acad. Sci. U.S.A. 86: 4132–4136. Wendel, J.F. and V.A. Albert. 1992. Phylogentics of the cotton genus (Gossypium): character-state weighted parsimony analysis of chloroplast-DNA-restriction site data and its systematic and biogeographic implications. Syst. Bot. 17: 115–143. Wendel, J.F., C.L. Brubaker, and A.E. Percival. 1992. Genetic diversity in Gossypium hirsutum and the origin of Upland cotton. Am. J. Bot. 79: 1291–1310. Yu, Z.H. et al. 2000a. Toward positional cloning of a major glandless gene in cotton. In Proceedings of the Beltwide Cotton Prod. Res. Conf. National Cotton Council America, Memphis, TN, CD. Yu, Z.H. et al. 2000b. Construction of a cotton BAC library and its applications to gene isolation. In Proceedings of the Plant and Animal Genome VIII, San Diego, CA, p. 146.

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CHAPTER 5 Sunflower Chao-Chien Jan and Gerald J. Seiler

CONTENTS 5.1 5.2

5.3 5.4

5.5

Introduction...........................................................................................................................104 Description and Crop Use....................................................................................................105 5.2.1 Botanical and Morphological Traits.........................................................................105 5.2.1.1 Basic Features ...........................................................................................105 5.2.1.2 Reproductive System.................................................................................105 5.2.1.3 Growth Habit and Plant Structures...........................................................106 5.2.2 World Production Area and Utilization ...................................................................108 Origin, Domestication, and Dispersion................................................................................109 Taxonomy and Germplasm Resources.................................................................................110 5.4.1 Taxonomy and Center of Diversity..........................................................................110 5.4.2 Germplasm Resources ..............................................................................................111 5.4.2.1 Ex Situ Collections....................................................................................111 5.4.2.2 Wild and Weedy Relatives ........................................................................114 5.4.2.3 Core Collection .........................................................................................116 5.4.2.4 Genetic Stocks...........................................................................................116 5.4.2.5 Germplasm Evaluation and Use ...............................................................116 5.4.2.5.1 Pathogens ................................................................................116 5.4.2.5.2 Insects......................................................................................117 5.4.2.5.3 Oil and Oil Quality .................................................................117 5.4.2.5.4 Protein .....................................................................................117 Cytogenetics .........................................................................................................................118 5.5.1 Helianthus Genomes ................................................................................................118 5.5.1.1 Euploidy and Aneuploidy .........................................................................118 5.5.1.2 DNA Content.............................................................................................119 5.5.1.3 Karyotype ..................................................................................................121 5.5.2 Microsporogenesis....................................................................................................122 5.5.3 Male Sterility ............................................................................................................124 5.5.4 Cytogenetic Stocks ...................................................................................................128

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Interspecific Hybridization .......................................................................................129 5.5.5.1 Hybridization Techniques .........................................................................129 5.5.5.2 Interspecific Hybrids among Helianthus Species.....................................131 5.5.5.3 Interspecific Hybrids between Wild Helianthus Species and Cultivated Lines ........................................................................................132 5.5.6 Mutagenesis ..............................................................................................................135 5.5.7 Male Sterility Induction ...........................................................................................135 5.5.8 Chromosome Doubling ............................................................................................136 5.5.9 Alteration of Fatty Acid Composition .....................................................................136 5.6 Germplasm Enhancement: Conventional Breeding .............................................................137 5.6.1 Breeding for End Use...............................................................................................137 5.6.1.1 Grain..........................................................................................................137 5.6.1.2 Protein .......................................................................................................137 5.6.1.3 Oil..............................................................................................................138 5.6.1.4 Oil Quality.................................................................................................138 5.6.1.4.1 Fatty Acid Composition..........................................................138 5.6.1.4.2 Tocopherol Composition.........................................................138 5.6.2 Breeding for Adaptation ...........................................................................................139 5.6.2.1 Plant Type..................................................................................................139 5.6.2.2 Phenology..................................................................................................140 5.6.3 Yield Potential and Stability ....................................................................................141 5.6.4 Improved Resistance to Biotic Constraints..............................................................141 5.6.4.1 Diseases .....................................................................................................141 5.6.4.2 Viruses and Bacteria .................................................................................144 5.6.4.3 Pests...........................................................................................................144 5.6.4.3.1 Insects......................................................................................144 5.6.4.3.2 Bird Depredation.....................................................................145 5.6.5 Improved Resistance to Abiotic Constraints............................................................146 5.6.5.1 Salt and Drought Tolerance ......................................................................146 5.6.5.2 Herbicide Tolerance ..................................................................................146 5.6.5.3 Soil Nutrition.............................................................................................147 5.7 Germplasm Characterization: Molecular Applications........................................................147 5.7.1 Genetic Diversity......................................................................................................147 5.7.2 Molecular Mapping ..................................................................................................148 5.7.3 Gene Mapping ..........................................................................................................149 5.7.4 BAC Library .............................................................................................................150 5.8 Conclusions and Prospects ...................................................................................................150 References ......................................................................................................................................151

5.1 INTRODUCTION Helianthus, the genus name of sunflower (Helianthus annuus L.) is derived from the Greek words helios, meaning “sun,” and anthus, meaning “flower.” The Spanish name for sunflower, girasol, and the French name, tournesol, literally mean “turn with the sun,” a trait exhibited by sunflower until anthesis, after which the capitula (heads) face east (Fick, 1989). Sunflower is a relatively new crop among the world field crops and is unique in several aspects. It is one of a few crops that have their origin in North America. Sunflower is further unique in that it has been bred for distinctly different uses: as an oilseed crop, edible confection, birdseed, and, to a much lesser extent, an ornamental for home gardens and the cut-flower industry. It is grown as an oilseed crop worldwide in temperate and subtropical climates.

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Botanical and Morphological Traits

5.2.1.1 Basic Features Sunflower is distinguished from other cultivated crops by its single stem and its large conspicuous inflorescence. The inflorescence (head) is of major interest to agronomists and plant breeders because its diameter influences seed size and the percentage of fertile disk flowers (seed set), which ultimately determine seed yield. When sunflower is in full flower, it is one of the most photogenic crops because of its large inflorescence with showy yellow-orange ray flowers. Flowering sunflower has served as an inspiration for artists, poets, and business promotions. 5.2.1.2 Reproductive System The inflorescence is a capitulum or head characteristic of one of the largest families of flowering plants, the Asteraceae. It consists of an outer whorl of showy and generally yellow ray flowers and from 700 to 3000 disk flowers in oilseed hybrids and up to 8000 disk flowers in non-oilseed hybrids (Pustovoit, 1975). The disk flowers are arranged in arcs radiating from the center of the head and are perfect flowers that produce seeds. Involucral bracts or phyllaries, which vary in form and size, surround the head. The showy flowers on the outer whorl of the head have five elongated petals united to form strap-like structures, which give them the name ray or ligulate flowers. Ray flowers are usually golden yellow, but may be pale yellow, orange-yellow, or reddish. Variation in petal color has been discussed by Cockerell (1912, 1918) and Fick (1976). Ray flowers are normally sterile, having a rudimentary pistil and vestigial style and stigma, but no anther. Mutants occur producing more than the normal numbers of ray flowers, or occasionally none. The remaining flowers covering the large discoidal head are called disk flowers. A single disk flower is often referred to as a floret. Each floret is subtended by a sharp-pointed, chaffy bract, a basal ovary, and two pappus scales (often considered a modified sepal). The disk flower is perfect (contains both a stamen and pistil). The corolla of each floret is composed of five fused petals, except at the tip. Inside the corolla tube, five fused anthers form a second tube with separate filaments attached to the base. Enclosed in the anther tube is the style, which terminates distally in a bi-lobed stigma that curls outward above the anther tube. The receptive surfaces of the stigma are in close contact in the bud stage before the flowers open. At anthesis, the outer whorl of disk flowers opens first, at about the time that the ray flowers open from their folded position against the buds of the disk flowers. Immediately after this stage is reached, the anther locules dehisce, releasing pollen inside the anther tube. An elongation of the lower portion of the style pushes the two-lobed pubescent stigma up the anther tube. The stigma is not receptive at this stage because the two lobes are held together covering the inner receptive surface. The stigma appears at about 1700 hour of the same day, and by the following morning it is fully emerged, with receptive surfaces exposed. At this time, the staminal filaments lose turgidity and the anther tube begins to recede into the corolla. The beginning of flowering of disk flowers has been described as the R-5.1 stage of sunflower development (Schneiter and Miller, 1981). One to four rows of disk flowers open successively daily for 5 to 10 days. The flowering period is prolonged if heads are larger, or if the weather is cool and cloudy. The stigmas remain receptive for up to 4 or 5 days. Floral nectaries, which are believed to play a role in attracting honeybees (Apis mellifera L.), occur in disk flowers (Sammataro et al., 1985). They are located at the base of the style directly above the ovary. Sunflower is the only Asteraceae in which the cytoplasmic male-sterile (CMS) system is known. Cytoplasmic male sterility is sporophytic in sunflower (Pearson, 1981). Sunflower plants that are CMS usually have anthers one half the normal length, which do not project from the corolla

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(Leclercq, 1969). Anthers in male-sterile lines are fused only at their bases, and not at their tips. In some lines, anthers are of normal length and project from the corolla, although devoid of pollen. Female flowers of male-sterile plants appear normal and are fertile when pollinated. The achene, or nut-like fruit of the sunflower, consists of a seed, often called the kernel, and adhering pericarp, usually called the hull. Achenes mature from the periphery of the whorl to the center. As achenes mature, the withered calyx, corolla tube, anther, stigma, and style drop off at the point of their attachment. The achenes usually are largest on the periphery of the head and smallest at the center. Achenes develop hulls whether they are fertilized or not. Seed development can be detected by the plumpness of the achene and uniform dense color of the pigmented layer of the pericarp when it is a characteristic of the female parent (Dedio and Putt, 1980). These characteristics are usually visible at about 20 days after fertilization. Empty achenes are frequently somewhat pinched in appearance. Disk flowers in the center of the head in some breeding lines fail to produce filled achenes, and the mature unfilled achenes may appear chaff-like. The lack of achene filling in the center of some heads is influenced by both genotype and the environment. The dimensions of achenes vary from 7 to 25 mm long and from 4 to 13 mm wide. The achenes of oilseed sunflower are usually black. Achenes are much smaller in the wild species, where they are 2 to 7 mm long and generally 1 to 2 mm wide. The weight of 100 achenes of cultivated sunflower varies from 4 to 20 g. The weight of an individual achene varies from 40 to 400 mg. Lengthwise, the achenes may be linear, ovoid, or almost round, and in cross section they may be flat to almost round. Large achenes usually have thick hulls and relatively small kernels. Small achenes, in contrast, usually have thin hulls tightly fitting around the kernel. Thinner-hulled achenes usually have a higher oil content than the thicker-hulled achenes. 5.2.1.3 Growth Habit and Plant Structures Cultivated hybrid sunflower stems are typically nonbranched annual plants. Branched types that possess recessive genes for branching are frequently used as parental male breeding lines or pollinator lines in commercial seed production. Stem dimension and development are influenced by environment and by plant population. Branching can also be influenced by the environment, especially when the terminal buds are injured early in phenological development. Many degrees of branching occur in sunflower, ranging from a single stem with a large solitary inflorescence in cultivated types to multiple branching from axils of most leaves on the main stem in the wild species. Branch length varies from a few centimeters to a distance longer than the main stem. Branching may be concentrated at the base or top of the stem or spread over the entire plant. Generally, heads on branches are smaller than heads on the main stem. Occasionally, some firstorder branches have a terminal head almost as large as the main head. In most wild species, the head on the main stem blooms first, but generally is no larger than those on the branches. Studies on the genetics of top branching have shown that it is dominant over nonbranching and is controlled by a single gene (Putt, 1940; Shull, 1908). Likewise, Hockett (1956) reported that top branching in cultivated sunflower is controlled by a single dominant gene, but branching in wild species is controlled by duplicate dominant genes. Putt (1964) reported finding a recessive gene for branching, which is a desirable feature in inbred lines used as pollinators in single-cross hybrids. Ross (1939) reported a high negative correlation (r = –0.71) between the number of branches and achene yield. Stem (plant) height of commercial sunflower cultivars varies from 50 to over 500 cm, and stem diameter from 1 to 10 cm. Rare plant types of 12 m tall were reported by Dodonaeus and cited by Cockerell (1915). Cultivars used for forage are usually tall and late flowering. Long-season oilseed hybrids of the southern hemisphere are taller than hybrids of the northern hemisphere. A generally accepted ideotype of productive sunflower is a medium plant height of 160 to 180 cm (Skoric, 1988). Commercial hybrids with a height of 120 to 150 cm have been developed and are referred to as semidwarf hybrids, while dwarf sunflowers are 80 to 120 cm tall (Schneiter, 1992).

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Stem pubescence of cultivated sunflower is variable. It varies from glabrous to densely pubescent. Stem length is determined by the number and length of internodes. Both tall and short plants with many internodes will have thick stems because of the positive association between numbers of internodes and stem thickness. Stems that are short because of fewer internodes will be thinner (Knowles, 1978). Sunflower head angle varies, although during flowering it is usually vertical (relative to the soil surface) and faces east. As the seeds develop, the heads usually nod and at maturity hang face downward. The face of a head that is parallel to the ground will show sun damage if the head is exposed to the sun during seed development. Exposed disk flowers and achenes will turn brown and kernels fail to develop. Heads facing upward suffer severe sun damage in southern latitudes. Increasing the head angle may position heads so they are less exposed to predation by birds (Rudorf, 1961; Parfitt, 1984; Parfitt and Fox, 1986; Seiler and Rogers, 1987; Gross and Hanzel, 1991). The head angles (from horizontal) in sunflower descriptors are given as 0 = horizontal, 45, 90, 135, 180 (which is an inverted head facing the ground), and 225° (head facing the stem) (IBPGR, 1985). Information on development of sunflower roots is limited. Early studies by Weaver (1926) on the cultivar Russian at Lincoln, NE, found that a strong central taproot penetrates the soil to a depth of 150 to 270 cm. Pustovoit (1967) reported root depths of 4 to 5 m in cultivars with a growth period of 100 to 110 days. Recently, root depth of 2 m or more has been frequently reported (Sadras et al., 1989; Gimenez and Fereres, 1986; Jones, 1984). Early in plant development, numerous strong lateral roots originate from the enlarged taproot in the top 10 to 15 cm of the soil. The lateral roots spread widely, from 60 to 150 cm, and develop in the upper 30 cm of soil. Root length density (RLD; cm2) in sunflower decreases exponentially with depth, with RLD up to 10-fold greater in the 0- to 0.2-m soil layer than in deeper soil (Connor and Sadras, 1992). If lateral roots turn downward, they do not penetrate beyond 90 to 120 cm. The roots diffuse almost completely through the top 60 cm of the soil. Roots of sunflower tend to be randomly distributed in the soil (Bennie et al., 1987). The rooting system of sunflower can be considered explorative, i.e., a large volume of soil is explored with a combination of thick and thin roots, low specific root length, and low root length density, as opposed to exploitative, i.e., root systems characterized by predominantly fine roots, high specific root length, and large root length density (Boot, 1990). Adventitious roots may be complementary to primary root systems and may function in plant anchorage and in water absorption and conductance. The leaf consists of the blade (lamina) and a stalk-like part, the petiole, which connects it to the stem. Leaves in sunflower are rarely sessile (without a petiole), except in some wild species. Leaves show their specialization as a photosynthetic structure in the expanded flat form of the blade. They are the receptors of radiation for conversion of light energy to food energy. The leaf is highly variable in both structure and function. In sunflower, as seedlings emerge from the soil, cotyledons unfold and reveal the first pair of true leaves at the top of the shoot. Leaves are produced in opposite alternating pairs, and after five opposite pairs appear, a shorter form of alternate phyllotaxy develops (Palmer and Phillips, 1963). The number of leaves on single-stemmed plants may vary from as few as 8 to as many as 70 (Knowles, 1978). For Native American cultivars, the number of leaves varies from 25 to 34 (Takami et al., 1981). The open-pollinated cultivar VNIIMK has 28 leaves (Rawson et al., 1980), and inbred and dwarf lines have from 23 to 26 leaves (Miller and Hammond, 1991). There appears to be some association between the number of leaves and time to maturity for plants with numerous leaves (Knowles, 1978). Plants with numerous leaves are also usually late maturing. The number of leaves on a plant and their expansion, size, shape, and duration can be greatly influenced by environmental factors. The surface of the leaf can be flat, concave (cupped), or convex in cross section. The leaf surface generally has some type of pubescence. It may be scabrous (having short projections or barbs on the outer wall of epidermal cells that give a rough texture), hispid (having short stiff hairs), or hirsute (having long silky hairs), and occasionally, the leaf surface is glabrous.

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There is considerable variation in leaf size, shape of the entire leaf, shape of the tip, base, margin, surface pubescence, and petiole characteristics. The length and width of leaves vary with the height of the stem. Leaf descriptions for cultivar identification should be standardized for specific location on the stem. The leaf blade is generally the largest in the mid-region of the plant. Length is measured from the tip to the base of the blade, and width at the widest part of the leaf. The ratio of length:width may be a useful criterion for cultivar description. Generally, the cordate (heart-shaped) leaf is the most common form found in cultivated sunflower. Extra floral nectaries that exude copious quantities of nectar are found on leaf blade margins and petioles. They are heavily browsed by entomophagous insects (Rogers, 1985). 5.2.2

World Production Area and Utilization

The world production of sunflower is estimated to be 21 million ha in 60 countries (USDA, 2005a). It is the second largest hybrid crop, second only to maize, and the fifth largest among the oilseed crops, after soybean, rapeseed, cottonseed, and groundnut. The largest producer of sunflower seeds is the former Soviet Union (FSU), consisting mainly of Russia and Ukraine, which produced 9.37 million ha of sunflower in 2003, followed by the European Union, consisting mainly of France, Hungary, Italy, and Spain, which produced 2.43 million ha (USDA, 2005a). Other European countries, Bulgaria, Romania, and Serbia and Montenegro, produced 1.96 million ha, followed by Argentina with 1.83 million ha. China produced 1.17 million ha, followed by the U.S. with 0.89 million ha. Sunflower seed production in the world increased by 24% (or 5 million metric tons) between 1993 and 2003. However, sunflower seed production declined in terms of world market share of the five major oilseeds during the same time frame, 1993 to 2003. In 2003, sunflower seed accounted for 10% of the world’s edible plant-derived oil (Kleingartner, 2004). The achene was originally used directly as food and crudely extracted oil. Native Americans had selected a tall, single-headed variety by the time European explorers reached North America in the 16th century. While sunflower was not a staple of their diet as were maize, beans, and squash, it nonetheless was cultivated by many tribes from eastern North America throughout the Midwest and as far south as northern Mexico (Putt, 1997). The Native American also used sunflower hulls as a source of dye, leaves for herbal medicine, and pollen in religious ceremonies. Records indicate that the Spanish introduced the sunflower into Europe in the early 1500s. Early English and French explorers subsequently introduced it to their respective countries. Sunflower was initially grown as an ornamental plant. From western Europe, sunflower spread along the trade routes to Egypt, Afghanistan, India, China, and Russia. By the early 1700s, sunflower seeds were eaten as a snack, and in 1716, the first patent for the use of sunflower oil (for industrial uses) was filed in England (Putt, 1997). The most significant boost for the sunflower as a crop, however, came from the Russian Orthodox Church. Lenten regulations prohibited the consumption of many oily foods, but since sunflower seed was not specifically listed, the seed and oil became a staple diet item in the FSU. Early cultivation of sunflower was primarily for livestock silage and seed for poultry. By the second half of the 20th century, improved Russian varieties with oil contents of 450 to 550 g/kg were available. The discovery of cytoplasmic male sterility (CMS) by French scientists laid the foundation for the development of sunflower hybrids in the early 1970s. Hybrid sunflower, with higher yields and oil content and more uniformity than open-pollinated varieties, provided the last great impetus in establishing sunflower as a global crop. Sunflower derives most of its economic value from the extracted oil, with the remaining value from the meal. The achenes of oilseed sunflower are usually black. The primary use of sunflower oil is as a salad and cooking oil, and as a major ingredient in some margarine and shortening products. Sunflower oil traditionally has been considered a polyunsaturated oil because of its relatively high content of linoleic acid (680 to 720 g/kg concentration). It is moderately low in saturated fat, which increases the risk of coronary diseases. The highly polyunsaturated nature of

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sunflower oil makes it an attractive product for health-conscious consumers who desire a diet that maximizes the polyunsaturated-to-saturated fat ratio, which is considered beneficial in reducing the risk of cardiovascular disease (Mensink et al., 1994; Willett, 1994). In the U.S., the sunflower industry has developed a mid-oleic (550 to 700 g/kg) sunflower oil called NuSun® (National Sunflower Association, Bismarck, ND) that possesses a significant advantage over several other popular oils, such as soybean and canola, because it does not have to be hydrogenated prior to its use as a frying oil, and therefore has negligible trans fatty acids. This oil offers desirable frying and flavor characteristics, increases the life span of the heated oil, and confers a healthful fatty acid composition containing an adequate level of heart-healthy polyunsaturated fatty acids, yet is free of hydrogenated trans fatty acids. In addition, the increased oleic acid content has the added benefit of slightly lowering saturated palmitic and stearic fatty acid concentrations. Sunflower oil is not commonly used for industrial purposes because of its generally higher value, compared to other oilseeds. However, it is used to some extent in some paints, varnishes, and plastics because of its good semidrying properties, without the yellowing problems associated with oils high in linolenic acid. Sunflower oil is also used in the manufacture of soaps and detergents. Along with other vegetable oils, it has potential value for the production of adhesives, agrichemicals, surfactants, additional plastics and plastic additives, fabric softeners, synthetic lubricants, and coatings. Actual use will depend to a large extent on its price, relative to that of petroleum and petro-based chemicals. Non-oilseed or confectionery sunflower usually has very large black with white-striped achenes; i.e., those achenes that pass over a 7.9-mm round hole sieve are used as a confection or snack food, roasted, and salted. Sunflower kernels are also used in the baking industry, as a condiment for salads, and other foods. Non-dehulled or partially dehulled sunflower meal can be substituted successfully for soybean meal of equal protein percentage in feeding ruminant animals. Partially or completely dehulled sunflower meal is desirable for feeding swine and poultry. Achenes are also used for feeding birds and in small animal feed. Sunflower, especially the cytoplasmic male-sterile type, which does not shed pollen, has been adapted for the ever-increasing cut-flower market. Several garden varieties have been developed and are marketed as ornamental sunflower with varying flower types and disk and petal color variations.

5.3 ORIGIN, DOMESTICATION, AND DISPERSION During the past 50 years, Heiser (1951, 1954, 1965, 1978, 1985, 1998) developed the following scenario for the origin and development of the domesticated sunflower from its progenitor, wild H. annuus L. Prior to the arrival of mankind in the New World, H. annuus was restricted to the southwestern U.S. Wild H. annuus was used by Native Americans for food, and due to its association with humans, it became a camp-following weed and was introduced eastward. This weedy sunflower was subsequently domesticated in the central U.S. and carried to the east and southwest. There is strong archaeological support for the origin of the domesticated sunflower from the central U.S. Large achenes (>7 mm in length) have been found at several archaeological sites in the central and eastern states, whereas only wild sunflower achenes have been found in sites from the Southwest and Mexico. Until recently, sunflower achenes from the Higgs site in eastern Tennessee (2850 B.P.) and the Marble Bluff Rock shelter in northwest Arkansas (2843 B.P.) were the earliest evidence of domesticated sunflower (Brewer, 1973; Crites, 1991; Ford, 1985). However, carbonized sunflower achenes more recently recovered from the Hayes site in middle Tennessee have yielded a radiometric date of 4625 B.P., extending the earliest record of the domesticated sunflower by 1400 years (Crites, 1993). The possibility that domesticated sunflower may have independent origins in Mexico and the southwestern U.S. has also been considered (Heiser, 1985, 1998). However, until recently, the presence

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of domesticated achenes in archaeological records outside of the U.S. was lacking. The morphological similarity among Native American varieties of the domesticated sunflower, and the virtual monomorphism for isozymes and chloroplast DNA in cultivated lines did not support the multiple origin hypotheses (Rieseberg and Seiler, 1990). Recent evidence from an archaeological site near San Andres, Tabasco, Mexico, indicates that a second or concurrent center of origin for domestication of sunflower may exist (Lentz et al., 2001). A carbonized achene from that site was dated at 4130 B.P. Molecular techniques have been used to search for the origin of cultivated sunflower. Arias and Rieseberg (1995) used randomly amplified polymorphic DNA (RAPD) loci to investigate the origin and genetic relationships of domesticated sunflower and its wild relatives. RAPD data supported the origin of the domesticated sunflower from wild H. annuus; however, because of the high identity between the two species, little information was provided regarding the geographic origin of the domesticated sunflower. Cronn et al. (1997), using allozyme variation, concluded that domesticated sunflowers form a genetically coherent group and that wild sunflowers from the Great Plains may include the most likely progenitor of domesticated sunflower. Systematic data do support the southwestern U.S. as a site of origin for annual sunflowers, including H. annuus (Heiser et al., 1969, Rieseberg et al., 1991). Quantitative trait loci (QTL) controlling phenotypic differences between cultivated sunflower and its wild progenitor were investigated by Burke et al. (2002b). They concluded on the basis of the directionality of QTL that strong directional selection for increased achene size appears to have played a central role in sunflower domestication. None of the other traits showed similar evidence of selection. The occurrence of the numerous wild alleles with cultivated-like effects, combined with the lack of major QTL, suggests that sunflower was readily domesticated. There is less agreement about the original geographic range of wild H. annuus or the identity of the form from which H. annuus is derived (Asch, 1993). Asch (1993) suggests that H. annuus originated as a colonizer of natural disturbances, and that bison (Bison bison Skinner and Kaiser) created extensively disturbed habitats suitable for colonization by sunflower. Bison may also have served as a dispersal agent for sunflower, by transporting sunflower achenes trapped in matted hair. He also suggested that predomestication events generated a wide distribution for wild H. annuus throughout the Midwest prior to the arrival of mankind on the scene, and that it was this midwestern form of H. annuus that actually gave rise to the domesticated sunflower. Unfortunately, no archaeological evidence exists to either refute or support such hypotheses, and archaeological records tell us nothing about the prehuman geographic distribution of wild H. annuus. The domesticated sunflower was introduced to Europe in the early 16th century, perhaps initially by a Spanish expedition in 1510 (Putt, 1997). Sunflower quickly spread throughout Europe, where it was cultivated as a novelty or as ornamental plants. Only upon the introduction of the domesticated sunflower into the FSU in the 18th century was its potential as an oilseed crop recognized. Active selection for high seed-oil content began in 1860 (Heiser, 1976). Sunflower historians generally concur that the present cultivated sunflower in North America comes from materials reintroduced from the FSU. Most references indicate the date as the latter part of the 19th century. By 1880, the Mammoth Russian cultivar was available from seed companies in the U.S. (Beard, 1981). Another highly likely route of reintroduction of sunflower from the FSU to North America was via immigrants bringing small quantities of seeds with them. Mennonites who settled in Canada about 1875, immigrating from the FSU, brought sunflower seed with them for roasting and eating whole (Putt, 1997).

5.4 TAXONOMY AND GERMPLASM RESOURCES 5.4.1

Taxonomy and Center of Diversity

The identification of sunflower species has long been problematic. Heiser et al. (1969) concluded that the greatest contribution of sustained efforts to understand sunflower taxonomy was

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not to provide an easy way to identify sunflower species, but rather to provide explanations for why distinguishing species is so difficult. The taxonomic complexity of the genus Helianthus stems from many different factors. Natural hybridization and introgression between many of the species result in morphological intergradation between otherwise distinct forms. Polyploidy in the perennial species also contributes to the complexity of species classification in Helianthus. This has led to various taxonomic treatments of the genus. There are still specimens, variously of hybrid origin or growing in unusual conditions or incompletely collected, that defy certain placement into a single species (Schilling, 2004). Since many of the species are wide-ranging geographically, they exhibit extensive phenotypic variation, which appears to include both heritable and nonheritable (environmental) components. Many species are also genetically quite variable, making rigorous identification and classification difficult. The genus Helianthus has been considered to comprise from as few as 10 species to more than 200. Linnaeus (1753) originally described nine species in the genus. Asa Gray (1889) recognized 42 species in North America. In the early 20th century, Watson (1929) accepted 108 species, 15 of them from South America. Heiser et al. (1969) recognized 14 annual species and 36 perennial species from North America in three sections and seven series, as well as 17 species from South America. Subsequently, Robinson (1979) transferred 20 perennial species of South American Helianthus to the genus Helianthopsis. The taxonomic classification of Helianthus by Anashchenko (1974, 1979) was a radical departure from all previous schemes. He recognized only 1 annual species, H. annuus (with 3 subspecies and 6 varieties), and only 9 perennial species with 13 subspecies. Schilling and Heiser (1981) proposed an infrageneric classification of Helianthus, using phenetics, cladistics, and biosystematic procedures, that places 49 species of Helianthus in four sections and six series (Table 5.1 and Table 5.2). The classification of Schilling and Heiser (1981) is presented herein with the following six modifications. First, the sectional name Atrorubens used by Anashchenko (1974) has taxonomic priority; thus, the section Divaricati E. Schilling and Heiser is replaced by section Atrorubens Anashchenko. Second, Helianthus exilis is recognized as a species, as opposed to an ecotype of H. bolanderi, due to recent information that has shown it to be morphologically and genetically distinct (Oliveri and Jain, 1977; Rieseberg et al., 1988; Jain et al., 1992). Third, the species name H. pauciflorus has priority over H. rigidus and is treated accordingly herein. Fourth, Viguiera porteri has been transferred to Helianthus porteri (Pruski, 1998; Schilling et al., 1998). Fifth, Helianthus verticillatus has recently been rediscovered and redescribed and is now recognized as a species (Matthews et al., 2002). Sixth, Helianthus niveus ssp. canescens has been transferred to Helianthus petiolaris ssp. canescens (Schilling, 2004). This brings the number of species to 51, with 14 annual and 37 perennial species (Table 5.1 and Table 5.2). 5.4.2

Germplasm Resources

5.4.2.1 Ex Situ Collections Genetic resources of a crop consist of the total pool of genetic variability that exists in the crop species or within species with which the crop plant is sexually compatible (Holden et al., 1993). For the domesticated sunflower, this includes most species of Helianthus. Sunflower germplasm resources can be categorized as in situ resources (i.e., wild populations and landraces) or ex situ resources (accessions preserved in seed banks). The genus Helianthus, besides constituting the basic genetic stock from which cultivated sunflower originated, continues to contribute specific characteristics for cultivated sunflower improvement. However, there is a continued need to collect, maintain, evaluate, and enhance wild Helianthus germplasm for future utilization in cultivated sunflower. The genetic diversity of the wild species can make a significant contribution to sunflower in developing countries by providing genes for resistance (tolerance) to pests and environmental stresses, allowing the crop to become and remain economically viable.

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Table 5.1 Infrageneric Classification of Annual Helianthus Species Sectiona

Species

Helianthus

H. annuus L. H. anomalus Blake H. argophyllus T. and G. H. bolanderi A. Gray H. debilis ssp. debilis Nutt. ssp. cucumerifolius (T. and G.) Heiser ssp. silvestris Heiser ssp. tardiflorus Heiser ssp. vestitus (Watson) Heiser H. deserticola Heiser H. exilis A. Gray H. neglectus Heiser H. niveus ssp. niveus (Benth.) Brandegee ssp. tephrodes (Gray) Heiser H. paradoxus Heiser H. petiolaris ssp. canescens (A. Gray) E.E. Schilling ssp. fallax Heiser ssp. petiolaris H. praecox ssp. hirtus Heiser ssp. praecox Englm. and A.Gray ssp. runyonii Heiser H. agrestis Pollard H. porteri (A. Gray) J. F. Pruski

Agrestes Porteri a

Common Name

Chromosome Number (n)

Prairie Anomalous Silver-leaf Bolander’s, serpentine

17 17 17 17

Beach Cucumber- leaf Forest Slow-flowering Clothed Desert Serpentine Neglected

17 17 17 17 17 17 17 17

Snowy Ash-colored, dune Pecos, puzzle, paradox

17 17 17

Gray

17

Deceptive Prairie

17 17

Texas Texas Runyon’s Rural, southeastern Confederate daisy, porter’s

17 17 17 17 17

Schilling and Heiser, 1981; Schilling, 2004.

The U.S. National Plant Germplasm System (NPGS) sunflower collection is maintained at the North Central Regional Plant Introduction Station (NCRPIS) in Ames, IA. Sunflower comprises about 8% of the total accessions held at the NCRPIS at Ames. The collection contains 37 perennial species, 14 annual species, and the cultivated species, Helianthus annuus. Although sunflower originated in North America and is well distributed across North America, a number of Helianthus species have restricted ranges. Nonetheless, since the present germplasm collection does not contain representative genetic variability of the genus Helianthus, additional populations of several species, particularly those that rare, endangered, or threatened, need to be collected. The history of exploration for wild sunflower species in the U.S. and Canada has been reviewed by Seiler and Gulya (2004). The NPGS sunflower collection is a diverse assemblage of 3860 accessions from 59 countries: 1670 cultivated H. annuus accessions (43%), 1006 wild H. annuus accessions (26%), 430 accessions representing 11 other wild annual Helianthus species (11%), and 754 accessions representing 37 perennial Helianthus species (20%). This collection is one of the largest and most genetically diverse ex situ sunflower collections in the world, and it is vital to the conservation of Helianthus germplasm. The mission of the NCRPIS is to conserve genetically diverse crop germplasm and associated information, to conduct germplasm-related research, and to encourage the use of germplasm and associated information for research, crop improvement, and product development. In 2003, 1485 items representing 1007 different accessions, 26% of the accessions in the collection, were distributed. More than half of the distributed items (58%) were wild Helianthus accessions. Sixteen percent of the distributed items were sent to researchers outside the U.S. (Marek et al., 2004).

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Table 5.2 Infrageneric Classification of Perennial Helianthus Species Sectiona Ciliares

Seriesa Ciliares

Ciliares

Pumili

Atrorubens

Corona-solis

Atrorubens

Microcephali

Atrorubens

Atrorubentes

Atrorubens

a

Angustifolii

Species H. arizonensis R. Jackson H. ciliaris DC. H. laciniatus A. Gray H. cusickii A. Gray H. gracilentus A. Gray H. pumilus Nutt. H. californicus DC. H. decapetalus L. H. divaricatus L. H. eggertii Small H. giganteus L. H. grosseserratus Martens H. hirsutus Raf. H. maximiliani Schrader H. mollis Lam. H. nuttallii ssp. nuttallii T. and G. H. nuttallii ssp. rydbergii (Brit.) Long H. resinosus Small H. salicifolius Dietr. H. schweinitzii T. and G. H. strumosus L. H. tuberosus L. H. glaucophyllus Smith H. laevigatus T. and G. H. microcephalus T. and G. H. smithii Heiser H. atrorubens L. H. occidentalis ssp. occidentalis Riddell H. occidentalis ssp. plantagineus (T. & G.) Heiser H. pauciflorus ssp. pauciflorus H. pauciflorus ssp. subrhomboides (Rydb.) O. Spring H. silphioides Nutt. H. angustifolius L. H. carnosus Small H. floridanus A. Gray ex. Chapman H. heterophyllus Nutt. H. longifolius Pursh H. radula (Pursh) T. and G. H. simulans E.E. Wats. H. verticillatus Small

Common Name Arizona Texas blueweed Alkali Cusick’s Slender Dwarfish California Ten-petal Divergent Eggert’s Giant Sawtooth Hairy Maximilian Soft, ashy Nuttall’s Rydberg’s Resinous Willowleaf Schweinitz’s Swollen, woodland Jerusalem artichoke Whiteleaf Smooth Small-headed Smith’s Purple-disk

Chromosome Number (n) 17 34, 51 17 17 17 17 51 17, 34 17 51 17 17 34 17 17 17 17 51 17 51 34, 51 51 17 34 17 17, 34 17

Fewleaf, western

17

Fewleaf, western

17

Stiff

51

Stiff Odorous Narrowleaf, Swamp Fleshy Florida Variable leaf Longleaf Scraper, rayless Muck, imitative Whorled

51 17 17 17 17 17 17 17 17 17

Schilling and Heiser, 1981; Schilling, 2004.

From 1976 to 1996, 10,000 samples of wild sunflower were distributed to 300 researchers in 30 countries. These accessions have become the basis of wild species research programs in Argentina, France, Italy, Spain, Germany, Bulgaria, Romania, Czechoslovakia, Hungary, Russia, Yugoslavia, India, China, and Mexico. Notable is the collection at the Institute of Field and Vegetable Crops, Novi Sad, Serbia and Montenegro, which contains 39 of the 51 wild species (IBPGR, 1984;

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Cuk and Seiler, 1985). The wild species collection of the Dobroudja Agricultural Institute (DAI) at General Toshevo, Bulgaria, is also notable, containing 428 accessions representing 37 of the 51 species of Helianthus (Christov et al., 2001). The wild species collection maintained at INRA, Montpellier, France, has more than 600 accessions of 45 of the 51 wild sunflower species (Serieys, 1992). The Institudo de Agricultura Sostenible (CSIC), Cordoba, Spain, maintains 44 annual and perennial accessions of Helianthus (Ruso et al., 1996). Preservation of genetic diversity within wild sunflower populations in their native habitat is critical because we lack the necessary resources to preserve locally adapted sunflower populations of all wild species in gene banks. Unfortunately, the long-term outlook for survival of many sunflower species is not promising; some species already are rare and endangered or, in the case of H. nuttallii subsp. parishii, probably extinct. The U.S. Department of Interior, Fish and Wildlife Service, has listed several species as endangered and threatened under the criteria of the Endangered Species Act of 1973. The threatened species include the annual H. paradoxus (Pecos, puzzle, paradox sunflower) and perennial H. eggertii (Eggert’s sunflower), while H. schweinitzii (Schweinitz’s sunflower) is listed as endangered. Other taxa that are rare and should be given special attention include the annuals H. anomalus, H. deserticola, H. exilis, and H. niveus subsp. tephrodes, as well as the perennials H. laevigatus, H. carnosus, and H. smithii. Helianthus verticillatus (whorled sunflower) is currently listed as a candidate species for federal protection. The primary obstacle for long-term preservation of wild sunflower populations is human activity. For example, the marshy habitat of H. nuttallii subsp. parishii in southern California has been completely eliminated and replaced by urban development. Also, the widening of highways and their rights-of-way in Texas has impacted populations of H. paradoxus and H. praecox subsp. hirtus, and mining activities in California have destroyed several populations of H. exilis. In addition to the extinction of populations by development, their disturbance by humans can lead to hybridization between widespread species and the resulting recent introduction of more widespread congeners (Rieseberg, 1991). Not only are the hybrid plants likely to be less fit than locally adapted populations, but populations of rare species may be genetically “swamped” out of existence by populations of the numerically larger introduced species. It is noteworthy that the common sunflower, H. annuus, occurs sympatrically and hybridizes with several rare annual sunflowers (e.g., H. paradoxus, H. anomalus, and H. deserticola), possibly posing a threat to their existence or genetic integrity. Additional potential threats to the preservation of rare sunflower populations include their small population sizes and subsequent loss of genetic diversity. Isozyme analyses of populations of annual sunflowers revealed a strong positive correlation between genetic diversity and geographic range (Rieseberg et al., 1991). In fact, 8 of the 11 narrow endemics had less genetic diversity than their more widespread congeners. In particular, very low levels of genetic diversity were observed for H. paradoxus, H. deserticola, H. debilis subsp. tardiflorus, and H. debilis subsp. vestitus. Although these values may be cause for concern, it should be pointed out that relationships among genetic diversity, fitness, and evolutionary potential are not yet well understood. 5.4.2.2 Wild and Weedy Relatives Wild relatives of crop plants typically are genetically much more diverse than related cultivated lineages. Genetic diversity is thought to contribute to long-term preservation of species by allowing them to adapt quickly to changes in their environment. Diversity in germplasm is also critical to crop breeding programs, but to date it has not been fully exploited (Harlan, 1976). Although many germplasm introductions appear to have no immediate use in breeding and genetic programs (Burton, 1979), they may contain unidentified genes that will protect crops against new pests in the future. Thus, if new pests or environmental stresses vary beyond normal limits of tolerance, productivity decreases and a search for germplasm with greater resistance to the stresses is initiated. Hopefully, the present germplasm collection will contain the necessary germplasm. Although we cannot predict with acceptable levels of confidence the occurrence, severity, or even the nature of

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future stresses, germplasms with as wide a range of genetic diversity as possible must be developed for breeding programs (Jones, 1983). Collection of germplasm not only serves a valuable purpose in preserving germplasm, but also provides valuable information about the diverse habitats occupied by wild sunflowers and associated species. This information is particularly important for the genus Helianthus because of the coevolution of its species and associated native insects and pathogens. Knowledge of a particular habitat and adaptations of a species occurring therein can often help to identify potential sources of genes for a desired trait. Based on the habitat of a species and its immediate environment, selection of potential species for a particular characteristic may become easier, more accurate, and more efficient. A widely acknowledged risk associated with transgenic crops is the possibility that hybridization with wild relatives will transfer fitness-related transgenes to persist in wild populations (Armstrong et al., 2005). If wild populations acquire transgenes for resistance to diseases, herbivory, environmental stress, or commonly used herbicides, they could become more abundant in their natural habits or invade previously unsuitable habitats. Since wild sunflower species are native to the major sunflower production areas of North America, there is a concern about the flow of genes from the cultivated crop to the wild species. Hybrids between cultivated and wild annual sunflower (H. annuus) are frequent. As high as 42% of progeny from wild plants near cultivated fields are hybrids, and cultivar genes have been shown to persist in the wild populations at least five generations, and in certain areas up to 40 years (Linder et al., 1998). Moreover, there was morphological evidence of hybridization in 10 to 33% of the populations surveyed within a given year. Burke et al. (2002a) indicate that the opportunity for crop–wild hybridization exists throughout the range of sunflower cultivation, where approximately two thirds of all cultivated fields surveyed occurred in close proximity to, and flowered coincidentally with, common sunflower populations. In these populations, the phenological overlap was extensive, with 52 to 96% of all wilds flowering coincidentally with the adjacent cultivar field. These findings indicate that crop–wild hybridization is likely in all areas where sunflowers are cultivated in the U.S. Crop-to-wild gene flow with species other than H. annuus is far less likely due to infertility barriers and nonoverlapping ranges. Although transgenes will often escape from cultivation, their rate of spread will be mainly governed by their fitness effects, not the migration rate. Thus, only highly advantageous transgenes will spread rapidly enough to have a substantial ecological impact. Therefore, research on the risks associated with transgene escape should focus on the fitness effects of the genes in question. Snow et al. (2003) concluded that wild sunflower plants containing a Bt-toxin gene (cry1Ac) specific to lepidopterans exhibited decreased lepidopteran herbivory and produced on average 55% more seeds than nontransgenic controls. Burke and Rieseberg (2003) examined the fitness effects of a transgene, an oxalate oxidase (OXOX) gene conferring resistance to Sclerotinia white mold. They concluded that the OXOX transgene will do little more than diffuse neutrally after its escape. Wild–crop hybridization has the potential to influence the evolutionary ecology of related wild/weed taxa such as sunflower, but little is known about the persistence or ecological effects of crop genes that enter wild populations via pollen movement. Snow et al. (1998) studied F1 wild–crop hybrids of sunflower and observed that F1 wild–crop hybrids had lower fitness than wild genotypes, especially when grown under favorable crop conditions, but the F1 barrier to the introgression of crop genes is quite permeable. High rates of hybridization and introgression have been reported between the cultivated sunflower and its wild progenitor, H. annuus. However, little consideration has been given to the possibility that other wild sunflower species may hybridize with cultivated sunflower. A closely related wild progenitor, H. petiolaris, was studied by Rieseberg et al. (1999) using selectable AFLP markers. They examined four sympatric populations of H. petiolaris and found a low rate of introgression ranging from 0.006 to 0.026, indicating that the H. petiolaris genome is differentially permeable to introgression and that escape is likely to be sporadic, occurring in some populations and not others, at different times. Thus, the risk assessment of wild H. annuus is of more immediate concern than that of H. petiolaris.

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Gene flow between crops and wild relatives has occurred for many years and contributed to the evolution and extinction of weed species. Resistance to imidazolinone (IMI) herbicides has been recently introduced in domesticated sunflower (Al-Khatib and Miller, 2000; Miller and Al-Khatib, 2002). Massinga et al. (2003) studied the gene flow of the IMI gene from domesticated sunflower to wild sunflower, concluding that domesticated sunflower outcrosses with common (H. annuus) and prairie (H. petiolaris) sunflower over distances typically encountered in the major sunflower production areas, and that backcross-resistant hybrids with wild parents are successful, further increasing the potential of spread of IMI-resistant feral sunflowers. 5.4.2.3 Core Collection Frequently, researchers are uncertain about the criteria or information needed to select germplasm materials needed for their specific research objectives. The assembly of a core subset of the cultivated sunflower collection may provide an efficient means of identifying useful genetic traits. This will enable researchers to sample the available diversity within the collection without testing excessively large numbers of accessions. A core subset of the cultivated sunflower collection was established by Brothers and Miller (1999). Twenty descriptors were used in the construction of the core subset. The sunflower core subset consists of 112 accessions (approximately 7% of the total available accessions) grouped into 10 clusters. Accessions within the same cluster should be more genetically similar than accessions between clusters. Accessions in the core subset represent 38 of the 57 countries of origin for the total cultivated sunflower collection. The core subset contains 2 ornamental accessions, 7 breeding lines, 12 landraces, and 91 cultivars. Researchers may initially use the core collection to determine traits of interest and, pending the results of their research, request additional accessions from one or more clusters to explore in depth at a later date. 5.4.2.4 Genetic Stocks There are 29 sunflower genetic stocks registered by the Crop Science Society of America. The first of these is a tetraploid (4x) of ‘Peredovik 21’ (Jan, 1992a). There are also four nuclear malesterile lines of HA 89 created by chemical mutation (Jan, 1992b). Two other nuclear male-sterile lines with markers for sterile and fertile plants were developed by Miller (1997). A series of altered saturated fatty acid genetic stocks developed by Miller and Vick (1999) contain low palmitic and low stearic acids in RHA 274, HA 821, and HA 382 backgrounds. Mid-range oleic acid (650 g/kg) lines have also been developed (Miller et al., 2002). Two other lines with reduced palmitic and stearic fatty acids have been developed (Vick et al., 2003). A series of herbicide-resistant genetic stocks have been developed. They include four genetic stocks having resistance to imazethapyr and imazamox (Miller and Al-Khatib, 2002) and two sulfonylurea (tribenuron)-tolerant stocks, SURES-1 and SURES-2 (Miller and Al-Khatib, 2004). A dwarf parental line near isogenic to RHA 271 (about half the height of RHA 271) has also been developed, and a dwarf parental line near isogenic to HA 89 (about one third the height of HA 89) has been developed by Velasco et al. (2003a). 5.4.2.5 Germplasm Evaluation and Use 5.4.2.5.1 Pathogens Wild sunflower species have been a valuable source of resistance genes for many of the common pathogens of cultivated sunflower. Helianthus annuus, H. petiolaris, and H. praecox are the major sources of genes for Verticillium wilt (Verticillium dahliae Kleb.) resistance (Hoes et al., 1973). These species plus H. argophyllus are also the major sources of resistance genes for downy mildew (Plasmopara halstedii (Farl.) Berl and deToni) and rust (Puccinia helianthi Schwein) in cultivated sunflower. Resistance genes for these pathogens occur frequently in the wild annual species

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(Tan et al., 1992; Quresh et al., 1993). Resistance to broomrape (Orobanche cernua Loefl.) has been observed in most of the wild perennial species (Fernández-Martínez et al., 2000). Early reports of broomrape resistance were from the FSU, where they developed cultivars Progress and Novinka using the “group immunity” breeding approach (Pustovoit and Gubin, 1974). Phoma black stem (Phoma macdonaldii Boerma) resistance has been reported in several perennial species: H. decapetalus, H. eggertii, H. hirsutus, H. resinosus, and H. tuberosus (Skoric, 1985). Phomopsis stem canker (Phomopsis helianthi Munt-Cvet. et al.) resistance has been found in perennials H. maximiliani, H. pauciflorus, H. hirsutus, H. resinosus, H. mollis, and H. tuberosus (Skoric, 1985; Dozet, 1990). Alternaria leaf spot (Alternaria helianthi (Hansf.) Tubaki and Nishihara) resistance was observed in perennials H. hirsutus, H. pauciflorus, and H. tuberosus (Morris et al., 1983). Rhizopus head rot (Rhizopus arrhizus Fischer) resistance was observed in perennials H. divaricatus, H. hirsutus, H. resinosus, and H. × laetiflorus (Yang et al., 1980). Powdery mildew (Erysiphe cichoracearum DC. ex. Meret) resistance was observed in annuals H. debilis subsp. debilis, H. bolanderi, and H. praecox (Saliman et al., 1982; Jan and Chandler, 1985). Sclerotinia (Sclerotinia sclerotiorum (Lib.) de Bary) head rot tolerance was observed in perennials H. resinosus, H. tuberosus, H. decapetalus, H. grosseserratus, H. nuttallii, and H. pauciflorus (Pustovoit and Gubin, 1974; Mondolot-Cosson and Andary, 1994; Ronicke et al., 2004). Sclerotinia root rot tolerance was observed in perennials H. mollis, H. nuttallii, H. resinosus, and H. tuberosus (Skoric, 1987). Sclerotinia mid-stalk rot tolerance was observed in annual H. praecox and perennials H. pauciflorus, H. giganteus, H. maximiliani, H. resinosus, and H. tuberosus (Skoric, 1987). 5.4.2.5.2 Insects Wild sunflowers are native to North America, where their associated insect herbivores and entomophages coevolved in natural communities. This provides the opportunity to search for insect resistance genes in the diverse wild species. Sunflower moth (Homoeosoma electellum (Hulst)) tolerance was observed in annual H. petiolaris and perennials H. maximiliani, H. ciliaris, H. strumosus, and H. tuberosus (Rogers et al., 1984). Stem weevil (Cylindrocopturus adspersus (LeConte)) tolerance was found in perennials H. grosseserratus, H. hirsutus, H. maximiliani, H. pauciflorus, H. salicifolius, and H. tuberosus (Rogers and Seiler, 1985). Sunflower beetle (Zygogramma exclamationis (Fabricius)) tolerance was observed in annuals H. agrestis and H. praecox and perennials H. grosseserratus, H. pauciflorus, H. salicifolius, and H. tuberosus (Rogers and Thompson, 1978, 1980). 5.4.2.5.3 Oil and Oil Quality Variability for oil concentration exists in the wild species. While oil concentration is lower in the wild species than in cultivated sunflower, backcrossing to cultivated lines quickly raises the oil concentration to an acceptable level. Annual H. anomalus has the highest oil concentration of 460 g/kg, the highest ever observed in a wild sunflower species, followed by H. niveus ssp. canescens with 402 g/kg, H. petiolaris with 377 g/kg, and H. deserticola with 343 g/kg. Perennial H. salicifolius had a concentration of 370 g/kg (Seiler, 1985; Seiler and Brothers, 2003). Cultivated sunflower generally contains 450 to 470 g/kg. Reduced concentrations of saturated palmitic and stearic fatty acids have been observed in a population of wild H. annuus, which had a combined palmitic and stearic acid concentration of 58 g/kg (Seiler, 1998). This is 50% lower than in oil of cultivated sunflower. A combined palmitic and stearic acid concentration of 65 g/kg was observed in a wild perennial species, H. giganteus (Seiler, 1998). 5.4.2.5.4 Protein The wild sunflower species may have potential to increase the protein content of seeds of the cultivated sunflower (Laferriere, 1986). Protein concentration of seeds is of interest for human or

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livestock consumption. Commercial sunflower meal has a protein concentration of approximately 440 g/kg (dehulled) and 280 g/kg (whole seed) (Doty, 1978). Protein concentrations ranging from 290 to 350 g/kg have been reported in a survey of 39 species of wild Helianthus (Pustovoit and Krasnokutskaya, 1975). Crude protein in whole seeds of perennial H. nuttallii ssp. nuttallii was 348 g/kg, while protein averaged 180 g/kg in wild H. annuus (Seiler, 1984). In other wild Helianthus species (6 perennials and 19 annual accessions) whole seed crude protein varied from 137 g/kg in H. neglectus to 305 g/kg in H. porteri (Seiler, 1986). The crude protein of whole seeds of annual species appears in general to be lower than that of perennial species (Seiler, 1984). Laferriere (1986) suggested that it is possible that the high protein concentration of seeds of wild species may be due to their smaller size. Sufficient variability for seed protein exists in the wild Helianthus species and seems to be useful in breeding programs with an objective to increase seed protein concentration.

5.5 CYTOGENETICS Cytogenetic studies of sunflower (H. annuus L.) and its interspecific hybrids have provided information about species relationships, improved the process of interspecific hybridization and gene transfer, and led to the production of valuable germplasm for the improvement of cultivated sunflower. Improved embryo culturing procedures enhanced interspecific hybridization for difficult crosses. Successful chromosome doubling of F1 interspecific hybrids restored fertility and facilitated amphiploid production. Progeny of chromosomally doubled cultivated lines resulted in autotetraploid, which led to the production of triploids, aneuploids, and trisomics after successive backcrosses. Over 70 cytoplasmic male-sterile (CMS) sources have been derived from wild Helianthus species and their restoration genes characterized. Mutagen-induced CMS and nuclear male-sterile (NMS) lines have been characterized. Genome designations are proposed based on cytological and molecular findings. Improved sunflower tissue culture methodology successfully produced haploid plants from wild Helianthus species, interspecific hybrids, and inbred lines, facilitating protoplast fusion, gene transfer, and sunflower transformation. In this section, sunflower genomes are discussed using DNA content, mitotic metaphase karyotype, and variation in chromosome numbers. Microsporogenesis is discussed in male-fertile, male-sterile, and interspecific hybrids. NMS, CMS, and fertility restoration genes are discussed. Interspecific hybridization between wild Helianthus species and cultivated lines is emphasized. Interspecific relationships are discussed utilizing both classical cytogenetics and molecular marker polymorphism. 5.5.1

Helianthus Genomes

5.5.1.1 Euploidy and Aneuploidy The genus Helianthus has a basic chromosome number of n = 17 and contains diploid (2n = 2x = 34), tetraploid (2n = 4x = 68), and hexaploid (2n = 6x = 102) species. Species classification of Schilling and Heiser (1981) classified Helianthus into 49 species, with 4 sections and 6 series, based on the measurements of 42 characters and interspecific F1 fertility. Based on this classification, the 13 annual species are diploid, and the 36 perennial species include 26 diploid, 3 tetraploid, 6 hexaploid, and 3 mixoploid species. H. ciliaris and H. strumosus have both tetraploid and hexaploid forms, while H. decapetalus contains diploid and tetraploid forms. Aneuploids can be the results of interspecific hybridization. Leclercq et al. (1970) obtained trisomic plants (2n + 1) in backcross progeny of H. tuberosus × H. annuus hybrids. Such progeny were resistant to downy mildew (Plasmopara halstedii), and they postulated that the extra chromosome came from the H. tuberosus genome.

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Aneuploids can be identified from progeny by their twin embryos. Gundaev (1971) suggests that twin embryos may have an intermediate number of chromosomes between the haploid and diploid number, and a continuous aneuploid series could be developed from such material. The maximum frequency of aneuploids was found in the group with 2n = 28. Many of the embryos studied displayed a chimeral structure with cells having 24 to 28 chromosomes. Trisomic plants were obtained after backcrossing interspecific hybrids H. petiolaris × H. annuus (Whelan, 1979) and H. maximiliani × H. annuus (Whelan and Dorrell, 1980; Whelan, 1982) with cultivated H. annuus. Multivalents were frequently observed in these F1 hybrids, indicating reciprocal translocation heterozygosity. The trisomics presumably originated from unequal disjunction of multivalents. Most trisomics were normal in appearance, while some had distinct morphological features. Jan et al. (1988) produced tetraploids, triploids, and aneuploids using the inbred line P21. Meiotic chromosome pairing of the autotetraploid P21 was reasonably normal with 28.16 bivalents, 0.85 univalent, and a small number of multivalents. Triploids were obtained by crossing tetraploids with diploid P21, and aneuploids by crossing triploids with the diploids (Table 5.3). The chromosome numbers of the 137 plants from reciprocal triploid × diploid crosses ranged from 2n = 34 to 2n = 47 + t. In general, plants with lower chromosome numbers were more prevalent when triploids were used as the pollen parents, while plants with higher chromosome numbers increased when triploids were used as the seed-bearing parent. These results suggest the effective transmission of extra chromosomes through male and female gametes, with the female gametes being better than the male gametes. However, for a rapid trisomic production requiring fast chromosome reduction, triploids need to be used as the pollen parents. These results suggest that diploid sunflower tolerates extra chromosomes well and a set of trisomic genetic stocks is possible. Pollen stainability, an indicator of viability, was above 90% for plants with 2n = 34 to 2n = 37. As chromosome numbers increased from 2n = 38 to 2n = 45 + t, pollen stainability decreased to about 40%, but an acceptable seed set level was still produced. A total of over 60 trisomic P21 plants with 2n = 35 have been obtained. The characterization of these trisomics is in progress, and the identity of the extra chromosome will be determined utilizing linkage group-specific restriction fragment length polymorphism (RFLP) probes and recently produced bacterial artificial chromosome (BAC) clones (Jan, unpublished data). In addition, a second set of trisomics from inbred line HA 89 is produced and will be characterized following the P21 trisomics. 5.5.1.2 DNA Content Studies of the DNA content of 22 diploid Helianthus species and subspecies indicated that 2C DNA increased continuously from 6.4 pg in H. neglectus to 12.02 pg in H. angustifolius, with an average increment of approximately 0.26 pg (Sims and Price, 1985). The DNA content of H. divaricatus (Div 115) was much higher, with 16.90 pg. The chromosome number and identification of accession Div 1115 was later determined to be H. hirsutus with 2n = 68, a tetraploid, which would explain the higher DNA content (Jan, unpublished data). However, it is still unknown why the annual diploid, H. agrestis with 25.91 pg, would have a DNA content as high as that of a hexaploid. More similar DNA contents were observed for closely related species than for more distantly related species, indicating little intraspecific variation. There were also indications that DNA content was related to chromosome size, which is clearly shown between the metaphase roottip chromosome preparations of H. agrestis and HA 89. Among 13 cultivated varieties and inbred lines, Michaelson et al. (1991) reported DNA amounts ranging from 6.01 to 7.95 pg. Using similar techniques, the 2C DNA contents of H. annuus lines were reported to be 4.9 pg (Bennett et al., 1982), 6.6 pg (Anderson et al., 1985), 8.8 pg (Ingle et al., 1975), and 6.1, 8.9, and 9.9 pg (Nagl and Capesius, 1976). Due to the lack of common standard checks, it is difficult to compare absolute amounts. Price et al. (2000) estimated a 2C DNA

44 100 98

1

201 × P21 202 × P21 205 × P21

No. of surviving plants Plant survival (%) Pollen stainability (%)

15 28

34

P21 × 202 P21 × 205

Cross

2 100 97

2

34+

30 97 98

4

26 1

35

22 96 91

4

19

36

10 83 91

5 1

4 2

37

8 57 68

2 3 8

1

38

3 50 68

1 5

39

2 40

5

40

0 0

1 2 18

10

1

0 0

1

Chromosome Number 41 41 + t 40 + t

9 75 45

10

1

1

42

3 38 34

8

43

1 100 44

1

44

0 0

2

44 + t

6 100 37

3

1

2

45

1 100 54

1

45 + t

1 100

1

47 + t

120

Table 5.3 Number of Plants with Different Chromosome Numbers, Plant Survival, and Pollen Stainability in the Progeny from Reciprocal Crosses between Triploid and Diploid P21 Plants (H. annuus)

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content of 7.3 pg for H. annuus, equivalent to twice the amount of the haploid sunflower genome of 3000 Mb (Arumuganathan and Earle, 1991). They also cautioned that sunflower leaves contain compounds that reduce nuclear propidium iodide fluorescence, which may partly explain the DNA variation. 5.5.1.3 Karyotype Classical karyotype describes each of the haploid chromosome sets of an organism based on features such as length of chromosome, ratio of arm lengths, position of centromere and secondary constrictions, and size and position of heterochromatic knobs. Karyotype analysis has provided useful markers in chromosome identification and the designation of chromosomes in many plant species. Most karyotypes of Helianthus species have been conducted with mitotic metaphase chromosome preparations in the 1970s and 1980s. Based on Levan et al. (1964), classification of chromosomes as median (m), submedian (sm), subterminal (st), or terminal (t) when the long/short arm ratio was in the range of 1.0 to 1.7, 1.7 to 3.0, 3.0 to 7.0, and 7.0 μ, respectively, was followed in all the literature cited. A summary of karyotype studies conducted on H. annuus and H. debilis (Raicu et al., 1976), H. mollis (Georgieva-Todorova et al., 1974), H. salicifolius (Georgieva-Todorova and Lakova, 1978), hybrids of H. annuus × H. hirsutus (Georgieva-Todorova and Bohorova, 1980), H. hirsutus and H. decapetalus (Georgieva-Todorova and Bohorova, 1979), cultivated H. annuus (Al-Allaf and Godward, 1977), and 12 Helianthus species (Kulshreshtha and Gupta, 1981) is shown in Table 5.4. In general, Georgieva-Todorova and Bohorova reported large chromosome sizes, with more chromosomes classified as m and sm, and two to four satellited chromosomes. On the contrary, Kulshreshtha and Gupta (1981) reported smaller chromosome sizes, chromosome types equally distributed among m, sm, and st, and only one or two satellited chromosomes. The large variation in chromosome condensation among studies of different authors was probably due to the differences in chromosome preparation. Perhaps the use of common cultivated H. annuus as a check will help resolve some of the problems when comparing total chromosome length of different genomes. Closely related tetraploid species H. decapetalus and H. hirsutus had remarkable similarity regarding most features of their karyotypes (Georgieva-Todorova and Lakova, 1978). The 2n = 30 H. mollis studied by Kulshreshtha and Gupta (1981) was much less than the basic set of 2n = 34 and was presumably from roots of a nonviable plant. Also, H. californicus is expected to be hexaploid with 2n = 102. H. californicus with 2n = 34 may have been a misidentified species. Speciation of Helianthus was shown to have involved chromosome exchanges such as translocation and inversion (Chandler et al., 1986), as well as the obvious polyploidization for the tetraploid and hexaploid species. We would expect a continuous variation in DNA and total chromosome length in diploid species, and multiples of single or combinations of the diploids for tetraploids and hexaploids. Characteristic karyotypes for individual Helianthus species were shown to be useful for studying species relationships and tracing evolutionary changes. However, due to the relatively small size and large number of Helianthus chromosomes, it is extremely difficult to distinguish similar chromosomes using only the mitotic metaphase. Other useful techniques such as karyotyping using meiotic pachytene chromosomes and the C- or N-banding techniques, need to be evaluated for Helianthus species. Pachytene chromosome karyotyping has played a major role in identifying individual trisomics for maize (Zea mays L.) (Rhoades and McClintock, 1935), tomatoes (Lycopersicon esculentum Mill.) (Rick and Barton, 1954; Rick et al., 1964), and rice (Oryza sativa L.) (Khush et al., 1984). The use of the the Giemsa C- and N-banding techniques enabled Singh and Tsuchiya (1981a, 1981b, 1982a, 1982b) to successfully clarify the identification of a whole set of barley (Hordeum vulgare L.) trisomics. Zeller et al. (1977) used C-banding to help identify six of the seven possible trisomics of rye (Secale cereale L.).

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Table 5.4 Karyotype Comparisons of Helianthus Species

Species

Chromosome Length (µm) Range Total

Chromosome Type Total sat m sm

Arm Ratio st

References

H. annuus (cultivar Record) H. debilis H. annuus (Bulgarian) H. mollis

3.76–5.15

73.83

3

10

3

4

1.08–5.34

Raicu et al., 1976

5.69–7.91 Small

110.19 —

2 3

9 4

3 8

5 5

1.15–4.63 —

3.16–4.50

45.65

2

13

4

0

1.23–2.21

H. salicifolius

6.78–9.64

140.28

2

12

5

0

1.31–2.48

H. hirsutus

3.49–6.54

172.62

4

14

14

6

1.02–3.93

H. decapetalus

3.77–7.00

191.10

4

14

14

6

1.05–3.30

H. annuus

5.11–7.37

104.68

2

5

8

4

—

H. annuus × H. hirsutus, F1 H. hirsutus

3.65–6.66

131.47

3

8

14

4

—

3.82–6.54

172.03

4

14

14

6

—

H. angustifolius

1.5–3.9

41.1

1

6

6

5

1.00–2.22

H. annuus

1.7–4.8

51.9

1

5

10

2

1.00–1.21

H. argophyllus

1.7–4.1

49.5

1

9

4

4

1.00–2.88

H. californicus

1.0–5.0

49.5

1

7

5

5

1.00–3.25

H. debilis

2.0–52

55.2

1

7

6

4

1.00–1.90

H. divaricatus

2.2–4.9

52.0

1

9

3

5

1.00–3.25

H. lenticularis

2.4–5.8

58.8

1

6

7

4

1.00–2.77

H. maximiliani

1.1–3.8

37.1

1

5

6

6

1.00–3.33

H. mollis

1.9-4.6

42.8

1

5

4

6

1.00–4.50

H. pumilus

1.5–3.8

43.0

1

5

5

7

1.00–4.00

H. trachaelifolius

2.4–4.9

51.0

1

6

4

7

1.00–2.37

H. tuberosus

2.1–5.5

156.3

2

15

9

27

1.00–3.50

Raicu et al., 1976 Al-Allaf and Godward, 1977 Georgieva-Todorova et al., 1974 Georgieva-Todorova and Lakova, 1978 Georgieva-Todorova and Bohorova, 1979 Georgieva-Todorova and Bohorova, 1979 Georgieva-Todorova and Bohorova, 1980 Georgieva-Todorova and Bohorova, 1980 Georgieva-Todorova and Bohorova, 1980 Kulshreshtha and Gupta, 1981 Kulshreshtha and Gupta, 1981 Kulshreshtha and Gupta, 1981 Kulshreshtha and Gupta, 1981 Kulshreshtha and Gupta, 1981 Kulshreshtha and Gupta, 1981 Kulshreshtha and Gupta, 1981 Kulshreshtha and Gupta, 1981 Kulshreshtha and Gupta, 1981 Kulshreshtha and Gupta, 1981 Kulshreshtha and Gupta, 1981 Kulshreshtha and Gupta, 1981

5.5.2

Microsporogenesis

Helianthus species are suitable for meiotic studies using immature anthers. The disk flowers usually are numerous and develop sequentially from the periphery of the head toward the center. For the monocephalic cultivated types, a wedge containing most stages of meiosis can be carefully cut from a head using a sharp knife or scalpel without altering the normal development of the remainder of the head. Regardless of varying species or the size of head, anthers 1.5 to 2.0 mm in length are generally undergoing meiosis. Samples should be collected from 1100 to 1300 h on clear days. The time of day affects meiotic division, and clear days avoid chromosome clumping. The samples are immediately put in an ice-cold fresh Carnoy’s (6:3:1) solution for 24 h, followed by at least three changes of 70% ETOH before acetocarmin squashing. After fixing, samples can be stored in 70% ETOH in a refrigerator for several years.

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Table 5.5 Estimates of Minimum Chiasma Frequency per Cell for Several Helianthus Species and Interspecific Hybrids Female Parent H. mollis H. mollis H. mollis H. mollis H. mollis H. mollis H. occidentalis H. grosseserratus H. grosseserratus H. grosseserratus H. grosseserratus H. salicifolius H. divaricatus H. giganteus H. giganteus H. giganteus H. atrorubens H. annuus H. annuus H. argophyllus H. argophyllus H. decapetalus (4n) H. rigidus

Male Parent

H. divaricatus H. atrorubens H. occidentalis

H. mollis H. salicifolius

H. mollis

Pollen Number Chiasma Frequency Range Mean Fertility (%) of Cells 50 50 50 72 50 47 50 50 50 12 12 12 50 50 50 50 50 48

H. annuus 120

20–29 20–26 19–24 17–22 20–29 17–24 35–30 21–28 20–27

20–26 20–30 20–28 20–26 29–32 24–30

28.7 22.5 20.8 18.9 23.8 20.2 27.4 23.8 22.4 23.9 27.5 25.2 22.0 23.5 22.9 31.0 28.1 23.9 20.8 19.9 39.3 66.3

97.0 98.7 49.4 34.0 97.0 49.5 98.6 99.7 94.41

100.0 99.4 99.7 59.7 92.4 95.0

96.0 75.0

Source Jackson and Guard, 1957a Jackson and Guard, 1957a Jackson and Guard, 1957a Jackson and Guard, 1957a Jackson and Guard, 1957b Jackson and Guard, 1957b Jackson and Guard, 1957b Jackson and Guard, 1957a Jackson and Guard, 1957a Long, 1957 Long, 1957 Long, 1957 Jackson and Guard, 1957a Jackson and Guard, 1957a Jackson and Guard, 1957a Jackson and Guard, 1957a Jackson and Guard, 1957a Whelan, unpublished data Georgieva-Todorova, 1970 Georgieva-Todorova, 1970 Georgieva-Todorova, 1970 Georgieva-Todorova, 1974b Georgieva-Todorova, 1971

According to Whelan (1974), the first indication of the onset of meiosis is the development of the callose wall, an unbranched 1,3-linked glucan, surrounding each meiocyte during meiosis. In addition to the presence of the callose wall, the meiocytes develop a prominent nucleolus and the chromosomes are evident as chromatic threads throughout the cytoplasm. In zygotene, pairing of the chromosomes can be seen and synapsis appears to start near the centromere. Unfortunately, chromosomes tend to clump in both zygotene and pachytene, so individual chromosomes rarely can be identified. Similar behavior was also observed in H. rigidus (= pauciflorus) by GeorgievaTodorova (1971). In pachytene, the centromeres are clearly delimited on both sides by intensely stained chromatic regions of varying size. Numerous chromatic regions are also evident in the chromosome arms. Such features usually allow easy and accurate description of pachytene chromosomes. Improved techniques for reducing the clumping of pachytene chromosomes are greatly needed for future work on sunflower chromosome identification. Chromosome behavior in diplotene appears to be unusually complex and similar to that observed for tomato (Moens, 1964). The homologous chromosomes undergo a marked repulsion and chiasmata are frequently evident. The bivalents become relatively diffuse at this stage and may be incorrectly identified as zygotene. The clarity of the bivalents increases as diplotene progresses. Bivalent counts cannot be made or chiasma frequency estimated until late diplotene or diakinesis because of a tendency for several of the bivalents to remain clumped, giving the false impression that multivalents are present. A typical meiocyte in diakinesis contains 2- to 10-ring bivalents and 6- to 16-rod bivalents. Two of the bivalents are associated with the nucleolus. The observations on 163 such meiocytes of the cultivar P21 showed a mean chiasma frequency per cell of 21.8, with a range of 18.0 to 26.0 (Jan, unpublished). Georgieva-Todorova (1970) estimated 23.9 chiasmata in the cultivar Peredovik. Estimates for other Helianthus species are summarized in Table 5.5. By metaphase I, the nucleolus, which has been evident throughout the meiotic prophase, has disappeared and the intensely stained, highly contracted bivalents have become oriented on the

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metaphase plate. Sequential, not simultaneous, disjunction of the bivalents occurs in anaphase I. One to three of the bivalents, usually the rod bivalents, rapidly undergo disjunction, and the resulting chromatids begin moving to the poles. Three of the bivalents frequently can be seen undergoing disjunction after the other chromatids have migrated to the poles. Some meiotic abnormalities such as bridges and fragments or lagging chromosomes may occur in fully fertile cultivars, which are believed to be the result of spontaneous breakage and exchange, rather than the presence of a paracentric inversion (Lewis and John, 1966). During telophase, a nucleolus forms and the chromatids lose their regular structure, and by interphase, chromosome counts are no longer possible. In metaphase II, the chromatids again can be counted, and in those with median centromeres, the four arms of the diad frequently are evident. In anaphase II, disjunction of the chromatids occurs and four groups develop with 17 chromatids in each group. The chromatids lose their regular structure as the nucleoli develop. Cytokinesis of the simultaneous type occurs at the end of the second meiotic division, peripheral constrictions develop, and walls form from these inward constrictions. Normally, four potential pollen grains form and their arrangement in the tetrad are tetrahedral. Following cytokinesis, the callose wall surrounding each meiocyte since the onset of meiosis disintegrates, and the young pollen grains are released into the anther locule. As the pollen grain develops, two mitotic divisions of the nucleus occur to produce a trinucleate pollen grain at anthesis. The vegetative or tube nucleus is oval, and the two generative or sperm nuclei are linear and sperm-like. At maturity, the pollen grains are yellow-orange, spherical, covered with spines (echinate), and have three apertures. The germinating pollen tube emerges from one of these aperture regions of the outer wall layer or exine that are very thin. The diameter of the body of the pollen grain without the spines varies from 33 to 39 μ. The aborted grains appear smaller than normal and are only partially filled. 5.5.3

Male Sterility

Both types of male sterility, nuclear male sterility (NMS) and cytoplasmic male sterility (CMS), occur in sunflower. Nuclear male sterility is generally a result of a single recessive gene pair. Genetic tests of 10 NMS lines indicated control by five different genes, which were designated ms1 through ms5 (Vranceanu, 1970). Jan and Rutger (1988) isolated seven mutation-induced NMS lines, which later were confirmed to be controlled by four different genes, ms6 through ms9 (Jan, 1992d). The ms10 and ms11 genes were assigned to the French line B11A3 and the U.S. Department of Agriculture (USDA) line P-21VR1, respectively. The close linkage between the NMS gene and an anthocyanin gene facilitated the early elimination of fertile red-stemmed seedlings and made sunflower hybrid production feasible using the NMS system. Since Vranceanu (1970) identified five different NMS genes from ten lines, and an additional four from seven lines were identified by Jan (1992b), it may be assumed that a limited number of genes control the critical steps of the biosynthetic pathway leading to normal male and female differentiation. The ms8 gene controls developmental stages that affect both male and female organs. All other nine mutants affect only male organs of a flower. Besides phenotypic descriptions, detailed cytological descriptions of these NMS lines have been very limited. Nakashima and Hosokawa (1974) found that meiosis in the NMS P-21 plants with the ms11 gene developed normally prior to the tetrad stage. The tapetal cells remained intact and enlarged after spore formation, in contrast to their disintegration in normal anthers. As the anther matured, a sudden disintegration of the tapetal cells and undeveloped pollen grains occurred. Pirev (1968) also observed normal meiosis in another NMS line prior to tetrad formation. Pollen development stopped completely, however, at the uninucleate and binucleate stages. Sterile pollen grains were low in cytoplasmic and nuclear contents. As anthesis approached, the exine or outer wall of the pollen grain degenerated, the grains clumped, and dehiscence failed to occur. Georgieva-Todorova (1974a) obtained nuclear male steriles from hybrids of H. grosseserratus × H. annuus. One type had normal meiosis, and pollen abortion

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occurred after spore release from the tetrad; in the other, sporogenous tissue degenerated prior to meiosis. A single male-sterile cytoplasm, PET1, derived from H. petiolaris subsp. petiolaris (Leclercq, 1969), and the identification of dominant fertility restoration genes (Enns et al., 1970; Kinman, 1970; Vranceanu and Stoenescu, 1971) advanced sunflower production from the use of openpollinated cultivars to hybrid production 30 years ago. This source of cytoplasmic male sterility and a few fertility restoration genes, including the widely used Rf1 and Rf2 genes, have been used exclusively for sunflower hybrid production worldwide (Fick and Miller, 1997). A total of 70 CMS sources have been identified from progenies of crosses between wild Helianthus accessions and cultivated lines, from wild accessions grown in observation nurseries, or from induced mutation. Fertility restoration genes have been reported for 34 CMS sources, and detailed inheritance studies have been conducted for only 19 of the CMS sources (Serieys, 2002). A universal coding system was proposed by Serieys (1991a) to accommodate the ever-increasing number of CMS sources. This coding system is widely accepted among sunflower researchers. A cytoplasmic male-sterile source is coded by a three-letter abbreviation of the cytoplasm donor species or subspecies followed by a numerical number starting from 1, depending on the time of its discovery and its reaction to restoration testers. The classical French CMS cytoplasm (Leclercq, 1969) has been coded CMS-PET1, and Whelan’s CMS cytoplasm from H. petiolaris, CMS-PET2. A summary of presently known CMS sources is presented in Table 5.6. Because the same ecotype of H. petiolaris was used to develop CMS-PET1 and CMS-PET3, Serieys (1991a) suggested that CMS-PET1 was the same as CMS-PET3. Similarly, the ARG1 and ARG3 derived from one wild accession of H. argophyllus (Christov, 1990) could also be the same. Both ARG1 and ARG3 were shown to be totally male sterile without negative effects and had similar restoration patterns as those of CMS-PET1. The FAO subnetwork on sunflower genetics and breeding devotes a whole working group’s effort to the identification, study, and utilization of new CMS sources, in breeding programs led by Dr. M. Christov, DAI, General Toshevo, Bulgaria. Ten countries participated in evaluating 13 CMS sources for detailed studies. The results of the 1987 to 1990 study indicated that CMS-PET1, ANL1, ANN1, ANN2, ANN3, ANN4, BOL1, and MAX1 had complete male-sterile heads with degenerated anthers in all locations. CMS-ANL2, ANT1, PET1, and PET2 exhibited visible empty anthers or anthers containing nonstainable pollen grains, and CMS-GIG1 developed indehiscent anthers with 10 to 90% stainable pollen grains (Serieys, 1991b). Many CMS sources from wild H. annuus (ANN1 through ANN9) were discovered in fieldgrown populations. The variable male sterility expression of CMS-ANN8, from complete malesterile to dehiscent anthers with 16% stainable pollen grains, makes it less desirable. All other CMS lines from wild H. annuus were completely male sterile with degenerated anthers. Restoration genes were found for ANN2, 3, 4, and 7 using a set of 20 fertility restoration testers, plus malefertile plants of each respective wild species accession. Without exception, fertile plants of each accession provided restoration genes for their CMS counterparts. Further studies of crossing and backcrossing of those fertile plants with nonrestorers confirmed that PI 413178 (ANN2), PI 413180 (ANN3), PI 406647 (ANN4), and PI 413024 (ANN7) had only CMS cytoplasms with varying frequencies of fertility restoration genes. Iuoras et al. (1989) also found a single dominant restoration gene from the same accession that CMS-ANT1 was derived from. In addition, when CMS plants occur as segregants of interspecific backcrosses, the male-fertile segregants could be kept for use as possible restoration sources. Continuous backcrossing of these male-fertile segregants with nonrestoring cultivated lines will lead to the production of male-fertile and male-sterile isogenic lines differing by the restoration genes. Inheritance studies of fertility restoration of ANN2 and ANN3 indicated complete fertility restoration by single dominant genes (Jan, 1991). Serieys (1994) also reported complete male sterility and full fertility restoration by single dominant genes for CMS-ANO1, CMS-NEG1, and CMS-PRP1. The utilization of these CMS sources for potential hybrid production should be pursued.

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Table 5.6 Summary of Presently Known CMS Sources in Sunflower Common Designation KOUBAN INDIANA 1 VIR 126 397 517 519 521 NS-ANN-81 NS-ANN-2

FUNDULEA 1 AN-67 AN-58 AN-2-91 AN-2-92 CMS-G CMS-DP CMS-VL

HEMUS PEREDOVICK STADION PEREDOVICK PEREDOVICK VORONEJSKII HA 89 HA 89 HA 89 HA 89 HA 89 HA 89 ANOMALUS ARGOPHYLLUS ARGOPHYLLUS ARGOPHYLLUS ARG3-M1 ARGOPHYLLUS BOLANDERI DV-10 EXILIS EXI2 CMG2 CMG3 MOLLIS NEGLECTUS CANESCENS FALLAX PET/PET CLASSICAL CMS

Origin (Species) H. annuus lenticularis H. annuus lenticularis H. lenticularis H. annuus wild H. annuus wild H. annuus wild H. annuus wild H. annuus wild H. annuus wild H. annuus wild H. annuus wild H. annuus wild H. annuus texanus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. annuus H. anomalus H. argophyllus H. argophyllus H. argophyllus H. argophyllus H. argophyllus H. bolanderi H. debilis H. exilis H. exilis H. giganteus H. maximiliani H. maximiliani H. mollis H. neglectus H. niveus canescens H. petiolaris fallax H. petiolaris petiolaris H. petiolaris Nutt

Accession Code

INRA-397 INRA-517 INRA-519 INRA-521

PI 413024 PI 413043 PI 413158 E-067 E-058 E-002 E-002 PI 432513 V. gigant p. DP-1 08 p. 1 638/4 GT-E-126 ANN-2101 ANN-2108 ANN-2112 ANN-2141 CMS H CMS P-UZ CMS S CMS P-114 CMS P-92 CMS VO 481 It 45-1 139-1 491-1 515-1 555-1 3149 INRA-525 E-006 E-007 E-006 E-006 E-007 INRA-255 E-010 90 INRA-130 INRA-331

INRA-286 INRA-201 INRA-197 INRA-200 INRA-737

FAO Code ANL1 ANL2 ANL3 ANN1 ANN2 ANN3 ANN4 ANN5 ANN6 ANN7 ANN8 ANN9 AMT1 ANN10 ANN11 ANN12 ANN13 ANN14 ANN15 ANN16 ANN17 ANN18 ANN19 ANN20 ANN21 ANN22 MUT1 MUT2 MUT3 MUT4 MUT5 MUT6 MUT7 MUT8 MUT9 MUT10 MUT11 MUT12 ANO1 ARG1 ARG2 ARG3 ARG3-M1 ARG4 BOL1 DEB1 EXI1 EXI2 GIG1 MAX1 MAX2 MOL1 NEG1 NIC1 PEF1 PEP1 PET1

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Table 5.6 (continued) Summary of Presently Known CMS Sources in Sunflower Common Designation CMG1 PETIOLARIS BIS PET34 PRAECOX population PHIR-27 PRAECOX PPR-28 RUN-29 RESINOSUS 243 VULPE RIG-M-28 STRUMOSUS

Origin (Species) H. petiolaris Nutt H. petiolaris Nutt H. petiolaris H. petiolaris H. praecox H. praecox hirtus H. praecox praecox H. praecox praecox H. praecox H. resinosus H. rigidus H. rigidus H. strumosus

Accession Code

E-034 PET-2208 PRA-1827 E-027 INRA-678 GT-E-028 E-029 PI 835864 M-002 STRUM-56

FAO Code PET2 PET3 PET4 PET5 PRA1 PRH1 PRP1 PRP2 PRR1 RES1 RIG1 RIG2 STR1

Source: Serieys, H., Report on the Past Activities of the FAO Working Group “Identification, Study and Utilization in Breeding Programs of New CMS Sources” for the Period 1999–2001, 2002, pp. 1–54.

Table 5.7 CMS Sources and Their Fertility Restoration Genes FAO Code

Genetic Control of the Restoration

ANL1 ANL2 ANN2 ANN2 ANN14 MUT7 to 12 ARG3-M1 ANO1 ANT1 BOL1 EXI1 MAX2 NEG1 PEF1 PEP1 PET1 PET2 PRP1 RIG1, RIGx

Two complementary dominant genes Two complementary dominant Rf genes One Rf dominant gene + modifiers One or two complementary dominant genes + modifiers Single dominant Rf gene Single dominant Rf gene Two dominant Rf genes Single dominant Rf gene Single to two dominant complementary Rf genes Complex; two independent dominant Rf genes explain many segregations Two complementary dominant Rf genes Single dominant gene One dominant Rf gene Two (or three) complementary dominant independent genes Two independent complementary Rf genes One or two complementary dominant independent genes Two dominant Rf genes One dominant Rf gene Two complementary dominant genes

Source: Serieys, H., Report on the Past Activities of the FAO Working Group “Identification, Study and Utilization in Breeding Programs of New CMS Sources” for the Period 1999–2001, 2002, pp. 1–54.

Twenty-two cytoplasmic male-sterile mutants were produced by Jan and Rutger (1988) using chemical mutants. These lines were all stable with degenerated anthers and with similar reactions to restorers as observed for the CMS-PET1. These CMS sources have the potential of being quickly utilized for future hybrid sunflower production using the currently available restoration lines for the CMS-PET1 cytoplasm. It seems to be relatively easy to isolate stable CMS cytoplasms, but the identification of simple and completely dominant fertility restoration genes has been far less successful (Table 5.7). Paun (1974) investigated four CMS A-lines (CMS-PET1) with the corresponding fertile B-lines and observed two types of meiotic behavior. The sporogenous cells in two of the four CMS lines degenerated before meiosis and produced necrotic anthers with no pollen. Meiosis in the other two

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lines was normal to the tetrad stages, except that the frequency of sporogenous tissues undergoing meiosis was much less than normal. Pollen degeneration occurred following release of the spores from the tetrad. The premeiotic abortion of sporogenous cells was also reported in CMS-ANT1, where the complete absence of meiosis and the lack of visible anthers were observed (Vranceanu et al., 1986). Whelan and Dedio (1980) described CMS-PET2 and CMS-GIG1 plants that produced indehiscent anthers with white pollen grains, which failed to germinate in in vivo tests, as well as crosses with CMS-PET1-CM400. The CMS-MAX1 cytoplasm produced plants with vestigial anthers that contain few or no pollen grains. Siculella and Palmer (1988) compared physical organization and transcriptional properties of mtDNA from isonuclear fertile and CMS lines with PET1 cytoplasm. Their results indicated an extremely high similarity between the CMS-PET1 cytoplasm and the H. annuus fertile cytoplasm. The only difference detected was a 17-kb region of the genome reflected as two mutations, a 12-kb inversion and a 5-kb insertion/deletion. One endpoint of both rearrangements is located within or near atpA, the only mitochondrial gene whose transcripts differ between the fertile and CMS lines. A nuclear gene that restores fertility of CMS was found to specifically influence the pattern of atpA transcripts. They suggested that this CMS was caused by the rearrangement at the atpA locus. Further support of this was provided by Monéger et al. (1994), where CMS was associated with the insertion into the mitochondria DNA of an open reading frame (ORF) located 3' to the atpA gene, which led to the production of a 15-kDa novel protein responsible for the CMS phenotype. The product of the restoration gene was shown to act on the posttranscriptional level to destabilize this novel protein in a tissue-specific manner to restore fertility. To simplify the comparison between the male-sterile and male-fertile cytoplasms, it would be much easier to compare isogenic lines of the cytoplasmic genome instead of isonuclear lines as presently used. The commonly used comparison of isonuclear lines is the equivalent of comparing normal H. annuus cytoplasm with the CMS cytoplasm from wild CMS donor species. The two cytoplasms could differ by many DNA sequences, which will complicate the interpretation of CMSrelated differences. Cytoplasmic isogenic lines are being developed (Jan, unpublished data). It is believed that CMS HA 89 mutants are more likely to differ from the HA 89 by CMS-specific changes. Wild H. annuus accessions PI 413024, PI 413131, and PI 413158 were shown to possess CMS cytoplasm, male-fertile cytoplasm, and Rf genes. The comparison of the CMS and the malefertile cytoplasms of the same accession will also minimize the complications of the interspecific cytoplasmic differences and offer better interpretation for the CMS-related differences. Comparisons between the CMS T-cytoplasm of maize and its fertile revertant demonstrated the use of another good isogenic source material. In this study, a 13-kDa polypeptide was shown to play a controlling role in the CMS phenotype (Dixon et al., 1982; Dewey et al., 1986). This polypeptide was not observed in the male-fertile revertant. When Rf genes were introduced to restore fertility, the synthesis of the 13-kDa polypeptide was reduced. It was suggested that the restoration genes are involved in RNA processing, which led to the decrease in the synthesis of the 13-kDa polypeptide. 5.5.4

Cytogenetic Stocks

Induction of polyploidy has been accomplished successfully using colchicine. Heiser and Smith (1964) grew young seedlings for 8 h on filter paper saturated with a 2.0 g/kg solution of colchicine and obtained some chromosome doubling. Using this technique, they obtained tetraploids from the perennial hybrid cross H. giganteus × H. microcephalus. One plant was obtained from this cross, two from the reciprocal cross, and two others from H. maximiliani × H. decapetalus (2n). All five plants showed over 90% pollen stainability, but a somewhat reduced seed set. Most meiocytes had 1 to 3 quadrivalents, but an occasional one contained 34 bivalents. Helianthus decapetalus was the only diploid species successfully doubled. Meiocytes contained from one to five quadrivalents. Unfortunately, the plant died before pollen fertility could be determined. Colchicine-induced tetraploids of H. annuus have also been obtained (Dhesi and Saini, 1973). In diakinesis, 71% of

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the meiocytes contained 34 bivalents. The remaining 29% contained quadrivalents, trivalents, and univalents. About 67% of anaphase I meiocytes showed unequal division. Micronuclei and lagging chromosomes were also observed. Despite these meiotic abnormalities, pollen fertility was estimated to be 83.8%. The tetraploids had a greater range in pollen grain size, with the average size similar to that of diploids. Jan et al. (1988) subjected inbred lines P21 and HA 89 to a 5-h colchicine treatment at 1.5 g/kg, pH 5.4, with 20 g/kg dimethyl sulfoxide, resulting in a high frequency of chromosome doubling and the production of autotetraploid P21 and HA 89. Tetraploids had larger disk florets and larger pollen grains; otherwise, tetraploid plants were morphologically similar to their diploid progenitors. Tetraploidy in P21 was not stable, with plants having 2n = 4x = 65 to 70 chromosomes. Tetraploid plants of HA 89 had reduced vigor and did not produce seed. At diakinesis, tetraploid P21 plants had an average of 0.85 univalents, 21.12 open bivalents, 6.66 closed bivalents, 0.21 trivalents, and 2.74 quadrivalents per cell. The number of chiasma per chromosome pair in P21 was reduced from 1.50 for diploid to 1.32 for tetraploid plants. Pollen stainability in tetraploid P21 was less than 50%, and the plants produced an average of eight seeds per sib-pollinated head, about 1% of normal seed set. Reciprocal crosses of diploid and tetraploid P21 produced four triploid plants. Backcrossing triploids to P21 produced 137 plants with 2n = 34 to 47 + t. Thirty-one of these plants were trisomics having 2n = 35. Trisomic progenies of the 31 originally identified trisomic P21 plants (Jan et al., 1988) only displayed limited variation in morphological characteristics when grown in the greenhouse. Few trisomic groups appear to have distinctive features such as unusual plant height, flowering dates, leaf texture, and stem size. In order to quickly identify the first set of sunflower trisomics, future research will need to be focused on the mitotic metaphase and meiotic pachytene karotyping, supplemented by the fluorescence in situ hybridization (FISH) technique using linkage groupspecific RFLP and BAC clones. The same colchicine treatment of interspecific F1 hybrids also resulted in high frequencies of chromosome doubling and the production of amphiploids (Jan and Fernández-Martínez, 2002). The tetraploid amphiploids produced included crosses of P21 × H. bolanderi (Jan and Chandler, 1989), H. gracilentus × P21, H. grosseserratus × P21, H. cusickii × P21, H. mollis × P21, H. maximiliani × P21, and H. nuttallii × P21. These amphiploids have restored fertility and provide additional genetic diversity for the improvement of cultivated sunflower. They can be backcrossed with cultivated lines, without the use of embryo culture to produce progenies. An amphiploid of H. hirsutus × P21 represents the first hexaploid amphiploid in sunflower. Like the autotetraploid P21, the amphiploids were not completely stable. After sib pollination of 2n = 68 amphiploids, progenies could have 2n chromosome numbers from 64 to 71. The sib pollination of 2n = 102 H. hirsutus × P21 amphiploid produced progenies with 2n chromosome numbers from 94 to 103. Additional hexaploid amphiploids of H. hirsutus × P21 and H. strumosus × P21 were also produced. We expect that these interspecific amphiploids will enable us to establish a number of chromosome addition lines for genetic studies of specific chromosomes of both cultivated and wild Helianthus species. With the available amphiploids and some specific interspecific crosses, the potential exists to establish addition lines with HA 89 chromosome pairs in H. californicus, and the chromosome pairs of H. hirsutus, H. angustifolius, H. cusickii, H. gracilentus, H. grosseserratus, H. nuttallii, H. strumosus, and H. giganteus in HA 89. 5.5.5

Interspecific Hybridization

5.5.5.1 Hybridization Techniques Prior to the first embryo culture method developed by Chandler and Beard (1983), nearly all the interspecific crosses were conducted in a classical fashion. Complete emasculation is necessary when cultivated sunflower is used as the female parent. When wild Helianthus species are used as

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female parents, emasculation is only applied to the disk flowers expected to flower that day, followed by washing the pollen grains from the head in the morning. An isolation bag is used to prevent contamination, and pollination is performed in the late afternoon when the heads are dry and before the appearance of the pollen grains for the following day. Isolation bags are used after pollination until harvest to avoid seed loss due to shattering. All the annual Helianthus species, except H. agrestis, can be hybridized and F1s backcrossed with cultivated lines using the classical method. Direct crosses of cultivated lines with many perennial Helianthus species are also possible using conventional methods. Hybrids of H. mollis × H. annuus and H. strumosus × H. annuus (Heiser and Smith, 1964) and of H. decapetalus × H. annuus (Heiser et al., 1969; Georgieva-Todorova, 1984) have been reported. Hybrids of H. tuberosus × H. annuus (Heiser et al., 1969; Atlagic et al., 1993), H. annuus × H. hirsutus (Georgieva-Todorova, 1984), and H. rigidus × H. annuus (Vranceanu and Iuoras, 1988) have also been successful. A single hybrid plant of H. laciniatus × H. annuus was obtained by Jackson (1988), but did not produce backcross progenies. Atlagic (1990) summarized five interspecific hybrids involving crosses of perennial species H. hirsutus, H. laevigatus, H. rigidus (= pauciflorus), H. tuberosus, H. maximiliani, and H. nuttalii with cultivated sunflower. All these interspecific crosses, including some rather difficult cross combinations, were accomplished without the use of the embryo rescue technique. Whelan (1978) used wild H. annuus as an intermediate parent or bridge to produce the first hybrids obtained between this species and H. giganteus and H. maximiliani. Direct hybridization with the cultivar Krasnodarets gave a single, highly sterile hybrid with H. giganteus. Repeated pollinations with or without subsequent embryo culture failed to give backcross progeny. Pollinating both H. giganteus and H. maximiliani with wild H. annuus pollen, however, produced three and four hybrids, respectively. These hybrids subsequently produced a small quantity of seed when pollinated with Krasnodarets pollen (Whelan and Dedio, 1980). The development of a two-step embryo culture procedure by Chandler and Beard (1983) greatly facilitated interspecific hybridization. They successfully produced 53 interspecific cross combinations without the exhaustive effort of endless pollination, and 21 of these combinations had not been previously produced. Three- to 7-day-old embryos were first cultured on a solid growth medium in petri dishes with Gamborg’s B5 salts (Gamborg et al., 1968) supplemented with vitamins, amino acids, NAA (α-naphthaleneacetic acid), and 120 g/kg sucrose. After 1 to 2 weeks, the enlarged embryos were transferred to a liquid germination medium in test tubes with B5 salts plus 10 g/kg sucrose. The young seedlings with roots and shoots were transplanted into soil in the greenhouse. Jan and Chandler (unpublished data) further modified the original procedure for culturing difficult hybrid embryos of wild perennial Helianthus species with cultivated H. annuus by adding vitamins, increasing sucrose to 20 g/kg, and the conversion from liquid to a solid medium with 0.7% agar. In addition, both growth and germination media were adjusted to pH 5.5 with 2(N-morpholino)ethanesulfonic acid (MES) buffer. Using these modified media, 18 perennial species × H. annuus hybrids were established in one season, and many of them represented the first hybrid combinations ever produced (Jan, 1988). This modified germination medium has also been effectively used to culture young backcross embryos for the acceleration of generations, using 10- to 12-day-old embryos. Kräuter et al. (1991) cultured 0.2- to 1.5-mm small embryos on B5 medium with 90 g/kg sucrose, and embryos >1.5 mm on a modified MS (Murashige and Skoog, 1962) medium with 10 g/kg sucrose. When these embryos reached the size of 2 to 3 mm, they were transferred to MS medium for germination. Using this method, they obtained 33 interspecific hybrid combinations with an overall success rate of 41%. Using cultivated sunflower embryos of varying sizes, Espinasse et al. (1985) concluded that a high sucrose concentration of 90 g/kg and low nitrogen content were required for culturing small young embryos less than 2 mm in size. Similarly, after evaluating responses of young embryos of cultivated sunflower on various media modifications, Denat et al. (1991) concluded that a low level of calcium chloride and high levels of sucrose, potassium chloride, and nitrogen were beneficial for early development of young embryos. Several difficult interspecific

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hybrids were produced using their modified media, including crosses of perennial diploid species H. maximiliani, H. mollis, H. pumilus, and H. salicifolius with cultivated H. annuus. As suggested by Dewey (1980), induced polyploidy could also serve as a bridge for interspecific gene transfer in sunflower. Jan and Chandler (1989) successfully doubled chromosomes of P21 × H. bolanderi F1 hybrids and increased the seed set on doubled heads. Chromosome doubling, using colchicine applied to apical meristems of young seedlings, has also been demonstrated in cultivated sunflower (Dhesi and Saini, 1973; Gupta and Roy, 1979; Jan et al., 1988) and in diploid perennial species and their interspecific hybrids (Heiser and Smith, 1964). Jan (1988) reported success of a modified colchicine chromosome-doubling technique on 19 embryo-cultured wild × cultivated interspecific hybrids, and its positive effect on a backcross seed set. For the colchicine treatment, each peat pot was wrapped in aluminum foil with the extruding seedlings inverted with the apical meristem submerged in a 1.5 g/kg colchicine solution with 20 g/kg DMSO (dimethyl sulfoxide) for 5 h in the dark. Chromosome doubling of each head was verified by pollen grain size and stainability (Alexander, 1969). Chromosome doubling increased pollen grain size and stainability of interspecific hybrids. The increased pollen grain size directly reflected chromosome doubling and provided a reliable criterion for classifying treated plants. Chromosome doubling restores normal fertility of amphiploids by providing an identical pairing partner for each chromosome. However, this increased fertility is likely to reduce the enforced interspecific chromosome pairing and gene exchanges during meiosis when an F1 head is not chromosomally doubled. It would be helpful if the researchers could backcross onto both doubled and nondoubled heads, and at the same time intercross doubled heads for amphiploid production. More cytological evaluations are needed to compare the efficiency of interspecific gene transfer with or without the assistance of chromosome doubling of F1s. Without chromosome doubling, we may expect very low BC1F1 seeds and a high frequency of weak BC1F1 plants. With chromosome doubling, due to preferential pairing of H. annuus chromosomes during meiosis, we may expect a reduced pairing of H. annuus chromosomes with chromosomes of wild Helianthus species. 5.5.5.2 Interspecific Hybrids among Helianthus Species A large number of earlier interspecific hybridization studies were focused on species relationships, with discussions mostly about the hybrid F1 seed set, pollen fertility, meiosis abnormality, and the seed set of further crosses (Jan, 1997). Only a few selected studies will be reviewed. The presence of a paracentric inversion in H. petiolaris × H. annuus hybrids (Heiser, 1947) was questioned (Heiser, 1961), but Whelan (1979) frequently noted a bridge and fragment in such hybrids, which is evidence of this chromosomal abnormality. Heiser et al. (1969) suggested that H. neglectus differs from H. petiolaris by a single translocation and from H. annuus by several translocations. Heiser and Smith (1960) suggest that H. × multiflorus, a cultivated ornamental, is a hybrid between H. annuus and H. decapetalus (4n). The hybrid is a sterile triploid with 17 bivalents and 17 univalents usually occurring in meiosis. The cross of H. decapetalus (4n) with H. debilis subsp. cucumerifolius and H. praecox (H. debilis subsp. hirtus) also produced almost all sterile triploids (Heiser and Smith, 1964). Georgieva-Todorova (1974b) reported 96% normal pollen in the tetraploid race, but only a 10 to 12% seed set. In diakinesis, 31% of the cells contained a quadrivalent or univalents. Bridges were seen in 24% of anaphase I meiocytes, and 33% of telophase II meiocytes contained micronuclei. Hybridization with H. mollis produced completely femalesterile plants with 8% normal pollen. Meiocytes contained numerous univalents and related defects (Georgieva-Todorova, 1972). Chandler et al. (1986) studied chromosome differentiation using interspecific F1 hybrids among a large number of annual species. Percent pollen stainability of hybrids varied from 0.2% for H. bolanderi × H. argophyllus to 76.4% for H. neglectus × H. petiolaris, and the frequency of univalents in both diakinesis and metaphase I averaged 0.1 to 3.6 and 0.1 to 15.7, respectively. In addition, frequent multivalents in diakinesis and the formation of bridges and fragments in

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anaphase I were also observed, suggesting numerous structural differences among species in the form of chromosome translocation and paracentric inversion. Heiser et al. (1969) suggest that the most likely origin of the 4n forms of H. strumosus is from hybridization of H. divaricatus × H. grosseserratus, followed by chromosome doubling. Subsequent crossing between this 4n hybrid and various 2n species such as H. divaricatus, H. grosseserratus, and H. glaucophyllus should give 3n hybrids, and subsequently 6n hybrids if chromosome doubling occurred. The cross with H. annuus gave an almost sterile triploid hybrid (Heiser and Smith, 1964). Helianthus pauciflorus (rigidus) has two interfertile subspecies: subsp. pauciflorus and subsp. subrhomboides. The first subspecies crossed with H. tuberosus is thought to have given rise to H. × laetiflorus (Clevenger and Heiser, 1963; Heiser et al., 1969). Georgieva-Todorova (1971) studied meiosis and pollen fertility of H. pauciflorus for 2 years. Of the 25 meiocytes examined at diakinesis, only 12 contained quadrivalents and 2 had univalents. Chiasma frequency per cell was estimated at 66.3 or 1.3/bivalent, and over 95% of the meiocytes in subsequent division stages were normal. Pollen fertility in the 2 years was 76.8 and 73.8%. She suggests a genome formula of Ar1Ar1Ar2 Ar2 BrBr. However, she subsequently reported difficulty in obtaining hybrids with H. tuberosus (Georgieva-Todorova, 1972). Long (1963) suggests that H. grosseserratus may have originated from hybridization between H. giganteus and H. salicifolius. It was suggested by Heiser et al. (1969) that H. giganteus and H. mollis contributed two of the three possible genomes for the hexaploid H. resinosus. It is interesting to note that after crossing H. microcephalus with H. divaricatus, two of the three hybrids were fertile with 17 bivalents and the third was partially sterile with a quadrivalent, but both hybrids obtained from the reciprocal cross had 17 bivalents and were fertile (Smith and Guard, 1958). 5.5.5.3 Interspecific Hybrids between Wild Helianthus Species and Cultivated Lines In recent years, interest in interspecific hybridization has been greater for transferring useful genes from wild species into cultivated lines to develop prebreeding germplasms for future sunflower improvement. Characteristics such as disease and insect resistance, salt tolerance, drought tolerance, fatty acid variation, CMS, and fertility restoration diversity have been emphasized. By successful hybridization between H. petiolaris and H. annuus and backcrossing with H. annuus, Leclercq (1969) transferred the H. annuus genome into H. petiolaris cytoplasm and obtained the first cytoplasmic male-sterile plants. Whelan (1980, 1981) and Whelan and Dorrell (1980) used the same technique to obtain cytoplasmic male sterility conditioned by the cytoplasm for the three species, H. petiolaris, H. giganteus, and H. maximiliani. H. tuberosus × H. annuus hybrids have been used widely in the FSU as a source of disease resistance. Heiser and Smith (1964) reported producing hybrids that were vigorous, but almost female sterile. Pollen stainability varied from 12 to 53%. Meiosis of the hybrids had a mean of 31 bivalents, with the rest of the chromosomes appearing as univalents or multivalents. A chromosome bridge was observed in anaphase in only 1 of 50 cells examined. Kostoff (1939) frequently observed chromosome bridges in his material. Cauderon (1965) observed two types of meiosis in H. tuberosus × H. annuus hybrids. One type was strongly asyndetic, that is, many of the homologous chromosomes were not paired, and the other type was weakly asyndetic. However, both types showed meiocytes with 34 bivalents. Aneuploid progeny from such hybrids have been reported (Leclercq et al., 1970) following backcrossing with H. annuus. Due to the use of a single male-sterile cytoplasm for worldwide hybrid sunflower production and its consequence of genetic vulnerability, as shown by southern corn leaf blight caused by Bipolaris maydis race T in the early 1970s (Tatum, 1971), a large portion of the interspecific hybridization in sunflower has focused on the identification of new CMS sources and their fertility restoration genes. Of the total 70 CMS sources resulting from interspecific hybridization, 38 were derived from wild H. annuus and 23 from other wild annual species, and only 8 from wild perennial

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species. Extensive research is now focused on the identification of fertility restoration genes using both cultivated and wild species and evaluation of their inheritance. Rapid improvement of interspecific F1 meiotic abnormality and low fertility was also demonstrated by Whelan (1978, 1979) when he discovered CMS-PET2, G1G1, and MAX1. The H. petiolaris × H. annuus F1 meiosis indicated that the two parents differ by one paracentric inversion, had 10 of the 34 chromosomes involved in translocation, and had a low 5% pollen stainability. Similarly, F1 hybrids of H. maximiliani and H. giganteus crossed with wild H. annuus indicated that the parents differ by a minimum of three translocations and a paracentric inversion. Both paracentric inversion and multivalent formation were quickly eliminated after one or more backcrosses with cultivated lines. However, trisomic plants were identified among the backcrossed progenies, but the identity of these single extra chromosomes has not been clarified (Whelan, 1982). A single hybrid of H. laciniatus with H. annuus had 2.24% fertile pollen, but failed to produce a backcross seed set (Jackson, 1988). The two species were shown to differ by a minimum of eight reciprocal translocations and one paracentric inversion. Gene flow between these two species was considered impossible. Hybrids of H. annuus × H. hirsutus, H. annuus × H. decapetalus, and H. scaberimus × H. annuus were produced by Georgieva-Todorova and Lakova (1979), with meiotic diakinesis or metaphase of H. annuus × H. hirsutus and H. annuus × H. decapetalus having 7 to 34 and 9 to 17 univalents, respectively, and the rest bivalents. The fact that some of the bivalents were heteromorphic, open, and chromosomes paired end to end suggests that the bivalents did not possess complete homologous chromosomes, but were the pairing of partially homologous chromosomes from H. annuus and those from the tetraploid genomes. Backcrosses to H. annuus were not immediately successful. Later, Bohorova et al. (1981) secured BC1F1 plants of H. annuus (H. annuus × H. hirsutus) with the use of embryo rescue, and the BC1F1 of H. annuus (H. annuus × H. decapetalus) was produced from seeds. Without the use of embryo culture, crosses between H. annuus and the perennial Helianthus species H. maximiliani, H. giganteus, H. grosseserratus, H. salicifolius, H. californicus, H. mollis, H. divaricatus, and H. nuttallii were not possible (Georgieva-Todorova, 1984). Hybrids of H. annuus × H. resinosus (2n = 102) had stainable pollen from 0 to 50%, and meiotic diakinesis had 28 to 36 bivalents with 1 to 6 univalents (Georgieva-Todorova, 1983). The high number of bivalents suggests a high homology between the chromosomes from H. resinosus and those from H. annuus. In general, good pollen stainability is expected in the F1s of hexaploid Helianthus species crossed with H. annuus. Atlagic (1990) reported an average pollen stainability of 49.8, 40.9, and 64.6%, respectively, for the hybrids of H. annuus with H. pauciflorus, H. tuberosus, and H. laevigatus. As expected, hybrids of H. annuus crossed with tetraploid H. hirsutus had 17.8% pollen stainability, which is considerably higher than the results of Georgieva-Todorova and Lakova (1979), as well as those of Jan (1988). Further studies of H. laevigatus × H. annuus indicated quadrivalent and hexavalent formation, suggesting chromosome translocation differences between the two species (Atlagic and Skoric, 1999). It was also noted that H. laevigatus may carry Rf genes for the CMS-9 used in the study. Hybrids of H. annuus × H. simulans were obtained through conventional crossing, and their F1 plants had one to four quadrivalents in diakinesis, indicating chromosome differences by up to four reciprocal translocations (Prabakaran and Sujatha, 2004). Seiler’s (1991a, 1993) release of 12 interspecific germplasm lines derived from perennial accessions of H. hirsutus, H. resinosus, and H. tuberosus also supports the reasonably good fertility of H. annuus × hexaploid accessions and some selected H. annuus × tetraploid accessions. Partial hybridization between cultivated sunflower and perennial Helianthus species H. mollis and H. orgyalis has been reported (Faure et al., 2002). These hybrids resembled female parents in reciprocal crosses, and the possibility of partial hybridization was proposed where a very limited part of the male genome was transferred to the female genome. The hybrids were confirmed using molecular markers and had 2n = 34 chromosomes. The exact mechanism remains to be determined. Similar unexplainable rare gene transfer was also observed by Jan (2004) while screening amphiploids

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for Rf genes for a new CMS-GIG. A single male-fertile plant with 2n = 34 was obtained after pollinating CMS-GIG with the amphiploid of H. maximiliani × P21, and the restoration was under single dominant gene control. The other nine F1 hybrids of the same cross were also male fertile, but all with the expected 2n = 51 number of chromosomes. An unusual cytoplasmic-nuclear interaction causing plants with reduced vigor has been observed, and a single dominant gene was needed to restore normal plant growth (Jan, 1992c). Plants with reduced vigor segregated in backcross progenies while substituting the HA 89 nucleus into the cytoplasms of H. mollis, H. divaricatus, H. angustifolius, H. grosseserratus, and H. maximiliani. Similar vigor-reducing cytoplasmic effects were observed in BC3F1 progenies of CMS-RIGX and CMS-MAX2 backcrossed with the maintainer line HA 89 (Jan, unpublished data). The vigorreducing cytoplasmic effects also have been observed in progenies when backcrossing HA 89 into cytoplasms of H. hirsutus, H. occidentalis, and H. giganteus. This seems to suggest a common problem of using perennial Helianthus species as CMS sources: the requirement of both vigor restoration and fertility restoration genes for the hybrids will present more challenges and difficulties in the breeding process. With continuous backcrossing with HA 89 as the recurrent parent into the cytoplasms of five diploid perennial species, H. mollis, H. maximiliani, H. grosseserratus, H. divaricatus, and H. angustifolius, and selection for normal segregants, Jan (1992c) discovered the vigor-reducing effects of these cytoplasms, and a single nuclear vigor restoration gene was needed to restore the vigor. Allelic studies indicated that the five vigor restoration genes derived from the above five crosses are at the same locus of the HA 89 genome. In addition, segregation of normal and reducedvigor plants is commonly observed when backcrossing HA 89 into cytoplasms of other perennial Helianthus species. A considerable number of cultivated lines were also found to possess the same vigor restoration gene, and it was suspected to have been derived from H. tuberosus because of that species’ popular use in early breeding programs in the FSU. The widespread existence of vigor-reducing cytoplasms and the vigor restoration genes in perennial species could suggest a unique origin of the perennial Helianthus species, and also presents problems and requires extra effort when using some CMS sources for hybrid production. Our recent discovery of a different vigor restoration gene derived from H. giganteus suggested the existence of different vigor restoration genes in varying perennial Helianthus species compensating for specific cytoplasmic effects causing reduced vigor (Jan, 2003). Transferring genes from wild annual species into cultivated lines can be accomplished rather easily with conventional crossing and backcrossing. Seiler (1991b, 1991c) released 15 interspecific germplasm lines having genes from wild annual species, and 13 tolerant to sunflower downy mildew, using the conventional method of crossing and backcrossing. Jan and Chandler (1985a) transferred resistance genes for powdery mildew (Erysiphe cichoracearum) from H. debilis and rust (Puccinia helianthi) and downy mildew resistance genes from wild H. annuus into cultivated sunflower (Quresh et al., 1993; Quresh and Jan, 1993; Tan et al., 1992). Crossing cultivated sunflower with wild perennial Helianthus species often results in serious problems of early hybrid embryo abortion, as well as high levels of sterility in the F1 or BC1F1 generation. However, utilizing an embryo-culturing technique, 26 interspecific hybrids of wild perennials × cultivated line P21 were produced. Subsequent chromosome doubling of the F1s of diploid and tetraploid wild accessions crossed with P21 improved the backcross and sib-pollinated seed set drastically (Jan, 1988). Amphiploids of wild species utilizing H. gracilentus, H. pumilus, H. hirsutus, H. strumosus, H. maximiliani, H. nuttallii, H. mollis, and H. grosseserratus crossed with cultivar P21 have been produced by sib pollination of chromosomally doubled heads of each cross, and will be released when sufficient seeds are available. These amphiploids can be maintained by sib pollination, are relatively unstable, with variations of chromosomes deviating from the expected 2n = 68 or 102, have improved pollen stainability and larger pollen grains, and have improved backcross seed sets (Jan and Fernández-Martínez, 2002).

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Interspecific gene transfer facilitated by the chromosome doubling of extremely difficult diploid perennials × H. annuus and tetraploid × H. annuus crosses has been demonstrated. Positive results of gene transfer from H. hirsutus into cultivated sunflower have been obtained (Jan and Zhang, 1995). By monitoring the rust resistance genes of H. hirsutus, which is immune to the four North American (NA) rust races, the hexaploid amphiploid was backcrossed with H. annuus twice. The resulting triploid BC2F1s had a complete set of 34 chromosomes of H. annuus, plus 17 chromosomes from H. hirsutus, and were all resistant to the four NA rust races. Several BC3F1 plants had 2n = 36 or 37 chromosomes and were resistant to NA rust races 1 and 2, and further backcrossing resulted in many BC4F1 race 1- and 2-resistant plants with 2n = 34. More recently, Jan et al. (2002) produced four sunflower germplasms with resistance to broomrape (Orobanche cumana) race F, with resistance genes transferred from wild perennial Helianthus via interspecific amphiploids. In addition, interspecific amphiploids of perennial × cultivated have provided fertility restoration genes for the new CMS cytoplasms derived from H. giganteus (Jan, 2004), while no Rf genes were identified in cultivated lines. Surprisingly, Rf genes for this CMS were identified in four of the seven amphiploids tested. Chandler (1991) reviewed sunflower genomic relationships and came to the conclusion that there is little evidence of the existence of distinct genomes in Helianthus. The author’s observation of many interspecific hybrids tends to agree with Chandler’s statements. Even the most sterile interspecific hybrids involving diploid perennial species and cultivated H. annuus had satisfactory chromosome pairing (Jan and Chandler, 1985b). In order to utilize this high degree of chromosome similarity between cultivated lines and wild Helianthus species for interspecific gene transfer, the best approach would be to backcross without F1 chromosome doubling. Without chromosome doubling, maximum chromosome pairing between cultivated lines and the wild species will be achieved. With chromosome doubling, preferential chromosome pairing of identical chromosomes in each parent will reduce the interspecific chromosome pairing and gene exchanges. However, the latter approach may have the advantage of having improved backcross fertility, and the reduced degree of gene exchange will enhance the quick recovery of a recurrent parent genotype carrying the specific selected gene. This was demonstrated with the rust resistance gene transfer from H. hirsutus into cultivated line HA 89 via amphiploidization, where chromosomes from H. hirsutus demonstrated their ability to challenge the perfect pairing of H. annuus chromosomes and to incorporate the resistance genes into the H. annuus genome (Jan and Zhang, 1995). 5.5.6

Mutagenesis

Mutagens have the potential of generating new genetic variations, but often cause chromosome aberrations and defects in meiotic cell division. Gundaev (1971) reports that air-dried sunflower seed treated with 40 Gy of x-rays and moist, swollen seed treated with 10 Gy produced about equal frequencies of chromosomal aberrations. The frequency of abnormal anaphase and telophase divisions was about 20%. Georgieva-Todorova (1969) also observed cytological aberrations in anaphase following treatment of seed with x-rays. Chromosome bridges and fragments were the most common defect. Gamma-radiation from a cobalt-60 source has been reported to affect length and stainability of sunflower chromosomes (Kurnik et al., 1971). However, mutagenesis has been successfully applied to generate useful traits for the improvement of sunflower, which is especially important if the trait is not found in wild Helianthus species. 5.5.7

Male Sterility Induction

Streptomycin and mitomycin C were used by Jan and Rutger (1988) to induce male sterility in inbred line HA 89. Seed was presoaked in distilled water for 12 h and then soaked in solutions of 5, 50, and 500 mg/l mitomycin C and 5, 50, 500, and 5000 mg/l streptomycin for 40 h at 4°C.

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A total of 22 CMS and 7 NMS mutants were selected. All the mutants exhibited vigor and plant height comparable to HA 89. Their male sterility characteristics with degenerated anthers resembled those of CMS HA 89 with H. petiolaris cytoplasm (cmsPET1). These male-sterile lines, except NMS line 747, were completely male sterile and female fertile. The fertility of CMS lines can be restored by restoration genes for the CMS-PET1 cytoplasm. Yield trial evaluation using seven selected mutant CMS lines indicated no deleterious effect of the mutation treatment, and those CMS lines and their hybrids produced with commonly used restoration lines performed similarly to CMS HA 89 (Jan et al., 2004a). Inheritance studies of the seven NMS lines indicated that they represented mutations at four different loci (Jan, 1992b). No cytological abnormalities related to meiotic chromosome pairings were observed for all the male-sterile lines, except the developmental abnormality of NMS 747. 5.5.8

Chromosome Doubling

Induction of chromosome doubling has been accomplished using colchicine. Heiser and Smith (1964) grew young seedlings for 8 h on filter paper saturated with a 2.0 g/kg solution of colchicine and induced polyploidy from the hybrids H. giganteus × H. microcephalus, H. microcephalus × H. giganteus, and H. maximiliani × H. decapetalus (2n). Most meiocytes had one to three quadrivalents, but an occasional one contained 34 bivalents. H. decapetalus was the only diploid species successfully doubled. Meiocytes contained from one to five quadrivalents. Colchicineinduced tetraploids of H. annuus have also been obtained (Dhesi and Saini, 1973). In diakinesis, 71% of the meiocytes contained 34 bivalents. The remaining 29% contained quadrivalents, trivalents, and univalents. About 67% of anaphase I meiocytes showed unequal division. Micronuclei and lagging chromosomes were also scored. Despite these meiotic abnormalities, pollen fertility was estimated to be 83.8%. The tetraploids had a greater range in pollen grain size, with the average similar to that of diploids. Chromosome doubling in inbred line P21 and HA 89 was also accomplished by Jan et al. (1988), with a 5-h colchicine treatment at 1.5 g/kg, pH 5.4, plus 20 g/kg dimethyl sulfoxide. The resulting tetraploid plants morphologically resembled their diploid progenitors, but had larger disk florets and larger pollen grains, and plants with 2n = 4x = 65 to 70 chromosomes. Similarly, colchicine treatment of interspecific F1 hybrids also resulted in high frequencies of chromosome doubling and the production of amphiploids, which had restored fertility and can be utilized easily for sunflower improvement. 5.5.9

Alteration of Fatty Acid Composition

The critical starting point of modern-day high oleic and mid-oleic NuSun type of sunflower oil can be traced back to the mutation breeding effort at the All-Union Research Institute of Oil Crops of the FSU (Soldatov, 1976). The initial plant with 500 g/kg oleic acid was identified in the M3 generation after treatment with a 5 g/kg dimethyl sulfate solution. Eventually, the high oleic variety ‘Pervenets’ was produced that contained up to 800 g/kg oleic fatty acid. High levels of palmitic and stearic acid were achieved by Osorio et al. (1995) using mutagens sodium azide and ethyl methanesulfate and x-rays on mature seeds. The resulting mutant lines were selected from single M2 and M3 seeds. The mutant line CAS-5 has 250 g/kg palmitic, and CAS-3, CAS-4, and CAS-8 contained high stearic acid at 260, 110, and 100 g/kg, respectively. High palmitic acid content up to 400 g/kg was obtained by Ivanov et al. (1988) using gammaray irradiation on dry seeds. Fernández-Martínez et al. (1997) used x-rays on dry seeds of high oleic line BSD-42-3, which resulted in the production of CAS-12, having 300 g/kg palmitic acid, but at the expense of the oleic acid content, which was reduced from 880 to 560 g/kg. Research on the utilization of high palmitic and high stearic hybrids is in progress (Fernández-Martínez, personal communication) and is expected to have potential for both food and industrial applications.

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5.6 GERMPLASM ENHANCEMENT: CONVENTIONAL BREEDING 5.6.1

Breeding for End Use

5.6.1.1 Grain Since the sunflower phenotype is the result of an interaction between the genotype and the environment, it is necessary to define within a model the major yield-forming traits and the major environmental factors that affect them. Generally, characteristics that are associated with good vegetative plant growth are correlated with high yield. These include days from sowing to maturity, plant height, head diameter, stem diameter, leaf area per plant, achene number per plant, achene weight, and disease resistance (Fick, 1978; Skoric, 1988; Fick and Miller, 1997). A high degree of self-fertility is also considered important for high yield in many areas, especially where insect pollinator populations are limiting. It may be concluded on the basis of the studies conducted on sunflower thus far that achene number per head is an important trait that should not be neglected when developing inbred lines. When screening for combining ability, those combinations should be selected that express a high heterotic effect for a high number of achenes per head. Sunflower seed yield may be increased significantly by breeding for achene size, i.e., for higher achene weight and test weight. According to Morozov (1947), an increase in 1000-achene mass of only 1 g increases achene yield by 40 kg/ha. It is certain that the number of disk flowers, i.e., the number of achenes formed, should be included among the direct components of yield. Hybrids were introduced in the U.S. and Europe in the 1970s, yielding about 20% more than the open-pollinated cultivars that they replaced. Genetic improvements in seed yield since the 1970s have been estimated to be more than 1% per year in the U.S. (Fick, 1985), 1.17% in Romania (Vranceanu et al., 1988), and 1.25% in France (Bonari et al., 1992). Domestication and breeding for yield potential has favored plant types, but reduced the plant’s responsiveness to interspecific competition (Evans, 1993). Sunflower cultivars released in Argentina between 1930 and 1995 (Lopez Pereira et al., 1999a, 1999b) showed that changes in phenology and partitioning to yield components accounted for most of the variation in yield potential. 5.6.1.2 Protein Significant genetic variation exists for the protein concentration in the achenes. When breeding for protein concentration, the findings of Diakov (1986) showed that a breeder should give preference to genotypes in which the kernel yield (per ha) increases with the increase in protein concentration, while the oil concentration decreases. Another important problem in sunflower breeding for protein quality is the elimination of chlorogenic acid. Protein content of achenes and kernels has been reported to vary from 90 to 240 g/kg, and kernels from 240 to 400 g/kg (Fick, 1978). Breeding to improve protein content and amino acid balance of sunflower meal has received considerable attention. Compared with soybean meal, sunflower meal is generally lower in protein and lysine content, but higher in methionine (Fick and Miller, 1997). Selection for high protein usually results in lower oil content because of a negative correlation between the two traits. Breeding to improve protein content of sunflower kernels from about 240 to near 400 g/kg while maintaining acceptable oil content appears to be a realistic objective (Ivanov and Stoyanoova, 1978). Sunflower meal is used primarily as an animal food protein concentrate. Protein content varies with the hull content of the meal, ranging from 280 g/kg for meal with hulled seeds to as high as 420 g/kg for meal from completely dehulled seeds. Fiber percentage ranges from 14 to 28%. Color of the meal varies from gray to black, depending on the percentage of hulls and the heat and treatment during oil extraction. Sunflower protein ingredients derived from dehulled kernels have been evaluated extensively for use in human food both in Europe and in the U.S. (Lusas, 1982). Sunflower flour and protein concentrates and isolates show promise and are used to a limited extent in bakery

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products, infant formula, meat, and meat extenders. The sunflower meal can also have a greenish color due to the high concentration of chlorogenic acid. 5.6.1.3 Oil The oil extracted for the achene contributes about 80% of the total value of the oilseed sunflower crop (Fick and Miller, 1997). Oil content depends on both the percentage of hull and the oil concentration in the kernel. About two thirds of the increase in achene oil from past breeding and selection has resulted from the reduction in hull percentage, and about one third from an increase in kernel oil content (Gundaev, 1966). Oil accumulation in sunflower seed begins the day after flowering and continues until physiological maturity. The period of most intensive oil accumulation takes place between the 15th and 22nd days after the beginning of flowering. The oil concentration in sunflower may be reaching a plateau, but most breeders believe that selecting for higher oil content is still a very important and realistic objective. Oil concentration in seed is a quantitative trait affected by environmental factors, especially at the seed-filling stage and the genetic makeup of the hybrid. The heritability of oil concentration is relatively high, and progress has been made in increasing oil content in sunflower. Oil concentration in the achene depends on mean daily temperature and available moisture at achene filling, as well as the duration of filling. The accumulation of oil in seeds is intensified by a mean daily temperature below 25˚C, sufficient moisture, and the absence of diseases. 5.6.1.4 Oil Quality 5.6.1.4.1 Fatty Acid Composition Sunflower oil is composed of triacylglycerols that exist in the liquid form at room temperature and have a low melting point. Traditional sunflower oil is composed primarily of saturated palmitic and stearic, monounsaturated oleic, and polyunsaturated linoleic fatty acids, with oleic and linoleic accounting for about 87% of the total. There is an inverse relationship between oleic and linoleic acid that is highly influenced by the environment, especially high temperature effects on oleic acid during grain filling. The levels of palmitic and stearic saturated fatty acids are about 50 and 60 g/kg, respectively. This is considered moderate for an oilseed. Breeding to modify the oil quality of sunflower oil received little attention until Soldatov (1976) created a high oleic variety with up to 900 g/kg using mutation. High oleic oil has steadily gained a share of the sunflower market, especially for food and industrial purposes where a high level of oxidative stability is required. Breeders have also incorporated the high oleic trait into non-oilseed sunflower, where it has been shown to enhance the shelf-life of sunflower kernels (Hettiarachchy et al., 1989). In early 1995, the initial idea to redesign the traditional sunflower oil to contain a mid-level oleic acid content was suggested by representatives from the snack food and oil processing industry in the U.S. (Vick and Miller, 2002). The new oil, called NuSun, has 650 g/kg oleic acid, 260 g/kg linoleic acid, 46 g/kg palmitic acid, and 43 g/kg stearic acid. The NuSun oil has a longer shelf-life and increased stability during frying. Unlike soybean and canola oil, the linolenic acid concentration in NuSun oil is negligible, which eliminates the need for hydrogenation, resulting in the formation of trans fatty acids during processing. Trans fatty acids are associated with an increased risk of cardiovascular heart disease. NuSun oil is derived from sunflower hybrids produced by conventional breeding methods and is not a product of a genetically modified organism (GMO). 5.6.1.4.2 Tocopherol Composition Tocopherols (vitamin E) are the most important and powerful natural fat-soluble antioxidants that inhibit lipid oxidation in foods and biological systems. Since commercial interest in tocopherols

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and their antioxidative properties has increased in recent years, enhancing this component of sunflower oil has become a breeding objective. Alpha-tocopherol is the principal tocopherol in sunflower oil, usually representing over 900 g/kg of the total tocopherols. Since it is the weakest antioxidant in vitro, its partial replacement by other tocopherol forms, beta, gamma, and delta, is an important breeding objective in sunflower. Dolde et al. (1999) screened 12 genetically modified sunflower oils for total tocopherols with values ranging from 534 to 1858 μg/g, with alpha-tocopherol the predominant form and betatocopherol found in only two samples at very low levels. Tocopherol concentration does not appear to be related to saturated fatty acid concentration, so breeding for both can be accomplished at the same time. After evaluation of 252 sunflower varieties and inbred lines, Demurin (1993) developed the first high beta-tocopherol line, LG-15, with beta-tocopherol comprising 500 g/kg of the total tocopherols, and the first high gamma-lines, LG-17 and LG-24, with tocopherol contents of 950 and 840 g/kg gamma-tocopherol, respectively. He concluded that two nonallelic unlinked genes, Tph1 and Tph2, control tocopherol composition and are double recessive. A partial substitution of alpha-tocopherol by another tocopherol derivative with greater antioxidant action would improve the oxidative stability of sunflower oil. Velasco and FernándezMartínez (2003), after screening 952 germplasm accessions, developed T589 with 300 to 490 g/kg beta-tocopherol and T2100 with 880 to 940 g/kg gamma-tocopherol of the total tocopherol composition. The gene control of the tocopherols was a partially recessive gene and had alleles at a single locus. Two germplasm lines, T589 with tocopherol compositions of 340 to 542 g/kg betatocopherol and T2100 with 850 g/kg gamma-tocopherol have been released (Velasco et al., 2004b) New variations of the tocopherol profile have been created by the use of the chemical mutagen ethyl methanesulfate (EMS) and also by recombination (Velasco et al., 2004a). From a total of 2000 treated seeds of four ‘Peredovik’ accessions, IAST-540 was selected in M3 and IAST-1 in M5 with gamma-tocopherol content of 940 g/kg. Crosses between IAST-1 and T589 led to the production of IAST-5, with 700 g/kg alpha- and 300 g/kg beta-tocopherol content, and IAST-4 with respective alpha-, beta-, gamma-, and delta-tocopherol contents of 40, 30, 340, and 580 g/kg of the total tocopherol composition. 5.6.2

Breeding for Adaptation

5.6.2.1 Plant Type When developing sunflower hybrids, the basic goal is to maximize oil production per unit of area. Sunflower hybrids are genetically less diverse than varietal populations, and thus hybrids with unique adaptive characters are required for each agroclimatological region. For example, earliness is an important characteristic for northern regions of Europe. In breeding for earliness, it is necessary to shorten the period between budding and the beginning of flowering. According to Roath and Miller (1981), the sunflower ideotype for the U.S. would include high levels of pest resistance and improved achene and kernel characteristics. The ideotype that is adaptable to all environments may not exist, and simultaneously breeding for a large number of traits is difficult, but breeders should direct their breeding efforts in directions that maximize production for specific geographic regions. A generally accepted ideotype of a productive sunflower is a medium plant height of 160 to 180 cm. Plant phenotypes with a stem height of 120 to 150 cm have been developed and are referred to as semidwarf hybrids, while dwarf sunflowers are 80 to 120 cm tall. Shorter hybrids with better resistance to lodging may have advantages where heavy rains and strong winds occur during the growing season, especially during and after grain filling. The limiting factor for development of productive shorter hybrids is the limited source of dwarfing genes. A “stay green” trait has been associated with increased tolerance to some stem diseases. Stems of plants with this trait stay green well into physiological maturity. Fewer stem infections are

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observed on these plants. The negative aspect of this character is that the stem remains green at maturity, which is undesirable for harvesting. A medium-sized head (20 to 25 cm in diameter), thin and flat on the upper side, with achenes having good contact with the head, is considered optimum for high yield. Increasing head size beyond this point can decrease kernel yield (g/plant) and oil concentration in the achenes and may simultaneously increase the percentage of hull and the incidence of empty seeds. Head shape also determines the number of disk flowers, as well as the number of empty achenes in the center of the head. Head angle is also important depending on where the sunflower is grown. Heads that are inclined at 45° angles and that remain above the foliage may reduce the incidence of Sclerotinia head rot (Fick and Miller, 1997). A concave-shaped head, head inclination parallel to the soil surface, and a stalk angle that places the head away from the leaves and leaf petioles may discourage bird depredation. Heads that are parallel to the soil surface may prevent sunscald in production areas with high temperatures and intense sunlight. Sunflower hybrids should develop the maximum leaf area in the shortest possible time and should maintain it as long as possible. Vertical and horizontal arrangements of leaves on the stem should allow maximum absorption of sunlight, good CO2 uptake, and optimum air circulation inside of the canopy. In addition to assimilative area and leaf number per plant, leaf area index, which is the product of plant leaf area and plant populations, is also important. Harvest index (HI) is another measure of plant productivity. This is the seed yield divided by the total dry matter yield of the plant. Early varieties such as ‘Peredovik’ had an HI of about 0.20, while hybrid 894 has an HI of 0.40 and semidwarf hybrids have a 0.36 HI (Seiler, 1988). A change in HI is most easily obtained by reducing the length of the stem, a trait that increases the resistance to lodging. 5.6.2.2 Phenology The scales that have been devised to quantify the phenological development of single-headed sunflower include both microscopic (Marc and Palmer, 1978, 1981) and macroscopic criteria (Schneiter and Miller, 1981). Connor and Sadras (1992) describe the following phenostages: sowing, germination, emergence, floral initiation, floret production, last anthesis, and physiological maturity. Floral initiation is the first crucial step marking the end of leaf production and the transition from vegetative to reproductive development. Temperature and moisture play crucial roles in all growth stages. One of the more critical stages for moisture and temperature is the reproductive stage. Rawson et al. (1984) showed that an increase in temperature from 15 to 27°C reduced the duration of floral initiation to anthesis by 19%, from 47 to 38 days, with high radiation, and by 47%, from 59 to 31 days, with low radiation. Because achene number (per plant or per area) is a major determinant of yield, each stage in the formation and growth of florets establishes one component of the changing yield potential of the crop. Genotype and environment determines the rate and duration of achene filling, which continues from last anthesis to physiological maturity. Crop growth and mobilization of assimilates during this phase determine the extent of seed filling, and hence the degree of realization of potential yield. Fast dry-down after physiological maturity is a desirable character for rapid harvest in areas where climatic conditions frequently change rapidly. The number of days from planting to maturity varies widely among sunflower cultivars, with a range from 75 to 150 days (Fick, 1978). Cultivars that mature in less than 100 days are adapted to many northern regions, areas where drought may occur during the latter part of the growing season, and in double-cropping systems. Conversely, in areas with long and favorable growing conditions, hybrids that mature in 120 to 150 days often have a yield advantage. Medium-maturing cultivars (100 to 120 days) appear to be best suited for the temperate continental climates of the U.S.

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Yield Potential and Stability

High achene yield depends on many factors, including suitable agronomic characteristics, tolerance to stress environments, and resistance to diseases, insects, and other pests. Generally, characteristics that contribute to good vegetative plant growth are associated with high yield. Sunflower possesses much genetic variability for achene yield. It is a complex trait that depends on a number of components, which are greatly influenced by the environment. Because of the importance of environmental effects, the heritability for achene yields is relatively low compared with other agronomic traits. In sunflower, the final components of achene and oil yield per unit area (ha) are number of plants per ha (55,000 to 60,000), number of achenes per plant (more than 1500), test weight (45 to 50 kg/hl), 1000-achene weight (more than 80 g), low hull content (20 to 24%), high oil concentration in achenes (over 500 g/kg), medium height and maturity, an upright head, resistance to the prevalent diseases and pests, and tolerance to drought. If the values previously mentioned are met, more than 2000 kg of oil per ha can be obtained (Skoric, 1992). The number of achenes per head is determined by the number of disk flowers formed and the degree of self-compatibility. A high degree of self-fertility is also considered important for high yield in many areas, especially where insect pollinator populations are limiting. When screening for combining ability, those hybrid combinations that express a high heterotic effect for number of achenes per head should be selected. To achieve high achene yield per unit area, many breeders consider it essential to develop a genotype capable of providing more than 1500 achenes per head even when grown at high densities. A head size of 20 to 25 cm and shape (flat) are important in attaining this goal. Knowledge of relationships among individual yield components and yield and the heritability of each component would provide a better understanding of the inheritance of achene yield. It is apparent that both additive and nonadditive genetic effects are important in controlling yield of sunflower. Breeding methods such as recurrent selection for general combining ability, reciprocal recurrent selection, or reciprocal full-sib selection capitalize on the additive genetic variance present in sunflower lines (Miller and Fick, 1997). To test for the nonadditive portion of genetic variance, procedures that involve some form of test cross evaluation, such as recurrent selection for specific combining ability, would be more effective in improving seed yield. Yield components often associated with achene yield in sunflower are the number of achenes per head, achene weight, and head diameter. While most breeding efforts have centered on the improvement of oilseed hybrids, significant research has also been conducted on non-oilseed confection sunflower types. Important objectives in breeding non-oilseed types include large achene size, a high kernel-to-hull ratio, and uniformity in achene size, shape, and color. Globally, yields of sunflower have declined progressively since 1993 (Kleingartner, 2004). This is likely due to the shifting of production to geographic regions with less favorable environments, and not to the inability of breeding programs to increase yield potential. 5.6.4

Improved Resistance to Biotic Constraints

5.6.4.1 Diseases Diseases limit production in a majority of sunflower-producing countries. Sunflower is a host to a wide array of diseases that can cause serious economic damage in terms of yield and quality, with the fungal diseases the most numerous and economically serious. In the U.S., the major diseases of concern are downy mildew, rust, Sclerotinia head and stalk rot, and Phoma black stem. Verticillium wilt, Phomopsis stem canker, Alternaria leaf spot, Septoria leaf spot, charcoal stem rot, and Rhizopus head rot occur to a lesser degree. In Europe and adjacent Mediterranean countries, downy mildew,

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Sclerotinia head rot, Phomopsis, Botrytis gray rot, and charcoal rot are considered the most important diseases. Some diseases are important in only a few countries, such as Verticillium wilt in Argentina and white rust (Albugo) in South Africa. The relative severity of individual diseases varies widely, depending on climate and host cultivars. Breeding for resistance is considered the most effective means of control. Sources of resistance or improved levels of tolerance for most diseases are available among the cultivated sunflower and the wild species of Helianthus. Sunflower rust, a foliar pathogen, occurs in virtually all sunflower-growing regions. Genetic resistance to the prevailing North American races of rust has been identified in three wild annual species, H. annuus, H. petiolaris, and H. argophyllus (Jan et al., 2004b). Genes for rust resistance are frequent in the wild progenitors of the cultivated sunflower (Quresh et al., 1993). In most cases, rust resistance appears to be conditioned by single dominant genes. Downy mildew occurs in most countries where sunflower is grown, with the apparent exception of Australia. The pathogen is unique in that it infects the seedling roots to initiate a systemic, often terminal disease, while airborne spores cause only local lesions. Until recently, fungicide seed treatments, such as metalaxyl and mefonoxim, were used to control downy mildew, but the fungus has developed resistance to the chemicals. Downy mildew can be controlled by single, race-specific dominant resistance genes. Multi-race-resistant and single-race-resistant germplasms have been developed from wild sunflower species (Miller and Gulya, 1988; Tan et al., 1992; Jan et al., 2004c). Wild Helianthus annuus, H. petiolaris, and H. praecox are sources of single dominant genes for single race resistance, while H. argophyllus is the source of dominant genes for all known races of the fungus (Miller and Gulya, 1988; Miller et al., 2002). Sclerotinia wilt (white mold) causes the greatest losses to sunflower on a global basis. This is in part due to the wide host range of Sclerotinia sclerotiorum, a facultative parasite that attacks 360 species of plants. Cultivated sunflower is highly sensitive to this pathogen, whose attack is manifested in several forms with destruction of the root, stem, head, and seeds. So far there is no efficient and effective chemical control for the pathogen. It appears that Sclerotinia resistance is complex and controlled polygenically, involving many genes, each with small effects. This means that the breeding strategy needs to be quite different than those for other diseases. There are reports of identification of cultivated sunflower genotypes with low susceptibility or moderate resistance to Sclerotinia wild mold. Wild species have also been identified as a potential source of genes for Sclerotinia tolerance. Interspecific hybrids with perennial H. maximiliani (Maximilian’s sunflower) exhibited higher levels of resistance than head rot-resistant inbred lines (Cerboncini et al., 2002; Ronicke et al., 2004). Rashid and Seiler (2004) identified potential sources of Sclerotinia head and stem rot resistance in populations of perennial H. maximiliani and H. nuttallii from Canada. Perennial H. resinosus has been identified as a good source for resistance to Sclerotinia head rot by Mondolot-Casson and Andary (1994). The Sclerotinia disease complex appears to be very complicated. The prospect of finding a single dominant gene for resistance does not look promising, but progress is being made in the development of germplasm with increased tolerance to Sclerotinia head rot. Currently there are no commercial hybrids that possess a satisfactory level of resistance to Sclerotinia rot. Some progress has been made in increasing the resistance to mid-stalk Sclerotinia rot in cultivated sunflower. Kohler and Freidt (1999) indicated that progenies of interspecific crosses with H. mollis and H. tuberosus had increased levels of tolerance to mid-stalk white mold infection. Miller and Gulya (1999) developed four maintainer and four restorer oilseed lines with improved tolerance to midstalk Sclerotinia rot. Sclerotinia sclerotiorum generates substantial quantities of oxalic acid, which has been identified as one of the key components in the infection process. One strategy for resistance is to obtain plants that are resistant to free oxalic acid by engineering them to degrade it. A wheat (Triticum aestivum L.) oxalate oxidase gene (OXOX) has been identified and transferred into sunflower via transformation (Scelonge et al., 2000). A transgenic sunflower line, H. annuus cv. SMF3, constitutively expressed the wheat OXOX gene (Hu et al., 2003) and exhibited enhanced resistance against

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the oxalic acid-generating fungus Sclerotinia. This approach to white mold resistance in sunflower awaits further testing and commercialization. Verticillium wilt is caused by soilborne fungus that infects sunflower roots, causing wilting and leaf mottling. The disease is especially severe in Argentina. Resistance to the disease can be controlled by single genes; however, virulent strains of Verticillium wilt have been identified recently, prompting a search for additional resistance genes. Helianthus annuus, H. petiolaris, and H. praecox were the major sources of genes (V-1) for Verticillium wilt resistance prior to the recent identification of a new strain. Since there is no known chemical, biological, or cultural control of Verticillium on sunflower, it is imperative to determine whether resistance to the new strains exists in the known germplasm, both cultivated and wild species. Phomopsis brown stem canker was first discovered in sunflower in Yugoslavia in 1980 and and now is considered a serious problem in much of Europe (Mihaljcevic et al., 1982; Acimovic, 1984; and Skoric, 1985). Cuk (1982) reported that wild H. debilis and H. pauciflorus are potential sources of resistance to P. helianthi. Kurnik and Walcz (1985) reported resistance to stem canker in H. argophyllus, tolerance in two other wild species, and susceptibility in local populations of H. tuberosus. Skoric (1985) also reported tolerance in four inbred lines (two based on H. tuberosus, one on H. annuus, and one on H. argophyllus). Dozet (1990) observed a high degree of resistance in two populations of H. tuberosus. Cultivated hybrids developed from H. tuberosus and H. argophyllus have high field tolerance to Phomopsis brown stem canker (Skoric, 1985). Skoric (1985) hypothesized that the resistance may be controlled by two or more complementary genes. Resistance is associated with the stay-green stem character and with resistance to charcoal rot, Phoma black stem, and drought. Alternaria leaf spot causes losses in cultivated sunflower in the U.S. and other parts of the world. In warm climates with high rainfalls, it causes defoliation and reduces yield significantly (Sackston, 1981). All 21 annual taxa and 18 of 21 perennial species are susceptible to A. helianthi spores applied in a suspension. Perennial species H. hirsutus, H. pauciflorus subsp. subrhomboideus, and H. tuberosus appear to resist infection by A. helianthi (Morris et al., 1983). Lipps and Herr (1986) showed that 13 accessions of H. tuberosus had significantly less Alternaria leaf spot than commercial hybrids and concluded that the species is a potential source of resistance to leaf spot. Several wild annual species, H. praecox, H. × laetiflorus, H. debilis subsp. cucumerifolius, and H. debilis subsp. silvestris, had high levels of resistance to Alternaria and Septoria helianthi Ellis and Kellerm. in field evaluations (Block, 1992). Although potential sources of resistance to Alternaria have been identified, resistance genes have not been transferred to cultivated lines. Powdery mildew is a widely distributed pathogen of cultivated sunflower in warmer regions of the world (Zimmer and Hoes, 1978). This foliar disease is found mostly on senescing leaves and is generally not of major economic concern. Helianthus debilis subsp. silvestris, H. praecox subsp. praecox, and H. bolanderi and 14 perennial species exhibited powdery mildew tolerance in both field and greenhouse tests (Saliman et al., 1982). Not all populations of some perennial species are resistant; populations of H. grosseserratus and H. maximiliani showed differential reactions. Jan and Chandler (1985a) characterized resistance to powdery mildew from H. debilis subsp. debilis as incompletely dominant. They incorporated genes from this species into a cultivated background and have released a germplasm pool having the PM1 gene (Jan and Chandler, 1988). Rhizopus head rot is an important sunflower disease in arid regions. The Rhizopus pathogen complex consists of three species, with Rhizopus arrhizus the more prevalent and virulent species. Currently cultivated sunflower does not possess resistance to Rhizopus head rot. Yang et al. (1980) reported that 4 of 32 wild species and subspecies tested were resistant when inoculated with R. arrhizus and R. oryzae Went. The resistant sources were H. divaricatus, H. hirsutus, H. × laetiflorus, and H. resinosus. Further breeding will be needed to transfer the identified sources of resistance into cultivated sunflower. Phoma black stem is present in all sunflower-producing regions of the world. In some regions, such as the U.S., the appearance of the disease is late in the season, resulting in very little economic

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loss. So far, most genotypes of sunflower have exhibited susceptibility to the pathogen. It has been suggested that resistance could be associated with the stay-green stem character, and with resistance to Phomopsis stem canker. Under natural infection, wild sunflower species H. maximiliani, H. argophyllus, H. tuberous, and H. pauciflorus possess excellent resistance to Phoma black stem (Skoric, 1992). Charcoal rot (Macrophomina phaseolina (Tassi) Goid) attacks sunflower and other crops in warm climates on all continents. Interspecific lines based on H. tuberous have resistance to charcoal rot. Wild species H. mollis, H. maximiliani, H. resinosus, H. tuberous, and H. pauciflorus have also shown resistance. The number of genes and the inheritance of resistance to the pathogen have not been ascertained, although resistance appears to be dominant. Broomrape (Orobanche cumana Wallr.) is a parasitic weed that infects sunflower roots, causing severe crop losses in southern Europe and the Black Sea region. It has also been observed in Australia, Mongolia, and China and is generally associated with drier climates. Five resistance genes (Or1 through Or5) have been used successfully for broomrape control following the progression of races A through E. Since broomrape is a highly variable pathogen, the breakdown of resistance is a frequent phenomenon, and multiple sources of resistance are needed. Ruso et al. (1996) evaluated wild annual and perennial sunflower species’ reaction to Spanish races and found two annual species, H. anomalous and H. exilis, that had resistance, and all 26 perennial species were resistant. Recent studies indicated the development of a new broomrape race in Spain, designated race F, which attacks all commercial sunflower hybrids, overcoming the previously effective resistance genes (Domínguez et al., 1996). High levels of resistance to race F have been observed in populations of wild perennial sunflower (Fernández-Martínez et al., 2000). Jan et al. (2002) have released four race F-resistant germplasms, BR1 through BR4, which were derived from wild perennial sunflowers H. maximiliani, H. grossesserratus, and H. divaricatus. Fernández-Martínez et al. (2004) released four sunflower germplasms, K-96, L-86, P-96, and R-96, with resistance to race F based on cultivated sunflower from eastern Europe. Resistance to race F appears to be controlled by dominant-recessive epistasis, complicating the breeding by requiring the genes to be incorporated into both parental lines of a resistant hybrid (Akhtouch et al., 2002). Other germplasms have been released that have resistance to various races (other than race F) of broomrape, including seven germplasms based on cultivated sunflower from the FSU, Romania, and Turkey (Miller and Domínguez, 2000). 5.6.4.2 Viruses and Bacteria Bacterial foliar diseases, including apical chlorosis (Pseudomonas syringae pv. tagetis (Hellmers) Young, Dye, and Wilkie) and bacterial blight (P. syringae pv. helianthi (Kawamura) Young, Die, and Wilkie), generally have little economic impact on sunflower (Gulya, 1982). Sunflower can be infected by over 30 viruses, but viral diseases are generally of concern only in tropical or subtropical climates, such as India, where tobacco streak virus is a problem. In North America, sunflower mosaic virus and sunflower chlorotic mottle virus are rarely seen on sunflower, with only sunflower mosaic virus noted on wild sunflower in Texas, but sources of resistance are available from wild H. annuus to produce resistant hybrids if necessary (Gulya et al., 2002). 5.6.4.3 Pests 5.6.4.3.1 Insects Sunflower is utilized by a large array of insects as a source of food or shelter. Although a number of North America insect species colonize sunflower under cultivated conditions and become economic pests, many others are held in check by a variety of factors, including natural enemies.

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North America has the greatest problems with insect pests because the insect pests of sunflower have coevolved with their native sunflower hosts in natural communities. On other continents, insects are generally considered minimal or infrequent problems, and when they occur, they are generally caused by omnivorous insects such as aphids, plant bugs (Lygus spp.), and other nonsunflower-specific insects. In the major production area of North America, there are about 15 principal insect pests of cultivated sunflower, and of this total, about 6 are considered of major importance as potential economic pests from year to year (Charlet and Brewer, 1997). The insects of main concern include the sunflower beetle, the sunflower stem weevil, the red and gray seed weevils (Smicronyx fulvus (LeConte) and S. sordidus (LeConte)), the banded sunflower moth, Cochylis hospes Walsingham, the sunflower moth, and the sunflower midge (Contarinia schulzi Gagne). Host plant resistance is a pest management method that utilizes the plant’s own defense mechanism against the insect. The first step in this process is to identify sources of resistance. Since wild sunflowers are native to North America, where their associated herbivores and entomophages coevolved, there is an opportunity to search for insect resistance genes in the diverse wild species. Sunflower moth tolerance was observed in annual H. petiolaris and perennials H. maximiliani, H. ciliaris, H. strumosus, and H. tuberosus (Rogers et al., 1984). Stem weevil tolerance was found in perennials H. grosseserratus, H. hirsutus, H. maximiliani, H. pauciflorus, H. salicifolius, and H. tuberosus (Rogers and Seiler, 1985). Sunflower beetle tolerance was observed in annuals H. agrestis and H. praecox and in perennials H. grosseserratus, H. pauciflorus, H. salicifolius, and H. tuberosus (Rogers and Thompson, 1978, 1980). Charlet and Seiler (1994) found indications of resistance to the red sunflower seed weevil in several native Helianthus species. Interspecific germplasms using wild species as resistance sources have been created. In preliminary testing, Charlet et al. (2004) noted that germplasm derived from H. petiolaris had the lowest number of stem weevils. Among material tested in a banded sunflower moth evaluation nursery, germplasm derived from H. praecox subsp. hirtus had less than 2% damage. Germplasm that incorporated H. strumosus and H. tuberosus had very little red sunflower seed weevil damage in test plots. Breeding populations of promising germplasms are being developed for further testing. Identification and transfer of insect resistance genes from the wild species have been much less successful than genes for disease resistance. The reason for the lack of success is not clear, but may be related to the fact that the insect resistance genes do not appear to be simple dominant genes and are more difficult to transfer and maintain in the breeding process. 5.6.4.3.2 Bird Depredation Sunflower seed losses from bird feeding can be significant. Losses have been estimated between 2 and 5% of the crop in the U.S., but can be as much as 100% in concentrated areas. Birds ranging in size from sparrows (Passeridae) to large parrots (Psitticidae) are a constant problem for sunflower production on all continents. In the U.S., the migratory red-winged black bird (Agelaius phoeniceus L.) causes the most damage when fields are located next to breeding and nesting areas. In Europe, many different sparrows (Passer spp.) and doves (Streptopelia spp.) are the major problems, while in South America, parakeets and doves (Columbidae) predominate. The differences in tolerance to bird depredation reported have been based on morphological traits. The resistance has been associated with traits such as concave-shaped heads, heads that are parallel to the soil surface, long involucral bracts, a long head-to-stem distance, and achenes that are tightly held in the receptacle at maturity. Three bird-resistant germplasms, BRS-1 through BRS-3, have been developed with white hulls, long warping bracts, flat or concave heads, and neck lengths exceeding 23 cm (USDA, 2005b). Hanzel and Gulya (1993) also developed two birdresistant oilseed germplasm lines, NDBR1 and NDBR2, with the resistance conditioned by concaveshaped heads, downward-facing heads, and increased head-to-stem distance, with hybrids derived from these germplasms reducing bird depredation by more than 50%. Based on preliminary

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inheritance studies, they concluded that expression of the bird resistance traits appears to be largely determined by nonadditive genetic effects. Fox and Linz (1983) stated that purple-hulled genotypes high in anthocyanin appeared to avert or deter achene depredation by red-winged black birds, possibly because of aversions to the taste of anthocyanin pigment. 5.6.5

Improved Resistance to Abiotic Constraints

5.6.5.1 Salt and Drought Tolerance Several species of Helianthus are native to salt-impacted habitats. Interspecific germplasm derived from H. paradoxus has been identified with high salt tolerances, withstanding salt concentrations up to electrical conductivity (EC) = 24.7 d/Sm. It appears that one major gene controls salt tolerance, although a modifier gene may also be present, possibly recessive in control (Miller, 1995). Two salt-tolerant parental oilseed maintainer lines, HA 429 and HA 430, have been released (Miller and Seiler, 2003). Mechanisms enabling plants to survive stress have been favored during plant evolution, sometimes at the expense of plant productivity in the absence of the stress (Turner, 1979). In natural ecosystems, a plant’s ability to survive environmental stress is probably more important than high seed (grain) productivity. During the process of selecting plants for high seed yield, breeders may have inadvertently lost some drought survival mechanisms common in wild species, and could possibly benefit from the infusion of germplasm from the wild species and selection to enhance drought tolerance in cultivated sunflower. Stomatal responses have been suggested as potentially useful traits to consider in developing plants with improved water use efficiency. Evaluation of 19 perennial and 1 annual wild sunflower species indicated that perennial species had higher diffusive resistance, transpiration, and stomatal densities than annual species (Seiler, 1983). In all perennial species, stomatal densities were higher on the bottom leaf surface than the top surface, similar to cultivated sunflower, while it was opposite in the wild annual species. Blanchet and Gelfi (1980) evaluated stomatal resistance, leaf water potential, photosynthetic activity, leaf structure, and number of stomata. They concluded that H. argophyllus is the best candidate source for drought tolerance genes because its pubescent leaves reflect sunlight, reduce water loss, and exhibit low transpiration rates. H. niveus subsp. canescens was their second choice. 5.6.5.2 Herbicide Tolerance A wild population of annual H. annuus from a soybean field in Kansas that had been repeatedly treated with imazethapyr for seven consecutive years developed resistance to the imidazolinone and sulfonylurea herbicides (Al-Khatib et al., 1998). Resistance to imazethapyr and imazamox herbicides has great potential for producers in all regions of the world for controlling several broadleaf weeds. Several populations of wild sunflower (H. annuus and H. petiolaris) from the U.S. and Canada have been screened for resistance to these two herbicides. Eight percent of 50 wild sunflower populations had some resistance to imazamox, and 57% had some resistance to tribenuron in the central U.S. (Olson et al., 2004). In Canada, 52% of 23 wild H. annuus populations had some resistance to tribenuron (Miller and Seiler, 2005). Genetic stocks IMISUN-1 (oil maintainer), IMISUN-2 (oil restorer), and IMISUN-3 (confection maintainer) have been developed and released (Al-Khatib and Miller, 2000). Miller and Al-Khatib (2002) also released one oilseed maintainer and two fertility restorer breeding lines with imidazolinone herbicide resistance. Genetic stocks SURES-1 and SURES-2 with resistance to the sulfonylurea herbicide tribenuron have been developed and released by Miller and Al-Khatib (2004). In addition, the two herbicides may control broomrape in areas of the world where this parasitic weed attacks sunflower (Alonso et al., 1998).

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5.6.5.3 Soil Nutrition Cadmium, a toxic trace element that naturally occurs in all soils, can be absorbed easily from the soil and translocated to food crops. Confection sunflowers (non-oilseed types used for their kernels) are natural accumulators of Cd compared to other grains, leading to concerns about the consumption of sunflower kernels. High clay soils and those containing high chloride levels can produce sunflower kernels with cadmium levels of 1.33 mg/kg (dry weight), exceeding the levels established in northern Europe of 0.6 mg/kg. Li et al. (1995a) screened 200 genotypes, including USDA Agricultural Research Service germplasm lines, plant introductions from various countries, and interspecific germplasm lines for Cd accumulation. Concentrations varied from 0.31 to 1.34 mg/kg, indicating that selection for reduced Cd levels in sunflower kernels was feasible. Li et al. (1995b) assessed the importance of combining ability and heterosis in the inheritance of sunflower kernel Cd levels and concluded that additive genetic effects predominately influenced the expression of kernel Cd accumulation in the hybrids. Crosses among the accessions and lines were initiated and resulted in the development of lower Cd genetic stocks. These lines are in the process of being released and registered (Miller, 2005).

5.7 GERMPLASM CHARACTERIZATION: MOLECULAR APPLICATIONS Molecular markers provide an effective means for characterizing genetic variability and establishing phylogenetic relationships among cultivated and wild Helianthus species. Markers linked with both qualitative and quantitative traits and genes will facilitate marker-assisted selection, and eventually lead to the cloning and manipulation of desirable genes. 5.7.1

Genetic Diversity

Using restriction fragment length polymorphism (RFLP) markers on 17 sunflower inbred lines, Gentzbittel et al. (1994) reported a lower available genetic variability in cultivated sunflower than in other crops, suggesting that efforts to introgress new genes from wild sunflower species should be increased. However, Berry et al. (1994), also using RFLP on 24 inbred lines, indicated much greater nuclear DNA polymorphism, and a cluster analysis clearly separated maintainer and restoration lines, reflecting breeding strategies that maximize heterosis. Amplified fragment length polymorphism (AFLP) analysis has been used to fingerprint 24 public inbred lines to describe their genetic diversity and to place the lines into heterotic groups as the basis for creating single-cross hybrids (Hongtrakul et al., 1997). More recently, simple sequence repeat (SSR) markers have been developed for sunflower and used to characterize the genetic diversity among 16 elite inbred lines and among 19 elite inbred lines and 28 domestic and wild germplasm accessions, including Native American landraces (Paniego et al., 2002; Yu et al., 2002; Tang and Knapp, 2003). AFLP markers have also been suggested as a rapid and robust marker system with a high degree of resolution for studying the genetic diversity of Orobanche cumana (broomrape), a parasite on sunflower (Gagne et al., 2000). Hybrid sunflower production based on a single source of cytoplasmic male sterility (CMS), cmsPET1 (French cytoplasm) from H. petiolaris, and the Rf1 gene for restoration narrows the genetic base of the crop and increases its vulnerability to pests and environmental stress. New CMS sources and respective fertility restoration genes are continuously being identified, and several studies have examined the nature of CMS sources. Köhler et al. (1991) and Laver et al. (1991) suggested that a new open reading frame, orfH522, in the 3'-flanking region of the atpA gene was associated with the CMS phenotype. It was later demonstrated that the gene product of orfH522, a 16-kDa protein, differed between the male-fertile and male-sterile lines (Monéger et al., 1994;

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Horn et al., 1991). Further studies of the diversity of 28 male sterility-inducing and one male-fertile cytoplasm of Helianthus using nine mtDNA genes and three probes for the open reading frame (Horn, 2002) clearly distinguished CMS sources by their mtDNA organization and CMS mechanism. Molecular similarities among the different CMS sources, together with their reactions to various fertility restoration genes, provide useful guidelines in CMS source selection for developing hybrids with diverse genetic backgrounds. Genetic variability to mid-stem infection of Sclerotinia sclerotiorum (white mold) among interspecific sunflower hybrids using arbitrarily primed polymerase chain reaction (AP-PCR) was demonstrated by Köhler and Friedt (1999). Interspecific hybrid progenies exhibited substantial genetic distance from their parental sunflower inbred lines, and hybrids having reduced Sclerotinia infection were identified. Hu and Vick (2003) developed a new technique, target region amplification polymorphism (TRAP), which uses bioinformatics tools and expressed sequence tag (EST) database information to generate polymorphic markers around targeted gene sequences. This technique has been successfully used to construct a genetic linkage map in wheat (Liu et al., 2005) and in an analyses of molecular variability within a global collection of sunflower downy mildew (Plasmopara halstedii (Farl.) Berl and de Toni) (Chen et al., 2005). Xu et al. (2003) used TRAPs to characterize genetic stocks of tetraploid wheat (Triticum turgidum L.; 2n = 4, x = 28; AABB genomes) and found that a large number of chromosome-specific markers could be identified using this technique. 5.7.2

Molecular Mapping

Sunflower molecular maps have been constructed using RAPD, RFLP, AFLP, and SSR (Rieseberg et al., 1993; Berry et al., 1995; Gentzbittel et al., 1995, 1999; Jan et al., 1998; Gedil et al., 2001; Tang et al., 2002; Yu et al., 2003). Rieseberg et al. (1993) developed a linkage map of H. anomalus with 161 RAPD loci and one isozyme locus to study the hybrid origin and speciation of H. anomalus, based upon similarities to its tentative parental species H. annuus and H. petiolaris. The genetic markers were distributed into 18 linkage groups covering 2338 cM. This report demonstrated the utility of genomic mapping for studying the mode of speciation in sunflower. The RFLP map of Berry et al. (1995) used 213 probes on 289 F2 individuals of a cross between inbred lines HA 89 and ZENB8. The map identified 234 loci covering 1380 cM and 17 linkage groups. Gentzbittel et al. (1995), using three F2 and two BC1F1 populations, developed a consensus map with 157 probes mapped to 23 linkage groups. Jan et al. (1998) produced an RFLP map using 93 F2 individuals of RHA 271 × HA 234, with 20 linkage groups covering 1164 cM of the sunflower genome, based on 271 loci detected by 232 cDNA clones. A composite RFLP map, based upon seven F2 populations, covering 1573 cM, with 17 major linkage groups, and 238 loci was developed by Gentzbittel et al. (1999). An integrated RFLP-AFLP linkage map using 18 AFLP primer combinations, 58 RFLP probes of Berry et al. (1995), and 42 RFLP probes of Jan et al. (1998) was based on 180 F2 plants from the cross HA 370 × HA 372. This map differentiated17 linkage groups with an average density of 3.3 cM and a total length of 1326 cM (Gedil et al., 2001). An SSR genetic map was constructed using 94 RHA 280 × RHA 801 F7 recombinant inbred lines (RILs) and 408 polymorphic SSR markers (Tang et al., 2002). The map was 1368.3 cM long and had a mean density of 3.1 cM per locus. Additional SSR maps and their integration with previous maps were accomplished by Yu et al. (2003) using three mapping populations: RILs of RHA 280 × RHA 801, F2s of PHA × PHB, and F2s of HA 370 × HA 372. These maps crossreferenced linkage groups identified by the RFLP maps of Berry et al. (1995), Gentzbittel et al. (1995), and Jan et al. (1998), and the SSR map of Tang et al. (2002). The integration of these maps substantially increased the number of molecular markers available for molecular breeding and genomic research in sunflower. Bacterial artificial chromosome (BAC) and binary-bacterial artificial

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chromosome (BIBAC) libraries and linkage group-specific clones provide resources and tools essential for comprehensive research of the sunflower genome. 5.7.3

Gene Mapping

The fertility restoration gene Rf1 has been used extensively to restore fertility to cmsPET1, the only CMS source used in hybrid sunflower production. The Rf1 gene has been mapped to linkage group 6 in the RFLP map and the consensus map (Gentzbittel et al., 1995, 1999), and tightly linked RAPD and AFLP markers to the Rf1 gene have been identified by Horn et al. (2003). Analysis using 11 restorer and 9 maintainer lines of cmsPET1 further confirmed the existence of the tightly linked markers in all the restorers, but absent in the maintainers (Horn et al., 2003). Gandhi et al. (2005) mapped the self-incompatibility locus (S) to linkage group 17 of the maps of Tang et al. (2002) and Yu et al. (2003) using NMS 373 × ANN 1811 BC1 F1 plants. In addition, the quantitative trait loci for self-pollination and seed dormancy were mapped to linkage groups 3, 6, 11, and 15. Closely linked markers to these genes provide potential for retaining the high self-compatibility, good seed set, and short dormancy of the cultivated lines while introducing useful wild genes into cultivated populations. Quantitative trait loci (QTL) mapping of Sclerotinia mid-stem rot resistance was conducted using 117 SSR markers on 351 F3 families of a cross between resistant NDBLOS and susceptible CM625 (Micic et al., 2004). However, because their QTL only explained 24.4 to 33.7% of the genotypic variance for the resistance, and estimated effects at most QTL were small, Micic et al. suggested that the prospects of marker-assisted selection for Sclerotinia resistance are limited. A study of oil percentage QTL using 289 F2 plants and RFLP markers located six regions representing a total of 57% genetic variability (Leon et al., 1995). In another study by Leon et al. (2003), eight QTL on seven linkage groups were identified that accounted for 88% of the genetic variation for seed-oil concentration across four environments. Successful utilization of this linkage information in marker-assisted selection is expected in the future. Broomrape is among the most damaging pests of sunflower in Spain and the areas around the Black Sea (Fernández-Martinez et al., 2000). The recent shift from race E to F is threatening the continuing success of the crop in the area. Due to the unavailability of efficient screening techniques, both greenhouse and field, a marker-assisted breeding scheme would be very beneficial. The single dominant gene for resistance to broomrape race E has been mapped to the terminal position of linkage group 3, using SSR markers on 262 recombinant inbred lines of PHC × PHD (Tang et al., 2003). QTL mapping of broomrape resistance to both races E and F was conducted using a line, P-96, with dominant resistance to race E and recessive resistance to race F (Perez-Vich et al., 2004). Five QTL for resistance to race E and six QTL for resistance to race F were detected on 7 of the 17 linkage groups. It was suggested that resistance to broomrape in sunflower is controlled by a combination of qualitative race-specific resistance genes, affecting the presence or absence of broomrape, and a quantitative non-race-specific resistance, affecting the broomrape stalks. The mapping of downy mildew resistance genes has been more successful than any other group of genes in sunflower. The resistance is race specific and is controlled by single dominant genes. There are up to 10 resistance genes described, denoted Pl, carrying resistance to various downy mildew races and mapped to molecular maps. Downy mildew resistance genes Pl1, Pl2, Pl5, Pl6, Pl7, Pl8, and Plarg have been mapped to linkage groups of different maps (Mouzeyar et al., 1995; Roeckel-Drevet et al., 1996; Vear et al., 1997; Bert et al., 2001; Dussle et al., 2004), with their interrelationship established through the work of Yu et al. (2003). Vear et al. (1997) confirmed that Pl6, conferring resistance to all downy mildew races, is a cluster of genes, each providing resistance to specific races. Further molecular analysis using specific PCR-based markers suggested that the Pl6 locus contains at least 11 tightly linked genes, each giving resistance to a specific downy mildew race (Bouzidi et al., 2002). Therefore, these genes are more appropriately characterized as multigenic

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loci comprised of single genes or a cluster of genes resistant to sunflower downy mildew. Pl1, Pl2, Pl6, and Pl7 were mapped to linkage group 1, and Pl5 and Pl8 were mapped to linkage group 6, which corresponds to the linkage groups 8 and 13 of the map by Yu et al. (2003). The Plarg gene is solely mapped to the telomeric region of linkage group 1 of the map by Yu et al. (2003). 5.7.4

BAC Library

Genetic mapping of genes of importance has provided a means for gene isolation through positional cloning, which requires a large-insert DNA library. Large-insert BAC libraries have been widely used as probes for fluorescence in situ hybridization (FISH). Since FISH allows direct observation of a probe hybridization signal on a chromosome, it has become a useful technique for chromosome identification and physical genome mapping (Jiang and Gill, 1994; Dong et al., 1998). Gentzbittel et al. (2002) reported that the first BAC library in sunflower was constructed using HindIII in the pBeloBAC11 vector, had a four- to fivefold genome coverage, and had an average insert size of 80 kb. Another sunflower BAC library was constructed using the same enzyme and vector system, had a 1.9-fold genome coverage, and had an average insert size of 60 kb (Özdemir et al., 2004). More recently, two complementary BAC and BIBAC libraries were constructed from nuclear DNA of sunflower cultivar HA 89 (Feng et al., 2005). The BAC library was constructed with BamHI in the pECBAC1 vector, contains 107,136 clones, and has an average insert size of 140 kb. The BIBAC library was constructed with HindIII in the plant transformation-competent binary vector pCLD04541 and contains 84,864 clones, with an average insert size of 137 kb. The two libraries together contain a total of 192,000 clones and are equivalent to approximately 8.9 haploid genomes of sunflower (3000 Mb/1C), providing a greater than 99% probability of obtaining a particular clone from the libraries. To facilitate chromosome engineering and anchor the sunflower genetic map to chromosomes, single- or low-copy RFLP markers from each linkage group of the sunflower genetic map of Jan et al. (1998) were used to design pairs of overlapping oligonucleotides (overgos), which identified 195 BAC and BIBAC clones representing 19 linkage groups of the genetic map. The BAC and BIBAC libraries and linkage group-specific clones will provide the resources and tools essential for comprehensive genomic research for sunflower. 5.8 CONCLUSIONS AND PROSPECTS The world production of sunflower is estimated at 21 million ha in 60 countries. It is the second largest hybrid crop and the fifth largest oilseed crop. Sunflower cultivation continues to be pushed into lower-fertility soils and other marginal environments where drought and high or low temperatures continually take their toll on the yield per unit area. The challenge for the sunflower breeding community is to breed sunflowers adaptable to these marginal environments and at the same time increase seed yield. Sunflower hybrids have an extremely narrow genetic base, using only a single female parent for all hybrids in the world, making the crop extremely vulnerable to an impending disaster, as seen in maize in the 1970s. There remains a need to increase the genetic diversity of cultivated sunflower due to the marked reduction in genetic diversity during domestication. Wild species of sunflower have been a source of many genes for pest resistance, especially for diseases. They also served as the female parent for all hybrid sunflowers. Since wild sunflower and the sunflower crop are native to North America, associated pests have coevolved in natural communities, thus providing the opportunity to search for pest resistance genes in the diverse wild species. Significant progress has been made in increasing the collection and preservation of wild species, increasing the available genetic diversity for the improvement of the crop. Thus far, only a small portion of the available diversity has been used globally.

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Significant advances have been made in understanding the origin, domestication, and organization of the genetic diversity, characterization, and screening methods for abiotic and biotic stresses. Useful germplasms have been identified for many agronomic traits, and some molecular markers for indirect selection of favorable alleles and QTL are becoming more available. Alternative recombination and selection methods have also been developed. While favorable alleles for a few traits have been incorporated into improved germplasm and hybrids, genes to overcome a large number of production-limiting factors need to be introduced into otherwise high-quality, high-yield parental lines with desirable plant and adaptation traits. This will be possible by introgression of favorable alleles from alien germplasm, pyramiding favorable alleles and QTL for specific traits, and simultaneously improving the maximum number of traits. This will require a multidisciplinary team approach and a commitment to a long-term integrated genetic improvement program. Molecular biology has added to the scope of plant breeding in sunflower, providing an option to manipulate plant expressions. The process has barely begun, but there is great unrealized opportunity to address all aspects of crop production, utilization, and food value. Molecular markerassisted selection is beginning to be used. Similarly, more integrated linkage maps are becoming available. Mapping with molecular markers and investigation of genomes across related plant families will allow both targeted genetic modification and increased efficiencies in breeding for difficult-to-screen traits with marker-assisted selection. The future direction for sunflower may include the transfer of target genes from wild relatives into domesticated sunflower with improved genetic backgrounds adapted to local conditions. New sunflower hybrids will possess pest and disease resistance genes from distantly related or even unrelated plants and other organisms. Researchers will have to strive to combine the best conventional and modern molecular approaches to improve sunflower germplasm to keep sunflower an economically viable global crop.

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Singh, R.J. and T. Tsuchiya. 1981b. Identification and designation of barley chromosomes by Giemsa banding technique: a reconsideration. Z. Pflanzenzücht. 86: 336–340. Singh, R.J. and T. Tsuchiya. 1982a. Identification and designation of telocentric chromosomes in barley by means of Giemsa N-banding technique. Theor. Appl. Genet. 64: 13–24. Singh, R.J. and T. Tsuchiya. 1982b. An improved Giemsa N-banding technique for the identification of barley chromosomes. J. Hered. 73: 227–229. Skoric, D. 1985. Sunflower breeding for resistance to Diaporthe/Phomopsis helianthi Munt.-Cvet. et al. Helia 8: 21–23. Skoric, D. 1987. FAO sunflower sub-network report 1984–1986. In Genetic Evaluation and Use of Helianthus Wild Species and Their Use in Breeding Programs, D. Skoric, Ed. FAO, Rome, pp. 1–17. Skoric, D. 1988. Sunflower breeding. J. Edible Oil Ind. 25: 1–90. Skoric, D. 1992. Achievements and future directions of sunflower breeding. Field Crops Res. 30: 195–230. Slabaugh, M.B., J.K. Yu, S.X. Tang, A. Heesacker, X. Hu, G.H. Lu, D. Bidney, F. Han, and S.J. Knapp. 2003. Haplotyping and mapping a large cluster of downy mildew resistance gene candidates in sunflower using multilocus intron fragment length polymorphisms. Plant Biotechnol. J. 1: 167–185. Smith, D.M. and A.T. Guard. 1958. Hybridization between Helianthus divaricatus and H. microcephalus. Brittonia 10: 137–145. Snow, A.A., P. Moran-Palma, L.H. Rieseberg, A. Wszlaki, and G.J. Seiler. 1998. Fecundity, phenology, and seed dormancy of F1 wild-crop hybrids in sunflower (Helianthus annuus, Asteraceae). Am. J. Bot. 85: 794–801. Snow, A.A., D. Pilson, L.H. Rieseberg, M.J. Paulsen, N. Pleskac, M.R. Reagon, D.E. Wolf, and S.M. Selbo. 2003. A bt transgene reduces herbivory and enhances fecundity in wild sunflowers. Ecol. Appl. 13: 279–286. Soldatov, K.I. 1976. Chemical mutagenesis in sunflower breeding. In Proceedings of the 7th International Sunflower Conference, International Sunflower Association, Paris, pp. 352–357. Takami, S., N.C. Turner, and H.M. Rawson. 1981. Leaf expansion of four sunflower (Helianthus annuus L.) cultivars in relation to water deficits. I. Patterns during plant development. Plant Cell. Environ. 4: 399–407. Tan, A.S., C.C. Jan, and T.J. Gulya. 1992. Inheritance of resistance to race 4 of sunflower downy mildew in wild sunflower accessions. Crop. Sci. 32: 949–952. Tang, S.X. and S.J. Knapp. 2003. Microsatellites uncover extraordinary diversity in native American land races and wild populations of cultivated sunflower. Theor. Appl. Genet. 106: 990–1003. Tang, S., J.K. Yu, M.B. Slabaugh, D.K. Shintani, and S.J. Knapp. 2002. Simple sequence repeat map of the sunflower genome. Theor. Appl. Genet. 105: 1124–1136. Tatum, L.A. 1971. The southern corn leaf blight epidemic. Science 171: 1113–1116. Turner, N.C. 1979. Drought resistance and adaptation to water deficits in crop plants. In Stress Physiology in Crop Plants, H. Mussell and R.C. Staples, Eds. John Wiley, New York, pp. 343–372. U.S. Department of Agriculture, Foreign Agricultural Service. 2005a. Oilseeds: World Market and Trade, Circular Series FOP 02-05. http://www/fas.usda.gov/oilseeds/circular/2005. U.S. Department of Agriculture, Agricultural Research Service, National Genetic Resources Program. 2005b. Germplasm Resources Information Network (GRIN). National Germplasm Resources Laboratory, Beltsville, MD, http://www.ars-grin.gov2/cgi-bin/npgs/html/acchtml.pl?1489758 (online database). Vear, F., L. Gentzbittel, J. Philippon, S. Mouzeyar, E. Mestries, P. Roeckel-Drevet, D. Tourvieille de Labrouhe, and P. Nicolas. 1997. The genetics of resistance to five races of downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.). Theor. Appl. Genet. 95: 584–589. Velasco, L., J. Domínguez, and J.M. Fernández-Martínez. 2004a. Registration of T589 and T2100 sunflower germplasm with modified tocopherols. Crop Sci. 44: 363. Velasco, L., J. Domínguez, J. Muñoz-Ruz, B. Pérez-Vich, and F.M. Fernández-Martínez. 2003. Registration of Dw 89 and Dw 271 dwarf parental lines of sunflower. Crop Sci. 43: 1140–1141. Velasco, L. and J.M. Fernández-Martínez. 2003. Identification and genetic characterization of new sources of beta and gamma tocopherol in sunflower germplasm. Helia 26: 17–24. Velasco, L., B. Pérez-Vich, and J.M. Fernández-Martínez. 2004b. Novel variation for the tocopherol profile in a sunflower created by mutagenesis and recombination. Plant Breed. 123: 490–492. Vick, B.A., C.C. Jan, and J.F. Miller. 2003. Registration of two sunflower genetic stocks with reduced palmitic and stearic acids. Crop Sci. 43: 747–748.

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Vick, B.A. and J.F. Miller. 2002. Strategies for the development of NuSun sunflower hybrids. In Lipid Biotechnology, T.M. Kuo and H.W. Gardner, Eds. Marcel Dekker, New York, pp. 115–128. Vranceanu, V. 1970. Advances in sunflower breeding in Romania. In Proceedings of the 4th International Sunflower Conference, International Sunflower Association, Paris, pp. 136–148. Vranceanu, A.V. and M. Iuoras. 1988. Hibridul interspecific dintre Helianthus rigidus Desf. si floarea-soarelui cultivata (H. annuus L.). Probl. Genet. Teor. Aplic. 2: 109–119. Vranceanu, A.V., M. Iuoras, and F.M. Stoenescu. 1986. A contribution to the diversification of the CMS sources in sunflower. Helia 9: 21–25. Vranceanu, A.V. and F.M. Stoenescu. 1971. Pollen fertility restorer genes from cultivated sunflower. Euphytica 20: 536–541. Vranceanu, A.V., F.M. Stoenescu, and N. Pirvu. 1988. Genetic progress in sunflower breeding in Romania. In Proceedings of the 12th International Sunflower Conference, International Sunflower Association, Paris, pp. 404–410. Vulpe, V. 1972. Surce de androsterilitete le floara soarelni. Analele I.C.C.P.T. 38: 273–277. Watson, E.E. 1929. Contributions to a monograph of the genus Helianthus. Papers Mich. Acad. Sci. 9: 305–475. Weaver, J.E. 1926. Root hairs of sunflower. In Root Development of Field Crops, J.E. Weaver, Ed. McGrawHill, New York, pp. 247–252. Whelan, E.D.P. 1974. Discontinuities in the callose wall, intermeiocyte connections, and cytomixis in angiosperm meiocytes. Can. J. Bot. 52: 1219–1224. Whelan, E.D.P. 1978. Hybridization between annual and perennial diploid species of Helianthus. Can. J. Genet. Cytol. 20: 523–530. Whelan, E.D.P. 1979. Interspecific hybrids between Helianthus petiolaris Nutt. and H. annuus L.: effect of backcrossing on meiosis. Euphytica 28: 297–308. Whelan, E.D.P. 1980. A new source of cytoplasmic male sterility in sunflower. Euphytica 29: 33–46. Whelan, E.D.P. 1981. Cytoplasmic male sterility in Helianthus giganteus L. × H. annuus L. interspecific hybrids. Crop Sci. 21: 855–858. Whelan, E.D.P. 1982. Trisomic progeny from interspecific hybrids between Helianthus maximiliani and H. annuus. Can. J. Genet. Cytol. 24: 375–384. Whelan, E.D.P. and W. Dedio. 1980. Registration of sunflower germplasm composite crosses CMG-1, CMG-2, and CMG-3. Crop Sci. 20: 832. Whelan, E.D.P. and D.G. Dorrell. 1980. Interspecific hybrids between Helianthus maximiliani Schrad. and H. annuus L.: effect of backcrossing on meiosis, anther morphology, and seed characteristics. Crop Sci. 20: 29–34. Willett, W.C. 1994. Diet and health: what should we eat? Science 264: 532–537. Yang, S.M., J.B. Morris, and T.E. Thompson. 1980. Evaluation of Helianthus spp. for resistance to Rhizopus head rot. In Proceedings of the 9th International Sunflower Conference, International Sunflower Association, Paris, pp. 147–151. Yu, J.K., J. Mangor, L. Thompson, K.J. Edwards, M.B. Slabaugh, and S.J. Knapp. 2002. Allelic diversity of simple sequence repeats among elite inbred lines of cultivated sunflower. Genome 45: 652–660. Yu, J.K., S.X. Tang, M.B. Slabaugh, A. Heesacker, G. Cole, M. Herring, J. Soper, F. Han, W.C. Chu, D.M. Webb, L. Thompson, K.J. Edwards, S. Berry, A.J. Leon, M. Grondona, C. Olungu, N. Maes, and S.J. Knapp. 2003. Towards a saturated molecular genetic linkage map for cultivated sunflower. Crop Sci. 43: 367–387. Zeller, F.J., G. Kimber, and B.S. Gill. 1977. The identification of rye trisomics by translocations and Giemsa staining. Chromosoma (Berlin) 62: 279–289. Zimmer, D.E. and J.A. Hoes. 1978. Diseases. In Sunflower Science and Technology, Agronomy Monograph 19, J.F. Carter, Ed. CSSA, ASA, and SSSA, Madison, WI, pp. 225–262.

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CHAPTER 6 Safflower (Carthamus tinctorius L.) Vrijendra Singh and N. Nimbkar

CONTENTS 6.1 6.2

6.3 6.4

6.5 6.6

6.7

Introduction...........................................................................................................................168 Description and Crop Use....................................................................................................169 6.2.1 World Distribution and Production ..........................................................................169 6.2.2 Utilization .................................................................................................................169 6.2.3 Botany.......................................................................................................................170 6.2.3.1 Basic Features ...........................................................................................170 6.2.3.2 Reproductive System.................................................................................171 Centers of Origin..................................................................................................................173 Cytogenetics .........................................................................................................................174 6.4.1 Genomic Relationships.............................................................................................174 6.4.1.1 Species Classification................................................................................174 6.4.1.2 Reclassification of Carthamus ..................................................................175 6.4.1.3 Molecular Classification of Carthamus ....................................................176 6.4.2 Classical Cytogenetics..............................................................................................176 6.4.3 Molecular Cytogenetics............................................................................................177 Germplasm Resources ..........................................................................................................177 Germplasm Enhancement: Conventional Breeding .............................................................178 6.6.1 Breeding Methods ....................................................................................................178 6.6.1.1 Introduction and Pure Line Selection.......................................................179 6.6.1.2 Hybridization.............................................................................................179 6.6.2 Hybrid Breeding .......................................................................................................182 6.6.2.1 Single Recessive Genetic Male Sterility ..................................................182 6.6.2.2 Dominant Genetic Male Sterility..............................................................182 6.6.2.3 Cytoplasmic-Genetic Male Sterility .........................................................183 6.6.3 Breeding for End Use...............................................................................................183 6.6.3.1 Disease Resistance ....................................................................................183 6.6.3.2 Oil Content and Quality............................................................................184 6.6.3.3 Insect Resistance .......................................................................................185 6.6.3.4 Spineless Safflower ...................................................................................185 6.6.3.5 Resistance to Abiotic Stresses ..................................................................185 Molecular Genetic Variation ................................................................................................185 167

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6.8

Tissue Culture and Genetic Transformation ........................................................................186 6.8.1 Somatic Embryogenesis ...........................................................................................186 6.8.2 Somaclonal Variation................................................................................................187 6.8.3 Biotic Stresses ..........................................................................................................187 6.8.4 Abiotic Stresses ........................................................................................................187 6.8.5 Genetic Modification ................................................................................................187 6.9 Polyembryony and Apomixis in Safflower ..........................................................................188 6.10 Future Direction....................................................................................................................188 References ......................................................................................................................................189

6.1 INTRODUCTION Safflower (Carthamus tinctorius L.) — an oilseed crop — is a member of the family Compositae or Asteraceae. Carthamus is the latinized synonym of the Arabic word quartum, or gurtum, which refers to the color of the dye extracted from safflower flowers. The English name safflower probably evolved from various written forms of usfar, affore, asfiore, and saffiore to safflower. Safflower has been grown in India since time immemorial. It is mentioned as kusumba in ancient scriptures. Presently, in India it is most commonly known as kardai in Marathi and kusum in Hindi. In China it is known as hong hua. Safflower, a multipurpose crop, has been grown for centuries in India for the orange-red dye (carthamin) extracted from its brilliantly colored flowers and for its quality oil rich in polyunsaturated fatty acids (linoleic acid, 78%). Safflower flowers are known to have many medicinal properties for curing several chronic diseases, and they are widely used in Chinese herbal preparations (Li and Mundel, 1996). The tender leaves, shoots, and thinnings of safflower are used as pot herb and salad. They are rich in vitamin A, iron, phosphorus, and calcium. Bundles of young plants are commonly sold as a green vegetable in markets in India and some neighboring countries (Nimbkar, 2002). Safflower can be grazed or stored as hay or silage. Safflower forage is palatable, and its feed value and yields are similar to or better than those for oats or alfalfa. Thus, each part of safflower has a value attached to it. Safflower has high adaptability to low moisture conditions. Therefore, its production all over the world is mainly confined to areas with scanty rainfall. Carthamus has 25 species, of which only C. tinctorius is the cultivated type, having 2n = 24 chromosomes. Though the crop has tremendous potential to be grown under varied conditions and to be exploited for various purposes, the area under safflower around the world is limited largely due to the lack of information on its crop management and product development from it. The research and development on different aspects of safflower, despite its adaptability to varied growing conditions with very high yield potential and diversified uses of different plant parts, have not received due attention. This probably is the main reason for its status as a minor crop around the world in terms of area and production, compared to the other oilseed crops. However, interest in this crop has been rekindled in the last few years due to three major reasons: 1. A huge shortfall in oilseed production in countries having a sizable area with scanty rainfall, to which safflower is most suited. 2. The preference of consumers for healthy oil with less amounts of saturated fats, for which safflower is well known. 3. The medicinal uses of flowers in China and extraction of edible dyes from flowers have become more widely known.

The present chapter deals with distribution, production, utilization, species, genomic relationships, classification, germplasm resources, genetics, breeding, and biotechnology of safflower.

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6.2 DESCRIPTION AND CROP USE 6.2.1

World Distribution and Production

Traditionally, safflower has been grown for centuries from China to the Mediterranean region and all along the Nile valley up to Ethiopia (Weiss, 1971). Presently it is grown commercially in India, the U.S., Mexico, Ethiopia, Kazakhstan, Australia, Argentina, Uzbekistan, China, and the Russian Federation. Pakistan, Spain, Turkey, Canada, Iran, and Israel also grow safflower to a limited extent. Safflower acreage and production around the world have witnessed wide fluctuations in the past. Safflower seed production in the world rose from 487,000 MT in the year 1965 to 1,007,000 MT in 1975, and subsequently it decreased to 921,000 MT in 1985 (Anonymous, 2002). Mexico was the largest producer of safflower in the world until 1980, when it occupied an area of 528,000 ha with a production above 600,000 MT in the year 1979–1980. However, the area and production of safflower in Mexico decreased significantly in later years, becoming only 10% of the area and production recorded for the year 1979–1980 (Cervantes-Martinez, 2001). Commercial production of safflower in the U.S. was started in the 1950s, and the area rapidly increased to 175,000 ha mainly in the states of California, Nebraska, Arizona, and Montana. Presently it is grown over an area of 100,000 ha (Esendal, 2001). Safflower in China is presently occupying an area ranging from 36,000 to 55,000 ha, producing 50 to 80 MT seeds annually. Xinjiang is the largest safflower producer state, which accounts for 80% of total safflower production in China. Other safflower-producing states in China are Yunnan, Sichuan, Henan, Hebei, Shandong, Jiangsu, and Zhejiang (Zhaomu and Lijie, 2001). Presently, India is the largest producer of safflower in the world, followed by the U.S., Mexico, and China. The safflower area in India in the year 2004–2005 was estimated to be 387,000 ha, with a production of 154,000 MT of seed (Anonymous, 2004–2005). In India, Maharashtra and Karnataka states account for 72 and 24% of safflower area and production, respectively. The other safflowerproducing states are Andhra Pradesh, Orissa, Madhya Pradesh, Chattisgarh, and Bihar. Safflower production in India is mostly confined to rain-fed conditions during winter. 6.2.2

Utilization

Safflower in India since time immemorial has been grown apart from orange-red dye extracted from its brilliant florets, for getting high-quality edible oil rich in polyunsaturated fatty acids, which helps in reducing the cholesterol level in blood. Safflower oil is nutritionally similar to olive oil, as it contains high levels of linoleic or oleic acid. The monounsaturated fatty acid like oleic acid is also known to reduce low-density lipoprotein (LDL; bad cholesterol) without affecting high-density lipoprotein (HDL; good cholesterol) in blood (Smith, 1996). Safflower oil is highly stable, and its consistency remains the same at low temperatures, thereby making it suitable for application in frozen/chilled foods (Weiss, 1971). Safflower oil is also better suited to hydrogenation for margarine production than are soy or canola oils (Kleingarten, 1993). Safflower oil is nonallergenic, and therefore suitable in injectable medications (Smith, 1996). Safflower is considered to be ideal for cosmetics and is used in ‘Macassar’ hair oil and Bombay ‘Sweet Oil’ (Weiss, 1971). Safflower oil is preferred for the paint and varnish industry owing to its specific properties of absence of linolenic acid, presence of high linoleic acid, low color values, low free fatty acids, low unsaponifiables, and no wax, which make the quality in paints, alkyd resins, and coatings beyond comparison (Smith, 1996). However, with the development of cheaper petroleum products and a shift to water-based paints, the use of safflower oil in the paint and varnish industry has been reduced drastically in recent times. In India, safflower oil is also used for lighting and manufacture of soap and to waterproof leather buckets (Weiss, 1971). Additionally, it is used to prepare roghan, which is used to preserve leather and as a glass cement. Charred oil is used for healing sores and in rheumatism (Weiss, 1971). It is also used in infant foods and liquid nutrition formulations.

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In addition to seed, safflower has been known and grown since ancient times for its brilliantly colored flowers, which were used to extract yellow and orange dyes for food and fabrics. With the advent of cheaper synthetic dyes like aniline, use of safflower flowers as a source of edible color gradually decreased to zero during the 20th century. However, recently interest in safflower flowers as a source of color for use in food is gaining importance owing to a recent ban on the use of synthetic colors in food in the European countries and elsewhere. The flowers are also reported to have medicinal properties to cure several chronic diseases, like hypertension, cardiovascular diseases, arthritis, spondylosis, and sterility in both men and women. Detailed information about clinical uses of safflower flowers has been given in the monograph on safflower written by Li and Mundel (1996). China produces approximately 1800 to 2600 MT of flowers annually to use them for extraction of dyes and in medicinal preparations (Zhaomu and Lijie, 2001). Flowers of nonspiny cultivar NARI-6 and nonspiny hybrid NARI-NH-1 have been reported to be rich in protein (10.4 and 12.86%), total sugars (7.36 and 11.81%), calcium (558 and 708 mg/100 g), iron (55.1 and 42.5 mg/100 g), magnesium (207 and 142 mg/100 g), and potassium (3992 and 3264 mg/100 g), respectively. All essential amino acids except tryptophan were present in safflower flowers (Singh, 2005a). A pleasant-tasting tea made with safflower flowers as its main ingredient has been developed in China (Li and Yuanzhou, 1993) and India (Singh, 2005a). With the commercialization of flowers as herbal health tea, extraction of dyes from them, and their use for medicinal purposes, the monetary returns to farmers from both seed and flowers are expected to grow to the extent of 141% of the monetary returns presently earned from the harvesting of seed alone (Sawant et al., 2000). Therefore, commercialization of safflower flowers will make the crop most remunerative among the crops grown in the winter season. This would certainly reverse the declining trend in area and production of safflower in India. Safflower oil meal is mainly used as animal feed. Safflower cake has the potential to be used as a human food if the bitter principles are removed (Nagaraj, 1995). Safflower cake in combination with all-purpose flour in 1:3 proportion was found to be highly suitable for manufacturing of proteinenriched biscuits with 22% protein in them (Singh and Abidi, 2005). Safflower leaves are rich in carotene, riboflavin, and vitamin C, and hence young seedlings and prunings are used as a green leafy vegetable in safflower-growing areas in India. Another important and interesting use of safflower seed has recently emerged by means of its genetic modification to produce high-value proteins as pharmaceuticals and industrial enzymes. SemBioSys — a Calgary-based (Canada) company — transforms safflower tissue genetically in order to get the proteins of interest to accumulate in the seed of the mature transgenic plant (Mundel et al., 2004). The process of transformation of safflower tissues follows the patented Stratosome™ Biologics system, which facilitates the genetic attachment of target proteins of interest to oleosin, the primary protein coating the oil-containing vesicles (oil bodies) of the seed. Such attachment permits the target protein to be purified along with the oil body fraction, which upon centrifugation floats to the surface of ground seeds/water slurry (van Rooijen et al., 1992). The purification process of the Stratosome system makes it more efficient than the other transgenic systems. The attachment of proteins to the oil bodies of safflower in the Stratosome Biologics system is expected to stabilize intracellular accumulation of foreign proteins, and also provide a useful attachment matrix and deliver benefits for end use applications. The commercialization of genetically modified safflower will further increase the acreage and production of this crop in the world. 6.2.3

Botany

6.2.3.1 Basic Features Safflower (Carthamus tinctorius L.) belongs to the family Asteraceae. Safflower plant can be described as a bushy, herbaceous annual possessing several branches, which are categorized as primary, secondary, and tertiary, with each terminating into a globular structure called capitulum (Figure 6.1).

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Figure 6.1

171

Single plant showing primary, secondary, and tertiary heads.

Stem and branches are encompassed with leaves having numerous spines. Safflower is mainly grown under dry land conditions as an oilseed crop. It produces white, shiny, and smooth seeds (fruits) called achenes, each weighing from 0.01 to 0.1 g. They may be with or without pappus (tufts of hair present on the seed) and are four sided, having thick pericarp. Initial growth after the germination of seed is slow in safflower. During the slow growing period, called the rosette stage, several leaves are produced at the stem base. The duration of the rosette stage in safflower varies from 20 to 35 days. After this stage, the stem elongates quickly and branches profusely. The branching habit in safflower is classified as narrow, with a <30° angle to the stem, spreading with a branch angle to stem of up to 75°. Branching habit in safflower is controlled both genetically and environmentally. Appressed branching is recessive to spreading types and is controlled both digenically (Fernandez-Martinez and Knowles, 1978; Singh, 2005b) and monogenically (Deokar and Patil, 1975). Each branch produces a globular flower capitulum, which is enclosed by tightly attached bracts. Safflower has a taproot system that elongates to 2 to 3 m in soils with adequate depth. The deep root system in safflower helps to extract the water and nutrients from much deeper layers of soil, compared to other crop plants, and thus makes it an ideal plant for rain-fed cropping systems. The flowering period in safflower lasts for a month, as the capitula based on primary branches flower first, followed by flowering of the capitula based on secondaries and tertiaries. Flowering in a capitulum begins in the outermost whorl of florets and proceeds centripetally over 3 to 5 days. The flower color in safflower is broadly grouped into four classes: 1. 2. 3. 4.

Yellow in bloom, turning to red on drying (Y-R) Yellow in bloom, turning to yellow on drying (Y-Y) Orange in bloom, turning to dark red on drying (O-dark R) White in bloom, turning to white on drying (W-W)

The first is the most prevalent (Figure 6.2). Safflower attains maturity in 30 to 35 days from the time when flowering ends. 6.2.3.2 Reproductive System Safflower, a member of the family Asteraceae, has a composite type of inflorescence, with each plant producing several flowering heads commonly called capitula. Each capitulum consists of

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Figure 6.2

Variability for flower colour in safflower exhibiting (from left to right) orange in bloom turning to dark red on drying (O-dark red); yellow in bloom turning to yellow on drying (Y-Y); white in bloom turning to white on drying (W-W); yellow in bloom turning to red on drying (Y-R).

Figure 6.3

Safflower capitulum having several flowers.

several flowers, with the number ranging from 20 to 250 (Figure 6.3). Flowers are enclosed by bracts in circular order. The disk flowers are attached to a flat or convex receptacle. In addition to the flowers, hairs or bristles are interspersed in between the flowers in a capitulum. A safflower flower is composed of petals that are attached to a corolla tube (Figure 6.4). The corolla tube in turn is attached at its base to an inferior ovary. Five fused anthers are attached to the corolla tube and surround the style and stigma. The corolla tube is 1.8 to 3 cm long and the five petal lobes are 6.5 to 8.5 mm long. Anther tube is 5 to 7 mm in length. The stigma, which is surrounded by five fused anthers, projects beyond the top of the anther tube by 5 to 6 mm. The inferior ovary in each

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Figure 6.4

173

Safflower florets showing petals, stigma, anther, carolla tube, and ovary.

flower develops into a single-seeded fruit called an achene, which is commonly known as seed. The pollen of safflower is yellow. Pollination occurs as the style and stigma protrude out of the fused anther tube. Unpollinated stigma remains receptive for several days. Outcrossing in safflower has been reported to range from 0 to as high as 59% in different genotypes in India (Patil et al., 1987). The outcrossing in genetic male-sterile lines in safflower is reported to be 100%, as no difference in seed yield of male-sterile and male-fertile plants under open pollination was observed (Singh, 1996). Safflower pollen is transferred by insects and not by wind. The most prevalent pollinator agent in safflower is honey bees. Bees visit the safflower flowers for both pollen and nectar. Each safflower capitulum produces 15 to 60 seeds.

6.3 CENTERS OF ORIGIN Vavilov (1951) proposed three centers of origin for cultivated safflower (Carthamus tinctorius L.). One in India (his center II) was based on variability and ancient culture of safflower production. A second center was proposed in Afghanistan (his center III), which was based on safflower diversity and proximity to wild species. A third center of origin, in Ethiopia (his center VI), was primarily based upon the presence of the wild safflower species in the area. The centers of safflower origin as proposed by Vavilov were reported by Kupzow (1932) in Russia after carrying out a detailed investigation of the safflower collections made in many areas. However, contrary to the above, Ashri and Knowles (1960) and Hanelt (1961) indicated the center of origin to be in the Near East. This assumption was based on the similarity of cultivated safflower to two closely related wild species: C. flavescens reported from Turkey, Syria, and Lebanon and C. palaestinus found in desert areas of western Iraq and southern Israel. Knowles (1969) described the safflower centers of cultivation as the “centers of similarity,” and not as the centers of origin or diversity, as there is a conspicuous similarity between the types existing in some or most of the centers. These centers are: 1. Far East (Vavilov’s center I — Chinese): China, Japan, and Korea 2. India–Pakistan (Vavilov’s center II — India): India and both West and East Pakistan (East Pakistan is now Bangladesh) 3. Middle East (Vavilov’s centers III and IV — Central Asiatic and Near Eastern): Afghanistan to Turkey, southern USSR to the Indian Ocean 4. Egypt (Vavilov’s center V — Mediterranean): Bordering the Nile north of Aswan. 5. Sudan (the southern reach of Vavilov’s center V): Bordering the Nile in northern Sudan and southern Egypt 6. Ethiopia (Vavilov’s center VI — Ethiopian) 7. Europe (western portion of Vavilov’s center V): Spain, Portugal, France, Italy, Romania, Morocco, and Algeria

Distinguishing characteristics of safflower from different centers of similarity are presented in order of decreasing frequency in Figure 6.5.

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Fl. color Head size

r int sp, spls

o, w, r sm, int sp

int

r, o, y, w

o, y, w, r

int, large

large, int

spls

sp, spls

few tall

India-Pak

sp

int

many

sp, spls

int

Middle East

int Egypt

sh,

Branching

int tall

int Sudan

Spines

Height

few

sh Far East

sm, int

sm

r

sp

many tall

y, o

o, r, y, w

Ethiopia

int Europe

Abbreviations : sh = short; int = intermediate; sp = spiny; spls = spineless; sm = small; r = red; w = white; o = orange; y = yellow (Modified from Knowles, 1969).

Figure 6.5

Distinguishing characteristics of safflower from different centers of similarity presented in order of decreasing frequency.

6.4 CYTOGENETICS 6.4.1

Genomic Relationships

6.4.1.1 Species Classification The genus Carthamus consists of 25 species, distributed worldwide. Among the 25 safflower species, the cultivated safflower grown around the world is only Carthamus tinctorius L., containing 12 pairs of chromosomes (Patel and Narayana, 1935; Richharia and Kotval, 1940; Ashri and Knowles, 1960; Kumar et al., 1981). Safflower has four chromosome numbers, viz., 2n = 20, 24, 44, and 64 (Ashri and Knowles, 1960). Based upon the four classes of chromosome numbers, the genus was categorized into four sections and the basic chromosome numbers (x) were indicated as 10 and 12 (Ashri and Knowles, 1960), and not 8 and 12 as suggested by Darlington and Wylie (1956). These sections as per Ashri and Knowles (1960) are: 1. Section I (2n = 24): Section I includes the annual species C. tinctorius L., C. palaestinus Eig, and C. oxyacantha M. Bieb. All three species cross readily and produce fertile hybrids, show high pairing between chromosomes, and are closely related (Ashri and Knowles, 1960). The possibility of natural gene transfer between C. tinctorius and its wild relatives C. palaestinus and C. oxyacantha appears to be very low since they are grown in different regions and seasons. C. oxyacantha is found from northwestern India to Iraq, and C. palaestinus is grown in southern Israel only. C. tinctorius is cultivated in much of India, in a few areas of Pakistan, over much of northern and central Iran, and in a few places in Jordan, Syria, Turkey, and Israel (Ashri and Knowles, 1960). C. oxyacantha is proposed to be the wild ancestor of the cultivated safflower (Bamber, 1916; Deshpande, 1952; Ashri and Knowles, 1960). The species of section I are described as pubescence minor or none; outer involucral bracts green, ovate to linear; inner bracts entire at apex; florets not saccate; corollas yellow, orange, red, or white; pollen grains yellow; pappus none or chaffy (Ashri and Knowles, 1960). 2. Section II (2n = 20): This section represents C. alexandrinus (Boiss. & Heldr.) Bornm., C. tenuis (Boiss & Blanche) Bornm., C. syriacus (Boiss.) Dinsm., and C. tenuis. All these species are found on the eastern side of the Mediterranean Sea. All species produce blue or pink flowers, and the first three are similar morphologically. C. glaucus is distinct from the others, as it has a larger head and bracts that are ovate rather than linear. Other species in this group are C. boissieri Halacsy, C. dentatus Vahl (genome, A1A1), C. leucocaulos Sibth. and Sm. (genome, A2A2), C. glaucus

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3.

4.

5.

6.

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subsp. anatolicus (Boiss.) Hanelt and subsp. glandulosus Hanelt, C. ambiguus Heldr., C. nitidus Boiss., C. rechingeri Davis, C. ruber Link, and C. sartori Held. Ashri and Knowles (1960) indicated that the species of sections I and II are not closely related. Artificial hybrids between species of the two sections were readily achieved, but all hybrids were sterile, as they showed very low pairing between the chromosomes of the involved species. This revealed that no exchange of genetic material between these two sections had occurred. Section III (2n = 44): This section consists of only one species, C. lanatus L., with 22 pairs of chromosomes. It occurs naturally in Portugal, Spain, Morocco, Greece, and Turkey. It was initially assumed that this species is the product of hybridization of species of section I and II, followed by chromosome doubling. C. lanatus on crossing with species of section I showed low pairing between chromosomes, suggesting that the species of section I were not involved in the ancestry of C. lanatus. However, C. lanatus showed good pairing with the chromosomes of species of section II, indicating that some species in section II contributed 10 pairs of chromosomes to C. lanatus (Ashri and Knowles, 1960). Section IV (2n = 64): Section IV comprises two species: C. baeticus (Boiss. & Reuter) Nyman and C. turkestanicus Popov. C. baeticus has 32 bivalents at Metaphase I, suggesting that it is an allohexaploid having three different genomes: one 12-chromosome genome and two separate nonhomologous genomes of 10 chromosomes. The hybrids between C. baeticus × C. lanatus showed perfect pairing of 22 chromosomes, indicating that C. lanatus (or a species closely related to it) is one of the ancestors of C. baeticus (Ashri and Knowles, 1960). C. glaucus M. Bieb. with 2n = 20 chromosomes is considered the progenitor of C. turkestanicus. C. baeticus spread to the eastern Mediterranean, North Africa, and Spain. C. turkestanicus is found in west Asia, east to Kashmir, and in Ethiopia. It has 22 pairs of chromosomes in common with C. baeticus and also has white pollen, and its similarity in appearance to C. lanatus in Thrace suggests considerable gene exchange between the two species (Khidir and Knowles, 1970). Carthamus species with 2n = 22: The only species with 11 pairs of chromosomes, viz., C. divaricatus (Beg. & Vaccari) Pamp., is found in a very limited area in Libya (Knowles, 1988). It produces yellow, purple, or white flowers with yellow pollen. It is self-incompatible and crosses readily with species possessing 10 pairs of chromosomes, but produces partially fertile hybrids. It also crosses with C. tinctorius, but produces sterile progeny. Other Carthamus species with 2n = 24: C. arborescens L. and C. caeruleus L., each having 12 pairs of chromosomes, possess distinct morphological characteristics and do not cross with any other Carthamus species (Ashri and Knowles, 1960); therefore, they were not grouped in any of the four sections. C. arborescens is found in southern Spain and adjoining areas of North Africa. C. caeruleus is well established in the Iberian Peninsula and in North Africa. Despite its occurrence around the Mediterranean Sea, it is quite different morphologically from C. arborescens and from other Carthamus species. C. rhiphaeus Font Quer & Pau, another species with 12 pairs of chromosomes, appears to be morphologically closely related to C. arborescens (Ashri and Knowles, 1960). C. riphaeus is found in a small area of northern Morocco. C. nitidus Boiss., which was earlier classified to have 10 pairs of chromosomes by Ashri and Knowles (1960), has been reclassified subsequently as having 12 pairs of chromosomes (Lopez-Gonzalez, 1990). This species has been grouped into a separate section due to its isolation from other species.

6.4.1.2 Reclassification of Carthamus Lopez-Gonzalez (1990) proposed a new classification system for genus Carthamus that is based on anatomical characteristics, biogeographic distribution, and biosystematic information. In the new classification system genera Carthamus and Carduncellus are grouped along with two new genera, Phonus and Lamottea. The corresponding species of Phonus, Lamottea, Carthamus, and Carduncellus are Carthamus arborescens L., Carthamus caeruleus L., Carthamus tinctorius L., and Carduncellus monspelienium All. The species of Phonus, Lamottea, and Carduncellus are of perennial nature, and all have 24 chromosomes in them; however, the genus Carthamus is annual and has species with 2n = 20, 22, 24, 44, and 64 chromosomes, including various putative allopolyploid species. The geographical distribution of the reclassified genera indicated that Phonus occurs in Spain, Portugal, and northern Africa; Lamottea is distributed in the western Mediterranean regions; Carthamus

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is found in west and central Asia, as well as in the Mediterranean region; and Carduncellus is grown in the western European region of the Mediterranean, northern Africa, Egypt, and Israel/Palestine. The new genus Carthamus is further subdivided into three sections. 1. Section Carthamus, having 24 chromosomes, is composed of species C. curdicus Hanelt, C. gypsicola Iljin, C. oxyacanthus M. Bieb., C. palaestinus Eig, C. persicus Willd. ex Bioss., and C. tinctorius L. The grouping of C. nitidus Boiss. (2n = 24) along with the above species is questionable; therefore, the species is isolated from the rest of the genera to form a separate section. 2. Section Odontagnathius (DC.) Hanelt (including section Lepidopappus Hanelt) has 20 or 22 chromosomes and consists of the species C. boissieri Halacsy, C. dentatus Vahl, C. divaricatus Beg. & Vaccari (2n = 22 chromosomes), C. glaucus M. Bieb., C. leucocaulos Sibth & Sm., and C. tenuis (Boiss. & Blanche) Bornm. 3. Section Atractylis Reichenb. is indicated to have n = 11 chromosomes producing numerous polyploids, including the species C. lanatus L., C. creticus (C. baeticus (Boiss. & Reuter) Nyman), and C. turkestanicus Popov.

6.4.1.3 Molecular Classification of Carthamus Molecular classification of the genus Carthamus groups species into two sections (Vilatersana et al., 2005). 1. Section Carthamus: This section consists of the same species as are furnished above under section Carthamus proposed by Lopez-Gonzalez (1990). 2. Section Atractylis: This section includes sections Atractylis, Lepidopappus, and Odontagnathius as indicated in the classification suggested by Hanelt (1963), and it coincides with the old genus Kentrophyllum. The only possible point of dispute in this molecularly redefined section Atractylis is the inclusion of C. nitidus having chromosome number n = 12, as the rest of the members of the section have n = 11 and n = 10. The relationship between species with n = 10, 11, and 12, as indicated, is a priori disconcerting but was explained in terms of descending dysploidy (Vilatersana et al., 2000) with the exclusion of the hybridogenic basic number n = 32 from the dysploid series. Further study revealed that molecular analysis does not support the usually adopted subspecific treatment for C. glaucus ssp. alexandrinus (Hanelt, 1963), C. glaucus ssp. tenuis (Schank and Knowles, 1964), C. lanatus ssp. creticus, and C. lanatus ssp. turkestanicus (Hanelt, 1963). Instead, the study strongly favors specific treatment for them because the four purported species do not form groups together with the species to which they are subordinated. Only C. creticus was found to be associated with C. lanatus. But in this case, the subspecific treatment was already inadequate, as C. lanatus is one of the progenitors of the allopolyploid C. creticus (Khidir and Knowles, 1970), and on this basis, C. creticus cannot be treated as a subspecies of C. lanatus.

6.4.2

Classical Cytogenetics

Carthamus cytology has been extensively studied by Knowles and his coworkers in the early 1960s and 1970s. (Ashri and Knowles, 1960; Hanelt, 1963; Schank and Knowles, 1964; Harvey and Knowles, 1965; Khidir and Knowles, 1970; Estilai and Knowles, 1976; Estilai, 1977). However, most of these studies, as stated earlier, were carried out to determine chromosome numbers in different species and the extent of pairing in interspecific crosses to establish the genomic relationship among species. Attempts to assign genes to chromosomes are completely lacking in this crop, although Estilai and Knowles (1980) did identify one primary and one secondary trisomic in the progeny of an open pollinated triploid plant and used them for assigning genes to chromosomes in safflower. The identification of additional chromosomes was presumably not possible due to relatively short chromosomes of more or less equal size (Knowles and Schank, 1964). Though these aneuploids were reported to be morphologically different from diploid plants, no effort was made to relate their morphological differences to the presence of extra chromosomes, mainly

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because progenies from open-pollinated plants had diverse genetic background (Knowles and Schank, 1964). Somatic chromosomes of C. nitidus Boiss. were observed to be similar to those of C. tinctorius, except the sat-chromosome of C. nitidus, which was found to be larger. Kumar et al. (1981) reported three sat-chromosomes in C. tinctorius L., compared to one reported by Knowles and Schank (1964). Kumar et al. (1981) carried out detailed karyotype analysis, including measurements of chromosome length and arm ratios, to identify translocation homozygotes in safflower. This study showed the identification of 10 translocation homozygotes out of 42 translation heterozygotes isolated in the M1 generation of gamma-irradiated populations. The chromosomes involved in interchange homozygotes were 6-8 (line 58), 1-3 (line 66), 3-12 (line 123), 3-10 (line 131), 3-6 (line 153), 4-6 (line 186), 4-8 (line 197), 3-8 (lines 208 and 279), and 5-9 (line 290). Chromosome 3 (sat-chromosome) was involved in 6 of the 10 translocations, and thus the segment interchanges were not random. The karyological characterization of individual chromosomes and the association of genes to specific chromosomes are lacking; as a result, a genetic and linkage map has not been developed in safflower. 6.4.3

Molecular Cytogenetics

Fluorescence in situ hybridization (FISH) is very useful in the identification of chromosomes and provides the information required for integration of genetic and physical maps, for localization of repetitive DNA sequences on the chromosomes, and to assist in functional and structural analysis of chromosomes. FISH analysis in C. tinctorius using pCtkpnI-1 and pCtkpnI-2 repeated sequences simultaneously revealed that the pCtkpnI-1 sequence was exclusively localized at subtelomeric regions on most of the chromosomes; however, the pCtkpnI-2 sequence was distributed on two nucleolar and one nonnucleolar chromosome pairs (Raina et al., 2005). The pCtkpnI-2 sequence also constituted the satellite and the intervening chromosome segment between the primary and secondary constrictions in the two nucleolar chromosome pairs. The pCtkpnI-2 repeated sequence, showing partial homology to the intergenic spacer (IGS) of 18S 25S ribosomal RNA genes of an Asteraceae taxon (Centaurea stoebe), and the 18S 25S rRNA gene clustser were located at independent, but juxtaposed sites in the nucleolar chromosomes (Raina et al., 2005). The application of FISH to other species will unravel the phylogenetic and evolutionary pathways in Carthamus.

6.5 GERMPLASM RESOURCES In view of the large number of species in safflower, the crop has enormous diversity in the germplasm for different traits. Despite this, the exchange of genetic material between cultivated and allied species is lacking due to the sterile nature of hybrids (Ashri and Knowles, 1960). This indicates that there has been no exchange of genetic material between cultivated species and the species possessing other than 2n = 24 chromosomes. The instances of natural crossings between cultivated species C. tinctorius and its wild relative C. oxyacantha with 2n = 24 chromosomes have been observed near Isfahan in Iran and in the experimental field at the Abu Ghraib station near Baghdad in Iraq. However, the possibility of such crossing is very limited, particularly in India and Pakistan, as harvesting of C. tinctorius is over when C. oxyacantha starts to flower (Ashri and Knowles, 1960). No instances of natural crossing between C. tinctorius and C. palaestinus, the other wild relative with 2n = 24 chromosomes, has been reported, as they naturally grow in different geographic areas of the world. The introgression of genes from wild relatives to C. tinctorius has not received due attention in safflower because instances of such introgression are few. However, introgression of phytophthora root rot resistance gene from a wild composite of 12 species into cultivated safflower has been recorded (Rubis, 1981). Heaton (1981) suggested the use of C. lanatus

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for improvement of disease resistance in C. tinctorius, as a disease resistance ability similar to that of C. lanatus was observed in the alloploid of C. tinctorius × C. lanatus. Interspecific crosses between C. tinctorius and C. oxyacantha also showed highly resistant reactions to Alternaria leaf blight and powdery mildew, though C. oxyacantha was found to be susceptible to powdery mildew (Anonymous, 1976–1977). This suggests some evidence of natural crossing between C. tinctorius and its wild relatives. Presently, India maintains about 7316 germplasm accessions at the Germplasm Management Unit (GMU) of the Directorate of Oilseeds Research, Hyderabad-500030 (Anonymous, 2002). The germplasm collection consists of the accessions received from safflower centers of the All India Coordinated Research Project (AICRP), local collections from traditional and nontraditional safflower-growing areas and exotic collections. The entire germplasm maintained at GMU has been characterized, and promising genotypes for economically important traits have been identified for use in the breeding programs. The U.S. maintains 2288 safflower accessions collected from more than 50 countries, at the Western Regional Plant Introduction Station (WRPIS), located at Pullman, WA, which is a part of the U.S. National Plant Germplasm System (NPGS) and the U.S. Department of Agriculture (Bradley and Johnson, 2001). The accessions from the U.S. safflower collection are distributed to scientists worldwide upon request at no charge. Data on many descriptors have been gathered on a large number of accessions in the collection and have been entered into the Germplasm Resources Information Network (GRIN), and this information is easily available to Internet users (Bradley and Johnson, 2001). Of the 2288 accessions in the U.S. safflower germplasm, a core collection of 207 accessions was developed based on country of origin and morphological data (Johnson et al., 1993). To enhance characterization and to determine diversity, the core collection was evaluated for seven quantitative factors, which indicated considerable diversity within the core collection. Correlation analysis showed the strongest association between plant height and flowering (r = 0.62), which was followed by association between outer involucral bract (OIB) width and OIB length (r = 0.54). The evaluation of accessions as per their places of origin, which have been distributed in major geographical regions, namely, the Americas, Australia, China, East Africa, Europe, Japan, the Mediterranean, South-Central Asia, Southwest Asia, and Thailand, revealed significant differences among regions for all factors except OIB length and yield per plant. Among the regions, accessions from Southwest Asia were the most different from those of other regions, but those from South-Central Asia and East Africa grouped closely together. Thus, the core collection was indicated to have highly diverse germplasm, and it appears that agronomic traits could distinguish regional differences (Johnson et al., 2001). The evaluation in Mexico of 721 accessions from the world collection of safflower for Alternaria resistance and seed-oil content showed 84 accessions possessing tolerance to Alternaria and 37 accessions having good agronomic characteristics. An oil content of >32.1% was recorded, with the maximum being 42.03%. This study showed that the U.S. safflower collection can be used as a source of Alternaria resistance and high oil content for breeding purposes (Cervantes-Martinez et al., 2001). The Beijing Botanical Garden at the Chinese Academy of Sciences collected and evaluated safflower germplasm, including the world safflower collections, and characterized it for 33 characters (Li et al., 1993).

6.6 GERMPLASM ENHANCEMENT: CONVENTIONAL BREEDING 6.6.1

Breeding Methods

Breeding for high yield and stability has been the major thrust of safflower research in India and other safflower-growing countries of the world. Therefore, a majority of the work carried out in safflower is related to enhancement of seed yield. Safflower falls in the category of often cross-pollinated crops, but the methods adopted to breed self-pollinated crops in general have been followed to develop cultivars in it. Crop improvement

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methods used extensively to breed cultivars with improved yield and stability in safflower are described below. 6.6.1.1 Introduction and Pure Line Selection Introduction is the simplest method of crop improvement and has been used extensively since time immemorial across the continents. Establishment of safflower as a crop in the U.S., Canada, and Argentina is a result of introductions from India, Russia, Turkey, etc., in the beginning of the 19th century (Claassen, 1981), though the introduction of varieties in a new area only occasionally results in direct release of varieties for commercial production. In general, introduced varieties require a few cycles of adaptation, followed by selection and evaluation, before they are formally released for commercial production, since the plants of an introduced cultivar show varied reaction to the changed environment. Therefore, acclimatization of the introduced cultivar is necessary before the population is subjected to selection for identifying promising selections and subsequent evaluation to release as a variety. Selection is the most commonly used breeding method followed for cultivar development as far as safflower improvement in India is concerned. This can be realized from the fact that 17 of the 25 varieties developed so far for commercial cultivation in the country have been evolved by resorting to selection in the locals. The safflower varieties developed by using this method in India and abroad are as follows: India: N-630, Nagpur-7, N-62-8, A-300, Manjira, S-144, JSF-1, K-1, CO-1, Type-65, APRR-3, Bhima, HUS-305, Sharda, JSI-7, A-2, PBNS-12; U.S.: Nebraska-5, Nebraska-10 (N-10); Canada: Saffire (Hegde et al., 2002). The pure line selection from local cultivars of safflower resulted in the development of several germplasm lines with many desired traits in safflower. Safflower, as indicated earlier, possesses enormous diversity for different traits of economic importance; however, the proper utilization of this diversity is lacking due to it being a rain-fed crop of minor economic importance. The availability of untapped variability for different traits in safflower is the reason that many of the safflower cultivars produced in India have been developed by pure line selection, and until today, this method is regarded as the most effective for varietal development in safflower. 6.6.1.2 Hybridization Hybridization is practiced mainly to bring together the desirable traits of two or more varieties into one. Hybridization, in addition to generating variation for various attributes in F2 and subsequent generations, has proved to be of great use in unraveling the genetic makeup of different traits. This has helped in formulating proper methodologies to bring out the desired improvement in different crops. The selection of parents has an important role in determining the success of a crop improvement program. Joshi (1979) gave very useful suggestions for selecting the parents for hybridization in a self-pollinated crop, the important ones being (1) selection of the parents on the basis of their per se performance for seed yield and other desired traits, (2) consideration of extent of expression in yield components, (3) consideration of genetic diversity of the parents to bring desired genes of diverse origin together, and (4) identification of the best general combining parents as well as the best cross combination by following the diallel cross approach. Evaluation of F1s along with their parents and check cultivars is essential in order to assess the performance of hybrids compared to parents and checks in respect to yield and other desired traits. This enables the selection of the most promising genotypes for generation advancement. The F1s selected as per the method stated above are advanced to the F2 generation. The selected F2s may be grown in a large area to have a fairly large population size. This is done to get individual plants representing all possible gene combinations existing in a cross. This makes it possible to select transgressive segregates. Corresponding F1s and standard checks may also be planted along with F2s to generate information on

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inbreeding depression. Low inbreeding depression in F2 suggests the availability of a fair amount of additive × additive components of variance that get fixed through subsequent inbreeding. The selection of promising individual plants possessing traits of economic importance is practiced in desirable crosses in F2, and the selected plants are harvested and threshed separately for plant to progeny row evaluation. While selecting individual plants in F2, the information generated on inheritance of yield and its components and interrelationships among them, as summarized below, may be used as a guideline for the crop improvement program. Genetic studies on gene action in safflower exhibited the importance of nonadditive gene action for seed yield and the number of capitula per plant (Makne and Choudhary, 1980; Ramachandram and Goud, 1981, 1982b; Ranga Rao, 1983; Narkhede et al., 1992; Patil et al., 1992; Parameshwarappa et al., 1995; Ghorpade and Wandhare, 2001; Gadekar and Jambhale, 2003) and additive gene action for oil content, seed weight, and seed number (Makne et al., 1979; Deokar and Patil, 1980; Ranga Rao, 1982; Mandal, 1990). The importance of both additive and nonadditive gene actions for the above traits was revealed by Prakash and Prakash (1993) and Singh (2004). Flower yield in addition to seed yield exhibited the importance of both additive and nonadditive gene actions, with nonadditive predominant for flower yield in safflower (Singh, 2004). For exploitation of gene action and to increase the chances of getting desirable recombinants, breeding methods such as biparental mating followed by reciprocal recurrent selection and diallel selective mating may be adopted in safflower (Ramachandram and Goud, 1981; Ranga Rao, 1982; Parameshwarappa et al., 1984; Narkhede et al., 1992). The presence of a high degree of nonadditive gene action for seed yield suggested tremendous potential for exploitation of hybrid vigor for seed and oil yields in safflower (Makne and Choudhary, 1980; Ranga Rao, 1982; Narkhede et al., 1992). Correlation studies delineated that seed yield and oil content are the most important and complex traits, with direct selection for them hampered due to the existence of large genetic–environment interaction in safflower (Ranga Rao and Ramachandram, 1979). Ineffectiveness of direct selection for yield is also reported by Nie et al. (1988). The yield components, viz., number of branches per plant, seed weight per capitulum, and 100-seed weight, contributed the most to the seed yield either directly or indirectly (Nie et al., 1988; Patil et al., 1990; Singh et al., 2004). Capitulum diameter showed positive association with seed yield (Mathur et al., 1976; Makne et al., 1979; Mandal, 1990; Singh et al., 1993; Anjani, 2000). Seed yield per plant and number of capitula per plant exhibited positive association with oil yield per plant (Prakash and Prakash, 1993). Seed weight was negatively associated with oil content (Ranga Rao et al., 1977; Mandal, 1990; Patil et al., 1990). Hull content showed significant positive association with seed weight but was negatively related to oil content (Ranga Rao et al., 1977; Sangale et al., 1982; Mandal, 1990). The magnitude of negative association between seed weight and seed number is approximately equal to that of positive correlation between seed number and oil content (Ramachandram and Goud, 1982a; Patil et al., 1990). Seed weight per capitulum, which is an outcome of seed number and seed weight, was observed to be a direct component of seed yield (Ramachandram and Goud, 1982a; Ramachandram, 1985; Ghongade et al., 1993; Nie et al., 1993; Singh et al., 2004). Thus, various investigations suggest the importance of number of capitula per plant, yield per capitulum, and seed weight for seed yield improvement in safflower. The studies to correlate flower yield with its component traits in nonspiny hybrids indicated that flower yield was significantly and positively associated with the number of primary branches per plant, capitulum diameter, number of capitula per plant, number of flowers per capitulum, stigma length, petal area per flower, and seed yield per plant. Therefore, selection for traits showing positive association with flower yield will help in improving flower yield in safflower (Singh, 2004). Individual plant selections can be made at the maturity of the crop, as all the attributes indicated as important for improvement of seed and flower yields can be taken into consideration while selecting the individual plants. Selection for high oil can be carried out by following the thumbnail technique (Sawant, 1989–1990). In this method, the dry seeds obtained from the main capitulum are pressed hard by

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using thumbnail pressure. The seeds with high oil content in general contain thin hulls and are thus pressed easily, in contrast to seeds with thick hulls, which are not pressed at all with thumbnail pressure. Thus, the selection for high oil types can be done in the field. The segregating populations in F2 and in subsequent generations, depending upon the trait to be improved, are handled by one of the methods illustrated below: 1. Pedigree method: This method has been used most frequently to improve seed yield, oil content, and other desired traits in safflower. The standard pedigree method, usually used in self-pollinated crops, is followed in safflower and is described briefly below. In this method, the selection of plants having desired traits is carried out in F2 populations. About 5 to 10% plants of the F2 population of each cross are selected, harvested, and threshed separately to raise plant-to-progeny rows in the F3 generation. F3 progenies may be evaluated in a replicated trial along with the standard checks for early-generation selection of the promising progenies for seed yield and desired traits. Selected progenies are advanced to F4, F5, and F6 generations in subsequent years. Each generation is subjected to inter- and intraprogeny selection of promising types. The selected plants need to be selfed at every stage of the selection process, as this makes it possible to get homozygous progenies by the time they reach the F6 generation. Uniform and homozygous progenies may be considered for yield trial at this stage, and the most promising ones of them may be further subjected to individual plant selections. The individual plant progenies are further evaluated in replicated trials to identify the most outstanding lines for multilocation evaluation. Multilocation evaluation is necessary to know their adaptability to different agroclimatic conditions before the release of the most adaptable line. Safflower cultivars developed by the pedigree method in India and other countries, along with their years of release for commercial production, are as follows: India: A-1 (1969), Tara (1976), Nira (1986), Girna (1990), JSI-73 (1998), NARI-6 (2001), Phule Kusuma (2003); U.S.: Leed (1968), Sidwill (1977), Hartman (1980), Rehbein (1980), Oker (1984), Girard (1986), Finch (1986); Mexico: Sahuaripa 88 (1989), Ouiriego 88 (1989), San Jose 89 (1990); Canada: AC Stirling (1991), AC Sunset (1995) (Hegde et al., 2002). 2. Bulk population method: In the bulk method, the F2 and following generations are harvested in bulk to grow the next generation. The major benefit of the bulk population method is that natural selection exerts strong selective pressure on bulk populations favoring the high-yielding types. As a result, poor yielders and uncompetitive types are eliminated during the process of evaluation of six to seven generations and the population becomes nearly homozygous. In the F7 or F8 generation the selection of promising plants carrying desirable traits is carried out. These selections are harvested and threshed separately for evaluation of individual plant progenies in replicated trials, along with standard checks, to identify the most promising ones for multilocation testing. Another advantage of the bulk method is that a breeder can handle several bulk populations simultaneously, which is not feasible in other breeding methods. It is desirable to self bulk populations of safflower, as otherwise the high rate of cross-pollination can make the bulk population method ineffective due to the presence of a large number of heterozygous plants at the end of the F7 generation. 3. Single-seed descent method: This method has been used by Fernandez-Martinez and DominguezGimenez (1986) in Spain to develop five safflower cultivars: Tomejil (1986), Rancho (1986), Merced (1986), Alameda (1986), and Rinconda (1986). In this method, from F2 onwards, randomly selected single seeds from each plant are taken to increase every subsequent generation until F5 and F6. In F7, a large number of individual plants are used to raise individual plant progenies. The outstanding progenies out of these are then tested for yield and other desirable attributes in a replicated trial. 4. Backcross method: This method has been used successfully in the U.S. to breed safflower cultivars US-10 (1959), by incorporating resistance to root rot caused by Phytophthora drechsleri (Thomas, 1964), and UC-1 (1966) (Knowles, 1968) and Oleic Leed (1976) (Urie et al., 1979), by transferring high oleic acid trait to both of them. This method is generally practiced with traits controlled by oligogenes. These genes are to be incorporated from a donor parent into a widely adapted variety. To incorporate a specific trait from a donor parent to the recurrent parent (widely adapted variety), a series of backcrosses are made between the hybrid and the recurrent parent. In each cycle of backcrossing, backcross progenies possessing desired characters are crossed with the recurrent

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parent. Six to seven backcrosses are desirable to develop a genotype homozygous for all the genes controlling different traits in the recurrent parent and for the genes controlling the trait under transfer. Selfing of the selected plants of the last backcross generation possessing requisite traits produces homozygous progenies that are similar to the recurrent parent.

6.6.2

Hybrid Breeding

The often cross-pollinated nature of safflower, existence of high heterosis for seed and flower yield, presence of many traits of commercial importance, and presence of genetic male sterility (GMS) and cytoplasmic male sterility (CMS) systems make safflower a suitable candidate for exploitation of hybrid vigor in the crop. Reports of the existence of high heterosis for seed yield and other desired traits in safflower have attracted several workers since the 1970s to seek the simple and easy-to-use methods of commercial-scale hybrid seed production (Urie and Zimmer, 1970a; Karve et al., 1979). The identification of genetic male sterility sources in safflower (Heaton and Knowles, 1980; Joshi et al., 1983; Ramachandram and Sujatha, 1991; Singh, 1996, 1997) and development of agronomically superior genetic male-sterile lines in India have resulted in the development and release of spiny safflower hybrids DSH-129 and MKH-11 in 1997, the first nonspiny hybrid safflower NARI-NH-1 in 2001 (Singh et al., 2003a), and the spiny hybrid NARI-H-15 in 2005. These hybrids in general show a 20 to 25% increase in seed and oil yield over the national check A-1. India is the only safflower-growing country in the world to grow a hybrid safflower. In safflower, genetic as well as cytoplasmic male sterility systems are harnessed for the development of hybrid cultivars. However, the male sterility system used for the development of safflower hybrids in India is the GMS system. The GMS systems available in safflower are of both monogenic recessive and dominant nature. 6.6.2.1 Single Recessive Genetic Male Sterility The GMS sources in safflower controlled by single recessive genes are: 1. 2. 3. 4.

UC-148 and UC-149 GMS lines developed by Heaton and Knowles (1980) GMS lines developed by Ramachandram and Sujatha (1991) MSN and MSV series of GMS lines developed by Singh (1996) DMS male-sterile lines associated with dwarfness developed by Singh (1997)

These male sterility sources segregate in a ratio of 1 male-sterile:1 male-fertile plant. Male-sterile and male-fertile plants are identified at flowering by the presence of a pinched capitulum opening in case of male-sterile plants and a normal opening in case of male-fertile plants. In case of DMS lines, owing to a linkage between sterility and dwarfing genes, the sterile and fertile plants become obvious at 30 to 40 days after sowing. The male-sterile plants attain a height of only 5 to 10 cm at 30 to 40 days after sowing; however, the male-fertile plants attain a normal height of 20 to 25 cm. The height difference between the two types enables the roguing out of male-fertile plants at this stage, leaving behind a 100% pure stand of dwarf male-sterile plants. 6.6.2.2 Dominant Genetic Male Sterility Joshi et al. (1983) reported a dominant gene-controlled male sterility in safflower. Identification of sterile and fertile plants as in single recessive genetic male sterility is possible in this case too at the flowering of the crop. Owing to the dominant nature of the gene imparting male sterility, the hybrids and the male-sterile line in this system segregate in a ratio of 1 MS (male-sterile) to 1 MF (male-fertile) plant. The success of hybrids based upon this source is hindered due to the occurrence of 50% MS plants in the hybrid population, which affects the yielding ability of the hybrid adversely if honey bee activity is not adequate to give 100% seed setting in the male-sterile plants.

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6.6.2.3 Cytoplasmic-Genetic Male Sterility Cytoplasmic-genetic male sterility (CGMS) has been reported to be exploited for hybrid development in safflower (Hill, 1989). The evaluation of CMS hybrids carried out in comparison with the GMS-based hybrids in India revealed the seed yield of CMS hybrids to be only 50% that of the corresponding GMS hybrids. In addition, all the CMS-based hybrids segregated into sterile and fertile plants, thereby suggesting the lack of fertility restoration to the sterile cytoplasm (Singh et al., 2000). The commercialization of CMS-based hybrids is still awaited. In India, too, efforts are under way to develop a CGMS system in safflower at the Nimbkar Agricultural Research Institute (NARI), Phaltan (Singh et al., 2001a), and at the Directorate of Oilseeds Research, Hyderabad. The CGMS systems at NARI are being developed by following interspecific crossing and mutagenesis with streptomycin. Both programs have resulted in development of CMS in safflower. Genotypes causing 100% restoration of fertility to the sterile cytoplasm have been identified in both cases (Singh, 2005b). Efforts are being made to develop suitable maintainer genotypes that can maintain 100% male sterility in the sterile cytoplasm. 6.6.3

Breeding for End Use

In general, safflower around the world is grown under rain-fed conditions. Therefore, the incidence of disease and pest infestation is reported to be of low severity. However, under favorable conditions they may cause considerable damage to the crop, as had happened in India during 1997 to 1998, when the entire crop of safflower in the major safflower-growing states of Maharashtra and Karnataka was completely wiped out by an outbreak of Alternaria (Anonymous, 1997–1998). In view of the above, the major emphasis in safflower improvement has been laid on seed yield; however, to meet the requirements of local agroclimatic conditions, cropping patterns, and market requirements, safflower improvement has also been directed to breed cultivars resistant to diseases and pests, and improved oil content and quality. 6.6.3.1 Disease Resistance Safflower is attacked by many diseases caused by fungi, bacteria, viruses, or physiological disorders due to abiotic stresses. Patil et al. (1993) reported that safflower is recorded to be infested around the world by 57 pathogens, including 40 fungi, 2 bacteria, 14 viruses, and 1 mycoplasma. Of these, Alternaria leaf spot caused by Alternaria carthami and wilt caused by Fusarium oxysporum are the most devastating ones and can cause 13 to 49% losses and wipe out the entire crop in the region under conditions conducive to their development, as indicated above in the case of India. Breeding safflower for disease resistance is the most economical and convenient method for controlling major diseases in safflower. Mundel and Huang (2003) described in detail how to control major diseases of safflower by breeding and using cultural practices. The genetics and the mode of inheritance of disease resistance and tolerance in safflower have not been studied for most diseases (Li and Mundel, 1996). Though germplasm lines or cultivars showing partial or full resistance to some of the major diseases have been identified, the genetics has been determined only for a few. Karve et al. (1981) showed that resistance to each of the diseases, viz., Alternaria carthami Chowdhari, Cercospora carthami Sund and Ramak, Ramularia carthami Zaprom, Fusarium oxysporum Sehl. ex. Fries, Rhizoctonia bataticola Bult, and Rhizoctonia Solani Kuhn, is imparted by single dominant genes. Study of inheritance of wilt (Fusarium oxysporum) resistance in safflower revealed the control of inhibitory gene action in the expression of wilt resistance in safflower (Singh et al., 2001b). The source of resistance to wilt has been identified in the local germplasm lines (Sastry and Ramachandram, 1992). Breeding for wilt resistance in safflower following backcross resulted in the development of wilt-resistant genotypes giving an increase in

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seed yield to the extent of 31% over the national check A-1 (Singh et al., 2003b). Breeding safflower varieties for resistance to multiple diseases resulted in the development of germplasm line VFR-1. This line was derived from the breeding line Nebraska 4051, and it showed resistance to Verticillium wilt, Fusarium wilt and root rot, and Rhizoctonia root rot (Thomas, 1971). Australian safflower cultivar Sironaria, showing resistance to Alternaria blight and moderate resistance to Phytophthora root rot, has been developed by backcrosses (Harrigan, 1987, 1989). Safflower cultivars resistant to Alternaria blight, viz., Sidwill, Hartman, Oker, Girard, and Finch, have been successfully developed in the U.S. (Bergman and Riveland, 1983; Bergman et al., 1985, 1987, 1989a, 1989b). These cultivars have been derived from the crossing of existing cultivar AC-1 with mass-selected Alternaria-resistant line 87-42-3 in a disease nursery initiated in the early 1960s. Resistance to all prevalent races of root rot caused by Phytophthora drechsleri was incorporated into the cultivar Dart (Abel and Lorance, 1975). Mundel et al. (1985) reported the incorporation of Sclerotinia head rot (caused by Sclerotinia sclerotiorum (Lib.) de Bary) resistance into the first Canadian safflower cultivar Saffire by adopting mass selection. 6.6.3.2 Oil Content and Quality Safflower varieties released for commercial production in India in general possess low oil content of 28 to 32%, except HUS-305, NARI-6, and nonspiny hybrid NARI-NH-1, each of which contain 35% oil. Of late, development of high oil-containing varieties and hybrids with in-built resistance to diseases and pests has been emphasized in the national safflower improvement program in India. Many studies have shown a negative association between hull content and oil content in safflower (Ranga Rao et al., 1977; Sangale et al., 1982; Mandal, 1990). Therefore, reduction in hull content directly increases oil content. A number of genes for different hull types in safflower have been described: partial hull (par par) recessive to normal hull, which is inherited independently of thin hull (th th) and striped hull (stp stp) (Urie, 1981); gray-striped hull (stp2) (Abel and Lorance, 1975); and reduced hull (rh rh), with small dark blotches on the seed. Partial hull is recessive to reduced hull (Urie, 1986). However, normal hull is dominant or partly dominant to reduced hull, depending upon the normal hull genotype used in the crossing program (Urie and Zimmer, 1970b). Tremendous improvement in oil content of safflower seed has been achieved in the cultivars developed in the U.S. (Bergman et al., 1985; Rubis, 2001). Safflower cultivar Oker contains 45% oil (Bergman et al., 1985). A safflower line having oil content as high as 55% has been reported by Rubis (2001). The quality of any oil is determined by the fatty acid composition of the oil, and the oils rich in poly- or monounsaturated fatty acids are considered good, as they help in reducing the cholesterol level in blood. In view of the above, safflower oil is considered the best, as it contains very high amounts of polyunsaturated (linoleic acid, 70 to 75%) or monounsaturated (oleic acid, 70 to 75%) fatty acids. Safflower is reported to be the best example of a crop with variability for fatty acid composition in seed oil (Knowles, 1989). Standard safflower oil contains about 6 to 8% palmitic acid, 2 to 3% stearic acid, 16 to 20% oleic acid, and 71 to 75% linoleic acid (Velasco and FernandezMartinez, 2001). Variants to the above composition with increased stearic acid content (4 to 11% of the total fatty acids), intermediate oleic acid content (41 to 53%), high oleic acid content (75 to 80%), and very high linoleic acid content (87 to 89%) have been detected in the released materials (Fernandez-Martinez et al., 1993; Johnson et al., 1999). Velasco and Fernandez-Martinez (2001) reported the development of lines with modified fatty acid composition having high palmitic acid content (10.3% of the total fatty acids), medium or high stearic acid content (3.9 and 6.2%), high or very high oleic acid content (>78 and 86%), together with reduced levels of the saturated fatty acids palmitic and stearic acid (<5%), and very high linoleic acid content (>86%) combined with reduced palmitic and stearic acid content (<5%). The sources of high total tocopherol content (up to 400 mg kg–1 seed) and increased gamma-tocopherol content (up to 9.9% of the total tocopherols) were also identified by them.

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The genetics of oleic, linoleic, stearic, and palmitic acids in seed were studied by Futehally (reported by Knowles, 1989). The genetics of fatty acids in safflower revealed that production of oleic, linoleic, and stearic acids is controlled by three independent recessive genes, ol ol, li li, and st st, respectively. Knowles (1968) released the first high oleic (oleic acid = 78.3%) safflower variety ‘UC-1’ in 1966 in the U.S., which was followed by the release of ‘Oleic leed’ in 1976 by him and his colleagues (Urie et al., 1979). ‘Alameda’ and ‘Rinconada’ developed by Fernandez-Martinez and Dominguez in Spain in 1986 and ‘Montola 2000’ and ‘Montola 2001’, having >80% oleic acid, developed by Bergman in the U.S. are other high oleic acid-containing cultivars released for commercial production (Li and Mundel, 1996). All other safflower varieties released for commercial production in different countries are of high linoleic type (linoleic acid = 70 to 75%). The fatty acid profile, genetic variability for fatty acids and their genetic control, suggests that fatty acid composition in safflower can be altered as required. 6.6.3.3 Insect Resistance Aphid is the most common pest of safflower, causing up to 50% damage. Germplasm lines exhibiting a stable tolerance to aphids have been identified in safflower. Two wild species C. flavescens and C. lanatus, have been reported to be carrying genes for resistance against safflower fly (Kumar, 1993). Genetics of aphid resistance in safflower has been reported to be of both additive and nonadditive nature. However, the role of nonadditive gene action was found to be predominant (Singh and Nimbkar, 1993). 6.6.3.4 Spineless Safflower Safflower in general is a spiny crop. However, the entire production of safflower in China is under spineless cultivars. Safflower production world over, barring China, is under spiny cultivars. Safflower production has been largely handicapped owing to its spiny nature, especially in nontraditional areas and in areas where mechanized cultivation has not yet been introduced. In India, too, safflower production is dominated by the spiny cultivars. Though spineless cultivars CO-1 and JSI-7 were available, because of their poor yielding ability, compared to spiny cultivars, they could not command a sizable safflower area. Recently, the nonspiny variety NARI-6 and nonspiny hybrid NARI-NH-1 (Singh et al., 2003a) were released, in 2001 and 2002, respectively, for all India production. The yield levels of the two cultivars are at par with their spiny counterparts, and they are reported to have better tolerance to foliar and wilt diseases than the spiny ones. Therefore, these cultivars are becoming very popular among the farmers in safflower-producing states in India. 6.6.3.5 Resistance to Abiotic Stresses Safflower is a rain-fed crop but, in general, suffers severely due to moisture stress. Studies with regard to abiotic stresses and genetic control of tolerance to them in safflower are largely lacking.

6.7 MOLECULAR GENETIC VARIATION The use of molecular techniques to study genetic variation in safflower has been initiated recently, and a very limited amount of information is available. Isozyme genetic markers to identify hybrid individuals from safflower populations (Carapetian and Estilai, 1997) and to study divergence in 89 accessions that originated from 17 countries have been used in safflower. The latter study revealed that materials from East Asia had the maximum estimates for both mean allele frequency and mean gene diversity. It was also indicated that the accessions from India showed high diversity; however, the accessions from Turkey were found to be closely related to those from the other

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Middle East countries. The accessions of unknown origin showed more resemblance to those from India, Turkey, and Middle East than to accessions from Europe and the U.S. (Zhang, 2001). The randomly amplified polymorphic DNA (RAPD) technique employed to detect genetic diversity of 28 safflower genotypes, including Iranian landraces, and several wild and exotic genotypes showed that the clusters based on RAPD markers correlated fairly well with a classification scheme based on morphological traits. Thus, the RAPD method appears to have great promise for the classification of safflower germplasm, identification of safflower landraces and its wild relatives, and their subsequent use in breeding of improved cultivars (Yazdi-Samadi et al., 2001). DNA fingerprinting of Indian safflower cultivars employing RAPD, Inter-Simple Sequence Repeat (ISSR) and amplified fragment length polymorphism (AFLP) markers indicated AFLP to be the most informative in discriminating between all 14 safflower cultivars (Sehgal and Raina, 2005). The study further identified specific markers for each cultivar, and cultivar NARI-2 was reported to contain the maximum number of diagnostic markers, followed by cultivars HUS-305, Bhima, and JSI-7. These four cultivars were identified as the probable source of new and novel alleles and were expected to be of great importance in breeding new cultivars (Sehgal and Raina, 2005).

6.8 TISSUE CULTURE AND GENETIC TRANSFORMATION 6.8.1

Somatic Embryogenesis

Safflower has attracted very little attention as far as tissue culture and genetic transformation are concerned. Initial efforts in safflower were directed to develop suitable culture conditions for whole plant regeneration. It has been demonstrated that regeneration frequencies are very high, and regeneration is possible through embryogenesis and organogenesis pathways. The mode of regeneration in general is through direct or indirect organogenesis (George and Rao, 1982; Tejovathi and Anwar, 1987, 1993; Sujatha and Suganya, 1996; Nikam and Shitole, 1999; Walia et al., 2005). Direct somatic embryogenesis from cotyledonary leaves has been produced (Mandal et al., 1995). Young safflower tissues, including roots, have been found suitable for in vitro regeneration as evidenced by the simple media requirements, and most of the studies have used Murashige and Skoog’s salts (1962) as the basal media. Studies are lacking for the enhancement of rhizogenesis in in vitro generated/multiplied shoots, and this has been considered a problematic area that is reducing the overall efficiency of whole plant regeneration. Survival of regenerated plants in soil has been reported to be as low as only 20% of transferred rooted plants of the var. Centennial (Baker and Dyer, 1996). Likewise, in variety Bhima, 34% of transferred plants showed success (Nikam and Shitole, 1999). This indicates that safflower needs more intensive and systematic experimentation to develop an effective method for supporting qualitative as well as quantitative rhizogenesis to enable large-scale transplantation of in vitro regenerated plants to the field. Production of haploids from anther or pollen culture, followed by chromosome doubling to produce homozygous diploids, has become an important tool to supplement the traditional breeding program in several crops. Use of haploids in development of cultivars is completely lacking in safflower. However, researchers at the Department of Genetics, Osmania University, Hyderabad, India, have developed reliable and reproducible protocols for whole plant regeneration from anther culture in safflower. Various parameters related to genotype, cold pretreatment (3 to 7°C) for 0 to 7 days — 15 days for immature flower buds, culture media (MS, Nitsch and Nitsch, Gamborg’s, Chaleff’s, Linsmaier and Skoog), growth regulators, growth adjuvants (abscicic acid, coconut milk, casein hydrolysate), and physiological condition of anther donor plants grown in field and in greenhouse have been evaluated for haploid production from anther culture in safflower. Cytological investigations of anther-regenerated plants revealed two ploidy levels, with haploids accounting for 64% (Prasad et al., 1991). Production of haploids could be followed by standard

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techniques to double the chromosomes of haploids by using colchicine or nitrous oxide treatments to get homozygous diploids. 6.8.2

Somaclonal Variation

Somaclonal variation as a source for inducing genetic variability has been demonstrated in safflower (Seeta et al., 2000). Somaclones generated from cotyledon explants of safflower genotype Manjira showed an enormous variation for both qualitative and quantitative traits and were found to breed true. Variants for plant height, leaf shape, plant type, flower color, seed shape, seed-oil content, days to flowering, days to maturity, number of capitula per plant, and types of fatty acids were recorded. 6.8.3

Biotic Stresses

Suganya et al. (1997) highlighted the applicability of tissue culture technique to select calluses exhibiting resistance to Fusarium oxysporum f. sp. carthami. 6.8.4

Abiotic Stresses

Evaluation of NaCl tolerance in safflower callus cultures by the repeated transfer of selected clones to the NaCl-rich medium enabled identification of salt-tolerant cell lines (Nikam and Shitole, 1997). Induction of high variability for qualitative and quantitative traits through tissue culture indicated that tissue culture can be suitably utilized to detect spontaneous somaclonal variants with improved tolerance to abiotic stresses. 6.8.5

Genetic Modification

Genetic engineering has become an important tool for crop improvement since it provides means of introduction of genes from diverse sources without changing the phenotype and agronomic performance of the transformed plant. Agrobacterium tumefaciens-mediated transformations produced transgenic safflower by using the cultivar Centennial (Ying et al., 1992). Efficient callus formation from cotyledon, stem, and leaf explants was observed. Transformation and integration of transgenes was confirmed with the application of β-glucuronidase (GUS) assay and DNA hybridization in kanamycin-resistant calli and GUS assay in regenerated shoots. A protocol for transformation and regeneration of safflower has been developed (Orlikowska et al., 1995). On the formation of leafy structure, it was transferred to elongation medium containing geneticin. After, elongation shoots were detached from the explant tissue and transferred to the same medium. Only transferred shoots that remained healthy were transferred to the rooting medium. A protocol for transformation using safflower embryo has been developed by Rohini and Rao (2000). In this method the embryo axes of germinating seeds with one of the cotyledons removed were pricked with a sterile sewing needle at the cotyledonary node and infected by gentle agitation for 10 min in a suspension of Agrobacterium tumefaciens. Following 24 h of co-cultivation and decontamination with cefotaxime for 1 h, they were placed on soilrite moistened with water to allow germination. Later, the seedlings were transferred to soil in pots where they grew into normal healthy plants in the greenhouse. The histochemical assay of a uidA gene followed by polymerase chain reaction (PCR) amplification of uidA and nptII marker genes and Southern analysis of T0 and T1 plant DNA indicated that the frequency of transformation was 5.3% in safflower ‘A-1’ and 1.3% in ‘A-300’. Matern and Kneusel (1993) attempted to introduce resistance to leaf blight caused by Alternaria spp. in safflower. They identified and cloned the brefeldin A-esterase gene to introduce resistance to leaf blight, but transformation of safflower with the isolated brefeldin A-esterase gene was

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unsuccessful. Mundel et al. (2004) reported that a Calgary-based company, SemBioSys Genetics, Inc., genetically transformed safflower tissue to produce the modified protein of interest in the seeds. It is indicated that transgenic safflower has a fair chance of getting permission for commercial cultivation in North America, as it has relatively low acreage and no weedy relatives with whom it can cross to produce fertile hybrids. In addition, safflower has several inherent agronomic qualities such as a low tendency to weediness, low seed dormancy, and large degree of self-pollination, which translates into a system that is quite easy to confine so that target products do not mingle with food or feed and thus make it a lower-risk production platform.

6.9 POLYEMBRYONY AND APOMIXIS IN SAFFLOWER Existence of polyembryony, like that in many plant species, has been observed in safflower (Singh et al., 2005). A safflower genotype D-149 with a tendency to produce twin seedlings at the rate of 0.6 to 10% in its different derivatives has been identified. Thirty percent of the twin seedlings were male sterile and did not show seed setting on crossing with fertile plants, as well as under open pollination, and thus were considered to have a changed ploidy level and are assumed to be haploid or triploid in nature. The haploid or triploid nature of plants from twin seedlings suggests the presence of polyembryony, since polyembryony is known to produce haploids/triploids and these in general are reported to be male sterile in nature. Efforts are under way to examine the feasibility of its utilization for cultivar development in safflower. Apomixis is yet to be confirmed in safflower, though preliminary studies carried out at the Nimbkar Agricultural Research Institute (NARI) at Phaltan in India indicate the possibility of existence of apomixis in safflower. Cytological and breeding investigations are under way at NARI to confirm the presence of apomixis in safflower.

6.10 FUTURE DIRECTION Information on genetic and linkage maps in safflower is completely lacking, and it needs immediate attention because this will help breeders to manipulate genes controlling different traits and to evolve new genotypes and cultivars with improved productivity and resistance to biotic and abiotic factors. The lack of homology between the chromosomes of cultivated safflower (C. tinctorius) with 2n = 24 and the species with other than 2n = 24 chromosomes has prevented introgression of desirable genes from the wild relatives to the cultivated types. Modern techniques like embryo rescue and other biotechnological tools may play an important role in overcoming such barriers. Development of a cytoplasmic-genetic male sterility system for hybrid breeding, a successful outcome of ongoing efforts to use polyembryony for varietal improvement, and confirmation of apomixis in safflower would augur well for further efforts to translate them into reality. Flower yield and pigment content of the flowers are the other traits that have gained economic importance recently, due to an increasing demand for safflower flowers as a source of natural food color in European and other Western countries and their use in medicines for curing several chronic diseases. No attention has been paid to improvement of these traits in safflower. The improvement in yield of flowers and pigments in flowers would certainly help in increasing total remuneration from the crop to the farmer. Genetic transformation of safflower to impart resistance to biotic and abiotic factors, in addition to development of seeds with altered fatty acid and protein profiles, is another area that has received very little attention. Conventional breeding techniques, though used for these purposes, have not been very successful. Therefore, genetic modification of safflower would be of enormous importance in improving productivity, production, and remuneration per unit area from the crop, which in turn would certainly help in increasing safflower area in the world.

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Singh, V. and N. Nimbkar. 1993. Genetics of aphid resistance in safflower (Carthamus tinctorius L.). Sesame Safflower Newsl. 8: 101–106. Singh, V., D.R. Rathod, M.B. Deshpande, S.R. Deshmukh, and N. Nimbkar. 2003b. Breeding for wilt resistance in safflower. In Extended Summaries. National Seminar on “Stress Management in Oilseeds for Attaining Self-Reliance in Vegetable Oils,” Hyderabad, India, January 28–30, 2003. ISOR, Directorate of Oilseeds Research, pp. 368–370. Smith, J.R. 1996. Safflower. AOCS Press, Champaign, IL, p. 624. Suganya, A., M. Sujatha, and K.R. Sastry. 1997. In vitro selection for resistance to Fusarium oxysporum Schlecht. carthami Klisiewiez and Houston in safflower. In Proceedings of the 4th International Safflower Conference: Safflower: A Multipurpose Species with Unexploited Potential and World Adaptability, Adriatica, Editrice, Bari, Italy, June 2–7, 1997. Corleto, A. and Mundel, H.H., Eds., pp. 305–308. Sujatha, M. and A. Suganya. 1996. In vitro organogenic comparison of different seedling tissues of safflower (Carthamus tinctorius L.). Sesame Safflower Newsl. 11: 85–90. Tejovathi, G. and S.Y. Anwar. 1987. Plant regeneration from cotyledonary cultures of safflower (Carthamus tinctorius L.). In Proceedings of the National Symposium “Plant Cell and Tissue Culture of Economically Important Plants,” Hyderabad, India. Reddy, G.M., Ed., pp. 347–354. Tejovathi, G. and S.Y. Anwar. 1993. 2,4,5-trichlorophenoxy propionic acid induced rhizogenesis in Carthamus tinctorius L. Proc. Ind. Natl. Sci. Acad. B59: 633–636. Thomas, C.A. 1964. Registration of ‘US10’ safflower. Crop Sci. 4: 446–447. Thomas, C.A. 1971. Registration of ‘VFR-1’ safflower germplasm. Crop Sci. 11: 606. Urie, A.L. 1981. Continued studies on inheritance of partial hull in safflower (Carthamus tinctorius L.). In Proceedings of the 1st International Safflower Conference, Davis, CA, July 12–16, 1981, pp. 264–271. Urie, A.L. 1986. Inheritance of partial hull in safflower. Crop Sci. 26: 493–498. Urie, A.L., W.F. Peterson, and P.F. Knowles. 1979. Registration of “Oleic Leed” safflower. Crop Sci. 19: 747. Urie, A.L. and D.E. Zimmer. 1970a. Yield reduction in safflower hybrids caused by female selfs. Crop Sci. 10: 419–422. Urie, A.L. and D.E. Zimmer. 1970b. Registration of reduced-hull safflower lines, reduced hull-1, -2, -3 and -4. Crop Sci. 10: 732. van Rooijen, G.J., L.I. Terning, and M.M. Moloney. 1992. Nucleotide sequence of an Arabidopsis thaliana oleosin gene. Plant Mol. Biol. 18: 1177–1179. Vavilov, N.I. 1951. The Origin, Variation, Immunity and Breeding of Cultivated Plants. Ronald Press Company, New York, 1951, 364 pp. Velasco, L. and J. Fernandez-Martinez. 2001. Breeding for oil quality in safflower. In Proceedings of the 5th International Safflower Conference, Williston, ND, and Sidney, MT, July 23–27, 2001. Bergman, J.W. and H.H. Mundel, Eds., pp. 133–137. Vilatersana, R., T. Garnatje, A. Susanna, and N. Garcia-Jacas. 2005. Taxonomic problems in Carthamus (Asteraceae): RAPD markers and sectional classification. Bot. J. Linn. Soc. 147: 375–383. Vilatersana, R., A. Susanna, N. Garcia-Jacas, and T. Garnatje. 2000. Karyology, generic delineation and dysploidy in the genera Carduncellus, Carthamus and Phonus (Asteraceae). Bot. J. Linn. Soc. 134: 425–438. Walia, N., A. Kaur, and S.B. Babbar. 2005. In vitro regeneration of a high oil-yielding variety of safflower (Carthamus tinctorius var. HUS-305). J. Plant Biochem. Biotech. 14: 1–4. Weiss, E.A. 1971. Castor, Sesame and Safflower. Leonard Hill Books, London, pp. 529–744. Yazdi-Samadi, B., R. Maali Amiri, M.R. Ghannadha, and C. Abd-Mishani. 2001. Detection of DNA polymorphism in landrace populations of safflower in Iran using RAPD-PCR technique. In Proceedings of the 5th International Safflower Conference, Williston, ND, and Sidney, MT, July 23–27, 2001. Bergman, J.W. and H.H. Mundel, Eds., p. 163. Ying, M., W.E. Dyer, and J.W. Bergman. 1992. Agrobacterium tumefaciens-mediated transformation of safflower (Carthamus tinctorius L.) cv. ‘Centennial’. Plant Cell Rep. 11: 581–585. Zhang, Z. 2001. Genetic diversity and classification of safflower (Carthamus tinctorius L.) germplasm by isozyme techniques. In Proceedings of the 5th International Safflower Conference, Williston, ND, and Sidney, MT, July 23–27, 2001. Bergman, J.W. and H.H. Mundel, Eds., pp. 157–162. Zhaomu, W. and D. Lijie. 2001. Current situation and prospects of safflower products development in China. In Proceedings of the 5th International Safflower Conference, Williston, ND, and Sidney, MT, July 23–27, 2001. Bergman, J.W. and H.H. Mundel, Eds., pp. 315–319.

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CHAPTER 7 Brassica Oilseeds Rod Snowdon, Wilfried Lühs, and Wolfgang Friedt

CONTENTS 7.1 7.2

7.3 7.4

7.5

7.6

7.7

Introduction...........................................................................................................................196 Description and Crop Use....................................................................................................197 7.2.1 World Production Area and Utilization ...................................................................197 7.2.2 Botany.......................................................................................................................199 7.2.2.1 Basic Features ...........................................................................................199 7.2.2.2 Reproductive System.................................................................................199 Germplasm Resources ..........................................................................................................199 Cytogenetics .........................................................................................................................202 7.4.1 Genomic Relationships among Diploid Species......................................................202 7.4.2 Classical Cytogenetics..............................................................................................202 7.4.3 Molecular Cytogenetics............................................................................................203 Genetic Mapping ..................................................................................................................204 7.5.1 Cytogenetic Maps.....................................................................................................204 7.5.2 Genetic Maps............................................................................................................205 7.5.3 Physical Maps...........................................................................................................207 7.5.4 Whole Genome Sequencing .....................................................................................209 Germplasm Enhancement: Conventional Breeding .............................................................210 7.6.1 Breeding Methods ....................................................................................................210 7.6.1.1 Cultivar Development ...............................................................................210 7.6.1.2 Hybrid Breeding........................................................................................211 7.6.2 Breeding for End Use...............................................................................................212 7.6.2.1 Protein Content and Meal Quality............................................................212 7.6.2.2 Oil Content and Quality............................................................................213 7.6.3 Yield Potential and Stability ....................................................................................214 7.6.4 Improved Resistance to Biotic and Abiotic Constraints..........................................214 7.6.4.1 Fungal and Viral Diseases.........................................................................214 7.6.4.2 Pests...........................................................................................................215 7.6.4.3 Abiotic Stress ............................................................................................215 Molecular Genetic Variation ................................................................................................216

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7.8

Tissue Culture and Genetic Transformation ........................................................................217 7.8.1 Generation of Doubled Haploids .............................................................................217 7.8.2 Sexual and Somatic Hybridization...........................................................................218 7.8.3 Genetic Modification ................................................................................................218 7.9 Interspecific and Intergeneric Hybridization........................................................................219 7.10 Future Direction....................................................................................................................221 References ......................................................................................................................................222

7.1 INTRODUCTION Brassica oilseed crops today deliver around 12% of the world’s edible vegetable oil (Figure 1.1). Oilseed rape, or canola (Brassica napus ssp. napus; genome AACC, 2n = 38) is today the most widely cultivated crop species in the crucifer family (Brassicaceae). The amphidiploid B. napus originated through spontaneous interspecific hybridization between turnip rape (Brassica rapa L., syn. campestris; genome AA, 2n = 20) and cabbage (Brassica oleracea L.; genome CC, 2n = 18), resulting in a genome comprising the full chromosome complements of its two progenitors. Because no wild B. napus forms are known, it is assumed that the species arose relatively recently, in the Mediterranean region, where both of its two parental species concurred. The closely related amphidiploid Brassica oilseeds B. juncea (L.) Czern (Indian or brown mustard; genome AABB, 2n = 36) and Brassica carinata L. (Abyssinian or Ethiopian mustard; genome BBCC, 2n = 34) arose in the same manner after crosses of black mustard (Brassica nigra, (L.) Koch; genome BB, 2n = 16) with B. rapa and B. oleracea, respectively. Figure 7.1 (See color insert following page 144) shows the Brassica triangle of U (1935), which describes the genomic relationships between the amphidiploid Brassica oilseed species and their diploid progenitors. Today, oilseed rape (B. napus) is the most heavily produced oilseed crop in Europe, and only soybean has a greater importance worldwide. Production of spring canola is dominated by North America (particularly Canada) and the northern provinces of China, whereas western Europe and central and southern China are the major producers of winter oilseed rape. On the other hand, the Brassica oilseeds exhibit an extremely broad adaptation to different agroclimatic conditions, with the more drought-tolerant B. juncea predominant on the Indian subcontinent and — along with B. napus — increasing in popularity in Australia. For B. juncea, various putative centers of origin have been identified, and the variation in morphotypes indicates that different subspecies of B. rapa were involved as independent genome donors in different regions (Prakash and Hinata, 1980). Indian mustard is widely grown throughout Asia. In Ethiopia, B. carinata is still the major oilseed crop, and due to its drought tolerance and resistance to pests and pathogens, this species is also growing in interest as a potential alternative oilseed crop within crop rotations in the dry areas of southern Europe. Cold-tolerant varieties of the diploid species B. rapa are grown in western Canada, where the early maturity can present an advantage over B. napus and other Brassica oilseeds. On the Indian subcontinent, the B. rapa ecotypes brown sarson, yellow sarson, and toria have regional significance. This chapter will concentrate on the production, breeding, and genetics of the most important Brassica oil crop, B. napus, but we will attempt to include information on the closely related mustard species where relevant. Details of germplasm resources for Brassica breeding will be presented, together with general information on the current status of genomic research relevant to Brassica oilseeds. Extensive information on production and use of Brassica oilseeds can be found in Kimber and MacGregor (1995), while breeding is covered in depth by Labana et al. (1993). A detailed overview of genetic and physical mapping in B. napus and its close relatives is available in Snowdon et al. (2006).

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Figure 7.1

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(See color insert following page 144.) The Brassica triangle, as described by U (1935), shows the genome relationships among the major Brassica vegetable and oilseed species. Brassica oilseeds show a wide range of seed colors, from yellow and light brown to dark brown and black forms. To date, most commercial varieties of winter oilseed rape and spring canola ( B. napus) are black seeded. However, yellow- and brown-seeded forms, as found in B. rapa, B. carinata, and B. juncea, contain less dietary fiber in the meal after oil extraction, and hence represent an interesting option for increasing the total value of the seed.

7.2 DESCRIPTION AND CROP USE 7.2.1

World Production Area and Utilization

Together, Brassica oilseeds have become a significant agricultural product during the past 30 years and are now the world’s third-leading source of both vegetable oil (after soybean and oil palm) and oil meal (after soybean and cotton). In 2004, the world production of the seven major oilseeds was around 361 million metric tons (MMT), with soybeans dominating (204 MMT), followed by rapeseed/canola, with 46 MMT (FAOSTAT, 2005; for current data, see http://faostat.fao.org/). The annual production of rapeseed was contributed mainly by China (13.0 MMT), the European Union member states (EU-25: 15.3 MMT), Canada (7.7 MMT), India (6.8 MMT) and Australia (1.5 MMT). Depending on species, type (winter vs. spring), cultivation techniques, and agricultural inputs, the world average yield of about 17.5 dt/ha covers a wide range, from 40 dt/ha in Western Europe, where predominantly winter rapeseed is produced, 1.8 in China, and 1.6 for spring canola in Canada, to 10 t/ha in India. Oilseeds generally are crushed to yield oil (40 to 45% in the case of canola) and residual meal, rich in protein, which is used mainly for animal feed. In contrast to soybean meal, rapeseed meal is not widely used for human consumption. Commodity rapeseed oil is now produced mainly from low erucic, low glucosinolate varieties (canola, or so-called 00 quality), but there are also high erucic cultivars and newer varieties with modified fatty acid composition furnishing specialty uses and niche markets. Whereas the sharp taste of erucic acid is not desired in European vegetable oils, the opposite is the case in India, where B. juncea oil containing erucic acid is a staple foodstuff.

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Figure 7.2

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(See color insert following page 144.) In comparison to other vegetable oils, low erucic acid Brassica oils (canola) contain very low concentrations of saturated fatty acids. Furthermore, rapeseed or canola oil contains a significant proportion of essential fatty acids, including high levels of α-linolenic acid, and it is the only commodity plant oil with an optimal 2:1 proportion of linoleic to α-linolenic acid. Because of this combination of positive attributes, canola oil is generally considered among the healthiest vegetable oils for human nutrition.

The nutritional advantages of canola as a cooking oil or margarine are rivalled only by olive oil, whereby rapeseed oil exhibits a higher content of essential fatty acids, especially α-linolenic acid, and is the only plant oil with an optimal 2:1 proportion of linoleic to α-linolenic acid. There is growing evidence that n-3 fatty acids play a role in the prevention and therapy of a number of chronic diseases, particularly in the reduction of the risk for coronary heart disease (Trautwein, 2001). As shown in Figure 7.2 (See color insert following page 144), rapeseed oil also contains the lowest proportion of saturated fatty acids of all the major vegetable oils, and the nutritionally significant monounsaturated fatty acid oleic acid comprises around 60% of the total fatty acids. The combination of all these factors makes rapeseed oil arguably the nutritionally most valuable vegetable oil. Rapeseed oil has many potential uses besides nutritional uses. Historically, the oil was used mainly for industry and for domestic lighting. As steam engines became widespread during the industrial revolution, rapeseed oil became a popular lubricant, because it was found to adhere to water-treated metal surfaces better than other lubricants. Rapeseed oil with high contents of erucic acid (22:1n-9) in particular has considerable advantages in specific applications due to the properties of this long-chain fatty acid. The special properties of high erucic acid rapeseed (HEAR) oil include high smoke and flash points, stability at high temperatures, durability, and the ability to remain fluid at low temperatures. The principal end use of HEAR is to produce erucamide, which is used as a slip additive in polyethylene and polypropylene manufacture to reduce surface friction and prevent adhesion between film surfaces. Erucamide is a relatively large complex molecule, making it difficult and expensive to produce synthetically from petrochemicals. HEAR oil is also used in printing inks and lubricants, and has a range of other applications (Lühs and Friedt, 1994; Piazza and Foglia, 2001). Methyl esters derived from rapeseed oil are today widely used as a diesel substitute (biodiesel). Commercial biodiesel production occurs in several countries worldwide. In Europe, low erucic rapeseed oil is the primary feedstock. In general, vegetable oil biodiesel fuels, either as a neat fuel or in blends with diesel, produce less smoke and particulates than pure diesel, have higher octane values, produce lower carbon monoxide and hydrocarbon emissions, and are biodegradable and nontoxic. The advantages and disadvantages of fat- and oil-derived alkyl ester diesel fuels with respect to fuel properties, engine performance, and emissions have been reviewed in detail by Graboski and McCormick (1998). The use of oilseed rape oils as industrial lubricants has considerable environmental benefits, because they are inherently biodegradable, of low ecotoxicity and toxicity toward humans, derived

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from renewable resources, and contribute no net carbon dioxide to the atmosphere. The cost of rapeseed oil falls in the range between mineral and synthetic oils. Furthermore, rapeseed oil has a high viscosity index, and the oil structure endures mechanical stresses well. Its low friction coefficient reduces heat development during use, and the freezing temperature is also very low. Due to its polarity, the oil adheres well to metal surfaces and provides good protection against corrosion. Because of these characteristics, it is not surprising that considerable efforts are directed toward increased use of rapeseed oil and oil derivatives in designing environmental fuels and lubricants (Piazza and Foglia, 2001). Furthermore, the thermoplastic properties and good biodegradation properties predispose rapeseed oil for use in bioplastic production. 7.2.2

Botany

7.2.2.1 Basic Features Brassica species display a wide range of morphotypes, including large leafy rosette forms, heading types, bulbous roots or swollen stems, and heavily flowering, oilseed-producing forms. This enormous morphological diversity is reflected in their diverse usage as vegetable, fodder, and oilseed crops. In the oilseed forms, a long vertical stem develops, either directly after germination or, in winter types, following winter vernalization, and shortly before the floral development lateral branches are formed. Flowering generally occurs in late spring, with pod development and ripening taking place over a period of around 6 to 8 weeks until mid-summer. Oilseed rape (B. napus) is cultivated in Europe and Asia predominantly as winter rapeseed, whereby in Canada, northern Europe, and Australia, only spring forms are suitable. The differentiation into winter and spring forms is governed by a genetic mechanism controlling the requirement for vernalization to promote the onset of flowering. Spring oilseed rape does not require vernalization and is not winter hardy; hence, the crop is sown in spring, and stem development begins immediately after germination. Winter oilseed rape, on the other hand, is sown in autumn and survives the winter in a leaf rosette form on the soil surface. 7.2.2.2 Reproductive System As members of the Brassicaceae (Cruciferae), the oilseed brassicas possess a Brassica-typical radial flower comprised of four petals in the typical crucifer cross form, alternating with four sepals. The inflorescence is racemose, with indeterminate flowering beginning at the lowest bud on the main raceme and continuing upward during the following days. The stigma is receptive from about 3 days prior until 3 days after the opening of the flower. The normally yellow flowers have one pair of lateral stamens with short filaments and four median stamens with longer filaments. Brassica anther sutures are introrse in the bud stage; however, the anthers of the four long stamens become extorse after the flowers open. In contrast to the majority of B. rapa and B. oleracea, its diploid progenitors, B. napus is a facultatively outcrossing species with a high degree of self-pollination. When insect pollinators are abundant, a greater proportion of cross-pollination can occur, although through targeted fertilization direction (e.g., using male-sterility systems for hybrid varieties; see below) it is possible to obtain up to 100% outcrossing. Brassica flowers possess two functional nectaries at the base of the short stamens, along with two nonfunctional nectaries at the base of the pairs of long stamens. The synocarpous ovary, consisting mainly of two but sometimes up to four carpels, develops after fertilization into a bivalved silique with a longitudinal septum. 7.3 GERMPLASM RESOURCES Due to the large number of closely related cruciferous crop species, an enormous variety of germplasm resources are available in international gene bank collections for evaluation and

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introgression of traits of agronomical interest into oilseed Brassica breeding material. Table 7.1 lists species and subspecies in the primary, secondary, and tertiary gene pools for identification and potential transfer of agronomic traits into Brassica oilseed species. As described later in this chapter, such resources have often been successfully used in particular for the identification and interspecific transfer of novel disease resistances to oilseed rape breeding lines. The sheer number and diversity of available Brassica gene bank accessions can make it difficult to identify relevant germplasm, however, even within the primary and secondary gene pools. Well-characterized core collections that represent as much as possible of the available diversity within a manageable number of genotypes are therefore a valuable resource for utilization of novel germplasm in breeding efforts. Within the framework of a recently completed European project, “Brassica Collections for Broadening Agricultural Use,” extensive collections of Brassica accessions from European gene banks were evaluated for morphological diversity and selected agronomic traits (van Soest et al., 2004). Within four international subgroups, the available gene bank material for B. oleracea, B. rapa, B. napus, and B. carinata was documented, characterized, evaluated, and rationalized to generate core collections representing as much as possible the range of available diversity useful for Brassica crop breeding. Although evaluated with different strategies and different general aims, the resulting core collections of B. oleracea (396 accessions; see Boukema et al., 2004) and B. rapa (100 accessions; see Pinnegar et al., 2004) are potentially very useful resources for screening of germplasm for novel resistance sources to be introgressed into oilseed rape by interspecific hybridization. Both core sets contain a tremendous variety of morphotypes and showed broad variation in selected disease resistances. Of more direct use for oilseed Brassica breeding, however, are the germplasm collections of B. napus (including genetically diverse spring and winter oilseed rape, fodder, vegetable, and swede forms) and the second amphidiploid species, B. carinata. Passport data for over 19,000 accessions are available in the ECP/GR Central Crop Database (BrasEDB: http://www.cgn.wur.nl/pgr/collections/brasedb/). From a total of 3787 entries for B. napus, a preliminary core collection of 200 accessions was selected, covering the different systematic groups, using various evaluation criteria (Poulsen et al., 2004). Particular emphasis was laid on the geographic origin of the material, with the collection selected to represent as much as possible the diversity present in all of the countries for which accessions were available. Minimum descriptors for numerous morphological characters were characterized, and together with data from agronomical evaluations, this information has been made available on the BrasEDB website. In order to facilitate the use of the core collection for oilseed rape breeding purposes, a number of relevant resistance and seed quality traits were evaluated in the preliminary core collection in field and greenhouse trials. These included tests for resistance to clubroot disease (Plasmodiophora brassicae Wor.), cabbage stem weevil, rape stem weevil, cabbage stem beetle, and field slugs. Two accessions were identified with resistance to the tested clubroot isolates, and a broad variation in susceptibility to infestation by the four pests was observed. Additionally, around 1100 accessions were analyzed for various seed quality characters, and together the morphological, resistance, and quality data were used to reduce the core collection to around 150 accessions that cover the broad variation in agriculturally important traits (Lühs et al., 2003; Poulsen et al., 2004). A subset of the preliminary core collection was also genotyped using simple sequence repeat (SSR) markers to evaluate the extent of genetic diversity in the material (Hasan et al., 2006). Collectively, the final core collection comprises a genetically diverse set of genotypes with wide variation for numerous traits of agronomic interest. Because commercial oilseed rape breeders were directly involved in the evaluation and selection of the material, these accessions represent a valuable resource for more detailed screening of further traits of interest with regard to introgression of novel germplasm into canola and oilseed rape breeding material (Poulsen et al., 2004). The B. carinata gene bank material was evaluated with particular emphasis on quality traits, which are of interest both directly, for the breeding of Ethiopian mustard as an alternative oilseed crop, and indirectly, for introgression of novel B. carinata germplasm into other Brassica oilseeds.

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Table 7.1 Selected Members of the Primary, Secondary, and Tertiary Gene Pools for Potential Transfer of Traits of Interest to Brassica Oilseed Crops Chromosome Number (n) 17 18 19 19 19

Primary Gene Pool Brassica carinata A. Braun (Ethiopian mustard) Brassica juncea (L.) Czern (Indian mustard, brown mustard) Brassica napus ssp. napus (oilseed rape, fodder rape) B. napus ssp. napobrassica (swede) B. napus ssp. napus var. pabularia (leaf rape, kale) Secondary Gene Pool

8 9 10

Brassica nigra Brassica oleracea (includes crop varieties, B. alboglabra, B. bourgeaui, B. cretica, B. hilarionis, B. incana, B. insularis, B. macrocarpa, B. montana, B. rupestris, B. villosa) Brassica rapa (includes wild and cultivated varieties) Tertiary Gene Pool

8 10 8 9 10 11 10 12 45 11 9 9 9 7 7 13 21 8 10 11 9 10 9 10 11 8 9 15 15 8 8 7 7 12 9 10 12 9 7 12 9

Brassica fruticulosa Brassica gravinae Brassica maurorum Brassica oxyrrhina Brassica repanda (includes B. desnottesii, B. nudicaulis, B. saxatilis) Brassica souliei (syn. B. amplexicaulis) Brassica tournefortii Coincya spp. (includes all species in the genus) Crambe abyssinica Diplotaxis acris Diplotaxis assurgens Diplotaxis berthautii Diplotaxis catholica Diplotaxis cossoniana Diplotaxis erucoides Diplotaxis harra (includes D. crassifolia, D. gracilis, D. hirtum, D. lagascana) Diplotaxis muralis [D. tenuifolia × D. viminea] Diplotaxis siettiana (includes D. ibicensis) Diplotaxis siifolia Diplotaxis tenuifolia Diplotaxis tenuisiliqua Diplotaxis viminea Diplotaxis virgata Enarthrocarpus ssp. (includes E. lyratus, E. pterocarpus, E. strangulatus) Eruca spp. (includes E. vesicaria, E. sativa, E. pinnatifida) Erucastrum abyssinicum Erucastrum canariense (includes E. cardaminoides) Erucastrum elatum [E. littoreum × E. virgatum] Erucastrum gallicum [E. leucanthum × Diplotaxis erucoides/D. cossoniana] Erucastrum nasturtiifolium (includes E. leucanthum) Erucastrum strigosum Erucastrum varium Erucastrum virgatum Onchophragmus violaceus Raphanus ssp. (includes R. raphanistrum, R. sativus, R. caudatus, R. maritimus, R. landra) Sinapidendron spp. (includes S. angustifolium, S. frutescens, S. rupestre) Sinapis alba (includes S. dissecta) Sinapis arvensis (includes S. allioni, S. turgida) Sinapis aucheri (syn. Raphanus aucheri) Sinapis flexuosa Sinapis pubescens (includes S. aristidis, S. boivinii, S. indurata)

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Table 7.1 (continued) Selected Members of the Primary, Secondary, and Tertiary Gene Pools for Potential Transfer of Traits of Interest to Brassica Oilseed Crops Chromosome Number (n) 8 8

Primary Gene Pool Trachystoma labasii Trachystoma spp. (includes T. aphanoneurum, T. ballii)

Note: The amphidiploid oilseed crops described at length in this chapter are divided into subspecies with common names given in round brackets. Secondary and tertiary gene pool species listed together are members of the same cytodeme, i.e., they share a single diploid chromosome number and are generally fully interfertile, while square brackets indicate amphidiploid taxa. For a complete list of genera, species, and subspecies in the tribe Brassiceae, along with exhaustive information on interspecific and intergeneric hybridization among Brassica crops and related species, see Warwick et al. (2000).

The 222 accessions investigated showed broad variation in seed storage components, including oil, protein, glucosinolates, dietary fiber, and fatty acid composition (Font et al., 2004).

7.4 CYTOGENETICS 7.4.1

Genomic Relationships among Diploid Species

The species of the Brassica triangle described by U (1935) (Figure 7.1) represent a unique group of closely related crops with intergenomic relationships that at first glance appear relatively straightforward: the three amphidiploid species B. napus (genome AACC; 2n = 38), B. juncea (AABB; 2n = 36), and B. carinata (BBCC; 2n = 34) arose via spontaneous interspecific hybridization among the respective diploids B. rapa (AA; 2n = 20), B. nigra (BB; 2n = 16), and B. oleracea (CC; 2n = 18). Hence, the three major oilseed species comprising the full chromosome complements of their respective progenitors, and comparative mapping studies have demonstrated that the chromosomes within the diploid genomes have remained more or less intact in their respective amphidiploids (Parkin et al., 1995; Sharpe et al., 1995; Axelsson et al., 2000). Underlying this simplicity, however, are the more complex structures of the respective diploid genomes, which themselves have been found to be more or less closely related ancestral polyploids with complex chromosomal rearrangements (Lagercrantz and Lydiate, 1996). Much knowledge regarding chromosomal homoeology on a subgenomic level has been obtained through investigations of chromosome pairing in hybrids among the Brassica diploid species and with closely related genera. Such analysis has shown that B. rapa and B. oleracea are extremely closely related species with highly homologous, only recently diverged genomes, a finding that has been confirmed by DNA sequence data. Whereas the Brassica A and C genomes exhibit a good degree of homology to Eruca sativa, Diplotaxis erucoides, and particularly Sinapis arvensis, the divergence from B. nigra apparently occurred much earlier and has resulted in a distinct lineage (Mizushima, 1972; Prakash and Chopra, 1991). 7.4.2

Classical Cytogenetics

The groundbreaking work in Brassica cytogenetics was done by Morinaga and U in the early 1930s (Morinaga, 1934; U, 1935). Through analyses of chromosome numbers and pairing in interspecific crosses, they found that amphidiploid Brassica species originate from diploid progenitors and contain the complete chromosome sets of their parental species. However, the difficulties associated with Brassica chromosomes as a cytological object — in particular their small size and lack of distinctive cytological landmarks — have limited the utilization of cytogenetics and prevented detailed karyological characterization of the chromosomes. Because of this, there is no clear

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chromosome nomenclature for Brassica oilseeds to date, and integration of chromosome landmarks with molecular genetic maps is only now becoming possible using fluorescence in situ hybridization (FISH), which enables the direct chromosomal localization of labeled DNA probes. The first detailed cytological descriptions of the Brassica somatic chromosome structure were published in 1960 by Röbbelen. Practical use of cytological techniques breeding, however, for example, the use of clearly identifiable chromosome introgressions as is performed in cereals, has not been possible in Brassica species due to the inability to clearly distinguish chromosomes by conventional means. 7.4.3

Molecular Cytogenetics

Fluorescence in situ hybridization (FISH) techniques offer the potential not only for more reliable chromosome identification in Brassica, but also in terms of the information they might be able to offer regarding the integration of genetic and physical maps, for ordering molecular markers and measuring physical genome distances, and for structural and functional chromosome analysis. FISH methods for the accurate localization of repetitive DNA sequences at the chromosomal subarm level, particularly ribosomal DNA sequences, have enabled the elucidation of molecular karyotypes for B. napus and its progenitor species and the identification of A and C genome chromosomes in the amphidiploid species (Fukui et al., 1998; Armstrong et al., 1998; Snowdon et al., 2002). FISH hybridization of bacterial artificial chromosome (BAC) clones to B. oleracea (Howell et al., 2002) and B. rapa (Jackson et al., 2000) chromosomes represents a first step toward integration of physical and genetic maps with the karyograms of the diploid species and their amphidiploid hybrid B. napus. Such techniques will play a key role in the selection of evenly spaced BAC clones from physical maps as seed BACs for the initiation of whole genome sequencing (see below). Total genomic DNA as a FISH probe (genomic in situ hybridization (GISH); see HeslopHarrison and Schwarzacher, 1996) is especially useful for diagnostic studies of the amount and integration of foreign chromatin in interspecific and intergeneric plant hybrids. Hybrids between high-yielding rapeseed cultivars and related species are relatively easily produced and have often been used to develop new lines containing introgressed traits like novel pest or disease resistances. Great advances in interspecific hybridization have resulted from the application of in vitro techniques for the generation of viable offspring from interspecific and intergeneric hybrids (Lühs et al., 2002). Identification of alien DNA in wide crosses has been achieved by quantification of chromosome content by flow cytometry (Sabharwal and Dolezel, 1993) and by tracing chromosome and DNA transfer using molecular markers. Visualization of alien chromatin in interspecific hybrids using in situ hybridization techniques, on the other hand, potentially allows the identification and tracing of chromosome additions and introgressions carrying traits of interest (Heslop-Harrison and Schwarzacher, 1996; Snowdon et al., 1997). For example, we have used GISH to characterize B. napus chromosome addition and introgression lines from sexual hybrids with Sinapis arvensis containing novel genes for resistance against blackleg disease (Snowdon et al., 2000), from interspecific crosses with oil radish (Raphanus sativus L.) exhibiting nematode resistance (Voss et al., 2000), and from asymmetric hybrids with Crambe abyssinica Hochst. ex. R.E. Fries exhibiting a high erucic acid content in the seed oil (Wang et al., 2004). The latter study used GISH in meiotic preparations to detect intergenomic chromosome pairing and chromatin exchange between B. napus and C. abyssinica chromosomes (Figure 7.3) (See color insert following page 144). The chromosomes of B. napus and C. abyssinica origin could be clearly discriminated by genomic in situ hybridization (GISH), and in meiotic cells intergenomic chromatin bridges were observed, confirming that chromatin transfer from the donor species to the rapeseed genome was occurring. Most of the progeny plants had a high pollen fertility and seed set, and some contained significantly greater amounts of seed erucic acid than the B. napus parent, demonstrating that intergeneric gene transfer can be successfully performed among distantly related crucifers and maintained in sexual

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Figure 7.3

(See color insert following page 144.) GISH analysis of mitotic and meiotic cells from sexual progeny of asymmetric B. napus × C. abyssinica hybrids. C. abyssinica chromosomes are labeled red with Cy3, whereas nonlabeled B. napus chromosomes are stained blue with DAPI. (A) Somatic metaphase chromosomes of a backcross progeny plant (2n = 50) with a full complement of 38 B. napus chromosomes, together with 12 additional chromosomes from C. abyssinica. (B to F) Meiosis of the same plant. (B) Pachytene stage with GISH signals confined to centromeric heterochromatin and the heterochromatic knob. (C, i) Diakinesis with 19 II from B. napus and 12 I from C. abyssinica. (C, ii and iii) In numerous cases, Cy3-labeled chromatin strands (arrows) were observed between C. abyssinica univalents and B. napus bivalents, indicating potential intergenomic gene transfer. (D) Early anaphase I showing late disjunction of C. abyssinica chromosomes. (E) Anaphase I showing bridging chromosome of C. abyssinica with Cy3-labeled chromatin strand extending to each pole (arrows). (F) Anaphase II showing laggards and unequal distribution of C. abyssinica chromosomes. (From Wang, Y.P. et al., Genome, 47, 666–673, 2004. Used with permission from the Genetics Society of Canada.)

progenies of the hybrids. Although such crosses can exhibit significant linkage drag and hence must be viewed as extremely basic material from a breeding perspective, such prebreeding is of great interest in terms of broadening the genetic variability for particular traits where novel genetic variation beyond the gene pool of the crop species is desirable.

7.5 GENETIC MAPPING 7.5.1

Cytogenetic Maps

In many important crops, particularly the major cereals, chromosome karyotypes based on classical cytogenenetic techniques like C-banding predated molecular genetic maps by many decades, meaning that an established chromosome nomenclature was often in place before genetic

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linkage groups were able to be identified by DNA markers. The availability of well-characterized sets of chromosome addition or substitution lines subsequently enabled a clear correlation of linkage groups, containing particular sets of genetic markers, to their corresponding chromosomes. In species with large, easily identifiable chromosomes, it is even possible to identify chromosome introgressions or translocations using classical banding techniques and to use these to breed for traits of interest carried on the respective introgressions. In crucifer species, on the other hand, it is extremely difficult to generate karyotypes in standard mitotic preparations, and even for experts it can be extremely difficult to clearly identify the chromosomes. C-banded Brassica karyotypes have been published (Olin-Fatih and Heneen, 1992), but rarely have these found practical application for breeding purposes or been aligned to genetic linkage maps. The first molecular karyotypes based on FISH techniques enabled more reliable identification of chromosomes (Fukui et al., 1998; Armstrong et al., 1998; Snowdon et al., 2002); however, these methods require special equipment and practical expertise not available in most labs, meaning that they are unlikely to ever find widespread use in Brassica breeding. Hence, in contrast to the situation described above for the major cereal crops, in Brassica the genetic map nomenclature has become broadly established and accepted before the cytogenetic map. It therefore makes sense that the karyotype nomenclature, instead of following classical cytological principles, should in the future be adapted with respect to the nomenclature system for the genetic map. This will become more feasible as genomic clones, from physical maps for the Brassica diploid species, which in turn are anchored to reference genetic maps, are physically localized on the respective chromosomes. Ultimately this will also enable the development of molecular karyotypes, aligned to genetic maps and following their nomenclature, not only for the Brassica diploids, but also for the amphidiploid oilseed crops. 7.5.2

Genetic Maps

The first complete linkage map for B. napus, published in 1991 by Landry et al. (1991), was based on restriction fragment length polymorphism (RFLP) markers in a segregating F2 population from a cross between the canola cultivars Westar and Topas. A total of 120 RFLP loci were mapped on 19 linkage groups covering a total of 1413 cM. This map gave the first indications of widespread locus duplication corresponding to the amphidiploid genome organization of B. napus, and the first evidence was observed for extensive rearrangements of the linear order of the duplicated loci. Comparisons of this map with the corresponding B. oleracea and B. rapa maps enabled the first detailed investigations of genome organization among the respective Brassica genomes. Furthermore, the fact that functional cDNA sequences were used as RFLP markers meant that the results were immediately relevant for applications in canola breeding. A further map was reported a short time later by Hoenecke and Chyi (1991), this time based on a cross between two breeding lines. In this case, 125 RFLP markers covering 1350 cM were mapped to 19 linkage groups. The first map developed from a doubled haploid (DH) population derived from a cross between a winter oilseed rape cv. Major and the spring Canola variety ‘Stellar’ (Ferreira et al., 1994). This population was used to localize quantitative trait loci (QTL) associated with the annual/biennial growth habit and for extensive studies of flowering time genes (e.g., Ferreira et al., 1995b; Osborn et al., 1997). The map positions for a subset of the mapped markers were compared with the locus ordering in F2 progeny from the same cross, and no significant differences could be established between the two maps. A total of 32% of mapped RFLPs were found to be duplicated, and conserved linkage arrangements provided evidence of intergenome homoeology between the B. napus A and C genomes. The first integrated B. napus map constructed using different segregating populations was developed by Sharpe et al. (1995). This study revealed considerable chromosome instability in one of the crosses, which involved a resynthesized (RS) rapeseed crossed with a normal rapeseed variety. Parkin et al. (1995) mapped this cross with 399 RFLP markers and discovered that the majority

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of loci exhibited disomic inheritance of parental alleles. This provided evidence demonstrating that the nuclear genomes of B. napus, B. rapa, and B. oleracea have remained essentially unaltered since the formation of B. napus. Hence, the linkage groups of B. napus correspond to the 10 A genome and 9 C genome chromosomes. Correspondingly, a linkage group nomenclature was suggested (Parkin et al., 1995; Sharpe et al., 1995) by which the 10 B. napus A genome chromosomes are named N1 to N10, and the 10 C genome chromosomes N11 to N19, corresponding to the respective linkage groups 1 to 10 (or A1 to A10) of B. rapa and 1 to 9 (or C1 to C9) of B. oleracea, respectively. This nomenclature is now widely accepted as the standard for B. napus mapping. A consensus map of B. napus that describes in detail the intergenomic homoeology between the respective A and C genome linkage groups was presented by Udall et al. (2005) based on RFLP polymorphisms in four crosses. Genetic mapping activities for B. carinata and B. juncea have not been as prolific as for B. napus; however, particularly for B. juncea, linkage maps have been used for gene localization, QTL analysis, and marker identification for major traits. Based on RFLP markers also mapped in other Brassica crops, Cheung et al. (1997) generated the first linkage map of B. juncea in 1997 and confirmed the genome duplications between the A and B genomes, and later used this map to localize markers linked to a gene for resistance to white rust (Cheung et al., 1998). The conservation of the progenitor genomes of B. juncea was further demonstrated through comparative mapping with RFLP maps of B. nigra and B. rapa (Axelsson et al., 2000). A high-density B. juncea map was developed by Pradhan et al. (2003) by integrating AFLP markers with RFLP loci, and in recent years the first reports of trait mapping in brown mustard have been published. A number of QTL for seed glucosinolates were localized on a B. juncea RFLP map by Mahmood et al. (2003), while Lionneton et al. (2002, 2004) mapped and compared major gene loci and QTL for numerous agronomical traits, including seed fatty acid contents, glucosinolates, seed coat color, flowering time, plant height, and 1000-seed weight. The two seed color gene loci in B. juncea were also mapped by Sabharwal et al. (2004) and Lakshmi Padmaja et al. (2005). The localization of QTL and SNP markers linked to the B. juncea FAE1 genes controlling erucic acid synthesis was reported by Gupta et al. (2004). A summary of some of the major mapping studies in B. napus and B. juncea and some of the traits investigated is given in Table 7.2. With the discovery of the polymerase chain reaction (PCR), the potential arose to greatly increase the marker density in existing genetic maps through amplification of highly polymorphic anonymous PCR fragments, first with randomly amplified polymorphic DNA (RAPD; Williams et al., 1990), and subsequently with amplified fragment length polymorphisms (AFLPs; Vos et al., 1995) and simple sequence repeat (SSR) markers. In the late 1990s, RAPD and AFLP markers began to be more broadly incorporated in new and existing Brassica genetic maps and provided relatively cheap and less work intensive alternatives for saturation of genome regions containing genes of interest. For example, bulked segregant analysis was used to identify RAPD markers linked to the restorer gene Rfo used in the ‘Ogura’Raphanus sativus cytoplasmic male sterility system (Delourme et al., 1994). Because of their robust nature, generally codominant inheritance, and relatively high level of polymorphism, SSR markers have become a valuable tool for genetic mapping; along with RFLPs, they have become the markers of choice for alignment of oilseed rape genetic maps from different crosses. The high abundance of microsatellites throughout the genome suggests that such sequences are also common within coding regions, which can make SSR markers particularly effective tools for marker-assisted selection and map-based gene cloning. As the quantity of Brassica SSR primer sequences available in the public domain (see www.brassica.info/ssr/SSRinfo.htm) increases, the use of SSR markers for mapping is becoming more widespread. Piquemal et al. (2005) produced a consensus map of B. napus, including 304 SSR loci covering a total of 2619 cM, using 574 F2 plants from an unbalanced diallel cross between six different parental lines. The number of publicly available Brassica microsatellite primers is increasing as a result of publicly funded international initiatives (see www.brassica.info/ssr/SSRinfo.htm); however, in comparison to other important crop species, relatively few markers are freely available to date, which

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Table 7.2 Summary of Some of the Major Linkage Mapping Studies in B. napus and B. juncea Species B. napus

B. juncea

Population Type

Markers Used

Application of Map

References

First B. napus linkage map Blackleg resistance, white rust resistance, growth habit, flowering time, winter tolerance, glucosinolate content, erucic and linolenic acid contents Comparative genome mapping

Landry et al. (1991) Ferreira et al. (1994, 1995a, 1995b, 1995c), Toroser et al. (1995), Thormann et al. (1996), Osborn et al. (1997), Kole et al. (2002b) Parkin et al. (1995), Sharpe et al. (1995), Parkin and Lydiate (1997) Ecke et al. (1995), Uzunova et al. (1995), Uzunova and Ecke (1999), Marwede et al. (2005) Foisset et al. (1995, 1996), Jourdren et al. (1996a), Pilet et al. (1998a, 1998b), Lombard and Delourme (2001) Parkin et al. (2005)

F2:F3 DH

RFLP RFLP, AFLP, isoenzyme

DH

RFLP

DH

RFLP, RAPD, SSR

Seed erucic acid, seed glucosinolates, oil content, tocopherol composition

DH

RAPD, AFLP, RFLP, isozyme, SSR, SCAR, Bzh gene

Dwarf gene, resistance to blackleg and light leaf spot, erucic acid content, linolenic acid content

DH

RFLP

DH

RFLP

DH

AFLP

DH

AFLP, RFLP

Detailed segmental alignment to A. thaliana chromosomes First B. juncea genetic linkage map; localization of white rust resistance gene Seed fatty acid content, glucosinolate content, seed coat color, flowering time, plant height, thousand-seed weight High-density map with >1000 markers

Cheung et al. (1997, 1998)

Lionneton et al. (2002, 2004)

Pradhan et al. (2003)

has hindered the effective integration of genetic maps produced by different groups worldwide. In 2005, a large set of B. napus SSR markers developed in the Celera AgGen Brassica Consortium involving 16 breeding companies from different countries is due to be released into the public domain (Piquemal et al., 2005), and other groups are also planning the release of further mapped SSR markers. The availablility in the public domain of robust, polymorphic, mapped SSR markers spanning the entire B. napus genome will without doubt assist the entire Brassica genetics community in consensus mapping and genome integration. Integration of consensus markers into existing and new genetic maps will considerably accelerate the progress of map and QTL alignment among diverse oilseed rape crosses, and hence will ultimately play a pivotal role in the correlation of candidate gene loci with important QTL. By integrating mapped SSR markers from reference maps developed from publicly available populations of B. rapa, B. oleracea, and B. napus into existing maps, it should ultimately be possible to integrate and align all B. napus maps and establish corresponding associations to newly developed physical maps and physical karyotypes. This will vastly increase the ability to exchange and compare information among different oilseed rape mapping populations. For example, a general comparison of QTL for agronomically important traits will be feasible among different crosses, giving a considerably broader overview of the genetic control of quantitative traits than has been available to date in individual populations. 7.5.3

Physical Maps

Over the past decade, considerable interest has developed in physical mapping of Brassica oilseed species. In many cases, much of the financial input for this work has derived from commercial interests, meaning that many of the resources that are being developed have not yet been made

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publicly available; however, as more and more public funding is invested into international Brassica genomics programs, the quantity of public genomics resources will begin to grow dramatically. Of particular note are the activities of the Multinational Brassica Genome Project (MBGP), which was founded recently by international Brassica researchers to coordinate Brassica genomics activities and pool resources to achieve common goals. The primary aim of this initiative is the provision of freely available genetic resources for Brassica genome analysis, including mapping populations, markers, genomic libraries, expressed sequence tags (ESTs), and genomic sequences. Included among the resources being developed within the MBGP are saturated genetic and physical maps for a B. rapa cross between inbred lines of the Chinese cabbage (B. rapa L. ssp. pekinensis) varieties ‘Chiifu’ and ‘Kenshin’. A group of international researchers has begun the construction of a high-density genetic map of this cross using AFLP, PCR-RFLP, EST, and SSR markers. At the end of 2004, the map comprised around 1000 markers with an average distance of around 2 cM; the aim is to saturate this map with up to 5000 markers during 2005. Because the ‘Chiifu’ inbred line is the genotype used for the complete sequencing of the B. rapa genome (see below), this map provides the opportunity for a direct alignment with the B. rapa physical map and annotation to the genome sequence. The anchoring of ESTs to the B. rapa genetic map, and of B. rapa genomic sequence tags to the Arabidopsis physical map, will in the foreseeable future provide a powerful new set of integrated data linking genetic and physical map information between the model and crop genomes. This information will be of enormous relevance to genome analysis in Brassica oilseeds. Although the physiology and developmental biology of Arabidopsis and Brassica are very similar, the genomes of Brassica species are much more complex than that of Arabidopsis thaliana, as a result of multiple rounds of polyploidy and genome rearrangement during their ancestry. The considerably larger number of genes in B. napus than in A. thaliana can make the identification of gene orthology relationships extremely difficult, and the presence in Brassica of multiple homologues of each gene in A. thaliana provides ample opportunity for divergence of gene function. Genome collinearity has been widely investigated in comparative genomic studies between the model crucifer A. thaliana, which possesses the most extensively studied higher plant genome, and the closely related Brassica crops. Numerous physical mapping and sequencing experiments have revealed considerable conservation of gene sequence and order between Arabidopsis and brassicas, although genome rearrangements are often considerably more complex than they appear at first glance. Nevertheless, around 80 to 90% homology is generally found between the exons of putative orthologous genes in Arabidopsis and Brassica (Schmidt, 2002), meaning that knowledge from Arabidopsis is highly relevant for gene isolation and characterization in Brassica crops. In comparative studies of genome regions flanking known genes, extensive collinearity between Arabidopsis and Brassica genome segments has been observed on a microsyntenic level. However, minor deletions, insertions, and translocations are relatively common in regions surrounding Brassica orthologues of Arabidopsis genes. On the other hand, the large-scale synteny over long chromosome stretches still often allows sequence information from markers flanking genes or QTL of interest in Brassica crops to be used to identify possible candidate genes from the corresponding chromosome regions in Arabidopsis. For example, different homoeologous regions in B. rapa and B. napus that contain various QTL influencing flowering time show significant collinearity to Arabidopsis chromosome sections containing a number of genes relevant to flowering time (Lagercrantz et al., 1996; Osborn et al., 1997; Kole et al., 2001). The use of Arabidopsis as a tool in marker development, map-based gene cloning, and candidate gene identification in Brassica crop species is complicated by the complex arrangement of the (ancestral) polyploid Brassica genomes. As the genome relationships between Arabidopsis and Brassica have been deciphered, however (e.g., Paterson et al., 2000; Schmidt et al., 2001; Parkin et al., 2005), the model plant has developed into the most important resource for gene isolation and characterization in Brassica crops. In recent years, it has become increasingly feasible to integrate genetic mapping with a candidate gene approach (Pflieger et al., 2001) using Arabidopsis

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resources and genome tools to identify gene loci involved in both simple and complex traits. The availability of detailed Arabidopsis sequence information enables the use of syntenic regions surrounding candidate genes for better characterization of orthologous locus copies. For example, BAC clones identified by gene-specific filter hybridization or PCR can be used as a basis for development of locus-specific SNP markers. These can be utilized for allele–trait association studies of relevant candidate genes (Snowdon and Friedt, 2004), or directly for integration of the gene loci in physical functional maps. As a participant in the MBGP, the Plant Biotechnology Centre (PBC) at Latrobe University in Bundoora, Australia, is involved in developing a set of bioinformatic tools for Brassica functional genomics (available at http://hornbill.cspp.latrobe.edu.au/brassica.html). Among other things, applications have been developed for the rapid discovery of SSR and SNP markers in Brassica species using data analysis of accumulated sequence data for primer design (Barker et al., 2003), and the data has been incorporated within an integrated gene annotation database, Brassica ASTRA, that includes modules for the gene ontology (GO) annotation of Brassica sequences and comparative mapping with A. thaliana. A leading role in the MGBP is being played by scientists from the Korean National Institute of Agricultural Biotechnology (NIAB) and the Chungnam National University in Daejeon, South Korea. In particular, this has involved the provision of BAC libraries from Chinese cabbage (B. rapa ssp. pekinensis), which together with high-density filters are freely available for use in the public domain, and an active participation in the ongoing sequencing and physical mapping activities (Yang et al., 2006). At the time of writing, a library of more than 10,000 cDNAs had already been developed, and it is planned to produce a public domain gene expression chip containing 100,000 ESTs within the next few years. More than 1000 molecular markers have been localized on a newly established B. rapa reference map, with alignment to the A genome chromosomes of B. napus and B. juncea. Together, this rapid accumulation of functional and physical genome orientation data and tools will revolutionize the study of Brassica genomes and further accelerate the current progress in the utilization of genome and map information for Brassica oilseed breeding. 7.5.4

Whole Genome Sequencing

The complete sequence of at least one of the Brassica crop species would be an extremely valuable resource for detailed physical and comparative genome analysis, gene isolation, and ultimately also for development of markers for practical breeding. Due to the extensive chromosomal and sequence collinearity among Brassica genomes, the sequence of B. oleracea or B. rapa could be used for physical genome analysis not only within these species, but also in the amphidiploid Brassica oilseed species. In recent years, considerable progress has been made in two different endeavors to sequence the complete genomes of the respective B. napus progenitor species. A total of 454,274 B. oleracea genomic sequence reads, with an average length of around 650 bp, have been generated from a joint whole genome shotgun-sequencing initiative between the Institute for Genomic Research (TIGR), Rockville, MD, and Cold Spring Harbor Laboratory, NY, funded by the U.S. National Science Foundation. The reads (available at http://www.tigr.org) cover some 295 Mbp (about 0.45x) of the B. oleracea genome, and around a quarter have a match to known proteins. Interestingly, however, only around 40% of the sequences appear to have a highquality match to the genome sequence of Arabidopis, although some 90% of the Arabidopsis proteome was represented in the B. oleracea sequence reads. As expected, matches to Arabidopsis sequences are very good in exons and reduced in introns, whereby the conserved regions generally extend into the introns somewhat (i.e., intron–exon splice sites are also conserved). According to Town et al. (2006), the B. oleracea sequence data not only provide an exciting new resource for analysis of Brassica genomes, but also will be extremely useful for improved gene annotation in Arabidopsis. First estimates using the B. oleracea sequence data to reprogram A. thaliana gene prediction models appeared to identify some 2000 to 5000 novel genes in the model genome. Very

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rough first estimates of the gene number in B. oleracea predicted a minimum number of around 41,000 genes based on the sequences available. A central part of the MBGP initiative has been the grounding of a multinational project aimed at sequencing the complete genome of B. rapa as a basic DNA sequence resource for Brassica A and C genome crops. The Brassica genome sequencing project aims initially to generate a fully oriented and ordered phase 2 sequence (meaning that it will contain some small sequence gaps and low-quality sequences) using BAC clones covering the 500-Mb genome of B. rapa ssp. pekinensis (Chinese cabbage). The genome sequence will be anchored to the MBGP reference genetic map and will be annotated with the help of the A. thaliana genome sequence. The sequencing program has been divided into three stages: Initially, an online Brassica information resource was established (http://www.brassica.info), along with an online portal for the MBGP (http://brassica.bbsrc.ac.uk/ and http://www.niab.go.kr). Databases have been constructed for the deposition of genetic (http://ukcrop.net/perl/ace/search/BrassicaDB) and physical mapping data (http://brassica.bbsrc.ac.uk/ IGF/index.htm and http://www.brassicagenome.org). Reference genome libraries have been produced for the B. rapa variety ‘Chiifu’ (Chinese cabbage, B. rapa ssp. pekinensis). The libraries ‘KBrH’ and ‘KBrB’, each consisting of 144 × 384 well plates, were generated using HindIII (‘KBrH’) and BamHI (‘KBrB’) digested genomic DNA, respectively. A total of 110,592 clones are available, providing 20-fold redundant genome coverage. International distribution centers for the two libraries have been set up at NIAB in South Korea and at the John Innes Centre, U.K. Reference DH and RIL populations for low- and high-resolution genetic mapping of the Chinese cabbage variety ‘Chiifu’ are under construction, and high-quality genetic maps for each population are being constructed using publicly available markers. The full set of 110,592 BAC clones in the reference libraries are currently being end sequenced, with data deposited in searchable public databases at TIGR, the Munich Information Centre for Protein Sequences (MIPS), and NIAB. The aim of the project is to deliver the flanking sequences of all available BACs during 2005. Using a computational tool developed by the Plant Biotechnology Centre of Latrobe University in Australia, the end sequences of the B. rapa BACs are comparatively mapped onto the Arabidopsis genome (Love et al., 2005). The publicly available C genome shotgun sequences have also been compared with the Arabidopsis genome sequence, meaning that comprehensive data will exist for navigation between homoeologous sequences in the genomes of B. rapa, B. oleracea, and A. thaliana. The second phase for the B. rapa sequencing project foresees an unambiguous genetic anchoring of around 1000 seed BACs to the Arabidopsis genome sequence using both end sequences, accompanied by the anchoring to one of the B. rapa reference genetic maps using single-locus SSR, SNP, or InDel markers. In the third and final stage, the actual genome sequencing will be performed, with division on a chromosome-by-chromosome basis, as in other multinational genome sequencing programs. Participants will begin by sequencing all seed BACs on their chromosome or defined subregion of a chromosome. All sequence data will be submitted to TIGR, MIPS, and NIAB as soon as phase 2 quality is achieved (see http://brassica.bbsrc.ac.uk/brassica_genome_sequencing_concept.htm).

7.6 GERMPLASM ENHANCEMENT: CONVENTIONAL BREEDING 7.6.1

Breeding Methods

7.6.1.1 Cultivar Development The majority of B. napus and B. juncea oilseed rape cultivars are pure lines derived from breeding schemes designed for self-fertilizing crops, i.e., pedigree selection or modifications thereof. For the largely self-incompatible B. rapa, on the other hand, the most common breeding strategy is recurrent selection, while synthetic cultivars are also used to a certain extent. Backcrossing has been successfully used to transfer simple inherited traits such as low erucic acid and

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glucosinolate content into adapted Brassica breeding material. Particularly, oilseed rape is one of the most amenable crop species to improvement through biotechnology. For instance, it is possible to reproducibly obtain haploid, and subsequently doubled haploid (DH), plants through anther or microspore culture (Friedt and Zarhloul, 2005; Weber et al., 2005). The principal advantage of the haploidy technique is the rapid fixation of segregating genotypes, occurring in lower frequency, in which recessive genes coding for specific traits are combined in the homozygous condition. Thus, utilization of microspore culture can allow a substantial acceleration of the breeding cycle. Due to the generally high response of B. napus genotypes, the use of DH production has become common practice in commercial breeding programs and has already resulted in numerous licensed cultivars (Friedt and Zarhloul, 2005). Besides haploid techniques, other prebreeding methods like wide hybridizations using embryo rescue techniques or protoplast fusion can also be used to create novel genetic variation (reviewed by Thierfelder et al., 1992; Inomata, 1993; Glimelius, 1999). However, once a useful property has been identified in a basic breeding stock, e.g., a mutant line or germplasm from a wild relative, it may take many years to accomplish the development of cultivars possessing this novel desirable trait. Marker-assisted selection has shown a significant impact on the efficiency of plant breeding routines such as backcrossing programs. In cases where conventional approaches have not been sufficient, further improvements can be achieved by genetic engineering (Murphy, 1995; Poulsen, 1996; Sharma et al., 2002). 7.6.1.2 Hybrid Breeding Although for many years the emphasis in oilseed rape breeding was strongly focused on openpollinating varieties, up to 30% heterosis for seed yield has been reported for B. napus (Schuster, 1969; Grant and Beversdorf, 1985; Lefort-Buson et al., 1987; Brandle and McVetty, 1989), and for both winter rapeseed and spring canola hybrid varieties have rapidly gained in importance over the past decade as effective systems for controlled pollination have been developed. The first restored winter rapeseed hybrids were released in 1995. In current European winter rapeseed material, yield improvements of up to 15% have been reported for F1 hybrids, compared to nonhybrid openpollinated varieties. This has led to a major increase in production of hybrid rapeseed in the leading producing countries. For example, although only 14 of the 53 approved German ‘00’ winter rapeseed cultivars listed by the German Plant Variety Office in 2004 were hybrids (Bundessortenamt, 2005), more than 50% of the 1.43 million ha of German winter rape in 2004/2005 were planted with hybrid varieties. Furthermore, in 2003/2004 the hybrid cultivar Talent replaced the open-pollenating ‘Express’ as the most widely cultivated winter oilseed rape variety in Germany, the first time a hybrid cultivar has achieved the top position. A similar situation is seen in the other major canola and rapeseed-producing countries. For example, over half of the more than 7 million ha of rapeseed now produced in China comes from hybrid varieties. One of the most important reasons for the upsurge in interest in hybrid varieties is that they tend to have higher yield stability and better adaptation to low-input cropping systems than conventional cultivars (Budewig and Léon, 2003; Friedt et al., 2003). Although numerous cytoplasmic male sterility (CMS) systems are available from different sources, their use in oilseed rape breeding is often inhibited by instability, the absence of suitable restorer or maintainer lines, or negative effects of the cytoplasm used to induce the male sterility. Environmental instability of the expression of nap male sterility means this system is unsuitable for hybrid production, and the ‘Polima’ pol) system was only made workable by the screening of huge numbers of lines in different environments (Bartkowiak-Broda et al., 1991), in order to identify stable maintainer genotypes. The monogenically inherited restorer genes for B. napus ‘Polima’ CMS can be readily introduced into elite lines, and pol is therefore now effectively used to produce registered F1 hybrid spring canola varieties in numerous countries. Male-sterile-inducing cytoplasm can also have negative effects on flower morphology, nectar production, or yield, and sometimes

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chlorophyll deficiencies also need to be overcome. In some cases, suitable B. napus restorer lines have been produced for B. tournefortii CMS (Banga et al., 1995; Stiewe et al., 1995a, 1995b). Restored F1 hybrids based on the ‘Ogura’ CMS system are under increasing production in France and other European countries, and hybrid cultivars based on the commercial Male-Sterility Lembke (MSL) system are currently among the best-selling winter oilseed rape varieties in Europe. 7.6.2

Breeding for End Use

In Brassica oilseeds, the occurrence of two components traditionally distinguished them from other major oilseeds. Both components, erucic acid and glucosinolates, were considered antinutritional for humans and animals. First, the seed oil formerly contained approximately 25 to 50% erucic acid (cis-13-docosenoic acid), referred to as high erucic acid rapeseed or mustard. It was claimed that erucic acid had an adverse effect on experimental animals when fed as a very large proportion of the diet. Second, the meal contained considerable amounts of glucosinolates, a group of chemically related thioglucosides, some of which had goitrogenic and other antinutrional properties. The situation changed after plant breeders successfully altered the chemical composition of Brassica oilseeds (for review, see Downey and Röbbelen, 1989). First, the introduction of oils virtually free of erucic acid enhanced their attractiveness as edible oil. Second, low erucic acid varieties with very low glucosinolate content in the seed additionally increased the potential of rapeseed meal as a protein supplement in animal feeds. Canola derived from B. napus and B. rapa is currently defined as having less than 2% erucic acid in the oil and less than 30 μmol total glucosinolates per gram of oil-free meal. Currently, a new standard is foreseen for Canadian canola that will demand an oil with less than 1% erucic acid and less than 18 μmol total glucosinolates per gram of seed (Canola Council of Canada, 2004). Seed from B. juncea will be included in this standard, provided the fatty acid profile of quality mustard oil meets the definition of canola. In order to improve the feeding value of mustard meal for livestock, low glucosinolate forms had to be developed, similar to those developed in B. napus and B. rapa (Love et al., 1991; Sodhi et al., 2002). 7.6.2.1 Protein Content and Meal Quality The gross seed composition of Brassica oil crop species varies widely, depending on both genetic (i.e., species, variety, cultivar) and environmental (e.g., temperature, water and nutrient supply) factors. The oil content ranges from 36 to 50% (on a dry matter basis), while the oil-free meal contains 33 to 48% protein (Salunkhe et al., 1992; Appelqvist and Ohlson, 1972; Arnholdt and Schuster, 1981). Although oil and protein content are negatively correlated, improvements can be achieved through selecting for the sum of the two seed components (Stefansson, 1983; Arnholdt and Schuster, 1981; Grami et al., 1977). Glucosinolates in the meal of oilseed rape (and other Brassica crops) still present several problems, however, as does the presence of antinutritive compounds like sinapic acid esters, phytates, phenolic acids, and the comparatively high dietary fiber content (approximately 15% of dry oil-free meal). Due to the small size of the seeds, the hull, accounting for about 10 to 20% of the seed weight, imparts most of the fiber content to the meal (Appelqvist and Ohlson, 1972; Anjou et al., 1977). Currently, the most promising method to genetically reduce fiber and hull content is to breed cultivars with a yellow or light-brown seed coat, as is found in B. carinata, B. juncea, and sarson subspecies of B. rapa. Since yellow seed coats are significantly thinner than brown or black ones, the development of pure yellow-seeded B. napus cultivars with agronomically acceptable performance still remains an important goal in quality breeding toward increased oil and protein content combined with better nutritional properties of the seed meal.

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7.6.2.2 Oil Content and Quality The improvement of oil content is an important goal due to the primary economic value of the oil component and its relatively high heritability (Grami et al., 1977), and has to date been quite successful due to the ease and speed with which oil content can be measured by nondestructive nuclear magnetic resonance (NMR) techniques. The value and suitability of rapeseed oil for nutritional or industrial purposes are determined by its fatty acid composition. The identification of naturally occurring zero-erucic mutants in both B. napus and B. rapa was the first discovery opening the era of mutant-derived quality improvement in oil crops (Stefansson and Hougen, 1964; Downey, 1964; Röbbelen, 1990). Canola-quality rapeseed low in saturated fatty acids and almost lacking nutritionally undesirable very long chain fatty acids meets all the requirements of a prime edible oil (Downey and Bell, 1990; Ackman, 1990; Trautwein, 1997). Despite the beneficial nutritional properties of α-linolenic acid (18:3n-3), the oxidative stability of the oil can be improved by decreasing the linolenate content from the average 10% to less than 3%, which results in enhanced shelf-life (Downey and Bell, 1990; Rakow et al., 1987; Pleines and Friedt, 1988, 1989). In the last three decades, improvements of the C18 fatty acid composition in rapeseed (B. napus) were achieved by selecting altered linoleate/linolenate genotypes after chemical mutagenesis. The fatty acid profiles of these lines indicated that nearly all of the linolenic acid was directed to linoleic acid and that the level of oleate increased only insignificantly (Röbbelen, 1990; Rakow, 1973; Rakow et al., 1987; Röbbelen and Nitsch, 1975; Röbbelen and Thies, 1980). In 1988, the first spring rapeseed cultivar, 'Stellar', which produces oil containing less than 3% linolenate, was released for commercial production in Canada, although its agronomic performance was less than satisfactory (Scarth et al., 1988). The most important factor influencing the biogenesis of the unsaturated fatty acids is the prevailing temperature during seed development (Pleines and Friedt, 1988, 1989). The development of canola cultivars with reduced levels of polyunsaturated fatty acids (PUFAs) accompanied by higher oleate content would produce a dietary oil with additional markets (Marsic et al., 1992). For industrial applications, a very high content of oleic acid (80 to 90%) is preferred, because this is most suitable for certain consecutive chemical reactions (Lühs and Friedt, 1994). To increase the oleic acid content to above 80% and, concomitantly, to lower the level of PUFAs, different breeding procedures have been utilized. These include mutagenesis applied to seeds (Auld et al., 1992; Rücker and Röbbelen, 1995; Schierholt et al., 2001) or microspore-derived embryos (Wong et al., 1991). Furthermore, relevant genes of Δ12 or Δ15 desaturases, responsible for the biosynthesis of 18:2n-6 and 18:3n-3, have been isolated, and subsequently, rapeseed has been genetically engineered, leading to either high oleic acid or high linoleic acid profiles (Hitz et al., 1995; Scheffler et al., 1997). Traditional varieties of Brassica oil crop species typically contain appreciable levels of longchain fatty acids like eicosenoic (20:1n-9), erucic (22:1n-9), and nervonic (24:1n-9) acids. Erucic acid synthesis is governed by one gene locus in the monogenomic species B. rapa (Dorrell and Downey, 1964; Jönsson, 1977) and B. oleracea (Lühs et al., 2000; Seyis et al., 2004). Consequently, two major gene loci contribute to erucic acid synthesis in the amphidiploids B. napus (Harvey and Downey, 1964; Jönsson, 1977), B. juncea (Kirk and Hurlstone, 1983), and B. carinata (Getinet et al., 1997). Multiple alleles occur at each locus, acting in a largely additive manner. Homozygous genotypes with various alleles produce levels of erucic acid ranging from less than 0.1 to about 60%. In the winter forms of B. napus, alleles are present that in a single dose give about 15 to 18% erucate (Jönsson, 1977). Because oleic acid operates simultaneously as the substrate for desaturation, it is assumed that in high erucic acid rapeseed at least two minor gene loci are also involved that control the desaturation of oleic acid to form linoleate and linolenate, respectively (Jönsson, 1977; Chen and Beversdorf, 1990).

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Yield Potential and Stability

Unlike soybeans, peanut, and most other oilseeds, breeding progress in Brassica oil crops has always been connected with drastic improvements of seed quality, followed by relatively quick acceptance by growers and the processing industry. However, research toward seed quality does not ignore the importance of high yields. In terms of morphological and agronomical characteristics, the yield of rapeseed is governed by the number of siliques per unit area, the number of seeds per silique, and the 1000-seed weight (Diepenbrock, 2000). Major advances in yield potential and stability have been achieved with hybrid cultivars, which generally exhibit an improved adaptability to abiotic constraints and relatively good yield stability also under conditions of nutrient deficiency. With new and improved male sterility systems and advanced knowledge regarding the basis and exploitation of heterosis, hybrid cultivars have brought major advances in yield potential and yield stability over the past decade and represent the most promising material for further improvements in the near future. On the other hand, further improvement of productivity must also account for agronomic parameters, such as early-maturing varieties, resistance to lodging and shattering, and resistance to weeds, insects, and particularly major diseases. 7.6.4

Improved Resistance to Biotic and Abiotic Constraints

7.6.4.1 Fungal and Viral Diseases A number of diseases lead to serious yield losses in Brassica oilseeds on a regional or global scale. In the following, we will present brief details on some of the more serious diseases of Brassica oilseeds; a detailed summary of the major diseases of Brassica oilseeds can be found in Rimmer and Buchwaldt (1995). Two of the most important diseases of B. napus are Sclerotinia stem rot (Sclerotinia sclerotiorum (Lib.) de Bary) and stem canker (Leptosphaeria maculans (Desm.) Ces. Et de Not., anamorph Phoma lingam (Tode ex. Schw.) Desm.), also known as blackleg disease. Blackleg is the most damaging disease of oilseed rape in Europe and Australia, and Sclerotinia stem rot is one of the most important diseases of oilseed rape in China, while both diseases play a significant role in Canada. Blackspot caused by Alternaria brassicae (Berk.) Sacc. and its close relatives is one of the most destructive fungal diseases affecting B. juncea and B. rapa worldwide, and simultaneous infection with white rust (Albugo candida (Pers. ex. Hook) Ktze.) and downy mildew (Perenospora parasitica (Pers. ex. Pers.) Fr.) is common on both B. rapa and B. juncea, particularly in northern India. White rust is one of the most important diseases of B. juncea in India. A number of additional diseases have significant local importance: Verticillium wilt caused by Verticillium longisporum (ex. Verticillium dahliae var. longisporum Stark) is a particular problem in affected areas of Sweden, Denmark, Great Britain, and the north of Germany, light leaf spot (Cylindrosporium concentricum Grev., anamorph Pyrenopeziza brassicae Sutton et Rawlinson) in northern parts of Europe, and clubroot (Plasmodiophora brassicae Wor.) in Scandinavian countries and the northern U.K., while in parts of China viral diseases can also cause substantial yield losses. Breeding for improved disease resistance has involved a range of strategies. In some cases, it has been possible, particularly in B. napus, to utilize natural variation to breed varieties with improved resistance, with the best examples being light leaf spot and blackleg disease. For blackleg, a broad and relatively durable quantitative resistance is available that has to date enabled relatively good control of this disease. For other diseases, like blackspot, Sclerotinia stem rot, white rust, and Verticillium wilt, availability of resistant lines is more limited. Cultural control methods, particularly rotation, are important means of controlling diseases such as clubroot and Sclerotinia, and good agronomic practice is necessary to limit the number of susceptible crops in the rotation (Walker and Booth, 2001). In many cases, the only option for development of resistant cultivars is the transfer of resistant germplasm from other Brassica species and related crucifers. This has been successfully achieved for resistance to clubroot and blackleg diseases, where resistance sources from B. rapa

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have been transferred to B. napus and resulted in the release of resistant cultivars. For more examples and details, see Section 7.9. For control of Sclerotinia stem rot, one option that has been pursued is the breeding of apetalous varieties (Buzza, 1983; Jiang and Becker, 2003; Zhao and Wang, 2004), which prevent the common infection pathway of ascospores entering the stem from senescing petals trapped in leaf axils. In recent years, some progress has also been made in transgenic approaches toward Sclerotinia control. The most well known viral diseases of Brassica oilseeds are turnip mosaic virus (TuMV), which infects all cultivated brassicas and many wild relatives; cauliflower mosaic virus (CMV); turnip yellows luteovirus (TuYV), also known as beet western yellows virus; and turnip yellow mosaic virus (TuYMV). TuYMV is spread by stem weevils, cabbage stem flea beetles, and other biting insects, while the other viruses are all spread by aphids. Because of the broad host range, control of the viruses can be difficult, and because little or no resistance is available in cultivars, control of the vector insects with pesticides is needed in heavily infested areas to prevent yield losses. In the case of TuYV, which in parts of Europe can infect entire oilseed rape fields and cause yield losses up to 20%, resistance sources have been identified in Chinese cabbage and transferred to breeding material via resynthesized rapeseed (Paetsch et al., 2003). 7.6.4.2 Pests A wide range of pests infest canola and other Brassica oilseed crops worldwide. For a detailed overview of the major insect pests in the main oilseed Brassica production areas of the world, see Ekbom (1995) and Alford et al. (2003). Generally, specific breeding for pest resistance has a low priority in comparison to disease resistance and yield improvement. For many pests, little resistance exists among current canola and rapeseed cultivars. On the other hand, a broad variation was observed by Poulsen et al. (2004) among genetically diverse gene bank accessions of B. napus tested for resistance to cabbage stem weevil (Ceutorhynchus quadridens (Panz.), syn. C. pallidachtylus Mrsh.), rape stem weevil (Ceutorhynchus picitarsis Gyll.), cabbage stem flea beetle (Psylliodes chrysocephala L.), and field slugs (Deroceras reticulatum (Mull.) or Deroceras agreste L.), four of the major pests infesting European rapeseed. Hence, the potential exists for introgression of resistance into breeding material if necessary. 7.6.4.3 Abiotic Stress One of the most important forms of abiotic stress for oilseed rape is cold stress in winter forms sown in autumn, which in particularly cold climates or harsh winters must possess a certain degree of freezing tolerance to survive the winter. Besides acclimatized and nonacclimatized freezing tolerance, winter survival may also be affected by genetic variation for other cold-regulated traits, like vernalization-responsive flowering time. Because biennial B. napus forms generally have a higher winter survival than annual forms, populations that segregate for resistance to cold stress can be generated by crosses between the two. Using the DH population produced by Ferreira et al. (1994) from the cross between the winter-hardy, freezing-tolerant winter rapeseed variety ‘Major’ and the cold-sensitive spring canola cultivar Stellar, different QTL for acclimatized and nonacclimatized freezing tolerance were detected (Teutonico et al., 1995). Using the same immortal population, Kole et al. (2002a) localized QTL for winter survival, freezing tolerance, and flowering time and compared the map positions with corresponding loci in B. rapa. The B. napus population was evaluated in multiple winters, and 6 of a total of 16 significant QTL for winter survival were detected in more than one winter. Some QTL for the different traits were found to co-localize both within B. napus and within B. rapa, suggesting that some alleles causing greater acclimated freezing tolerance and later flowering time also contributed to increased winter survival. Correspondence in the map positions of QTL between species provided evidence for allelic variation at homologous loci in B. rapa and B. napus. Interestingly, many of the DH lines were found to exhibit better

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winter survival than the winter-hardy parent ‘Major’, suggesting that favorable alleles were also contributed by the cold-sensitive parent. This was supported by results from QTL mapping, in which alleles increasing winter survival were detected from both parents. Because of the complex nature of this trait, breeding is difficult, and generally it is necessary to screen prospective lines over numerous winters to ensure reliable winter hardiness.

7.7 MOLECULAR GENETIC VARIATION In contrast to B. juncea, for which a large variety of distinct forms have enabled the identification of various putative centers of origin, for winter oilseed rape only three distinct local landraces are known. These evolved in different European climate zones, and hence display variation in vegetative growth and winter hardiness. The first cultivar released, 'Lembkes', was selected in Germany from a Mecklenburg landrace in the early 20th century and subsequently exploited extensively in French, Swedish, German, and Polish breeding programs. The genetic base of canola and oilseed rape is even narrower today, because the introduced double-low (00) quality again originates from single sources. Consequently, there is a need to introduce new genetic variation to breeding material, since most cultivars share a more or less common parentage (Thompson, 1983; Downey and Rakow, 1987). Compared to the narrow gene pool of present canola-quality oilseed rape breeding material, which severely limits the formation of heterotic pools, erucic acid- and glucosinolate-containing plant material represent a comparatively genetically divergent source for the development of heterotic rapeseed forms (Röbbelen, 1975; Thompson, 1983; Schuster, 1987). Because of the emphasis on oil quality, such material has found only limited use in practical rapeseed breeding in the past few decades. However, strong heterotic effects are observed in experimental crosses between material of distant geographical and genetic origin (Lefort-Buson et al., 1987; Brandle and McVetty, 1990), and efforts are increasing to develop new cytoplasmic-genetic male-sterile and restorer lines as the most promising system for the production of new hybrid cultivars. Following appropriate quality conversion, inbred lines and DH lines with a high genetic distance to existing 00-quality varieties have the potential to become an important resource for the development of high-performance pools with improved combining ability compared to existing 00-rapeseed material. Besides spring and winter oilseed rape types, B. napus is often also grown as a fodder crop or as green manure. Swede cultivars (B. napus ssp. napobrassica (L.) Hanelt) are also relatively common, particularly in Great Britain and Scandinavia, and a small number of rape kale vegetable forms (B. napus ssp. napus var. pabularia) are also known, predominantly in Asia. Owing to their generally unsuitable seed characters, however, in particular high contents of seed erucic acid, glucosinolates, and other antinutritive substances, fodder and vegetable rape forms have been generally overlooked for breeding of canola-quality oilseed cultivars in recent decades. This emphasis on specific oil quality traits has led to a considerable narrowing of the gene pool of elite breeding material in recent decades. On the other hand, genetically diverse material among vegetable and fodder rape represents a potentially valuable source for improved pathogen and pest resistance (Poulsen et al., 2004), and introduction of untapped germplasm into breeding lines also has the potential to improve heterotic potential. However, the construction of genetic pools, as used, for example, in maize hybrid breeding, has not been achieved for oilseed rape to date. Because of linkage drag for negative seed yield and quality traits associated with non-oilseed rape morphotypes, identification of exotic germplasm among the respective gene pools of winter and spring oilseed forms is of particular interest in this respect. Using SSR markers, we found considerable genetic variation in B. napus vegetable and fodder rape genotypes compared to the gene pools of conventional spring and winter oilseed material (Hasan et al., 2006). Similar extreme genetic variation, compared to conventional rapeseed cultivars, was also found in resynthesized rapeseed lines analyzed by (Becker et al., 1995) using allozyme and RFLP markers, and in other resynthesized rapeseed material, investigated by Seyis et al. (2003a) using AFLP markers. In the latter study, the genetic differences were correlated to heterotic yield

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potential in experimental hybrids (Seyis et al., 2006). Such exotic material must obviously be viewed from a long-term perspective with regard to use in oilseed rape breeding; however, genetic diversity analyses using molecular markers have the potential to identify novel genetic variation that might assist in future improvement of heterotic potential in B. napus. A promising new method for inducing, detecting, and utilizing novel nontransgenic mutants in crop plants is TILLING (targeted identification of local lesions in genomes; McCallum et al., 2000). The TILLING technology was developed for the detection of point mutations or small insertion–deletion events (indels) in genes, usually created through chemical mutagenesis using ethyl methanesulfonate (EMS). The basic principle involves the isolation of DNA from individuals of a mutant population, its mixture with DNA from a nonmutant individual, amplification by PCR of the target DNA, the de- and renaturation of the PCR products, and the treatment of these doublestranded DNA fragments with the enzyme CelI endonuclease (Oleykowski et al., 1998). CelI cleaves DNA strands specifically at mismatch sites, which will occur in the double-stranded PCR products after renaturation if there is a change in the sequence in this region of the DNA in the mutagenized individual. After the CelI reaction, the DNA fragments are separated, usually on a DNA sequencer, and the presence of a mutation can be detected through the appearance of shortened DNA fragments (created through the nuclease cleavage at the mismatch site). In analogy to the detection of induced mutations, this technique can also be applied to detection and distinction of natural variation in genes. The development of mutagenized TILLING populations for Brassica oilseed crops will in the future offer the possibility to generate and directly identify interesting mutants displaying novel genetic variation for specific traits (genes) of interest. Genotypes with altered expression of relevant genes can potentially be used directly for cultivar development, because they generally represent near-isogenic lines (i.e., more or less single-gene or single-trait mutants) of a homogeneous cultivar. Thus, particularly for traits with well-characterized gene expression pathways, like fatty acid biosynthesis, TILLING offers a unique technology for rapid generation of non-genetically modified (GM) variants with novel trait expression. Numerous commercial and public consortia are currently working on establishing TILLING populations and screening resources for oilseed rape, and this breeding option is certain to gain in importance in the coming decades.

7.8 TISSUE CULTURE AND GENETIC TRANSFORMATION 7.8.1

Generation of Doubled Haploids

Considerable progress has been accomplished in the cellular and molecular biology of Brassica species over the past few decades. Plant regeneration has been increasingly optimized via organogenesis and somatic embryogenesis using various explants, with tissue culture improvements focusing on the developmental stage of the explant, genotype, and media additives. The production of haploids and doubled haploid (DH) lines using microspores has now become a routine procedure for commercial Brassica breeders, a development that has considerably accelerated the production of homozygous lines. Haploidization followed by subsequent chromosome doubling has become an effective tool in Brassica oilseed breeding for the generation of homogeneous lines from early-generation material (for a recent review, see Friedt and Zarhloul, 2005). Haploid induction through anther culture has become a standard procedure since the first reports for B. napus in the mid-1970s (Thomas and Wenzel, 1975; Keller and Armstrong, 1978), and a few years later for B. rapa (Keller and Armstrong, 1979) and B. juncea (George and Rao, 1980). Haploids are generally generated by inducing microspore embryogenesis, which can achieve recovery of large numbers of embryos in suitable Brassica material. The success of embryogenesis is influenced by numerous factors, including various pretreatments, the composition of the culture medium, and the genotype, physical condition, and pollen developmental phase of the anther donor.

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Segregating DH populations have also played a major role in genetic mapping studies for Brassica oilseeds. The majority of the published mapping and QTL studies in oilseed rape were performed in DH lines, which in contrast to F2:F3 families have the advantage of being “immortal” populations that can be tested in unlimited environments and years for multiple traits. For example, the same DH population developed at the University of Wisconsin by Thomas Osborn and colleagues from a cross between the winter rapeseed variety ‘Major’ and the spring canola cultivar Stellar (Ferreira et al., 1994) has been used for mapping studies of a broad range of traits, including blackleg resistance (Ferreira et al., 1995a), white rust resistance (Ferreira et al., 1995c; Kole et al., 2002b), vernalization requirement (Ferreira et al., 1995b), flowering time (Ferreira et al., 1995b; Osborn et al., 1997), winter tolerance (Kole et al., 2002a), glucosinolate content (Toroser et al., 1995), and the contents of erucic acid and linolenic acid (Thormann et al., 1996). Similarly, the DH population developed by the INRA research institutes in France from a cross between the French dwarf winter rapeseed ‘Darmor-bzh’ and the Korean spring type ‘Yudal’ (Foisset et al., 1995, 1996) has been used to map numerous agronomically important traits and single genes, including the dwarf gene Bzh (Foisset et al., 1995), gene loci for erucic acid content (Jourdren et al., 1996a) and linolenic acid content (Jourdren et al., 1996b), and QTL for blackleg resistance (Pilet et al., 1998a, 1998b, 2001) and light leaf spot resistance (Pilet et al., 1998b). Compared to other immortalized populations, like recombinant inbred or single-seed descent lines, backcross lines, or chromosome introgression lines, DH populations are a comparatively cheap and rapid alternative that today can be produced at relatively high efficiency from the majority of oilseed rape crosses. 7.8.2

Sexual and Somatic Hybridization

Tissue culture techniques have also played a major role in the introgression of novel germplasm into the genomes of Brassica oilseed crops through interspecific hybrids with related crucifer species, or via resynthesis of the amphidiploid species by crosses among the diploid progenitors (Inomata, 1993; Lühs et al., 2002). Such crosses are generally inviable due to incompatibility, and can only be generated using ovuli culture or embryo rescue techniques (Inomata, 1993). Generation of fertile offspring is then achieved via chromosome doubling, whereby embryo rescue is often also required for the initial backcross generations derived from intergeneric hybrids. Somatic cell fusion has been used for many decades to develop interspecific and intergeneric hybrids for the transfer of traits of interest (particulary biotic resistances and male sterility systems) to oilseed Brassica crops, and crop improvement using somaclonal variation has also been achieved (Glimelius, 1999). Numerous examples for the successful use of these techniques for trait introgression, particularly into oilseed rape, are given below in Section 7.9. 7.8.3

Genetic Modification

Genetic engineering is considered a powerful tool for practical plant breeding, since the transfer of specific traits to a target genotype is possible without changing the phenotype and agronomic performance of the recipient plant. Oilseed rape is particularly amenable to Agrobacterium tumefaciens-mediated transformation, and during the last two decades, considerable progress has been achieved in the development of transgenic varieties (Poulsen, 1996). Soybean, cotton, corn, and canola are the four principal crops in which transgenic technology is utilized. After herbicidetolerant soybean and insect-tolerant Bt-corn and Bt-cotton, the fourth most dominant GM crop worldwide is herbicide-tolerant canola, which in 2003 was grown on 3.6 million ha, equivalent to 5% of the global transgenic production area. The widespread production of genetically modified crop plants in North America has not been continued in the European Union member states, however. This is mainly due to limited public acceptance and unclear administrative legislation (Friedt and Lühs, 1998). One of the major markets for transgenic canola is Canada, where today the vast majority of the crop comprises herbicide-tolerant varieties, and a significant proportion

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of the continually expanding production of oilseed rape in China is also comprised of genetically modified varieties. The first generation of transgenic canola varieties showed a strong emphasis on herbicide tolerance and hybrid breeding systems; however, efforts are increasing in the areas of genetically modified fatty acid biosynthesis and, to a certain extent, in the introgression of transgenic pest and disease resistance. The first glufosinate-ammonium-tolerant B. napus spring cv. Innovator was registered for production in Canada in 1995 (Oelck et al., 1995). Although weed control in canola is possible with available herbicides, multiple treatments with chemicals of different herbicide families are often required for control of all weeds. Certain cruciferous weeds, such as wild mustard (Sinapis arvensis L.) and stinkweed (Thlaspi arvense L.), are difficult to control, and the use of specialty herbicides for cruciferous weed control is sometimes required. In addition, the need to use a range of different herbicides for effective weed control increases production costs and the chemical load on soils. The availability of several types of herbicide-tolerant plants allows for rotation of herbicides, minimizing the risk of weeds becoming resistant to any particular one. Several varieties of transgenic herbicide-tolerant oilseed rape are grown and processed in the U.S., Canada, and China. In 2004, herbicide-tolerant varieties comprised more than 85% of the canola crop in Canada, with the majority of the herbicide tolerance being of transgenic origin (see http://www.canola-council.org/). Modification of the fatty acid composition to make rapeseed oil more competitive in various segments of the food and industrial oil markets has become an important objective of oilseed rape molecular genetics and breeding. One of the central objectives in this context is the genetic modification of the seed storage oil by maximizing the proportion of specific or functional fatty acids, in order to obtain tailor-made raw materials suited for various industrial purposes (Friedt and Lühs, 1998; Biermann et al., 2000). However, the quality of vegetable food products has increased in relevance for human nutrition in recent decades, with the advent of so-called functional foods. With regard to specific properties of such nutritive substances, genetic engineering offers the possibility to adapt plant storage lipids and secondary compounds (such as tocopherols and other vitamins) to meet specific nutritional and even therapeutic requirements (Leckband et al., 2002; Friedt et al., 2004; Kumar et al., 2005). Rapeseed oil is unique in having a large spectrum of usability and positive properties for food and nonfood applications. Genetic engineering of plant lipid biosynthesis in rapeseed has already led to commercialization, with transgenic varieties expressing genetically modified fatty acid patterns available since 1995 (Friedt and Lühs, 1998).

7.9 INTERSPECIFIC AND INTERGENERIC HYBRIDIZATION The genetic basis of oilseed rape breeding material can also be expanded by introgressing novel germplasm from resynthesized (RS) rapeseed produced by crossing genetically diverse genotypes of the diploid parents of the amphidiploid B. napus. This has the potential not only to increase genetic variability with a view to hybrid breeding or quality improvement (Seyis et al., 2003b, 2005), but also to broaden the genetic base with respect to pest and disease resistances, which in some cases is severely eroded in B. napus. For such interspecific hybridizations, biotechnological tools like embryo rescue techniques or protoplast fusion are vital to circumvent incompatibility barriers; however, the resulting genotypes are generally fully fertile and can be introduced directly into backcrossing programs to introgress the traits of interest into oilseed rape or canola breeding lines. Resynthesized rapeseed represents an interesting source of genetic variation for quality improvement in oilseed rape. For example, we have made crosses between B. rapa ssp. trilocularis (yellow sarson) and several selected cauliflowers (B. oleracea convar. botrytis var. botrytis) in order to create new oilseed rape germplasm with a high erucic acid content. The offspring displayed desirable variation in the content of major fatty acids, raising the possibility of producing breeding lines with an erucic acid content of 60% or even more (Lühs and Friedt, 1995). Furthermore, the high genetic

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distance of these lines from conventional cultivars also makes them potential candidates for improving heterosis (Seyis et al., 2003a, 2003b). In field trials at two locations, experimental hybrids based on these resynthesized lines gave a higher yield potential than check cultivars (Seyis et al., 2006). Interspecific crosses are also an important source of seed color variants for the breeding of light-seeded rape. Brown or yellow seeds are of particular interest for breeding of oilseed rape because of their association with a thinner seed coat that results in reduced dietary fiber content. This considerably improves the feed quality of rapeseed meal after oil extraction (Shirzagedan and Röbbelen, 1985; Slominski et al., 1994, 1999). Light seed color and low fiber content are considered to coincide because the biochemical pathways leading to lignin (fiber) and pigment synthesis have common precursors, such as p-cumarate (Theander et al., 1977; Whetten et al., 1998). Furthermore, the reduction in testa thickness in yellow-seeded oilseed rape has also been found to be associated with increased seed oil or protein content per dry weight (Xiao and Liu, 1982). A variety of different yellow-seeded rapeseed materials have been generated by interspecific crosses between yellowseeded B. rapa and brown-seeded B. oleracea (Schwetka, 1981) or B. alboglabra L. (Chen et al., 1988; Rahman, 2001, 2003). The yellow-seed trait has also been introduced to B. napus from B. chinensis L. (Liu et al., 1983), B. juncea (Rashid et al., 1994), and B. carinata (Rashid et al., 1994; Meng et al., 1998; Rahman et al., 2001, 2003). In studies on the genetics of yellow seed color and raw fiber content in crosses involving yellow-seeded lines from two genetically divergent B. rapa sources, we found that in each case, the trait was controlled by a major dominant gene along with either one or two epistatic loci (Badani et al., 2006). This finding, corroborated by QTL localization and segregation analyses, supports results published previously (Somers et al., 2001; Liu et al., 2005) for two further independent yellow-seed sources. Furthermore, we found that the major gene locus for yellow seed color co-localized with a significant QTL for reduced dietary fiber (Badani et al., 2006), suggesting a direct causal relationship between the two traits. Resynthesized rapeseed may also be a potentially valuable source of germplasm for development of resistance to Verticillium wilt. As mentioned previously, this disease causes grave yield losses in affected areas of Sweden, Denmark, Great Britain, and the north of Germany, and because accredited fungicides are not available to combat the persistent soil microsclerotia, the only current alternative for effective control of the disease in short crop rotations is the breeding of resistant cultivars. Very little resistance is available in either winter or spring rapeseed, however, necessitating a search for resistance sources in related species. Resistance transfer from B. oleracea to B. napus via resynthesized rapeseed has been reported recently (Happstadius et al., 2003), and in our own work we have identified further resistance donors in ongoing screening of diverse B. rapa and B. oleracea accessions (W. Rygulla, University of Giessen, Germany, unpublished results). In order to develop durable polygenic resistance to Verticillium wilt, we aim to combine resistances from B. oleracea and B. rapa in novel RS B. napus genotypes by interspecific hybridization, assisted by embryo rescue (ovule culture). After characterizing the resistance by genetic mapping, it should be possible, using marker-assisted backcrossing, to simultaneously transfer A and C genome resistance genes into elite rapeseed lines, as a starting point for the development of new varieties with combined resistance from the diploid progenitors. In some cases, resynthesized rape forms have already resulted in the successful release of cultivars carrying novel resistance genes from the diploid species. For example, Diederichsen and Sacristan (1996) successfully used protoplast fusion to transfer resistance to clubroot from B. oleracea to B. napus. Through advanced backcrossing, a race-specific resistance was subsequently transferred from RS rapeseed progeny to elite winter oilseed rape material, and the winter oilseed rape varieties ‘Mendel’ and ‘Tosca’ derived from this material were released in the early 2000s to specifically combat this disease in affected areas of Britain and Germany. In another example, Mithen and Magrath (1992) generated synthetic lines of B. napus carrying resistance to blackleg disease derived from B. rapa via embryo culture. The resistance was subsequently successfully integrated into spring canola, resulting in the release of the cv. Surpass in the late 1990s and subsequent efforts to introgress this resistance into winter oilseed rape material. Although this

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blackleg resistance from B. rapa has in the meantime been overcome by virulent L. maculans isolates in Australia, and the clubroot resistance from B. oleracea is also race specific and hence not durable without careful agronomic management, these examples nevertheless demonstrate the potential utility of B. oleracea and B. rapa for the identification and combination of novel resistance genes to important oilseed rape pathogens. Other Brassica species and even less closely related genera are also important as potential sources of disease resistance for oilseed rape breeding. A prime example for this is the use of interspecific and intergeneric hybrids as a source for new resistance against blackleg disease. The genetic basis of blackleg resistance in B. napus in European cultivars originates, for the most part, from the French cultivar Jet Neuf, which possesses a partial, polygenically controlled adult plant resistance not expressed at the seedling stage (Cargeeg and Thurling, 1980). In contrast, all Brassica species containing the B genome exhibit an absolute and stable resistance to most of the aggressive pathogen isolates studied to date. B genome resistance is mono- or oligogenically controlled (Rimmer and van den Berg, 1992; Dixelius, 1999) and efficient from the seedling stage onwards. Thus, B genome donors like B. nigra and B. juncea have been extensively used as a genetic pool in an attempt to develop resistant oilseed rape (Roy, 1978; Sacristán and Gerdemann, 1986; Sjödin and Glimelius, 1989; Chèvre et al., 1996; Struss et al., 1996; Plieske et al., 1998; Dixelius, 1999). On the other hand, some aggressive isolates of the pathogen have been shown to overcome the resistance of B. juncea (Purwantara et al., 1998; Winter et al., 1999). Leptosphaeria maculans exhibits a broad variation in virulence, giving it the potential to adapt quickly to a given resistance (Kuswinanti et al., 1999). Generation of durable resistance therefore necessitates the application of a broad spectrum of resistance sources in oilseed rape breeding. For this reason, interspecific and intergeneric transfer of blackleg resistance from wild crucifers is an interesting alternative, and in recent years, progress has been made to introgress resistance into oilseed rape from different sources, including Sinapis arvensis L. (Snowdon et al., 2000; Winter et al., 2003) and Coincya monensis (L.) Greuter & Burdet ssp. recurvata (All.) Leadley (Winter et al., 2003). Intergeneric hybridization has also been applied for the transfer into B. napus of resistance to beet cyst nematodes (Heterodera schachtii Schm.) on Raphanus sativus addition chromosomes (Thierfelder and Friedt, 1995; Voss et al., 2000; Peterka et al., 2004), while Klewer et al. (2003) used sexual and somatic hybridization in an attempt to transfer resistance to Alternaria blackspot to B. napus from Brassica elongata Ehrh., Sinapis alba L., Diplotaxis tenuifolia L., and D. erucoides L. Such broad intergeneric hybrids cannot be achieved without tissue culture techniques to overcome incompatibility barriers; however, a successful transfer of the desired trait is often achieved. The prerequisite for this is that intergenomic chromosome recombination takes place in early backcross generations before the loss of nonhomologous donor chromosomes. The example in Figure 7.3, which shows the use of meiotic GISH to detect intergenomic chromatin transfer in asymmetric hybrids between Crambe abyssinica and B. napus, demonstrates that homoeologous gene transfer is feasible even between distantly related crucifer species. Hence, the entire Brassicaceae family can theoretically serve as potential gene donors for the identification and introgression of novel germplasm for Brassica oilseed breeding, and future cultivars could potentially express novel traits currently not present in the available breeding material.

7.10 FUTURE DIRECTION The growing collection of physical genome resources and genomic sequence data for Brassica crop species, combined with the available information from the model crucifer Arabidopsis, is opening the horizon for increasingly detailed annotation and navigation between the Arabidopsis and Brassica genomes. This will in the coming decade undoubtedly continue to accelerate our everincreasing understanding of the genetic functionality underlying complex genetic traits in Brassica oilseeds. The increasing availability of gene expression data, for example, based on DNA chip

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technologies, will also contribute to a better understanding of the genes involved in agronomically relevant traits and lead to new marker technologies for exploitation of allelic variation in breeding. In the coming years we can expect enormous advances in genome mapping and molecular breeding technologies based on high-throughput genotyping and whole genome sequencing, that will ultimately allow genetic research to advance from analysis of gene functions underlying traits of interest to a broader investigation of complete biosynthesis pathways underlying complex metabolic expression patterns. On the other hand, however, the available genetic resources and gene bank collections representing the gene pools of the Brassica species and their close relatives remain an indispensable resource for continued breeding success. Maintenance, characterization, and exploitation of Brassica germplasm collections are an important priority in order to further improve Brassica oilseeds as a major crop.

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CHAPTER 8 Sesame (Sesamum indicum L.) Amram Ashri

CONTENTS 8.1

8.2

8.3

8.4

8.5

8.6

Introduction...........................................................................................................................232 8.1.1 World Production......................................................................................................232 8.1.2 General Description..................................................................................................237 8.1.3 Genetic and Breeding Research Background ..........................................................237 Botany...................................................................................................................................238 8.2.1 Taxonomy .................................................................................................................238 8.2.2 Plant Structure and Growth Habit............................................................................240 8.2.3 Flowering and Floral Biology ..................................................................................241 8.2.4 Capsules and Their Anatomy ...................................................................................242 8.2.5 Seeds .........................................................................................................................244 Cytogenetics .........................................................................................................................244 8.3.1 Chromosomes: Description and Numbers ...............................................................244 8.3.2 Interspecific Relationships........................................................................................244 8.3.3 Origin and Domestication ........................................................................................247 Genetics ................................................................................................................................248 8.4.1 Genetic Studies.........................................................................................................248 8.4.2 Yield and Yield Components....................................................................................249 8.4.3 Genotype × Environment Interactions .....................................................................250 8.4.4 Heritability................................................................................................................250 8.4.5 Combining Ability ....................................................................................................251 8.4.6 Heterosis ...................................................................................................................251 8.4.7 Male Sterility ............................................................................................................252 Seed Retention......................................................................................................................253 8.5.1 General......................................................................................................................253 8.5.2 Monogenic Prevention of Seed Shattering ..............................................................254 8.5.3 Nonshattering Achieved by a Combination of Traits ..............................................255 8.5.4 Seed Retention Measurements .................................................................................256 Seed Composition and Quality ............................................................................................256 8.6.1 General......................................................................................................................256 8.6.2 Oil Content ...............................................................................................................256 8.6.3 Fatty Acids................................................................................................................257 231

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8.6.4 Proteins and Amino Acids........................................................................................259 8.6.5 Isozymes and Protein Bands ....................................................................................259 8.6.6 Lignans .....................................................................................................................260 8.6.7 Allergens...................................................................................................................261 8.6.8 Flavor and Taste .......................................................................................................262 8.7 Molecular Variation ..............................................................................................................262 8.7.1 General......................................................................................................................262 8.7.2 DNA Markers ...........................................................................................................262 8.7.3 DNA Cloning and Transfer ......................................................................................263 8.8 Germplasm Resources ..........................................................................................................263 8.8.1 Background...............................................................................................................263 8.8.2 Core Collections .......................................................................................................265 8.8.3 Wild Species .............................................................................................................266 8.8.4 Seed Dormancy and Storage ....................................................................................266 8.9 Breeding Objectives .............................................................................................................267 8.9.1 General......................................................................................................................267 8.9.2 Objectives .................................................................................................................267 8.9.3 Additional Considerations ........................................................................................269 8.10 Breeding Methods ................................................................................................................270 8.10.1 General......................................................................................................................270 8.10.2 Introduction...............................................................................................................270 8.10.3 Selection ...................................................................................................................271 8.10.4 Hybridization ............................................................................................................272 8.10.5 Induced Mutations ....................................................................................................273 8.10.6 Hybrid Cultivars .......................................................................................................275 8.11 In Vitro Techniques...............................................................................................................276 8.11.1 Tissue Culture...........................................................................................................276 8.11.2 Embryo Culture ........................................................................................................276 8.11.3 Protoplast Culture.....................................................................................................277 8.11.4 Anther Culture ..........................................................................................................277 8.12 Looking Ahead .....................................................................................................................277 Acknowledgments ..........................................................................................................................278 References ......................................................................................................................................278

8.1 INTRODUCTION 8.1.1

World Production

Sesame is a very ancient oilseed crop, one of the earliest domesticated crop plants. It is adapted to the tropics and subtropics, from 40°N to 40°S, but is more common in a narrower belt closer to the equator, mostly north of it. Sesame is typically a crop of the developing countries in the more southern latitudes. Table 8.1 summarizes the areas harvested, the yields, and the production of sesame seeds — at 5-year intervals — in the major producing countries and regions for the period 1988 to 2003. As shown, the world sesame area has been fairly constant, but there have been shifts within and between countries and regions. In 2003, the leading countries in acreage were India (30%), Myanmar (18%), Sudan (13%), and China (11%), with more than 70% of the total acreage between them. The highest in production in 2003 were China (28%), India (21%), Myanmar (13%), Sudan (4%), and also Uganda (3.7%). The national production figures reflect the mean yields. Thus, in 2003, India, with more than double the area of sesame of China (1,940,000

b

6695

Source: FAOSTAT.FAO.ORG; October 19, 2004. Calculated by the author as in Table 8.2.

6435

World

1958 4543 193 128

82.0 21.0 26.5 31.6 754.2 24.0 9.0 33.6 2217.0 30.0 28.0 46.4 35.5 991.9 129.4 73.1 1231.3 83.0 60.3 80.0 150.0 22.0 28.7 47.0 19.5

6027

2215 3578 234 169

79.7 22.0 20.1 42.0 630.9 22.3 39.8 49.7 1609.0 38.4 29.0 52.8 57.8 774.2 143.0 71.1 1404.1 90.0 61.9 69.0 179.0 28.0 45.6 25.0 30.3

6566

1846 4497 225 123

80.0 25.5 26.0 44.0 751.0 28.5 58.8 53.6 1940.0 40.0 26.0 44.2 44.2 1200.0 166.0 120.0 850.0 105.0 65.0 50.0 211 30.0 43.0 34.1 32.4

336

258 573 490 507

555 571 427 567 575 1149 583 728 279 657 386 668 428 270 400 405 165 329 544 479 444 — 459 374 378

1988

338

249 366 598 612

598 619 311 775 747 1292 400 706 254 633 429 351 638 239 402 441 142 349 544 375 500 500 500 575 582

426

291 500 557 538

616 591 645 786 1041 1174 552 638 328 695 448 525 548 382 462 452 187 400 583 493 430 607 599 400 576

Yield, kg/ha 1998 1993

448

331 485 665 549

613 608 577 864 1099 1228 662 605 320 750 385 539 511 325 452 513 144 391 615 440 521 600 674 387 570

2003

2163

468 709 159 79

45.2 4.0 7.9 18.0 404.5 14.0 3.5 25.8 681.8 19.0 7.8 52.4 35.0 170.0 36.0 10.1 194.0 27.0 27.3 45.0 36.0 — 68.3 10.7 5.9

1988

2265

487 1662 116 78

49.0 13.0 8.3 24.5 563.3 31.0 3.6 23.7 564.0 19.0 12.0 16.3 22.6 237.1 52.0 32.3 175.0 29.0 32.8 30.0 75.0 11.0 14.4 27.0 11.3

2564

644 1789 130 86

49.0 13.0 13.0 33.0 656.5 26.2 22.0 31.7 527.3 26.7 13.0 27.7 31.7 296.0 66.0 32.1 262.0 36.0 36.1 34.0 77.0 17.0 27.3 10.0 17.5

Production, 1000 Mt 1993 1998

2942

611 2179 150 68

49.0 15.5 15.0 38.0 825.5 35.0 38.9 32.4 620.0 30.0 10.0 23.8 22.6 390.0 75.0 61.6 122.0 41.0 40.0 22.0 110.0 18.0 29.0 13.2 18.5

2003

SESAME (SESAMUM INDICUM L.)

a

1811 2018 324 155

81.5 7.0 18.4 31.7 703.8 12.2 5.8 35.4 2447.9 28.9 20.2 78.4 81.7 628.5 90.0 24.9 1174.1 82.0 50.2 94.0 81.0 — 148.9 28.7 15.6

Area Harvested, 1000 ha 1988 1993 1998 2003

Africa Asia South America North and Central America

Regionb

Bangladesh Brazil Burkina Faso Central African Republic China Egypt Ethiopia Guatemala India Iran Kenya Korea, Republic of Mexico Myanmar Nigeria Pakistan Sudan Tanzania Thailand Turkey Uganda Uzbekistan Venezuela Vietnam Yemen

Country a

Table 8.1 Sesame Areas, Seed Yield, and Production by Major Countries and Regions, 1988–2003

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233

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ha vs. 751,000 ha), had a lower production than China (620,000 metric tons (Mt) vs. 825,000 Mt). As shown in Table 8.1, the reported sesame yields are usually low, even very low. The actual yields are higher, but much is lost due to shattering (see below). In 2003, the highest yields were in Egypt, where it is irrigated (1228 kg/ha), and China (1099 kg/ha). In none of the other countries did the yields approach 1000 kg/ha; in many they were considerably lower (Table 8.1). The quantities of exported sesame seeds and their values (Table 8.2) rose considerably over the years, reaching 800,000 Mt in 2003, valued at close to U.S.$542 million. The major exporting countries in 2003 were India (24%), Sudan (22%), China (13%), and Ethiopia (9%). It should be noted that sesame seed exports from Ethiopia, India, Myanmar, Pakistan, and other countries where the crop is rain fed are severely affected by the vagaries of the monsoons and rains. Thus, there are marked production and export shifts from year to year. Sesame seed imports from 1988 to 2003 are shown in Table 8.3. They rose markedly during this period, reaching a total of 770,000 Mt valued at U.S.$597 million in 2003. By far the largest amount was imported by Japan (19%), followed by China (13%), the Republic of Korea (11%), Turkey (9%), Egypt (5%), and the U.S. (5%). Generally speaking, the importing countries fall into two groups: those where sesame seeds are used for traditional foods (e.g., oil, tahini, sweets) and those where the seeds are used primarily in baked goods and as condiments. It is noteworthy that China and the Republic of Korea are major producers and large exporters as well as importers. Netherland and Singapore (not included in Table 8.2 and Table 8.3), which are large importers and exporters, serve as transit sites. Sesame has many agricultural advantages: it grows well in tropical to temperate climates, it can set seed and yield well under fairly high temperatures, it prefers well-drained soils of moderate fertility at pH 5.4 to 6.7, it can grow on stored soil moisture (wet to the depth of ca. 90 cm at planting time) without rainfall or irrigation, it is a good crop in the rotation since its taproots make the soil more permeable (farmers report 5 to 20% yield increase in subsequent crop (D.R. Langham, personal communication)), it can be grown in pure or mixed stands with diverse crops, and it is commonly grown with low inputs (Joshi, 1961; Weiss, 1971, 1983; Brar and Ahuja, 1979; Mazzani, 1983; Ashri, 1989b; Bennett and Conde, 2003). Although potentially sesame yields can be much higher, they are often low to very low (Table 8.1) since the crop is grown on less fertile soils, with limited or no inputs, and due to the seed losses. Thus, the poor yields, the high manual labor required for weeding, harvesting, and threshing, and the dependence on the monsoons’ patterns or other climatic factors render the crop less attractive and more risky, a situation that reconfirms itself. The other main factors for the low yields are the seed shattering noted above, lack of improved cultivars, mixed local landraces, poor-quality seeds, poor cultural practices and management, indeterminate growth habit leading to asynchronous capsule ripening, low harvest index, poor crop rotations, and losses due to diseases, pests, and weeds. Sesame seeds are used either decorticated or whole in sweets such as sesame seed bars and halva, in baked goods, or milled to obtain high-grade edible oil or tahini, an oily paste (Joshi, 1961; Weiss, 1971; Morris, 2002; Bedigian, 2004). The oil contains natural antioxidants known as lignans (see Section 8.6.6), which prevent rancidity and give it a long shelf-life. Mixing small amounts of sesame oil in other oil-containing products (e.g., peanut butter) prolongs their shelf-life. Traditionally, sesame oil has been used in home cosmetic preparations and has been reported to ease joint pains when used as an ointment (Weiss, 1971). It is used in the preparation of cosmetic creams and as a carrier for medicines. Sesame oil has been used in some areas for lighting and as a synergist for pyrethrum insecticides. Protein-rich flour can be made from the meal. However, in the traditional growing areas, sesame meal has been used to feed livestock and as manure. The stalks remaining after threshing are sometimes used as fuel; their use as roughage for farm animals is discouraged by their woody nature, although goats do feed on them.

b

Source: FAOSTAT.FAO.ORG; November 7, 2005. Calculated by the author.

427

World

a

123 231 4 67 69

Regionb

250

74 124 4 46 48 538

157 294 14 71 66

303

80 151 13 57 54

563

247 223 21 68 52

10.5 46.8 49.1 21.1 86.5 20.2 0 42.2 5.2 30.0 8.9 0.6 133.1 19.7 6.8 2.7 18.7 1.8

0.8 71.6 0.3 16.0 17.5 21.1 0 24.1 6.8 0.3 3.7 0.3 68.2 3.7 5.4 2.3 4.4 10.0

1.6 140.4 4.5 24.9 28.2 18.1 0 62.3 9.0 0.7 7.2 0.4 125.8 9.0 10.6 2.3 6.2 14.5

0.8 65.0 6.2 14.2 7.9 24.9 0 0.4 2.4 0.3 0 0 59.7 2.5 12.2 2.1 0 3.8

2.8 125.6 7.3 21.8 11.0 35.6 0 2.1 2.6 1.6 0 0 98.9 5.9 23.9 2.1 0 12.5

Africa Asia Europe South American and the Caribbean North and Central America

Burkina Faso China Ethiopia Guatemala India Mexico Mozambique Myanmar Nicaragua Nigeria Pakistan Paraguay Sudan Tanzania Thailand Turkey Venezuela Vietnam

Country

Quantity, Mt/1000

Value, $ millions

1993 Quantity, Mt/1000

Value, $ millions

Quantity, Mt/1000

1988

421

144 173 23 78 56

5.1 47.0 31.0 18.9 68.4 23.9 0 26.3 5.5 9.0 4.8 0.4 76.9 18.5 4.3 3.4 25.2 1.7

Value, $ millions

1998

Table 8.2 Sesame Seed Trade: Major Exporting Countries and Regions, Quantities and Values, 1988–2003a

800

322 382 42 16 37

24.0 104.4 71.3 20.4 189.1 11.4 5.3 41.8 2.1 24.2 46.0 7.2 172.0 18.0 13.4 4.2 4.7 3.9

Quantity, Mt/1000

542

156 294 45 2 36

6.6 67.1 31.3 20.3 76.7 12.4 3.3 24.8 1.2 9.0 20.4 4.6 74.0 9.3 8.9 5.1 4.1 2.3

Value, $ millions

2003

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SESAME (SESAMUM INDICUM L.) 235

b

Source: FAOSTAT.FAO.ORG; November 7, 2005. Calculated by the author.

370

World

a

23 262 37 1 37

Africa Asia Europe South American and the Caribbean North and Central America

Regionb

4.3 3.3 26.3 17.3 1.7 10.1 6.2 1.4 16.2 108.0 9.1 8.0 4.7 4.9 0.3 2.1 12.9 3.1 7.1 3.8 33.1 15.3

225

15 143 30 1 30

3.5 3.1 12.0 11.2 1.3 9.1 4.3 1.4 11.3 63.3 5.1 3.7 3.1 1.9 0.2 1.5 6.4 1.9 3.7 3.4 26.8 9.5

507

23 367 54 17 57

4.5 3.9 31.6 17.4 2.8 11.5 6.8 0.3 24.6 118.0 13.5 52.8 6.0 6.1 9.5 3.9 4.7 4.6 44.4 6.1 36.9 9.5

332

16 208 50 9 50

4.8 4.2 13.9 12.5 2.6 11.6 4.9 0.1 15.1 72.3 7.3 28.9 6.6 2.9 5.0 3.9 2.6 2.4 22.2 6.7 38.4 6.5

1993 Value, Quantity $ millions Mt/1000

600

48 380 96 16 66

6.1 5.7 41.4 38.4 3.9 16.8 19.6 0.4 15.3 140.9 10.8 54.0 9.1 8.6 11.4 7.1 17.8 6.6 2.7 7.2 47.4 1.4

501

40 285 94 11 71

6.7 7.0 21.9 33.0 4.5 16.7 16.0 0.3 17.7 113.9 7.9 45.1 6.5 3.6 6.7 7.3 12.9 4.5 16.5 9.1 55.6 1.4

1998 Value, Quantity, $ millions Mt/1000

770

53 516 128 4 62

5.5 6.3 98.3 38.5 5.8 18.2 21.7 5.9 25.6 149.4 12.8 81.3 14.4 7.4 14.3 8.8 10.6 8.7 66.1 6.0 37.3 11.4

597

35 378 114 4 60

5.2 7.0 63.8 24.2 6.8 17.4 18.8 2.2 19.9 114.2 9.8 68.7 11.2 4.2 10.8 7.6 9.2 6.7 43.1 6.9 39.3 5.8

2003 Value, Quantity, Mt/1000 $ millions

236

Australia Canada China Egypt France Germany Greece Iran Israel Japan Jordan Korea, Republic of Lebanon Malaysia Mexico Poland Saudi Arabia Tunisia Turkey U.K. U.S. Yemen

Country

1988 Value, Quantity, $ millions Mt/1000

Table 8.3 Sesame Seed Trade: Major Importing Countries and Regions, Quantities and Values, 1988–2003a

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SESAME (SESAMUM INDICUM L.)

Figure 8.1

8.1.2

237

Drying bundles of sesame plants and their threshing by beating over a cloth in Myanmar (Burma).

General Description

Sesame is an annual with a growing period of 70 to 150 days, more commonly 100 to 120 days. With good management, it is a good crop in the rotation. Its seeds are very small; hence, a well-prepared seedbed and shallow sowing are required to ensure good emergence and good stands. In the first few weeks after germination, the roots develop rapidly while the shoots develop slowly; therefore, weeds can be very problematic and can damage severely the stands of sesame. As the season advances, the plants grow more rapidly and can reach a height of 2 m. The plants have strong taproots that can penetrate through plow pans and hard pans. Sesame plants are indeterminate and develop leaves, flowers, and capsules as long as conditions (soil moisture, soil fertility, weather) permit. Thus, as the season progresses, the plants carry flower buds at the apex, as well as flowers, young capsules, and mature capsules toward the base, which shatter their seeds. Any harvest date is thus a compromise, aiming to maximize the proportion of nonshattering mature capsules and to minimize the proportion of shattering ones. On the other hand, being indeterminate, sesame plants can withstand dry or cold spells and resume flowering and seed setting when conditions improve. In most of the sesame-growing areas the plants are harvested by the traditional methods: they are cut above the ground or uprooted, bundled, and stacked upright to dry. Once the bundles dry, the capsules dehisce. Then the bundles are inverted over a smooth surface (cloth or polyethylene sheets, hardened floor), beaten, and shaken to ascertain that all the seeds were released (Figure 8.1). The seeds are collected, dried further if necessary, cleaned, stored, and marketed. In many of the traditional sesame-growing areas, yields of several growers are assembled into bigger lots by middlemen. There are some notable exceptions: in the U.S. (Texas, Oklahoma), Venezuela, and Australia, sesame fields are combined directly. In the past, in Venezuela, the plants were mowed with a binder, manually shocked, dried, and then combined or threshed with stationary threshers. In both Australia and Venezuela, seed losses due to shattering can be high. 8.1.3

Genetic and Breeding Research Background

Genetic and breeding improvement efforts in sesame have been limited and slow. A major reason is that it is a crop of the developing countries, and within these countries it is a crop of small holders. In many of the sesame-growing areas the research resources are limited, and other

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crops have higher priority, drawing funds and researchers to them. Sesame improvement has been further hampered by the lack of widely based germplasm resources until the last decade or two, short duration and lack of continuity of projects, limited exchange of materials and know-how, and lack of incentive for commercial companies to invest in it, perhaps because it is a self-pollinated crop grown mainly by small holders. So far, none of the international research centers has been mandated to conduct research on sesame, which is regrettable, especially since it is a traditional small holders’ crop in Africa and Asia. Some international research and cooperation efforts were initiated in the last two to three decades by the Crop Production and Protection Division of the FAO, Rome, which sponsored two conferences (Anon., 1981b, 1985a) and has been supporting the publication of the annual Sesame and Safflower Newsletter since 1985. Important contributions were made by IBPGR, now IPGRI, Rome, which issued a list of Descriptors for Sesame (Anon., 1981a) and a revised edition (IPGRI and NBPGR, 2004), and supported the collection and assessment of the germplasm of sesame (see Section 8.8.1). The Plant Breeding and Genetics Section of the Joint FAO/IAEA Division and the Technical Cooperation Division of the IAEA, Vienna, have implemented coordinated research projects dealing with sesame mutations and breeding in many developing countries (see Section 8.10.5). Important contributions were made by the Oil Crops Network established by the International Development and Research Center (IDRC) of Canada in East Africa and South Asia. The achievements in these research and cooperation programs show that much progress could be made with concerted efforts, and that it should be undertaken on a wider scale. The commercial nonshattering varieties developed by Sesaco Corp. in the U.S. for direct combined harvesting (see Section 8.5.3) serve as a further demonstration of the impact of long-term sustained breeding efforts. Actually, as will be shown in this chapter, sesame breeding can benefit from several attributes of the plant: it is easy to self its flowers or to emasculate and cross-pollinate them (Yermanos, 1980), the capsules contain many seeds (50 or more), each plant can produce hundreds of seeds, and two — and sometimes three — generations can be grown in a year. It is regrettable that the improvement of sesame has lagged behind other crops. Since other crops are becoming more remunerative, and due to a shortage of manual labor at peak harvest times, sesame is relegated to more and more marginal conditions. If this trend is unchecked, the process will continue and sesame production areas may decrease in the foreseeable future. Yet, in many sesame-growing regions, the crop cannot be replaced due to the lack of other suitable crops that can grow under the same conditions, including drier ones. Furthermore, sometimes sesame is grown because other crops are unsuccessful. A case in point is that of sesame in the lowlands of Eritrea (near Sudan), where it replaces sorghum, which is attacked by the prevalent parasitic Striga plants. Similarly, sesame growing has been proposed as a way to overcome Striga damages in pearl millet fields in the West Africa Sahel (Hess and Dodo, 2004). The smaller range of crops and cultivars reduces biodiversity, increases genetic vulnerability, and reduces the options of the growers, processors, and consumers. It is generally recognized that breeding-improved cultivars with higher yield potentials is the key that can lead to better practices, improved performance, higher yields, better returns, and higher inputs. Breeding-adapted and more productive sesame cultivars were widely recognized by several expert consultations as critical to enhanced sesame growing (Anon., 1981b, 1985a, 1985b; Ashri, 1987, 1998; Van Zanten, 2001). This is especially true in developing countries, where low inputs prevail. Breeding-improved cultivars may be the only available means to reverse the trend there.

8.2 BOTANY 8.2.1

Taxonomy

The genus Sesamum, belonging to the Pedaliacea family, is the biggest of sesame’s 16 genera. Its taxonomy has not been well studied over the years. In a review a few years ago, Ashri (1998)

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239

Table 8.4 Revised List of Sesamum Species and Their 2n Chromosome Numbers 2n = 26 S. alatum Thonn. S. capense Burm. f. S. indicum L. S. malabaricum Burm.

2n = 64 S. radiatum Schum. & Thonn.

2n = 32 S. angolense Welw. S. angustifolium Engl. S. laciniatum Willd. S. latifolium Gillett S. prostratum Retz. 2n chromosome number unknown S. abbreviatum Merxm. S. calycinum Welw. ssp. calycinum. S. calycinum Welw. ssp. baumii (Stapf) Seidenst. ex. Ihlenf. S. calycinum Welw. ssp. pseudoangolense Seidenst ex. Ihlenf. S. capense Burm. f. ssp. lepidotum Schinz S. marlothii Engl. S. parviflorum Grabow-Seidenst. S. pedalioides Heirn S. rigidum Peyr. ssp. rigidum S. rigidum ssp. merenksyanum Ihlenf. & Seidenst. S. schinzianum Aschers. ex. Schinz S. triphyllum Welw. ex. Aschers. S. triphyllum Welw. ex. Aschers. var. grandiflorum (Schinz) Merxm. Source: IPGRI and NBPGR, Descriptors for Sesame (Sesamum spp.), IPGRI, Rome; NBPGR, New Delhi, 2004.

concluded that the taxonomy and cytogenetics of the genus required more definitive investigations. This conclusion arose from the fact that some species were switched back and forth from species to subspecies to synonyms and between the Sesamum genus and the related Cerathoteca genus. Other genera considered related to Sesamum are Anthadenia and Volkameria (Kobayashi, 1991). Furthermore, various numbers of species were reported for the genus, such as 36 by Joshi (1961), who based it on the Index Kewensis and Supplements, 34 by Nayar and Mehra (1970), and 37 by Kobayashi (1991). Additional taxa have been described since then, such as S. mulayanum Nair (Mehetre et al., 1993), actually S. malabaricum, and S. indicum var. sencottai and var. yanamalai (Devarathiman and Sundraresan, 1990) in India. The degree of confusion is exemplified by the fact that there are 15 synonyms for S. indicum, including three in other genera, seven synonyms for S. radiatum, and fewer synonyms for many of the other species (IPGRI and NBPGR, 2004). Furthermore, the chromosome numbers of many of the species are not known, and there are only limited findings on their interspecific relationships (see Section 8.3.2). The required in-depth study was undertaken by Bedigian (in preparation). Her proposed revision of the genus is summarized in the list of species in the 2nd edition of the Descriptors for Sesame (IPGRI and NBPGR, 2004). The revised list contains 24 taxa, including 6 subspecies and 1 variety (Table 8.4). Only one of the species is cultivated: S. indicum. It should be noted that the 17th International Botanical Congress held in Vienna in 2005 decided to conserve S. indicum, rather than S. orientale (Anon., 2005). A few other species are harvested and eaten occasionally, mainly in times of famine or food shortages: S. angustifolium, S. calycinum ssp. baumii, S. malabaricum, and S. radiatum. The distribution areas of the wild species are not well documented. Nearly all of the wild species are endemic to Africa. Two species, S. laciniatum and S. prostratum, grow in Africa and India, while S. malabaricum grows only in India. Three species are found in Africa and the East Indies: S. marlothii, S. schinzianum, and S. triphylum. Finally, S. radiatum grows in Africa and Sri Lanka. Kobayashi (1991) noted also one species in Crete.

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Figure 8.2

Indeterminate growth habit in sesame, Israel variety ‘No. 45’ (Dt45/Dt45): (A) with leaves removed; (B) with leaves. Note the progressive development and position of capsules and flowers.

The confused taxonomical situation led to many duplications and synonyms. Consequently, much care is required in comparing cytogenetic and other findings of different authors in different locations and periods. 8.2.2

Plant Structure and Growth Habit

Sesame is an annual plant that can reach a height of 2 m, although varieties that are 1.00 to 1.40 m tall are more common. It is indeterminate, producing leaves, flower buds, flowers, and capsules continuously, as the season progresses and growing conditions permit. Later in the season, while there are still young flower buds close to the shoot apex, at the base of the plants the early capsules mature, may dehisce, and shatter their seeds (Figure 8.2). Thus, modifications of the growth habit and seed retention are major objectives in sesame improvement and will be dealt with below in more detail. The varieties and germplasm accessions of sesame vary markedly in their branching pattern. Some varieties have many branches, others have few, and still others are unbranched, i.e., uniculm. There is variation for the location of the branches — whether they grow from the base of the plant or in the upper part. Certain branching patterns are associated with germplasm from certain traditional growing areas. The plants are usually erect or semi-erect, but prostrate habit is also found. The first true leaves are usually small and entire, then they increase in size, with the fourth or fifth leaves being the largest; they are flat and sometimes tri-lobed. As the season progresses, the newly formed leaves become smaller, ovate to lanceolate, and pointed with entire to serrate margins. Leaf color varies: in some varieties/accessions they are lighter green, in others they are darker graygreen, and in some there is reddish anthocyanin pigmentation, expressed especially in the petioles and the stems. In the indehiscent idid mutants the cotyledons are concave and there are irregular enations on the underside of their leaves. Sesame stems may be round or square in cross section; they are erect, usually pubescent and woody, and are not palatable to animals. Sesame is sown in the tropics two or three times a year and in the temperate zone when the soil temperature reaches 15˚C, preferably 20˚C, when other warm-weather crops, such as cotton or peanuts, are planted. Optimum growth is obtained in the range of ambient temperatures of 25 to 35°C. The length of the growing period varies with varieties, locations, and other conditions, such as temperatures, available moisture, and photoperiod.

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8.2.3

241

Flowering and Floral Biology

Flowering, pollination, and hybridization of sesame have been reviewed by Joshi (1961), Weiss (1971, 1983), Yermanos (1980), and Ashri (1985, 1998). Sesame is an indeterminate plant with acropetal flowering, producing leaves, flowers, and capsules as long as conditions permit. As a result, later in the growing period, the plants carry fully mature capsules, immature ones, flowers, and buds. The flowers are borne on short pedicels in the leaf axils. A single dominant allele (T–) determines if there is one flower per axil or two or more flowers per axil (tt). When the flowers are solitary, the two lateral primordia produce nectaries. If there are two or more flowers (usually three), the laterals bloom a few days after the central one, and also mature a few days later. The flowers, which are monosymmetric (= zygomorphic), vary in size, color, and markings and are geniculate. The calyx is small, five-parted, and the corolla is tubular, campanulate, five-lobed, and about 3 cm long. The lower corolla lobe is longer and forms a lower lip. The corolla exterior and interior are pigmented with colors ranging from white to pink, violet, red, and maroon; nine colors are listed in the revised descriptors list (IPGRI and NBPGR, 2004). There are five stamens in each flower — four functional and one sterile — which are inserted at the corolla base. Temperatures below 15°C or above 40°C lead to pollen sterility, reduced fertilization, and lower seed set, but exceptions have been noted. The ovaries are superior; a single gene determines if they are bicarpellate (Tc/–) or tetracarpellate (tctc) (called also quadricarpellate). Each carpel has two locules; thus, their number per capsule can be four or eight, but at times different numbers are encountered. The fruit of sesame, which is a capsule, will be discussed fully below (see Section 8.2.4) because its traits have a major effect on the management of the crop and on its yield. The flowers usually open at dawn, and the pollen is shed shortly after and remains viable for about 24 h; on cloudy or cool days, the flowers may open up to 3 h after sunrise. The stigmas become receptive a day earlier and normally remain receptive for two more days if not fertilized. Emasculation is easily performed by pulling off the closed corolla from the buds on the day before opening. In genetic studies, the style is protected after emasculation by a short piece of drinking straw or a gelatin capsule or a small paper bag. In large-scale crossing blocks, where the focus is on generating much new variability, the styles are not covered, thus expediting the operations; little contamination was observed in such cases in the offspring (D.R. Langham, personal communication). Pollination is effected on the following morning, using anthers collected the day before and kept in Petri dishes overnight at room temperature. A detailed description of sesame hybridization procedures was given by Yermanos (1980). A better success rate is obtained when the plants selected for emasculation are in full bloom, not during the first few days of flowering and not toward the end of the period. With good handling, the crossing success rate is quite high, and each obtained capsule contains tens of hybrid seeds. Pfahler et al. (1996) in a study of 12 diverse genotypes of sesame for anther, pollen grain, and pistil characters found both genotypic and environmental variation for many of the traits studied. They concluded that these differences could lead to differential transmission of alleles by male and female gametes. Rehman et al. (2002) found that when mature sesame pollen grains were hydrated, they swelled in less than a second. They deduced that this instantaneous swelling could be related to potassium located at the 12 furrow aperture areas of the grains. In a study of the exudates of the stigmas and styles, Berlingeri and Jaurqui (2002) found that the stigmas secrete mainly lipids while the styles’ secretions contained high levels of carbohydrates. They related these differences to their different functions during pollen germination and pollen tube growth. Single gene effects on floral development were noted in the case of the indehiscence gene, Id:id. It is well known that the indehiscent idid plants have lower yields. Berlingeri and Jaurqui (1999) and Berlingeri et al. (1999) concluded that the low seed set of the idid plants resulted from the poor pollen load on the stigmas. They found that in the idid plants pollen production and

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viability and ovule number and viability were normal. However, the stigmas and the anthers were not as close as in Id/– plants due to their curved styles, which do not push through the anthers. Furthermore, in the idid plants the stigmas and their papillae were smaller. They also suggested that the bent styles might impede pollen tube growth, thereby reducing the seed set further. Sesame is classified as a short-day plant. However, long-term selection in regions with varying day length and light intensity produced genotypes with different photoperiod requirements. In areas where sesame is grown in two or three seasons during the year (e.g., India, Myanmar), cultivars with different photoperiod responses were developed. In such areas, the farmers’ fields often contain mixtures of photoperiod types. Significant interactions of temperature and day length with flowering rate were reported by Suddihiyam et al. (1992). Since the first genetic studies of photoperiod in sesame were reported (Rhind, 1935), it was investigated further by just a few authors. Kotecha et al. (1975) concluded that it is controlled by at least three loci, with lateness being dominant. Since photoperiodic response is a complex character, it can be expected that many genes would be involved, both additive (Chaudhary et al., 1977) and nonadditive, major genes and polygenes. This aspect should be considered especially in the introduction of germplasm lines from diverse geographical origins. Thus, for example, when Ethiopian cultivars were planted in Rehovot, Israel (31.5°N), in April (recommended planting time), they grew very tall (2 m) and began to bloom only in September, while the locally adapted accessions flowered in June and were harvested in August. Sesame is considered a self-pollinated crop, but this is mainly because pollinating insects prefer flowers of other species if available. Where insect activity is high, outcrossing can reach high levels. Yermanos (1980) reported that when his sesame plots were surrounded by other flowering crops, cross-pollination was under 1%, whereas in an isolated plot in a dry area in California with minimal other vegetation, outcrossing reached 68%. In Nigeria, Van Rheenen (1980) found different outcrossing rates between cultivars under the same conditions in isolated field plots: ‘Margo’ had 51.72% ± 21.04 natural cross-pollination, while two other varieties had 2.76% ± 1.94 and 9.02% ± 5.76 outcrossing. Several tall maize guard rows surrounding the test plots reduced cross-pollination markedly. Varying rates of cross-pollination were reported from various regions, depending on insect activity (Brar and Ahuja, 1979; Yermanos, 1980; Musa and Padilla, 1991). The high rates of outcrossing enhance heterozygosity and heterogeneity within populations, which may assist in maintaining the productivity of landraces and in varietal improvement. But, in breeding plots and germplasm collections’ multiplication, it can be expected that the populations will not remain true to type after several generations of open pollination. Therefore, in critical studies it would be prudent to isolate the plots or bag plants to ascertain self-pollination. Pathirana (1994) found that seeds formed early, in the middle, or late in the growing season showed somewhat different outcrossing rates. This depended on whether other lines flowered at the same time. Weiss (1971) considered an isolation distance of 180 to 360 m as adequate. High activity of insect pollinators in sesame is vital for the production of hybrid cultivars. Sesame flowers are visited by many insect species of several genera, with some more active than others (Patil and Viraktanath, 2000; Sachdeva et al., 2003). Bee attractants were effective in India in attracting higher numbers of pollinators (Patil et al., 2000). Male sterility was described in sesame (see below), while self-incompatibility has not been reported so far. 8.2.4

Capsules and Their Anatomy

The capsules that are borne in the leaf axils can vary in length from ca. 2 to 7 cm. As noted above, a single gene determines if there is one capsule per axil (T/–) or more (usually three) (tt). The capsules may be square or oblong with a shorter or longer tapered apex (beak). The capsules are usually bicarpellate (Tc/–) or tetracarpellate (tctc), with higher carpel numbers occasionally. The seeds are attached to the placentas in rows. A monogenic thin shell trait (paper shell) was studied by Culp (1960).

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Figure 8.3

243

(See color insert following page 144.) Bicarpellate green and mature (some dehisced) capsules.

When the capsules mature and dry, they dehisce at the sutures from the apex down and shatter their seeds (Figure 8.3) (See color insert following page 144). A recessive id/id indehiscent mutant was discovered in Venezuela (D.G. Langham, 1946). Ashri and Ladizinski (1964) found that splitting of the indehiscents’ capsules is prevented mainly by an increase in the number of mesocarp cell layers at the sutures. They concluded that capsule dehiscence, which is brought about by differential tensions in the capsule walls due to repeated moisture absorption and drying, is prevented by these extra cells. Day (2000a) studied in detail capsule anatomy in 32 sesame accessions: 14 indehiscent idid breeding lines and 18 commercial dehiscent varieties from diverse origins. He concluded that indeed the “only qualitative difference” between the dehiscent and indehiscent (idid) materials was that in the latter there were “many cell layers between the median vascular bundle and the epicarp” at the capsules’ sutures. Day (2000a) confirmed also that the differential tensions in the capsule caused splitting. Day (2000b) concluded that there was weak evidence to support the suggestion that the id/id phenotype was due to auxin physiology changes. Another single-locus recessive variant (gsgs) with closed capsules, termed seamless, was discovered in 1986 in the U.S. (Langham and Weimers, 2002). The capsules appeared to be monocarpellate, but were actually bicarpellate with no false septa. This genotype had good seed retention but was hard to thresh; its seeds were damaged during threshing; therefore, breeding efforts with it were discontinued (Langham and Weimers, 2002). As noted above, within each carpel there are two locules; thus, in a bicarpellate capsule there are four rows of seeds, and in tricarpellate and tetracarpellate capsules, six and eight rows,

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respectively. Complete or incomplete false membranes (septa) cover each carpel; they affect seed retention considerably. 8.2.5

Seeds

Sesame seeds are oval and small, with 1000 weight usually varying between 2 and 4 g. At 3.4% moisture the mean dimensions of the seeds in one study were 2.80, 1.69, and 0.82 mm for length, width, and thickness, respectively, and the mean geometric diameter, sphericity, surface area, and density were 1.56 mm, 0.56 mm2, 7.80 mm2, and 1224 kg m–3, respectively (Tunde-Akitunde and Akitunde, 2004). Kumar et al. (1998) found that seed weight, volume, and density were controlled by both additive and nonadditive gene action. The seed coat is thin and smooth to rough (partially or more). The coefficients of friction of the seeds were 0.39 on glass and 0.54 on plywood (TundeAkitunde and Akitunde, 2004). Seed coat color varies from white to shades of brown, red, olive, gray, and black; the descriptors list (IPGRI and NBPGR, 2004) gives 12 color states. Dormancy is rare in the cultivated species; it was reported only in the Mexican variety ‘Cola de Borrego’ (Ashri and Palevitch, 1979) and in ‘Hnanni 25/160’ from Myanmar (Beech and Imrie, 2001).

8.3 CYTOGENETICS 8.3.1

Chromosomes: Description and Numbers

The chromosomes of S. indicum, the cultivated species, are small, ranging in length from 0.66 to 1.80 μm, and the centromeres are median, submedian, and subterminal (Kobayashi, 1991). Zhan et al. (1987) reported that five chromosomes were median and eight submedian, one with a satellite, in contrast to earlier studies, which suggested that all the chromosomes had terminal centromeres. Giemsa banding in sesame was achieved in China, facilitating identification of each of the 13 chromosomes of the genome (Zhan et al., 1987). As noted above and shown in Table 8.4, the chromosome numbers in the genus were determined for 11 species only, about one half of the recognized taxa. The known chromosome numbers are 2n = 26 (S. indicum and the wild S. alatum, S. capense including the synonym S. schenckii, S. malabaricum (initially classified as S. orientale ssp. malabaricum)); 2n = 32 (S. prostratum, S. latifolium, S. laciniatum, S. angolense, S. angustifolium); and 2n = 64 (S. radiatum (including the synonym S. occidentale) and S. schinzianum). The basic chromosome numbers in the Sesamum genus are x = 8 and 13. The basic chromosome number in the related genera, Ceratotheca and Pedalium, is x = 8. The x = 13 number probably resulted from ancient polyploidy. The polyploid origin of all the Sesamum species, including the present-day diploid 2n = 26 ones, is supported by the secondary chromosome associations reported by Sengupta and Datta (2003b). They found in most of the cells, in diakinesis and first metaphase in the Indian cultivar B-67 and in the M2 and M3 of 21 induced mutants, up to five groups of two bivalents each. Their conclusion was that the 2n = 26 chromosome species have an alloploid origin and that the basic chromosome number at the time was x = 6 or 7 or 8. Other than a report on translocations (see below), no structural aberrations or numerical deviations have been reported in sesame so far, although structural changes were most probably induced by radiations. 8.3.2

Interspecific Relationships

The interspecific relationships of the species within and between the chromosome number groups were studied by several authors, whose findings are summarized in Table 8.5.

+ Fy F(z) F, S F, S N

N (F) + N N N N

F N + / F N

N (F) / / / / /

F N F + F N

F N F F + N

/ / / N / /

N (F) N N N N +

N (F) / / / / F

S. indicum S. alatum S. malabaricum S. capense S. laciniatum S. prostratum S. angolense S. radiatum S. schinzianum (2n = 64?) 2n = 64 2n = 32 2n = 32 2n = 32 2n = 26 2n = 26 2n = 26 2n = 26

Source: Nayar and Mehra, 1970; Biswas and Mitra, 1990; Kobayashi, 1991; J.I. Lee et al., 1991; Shi, 1993; Qu et al., 1994; Thangavelu, 1994; Nayar, 1995; Prabakaran, 1996; Kavita, 2001; Hirmath and Patil, 2002; Tarihal et al., 2003.

Note: F = viable hybrids; N = no seeds; S = sterile hybrids; / = no report found; ( ) = not clear; + = intraspecific; y = one viable hybrid only; z = fully fertile with S. indicum as female parent, and male sterile with S. malabaricum as female parent.

S. indicum S. alatum S. malabaricum laciniatum S. prostratum S. radiatum

Parents

Table 8.5 Cross Compatibility of Cultivated Sesame and Wild Sesamum Species in Interspecific Hybridizations

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Of the 2n = 26 chromosome species, S. indicum and S. malabaricum are closely related; their F1 hybrids have normal meiosis and are fertile (Kobayashi, 1991; Nayar, 1995; Bedigian, 1981, 2003). In crosses of S. malabaricum as female and S. indicum as male, the F1 hybrids were male sterile (Thangavelu, 1994; Prabakaran et al., 1995; Prabakaran, 1996); the reciprocal F1 hybrids were fully fertile. Similarly, crosses of S. indicum as female with S. malabaricum (as S. mulayanum) were successful, yielding partly fertile F1 hybrids, while the reciprocal crosses set no seeds (Biswas and Mitra, 1990). These reports indicate that there are nuclear-cytoplasmic interactions and differences between the two species. Kawase (2000) distinguished two ecotypes of S. malabaricum (which he called S. mulayanum), a ruderal weed type and an associated weed type. About 50% of the pollen grains of the F1s of the ruderal type () × S. indicum were sterile, and the plants had poor seed set. The reciprocals of the above, as well as the reciprocal hybrids of the second ecotype × S. indicum, had normal pollen fertility and seed sets. These findings supplement the above and indicate that there are cytoplasmic/genic differences between populations within S. malabaricum. In crosses of S. indicum × S. capense (as S. grandiflorum), 2n = 26, the F1 hybrids were partly fertile and F2 progenies were obtained; thus, the two species are related (Nayar and Mehra, 1970; Kobayashi, 1991). Reciprocal crosses of the cultivated species with S. alatum, 2n = 26, produced some seeds or capsules, but the seeds were not viable (Nayar and Mehra, 1970; Kobayashi, 1991; Kavitha, 2001). It would appear that S. alatum is more distantly related to S. indicum. Embryo rescue might lead to viable F1 hybrids between the two species. In the 2n = 32 chromosome group, fertile interspecific F1 hybrids and F2 were reported between S. prostratum and S. laciniatum (Joshi, 1961; Nayar and Mehra, 1970; Kobayashi, 1991). In reciprocal crosses of S. laciniatum and S. angolense, a few inviable seeds were obtained (Nayar and Mehra, 1970; Kobayashi, 1991). Embryo rescue might help obtain F1 hybrids in this combination. In the 2n = 64 chromosomes groups, Hiremath and Patil (2002) concluded that S. radiatum and S. schinzianum are close. Their hybrids have regular meiosis, but they differ by two translocations. The same authors studied the hybrids of S. radiatum (as S. occidentale) with S. schinzianum and found them to be fully fertile. Thus, the two species can be considered as one biological species. There is little knowledge regarding the relationships between the species across the three chromosome number groups. Of particular interest is the potential to introgress desirable genes from the various wild species into the cultivated one. Reciprocal crosses of S. indicum 2n = 26 with S. prostratum (2n = 32) and with S. laciniatum (2n = 32) produced sterile F1 hybrids (Nayar and Mehra, 1970; Kobayashi, 1991, Kavitha, 2001). Such sterile or semisterile F1 hybrids could still serve in the transfer of genes to cultivated sesame by pollinating them repeatedly with sesame pollen and backcrossing for several generations. In crosses of S. indicum (as female) with S. radiatum (as S. occidentale), 2n = 64, capsules were not obtained (Nayar and Mehra, 1970; Kobayashi, 1991). However, in reciprocal crosses of the cultivated species with S. radiatum, 2n = 64, a few capsules were produced with some viable but mostly inviable seeds (Nayar and Mehra, 1970; Kobayashi, 1991; Lee et al., 1991). Protocols for embryo culture for S. indicum and S. radiatum were developed by B.H. Lee et al. (1991) and then used to rescue embryos of S. indicum × S. radiatum; six F1 hybrids were obtained (of 1630 cultured embryos), while none survived in the reciprocal (of 653 embryos). Fertile F1 hybrids were obtained between one of four S. indicum cultivars used as the male parents and S. schinzianum, 2n = 64, following embryo rescue (Shi, 1993; Qu et al., 1994). Clearly, as in other procedures and other crops, the genotypes of the parents are also important. Thus, crosses should be attempted between several unrelated cultivars of the cultivated species and several divergent accessions of each of the wild species for every desired combination. Tarihal et al. (2003, 2004) concluded that in crosses of two varieties of sesame with two S. radiatum accessions (one as S. occidentale), the hybridization barriers were both pre- and postzygotic. They suggested that bud pollinations followed by embryo rescue could yield viable interspecific hybrids. Rajeswary and Ramaswamy (2004) attempted to cross S. alatum with four sesame

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cultivars. They found that applications of gibberellic acid (GA3;100 mg/l), naphthalene acetic acid (NAA; 15 mg/l), and kinetin (5 mg/l) to the flowers immediately after cross-pollination, and to the developing capsules, helped retain 5 to 10% of the capsule 7 days after pollination; none were retained in the controls. They observed some well-developed, normal-looking ovules in the retained ovules, which could be used for embryo rescue; the later fate of the ovules was not described. S. indicatum, an experimental amphidiploid with 2n = 58, was first produced by Ramanajan in 1942 by hybridizing S. indicum (2n = 26) (as S. orientale) and S. prostratum (2n = 32) and doubling the chromosome number of the F1 with colchicine (Joshi, 1961). Prabakaran (1996) doubled the chromosome numbers of the cultivated species and of S. alatum, but these 2n = 52 lines did not facilitate additional hybrid combinations. On the other hand, doubled F1 hybrids of S. indicum ∞ S. laciniatum and S. indicum ∞ S. prostratum could be backcrossed with S. indicum. Hundreds of crosses were made in South Korea between Ceratotheca triloba (as female) and S. indicum cultivars; many viable seeds were obtained, but most seedlings died very early, while the remaining survivors were sterile (C.W. Kang, personal communication). Falusi et al. (2001) obtained F1 hybrids of S. indicum × Ceratotheca sesamoides (2n = 32) that were nearly completely sterile due to meiotic irregularities. Still, intergeneric gene exchange with this species might be achieved by repeated pollinations of the hybrids with sesame pollen and several generations of backcrossing to cultivated sesame. In crosses of S. laciniatum (2n = 32) with Martynia annua (2n = 32) capsules with seeds were obtained (Thangavelu, 1994). In crosses of S. radiatum (2n = 64, also as S. occidentale) × M. annua (2n = 32) only capsules were produced (Thangavelu, 1994). Joshi (1961) described three colchicine treatment methods used in sesame: seed soaking (0.5% solution for several days), drops on the growing points of germinating seedlings (0.2 to 0.5% solution for 5 or 6 days), and coating axillary buds with 0.4 to 0.5% colchicine in lanolin emulsion. Recently, Zhang et al. (2001) found that autotetraploid sesame plants could be obtained by soaking the seeds in 0.3 to 0.5% aqueous solutions of colchicine for 24 h at temperatures lower than 28°C. Meiosis in the autotetraploids was normal, mostly quadrivalents and occasionally some divalents (II + II) were observed, and the pollen fertility was about 95%. The tetraploids had thicker stems; larger leaves, flower organs, and seeds; slower growth; and a lower seed set (Joshi, 1961; Zhang et al., 2001). Kamala and Sasikala (1985) obtained three morphological mutants following seed soaking in 0.1 to 0.4% colchicine solutions for 24 h. The newer tools of molecular genetics for establishing species relationships were employed by Nanthakumar et al. (2000) in Sesamum, using RAPD DNA markers and isoenzymes. The 2n = 26 species, S. indicum (cultivated) and S. alatum, proved to be closely related. Similarly, the 2n = 32 chromosomes species, S. laciniatum and S. prostratum, formed another cluster. S. malabaricum (2n = 26) was most closely related to the cultivated species and surprisingly also to the 2n = 64 species, S. radiatum (Nanthakumar et al., 2000). This last observation merits further examination; it could be that the latter species was mislabeled. 8.3.3

Origin and Domestication

As noted above, sesame is a very ancient crop, one of the oldest oil crops, if not the oldest. Charred sesame seeds, about 5000 years old, were found in archaeological excavations in Harapa (Pakistan). Sesame was known in antiquity also in Anatolia (Turkey) and Mesopotamia (now Iraq) (Bedigian, 2003, 2004), and its oil was used in food preparation, lighting, and personal grooming. Sesame is not mentioned in the Bible, but it was well known in the Hellenic and Roman eras in the Middle East. The origin of cultivated sesame, its progenitor, and its domestication have been debated for a long time. Over the years, two alternative centers of origin were proposed for sesame, Africa or the Indian subcontinent (Joshi, 1961; Weiss, 1971; Nayar and Mehra, 1970; Brar and Ahuja, 1979; Bedigian, 1981; Bedigian and Harlan, 1986; Bedigian et al., 1985; Nayar, 1995). The African origin proposal drew support from the recognition that most wild Sesamum species are endemic in Africa.

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Table 8.6 History of Development of Nonshattering Sesame Cultivars of the Sesaco Corporation in the U.S., 1982–2005 Varieties Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco Sesaco

1a 2–3 4 5–10 11 12 14–16 17 18–20 21–22 23 24 25 26 27 28 29

Years Grown

Shaker Shatter Resistance, %

1982 1983–1987 1984–1987 1984–1991 1988–1995 1986–1987 1989–1996 1994–2000 1994–1997 1995–1998 1998–2000 1998–2004 2001–2002 2002– 2002– 2004– 2005–

99 9–28 25 4–48 60 1 3–42 61 34–56 49–65 66 75 73 73 74 74 76

Seed Retention, % Upright Inverted

Harvest Method

100 59–82 96 66–91 — 76–89 89–90 100 78–100 100 — — — — — — —

Swathed Swathed Swathed, direct Swathed Swathed, direct Swathed Direct Direct, swathed Direct Direct, swathed Direct Direct, swathedb Direct, swathedb Direct, swathedb Direct Direct, swathedb Direct, swathedb

100 83–87 88 21–48 — 0–3 79–82 95 94–97 89–94 — — —

a

Indehiscent, idid. Swathed to fit with farming practices in Arizona and Oklahoma, not for seed shattering reasons. Source: Langham, 2001; U.S. Patent 6,781,031 B2, 2004; Langham personal communication, 2006. b

Support for the Indian subcontinent origin of sesame was based on its ancient cultivation there and on the presence, only in India, of the closely related species S. malabaricum. In a recent publication, Bedigian (2003) reviewed and marshaled convincing evidence that sesame was domesticated in India. Thus, cultivated sesame and S. malabaricum have 2n = 26 chromosomes and are cross-fertile in both directions. The cultivated materials in India and S. malabaricum have similar growth patterns and morphological attributes. Furthermore, the cultivated sesame and S. malabaricum contain two lignans (see Section 8.6.6 and Table 8.6): sesamol and sesamolin. The other species that contain both lignans are S. angolense, S. angustifolium, and S. calycinum (Bedigian, 2003). However, the first two species can be excluded, as they have 2n = 32 chromosomes; the number in the third species is unknown. Last, but not least, the affinity of S. indicum and S. malabaricum was confirmed by molecular studies of DNA markers. There remains the question of how a Sesamum progenitor arrived in India, when nearly all species of the genus are endemic in Africa. Bedigian (2003) proposes that it could have arrived there in the Gondwanaland period, before it broke up, or that the wild progenitor reached the Indian subcontinent with human migration and was subsequently domesticated there.

8.4 GENETICS 8.4.1

Genetic Studies

The pioneering studies by D.G. Langham in Venezuela (1945a, 1945b, 1946, 1947a, 1947b) on sesame breeding and the genetic control of different traits were seminal. His findings and those of others on the mode of inheritance of various characters were reviewed by Joshi (1961), Weiss (1971, 1983), Brar and Ahuja (1979), and Ashri (1998). The review of Brar and Ahuja (1979) is very detailed, useful, and still timely. Unfortunately, few additional inheritance studies were reported in the elapsed time, except for findings on combining ability, yield components, heterosis, and heritability (see below). Of the 71 traits reviewed by Brar and Ahuja (1979), monogenic control was reported for 51, digenic for 15, and polygenic for 5. In a few cases, both monogenic and digenic controls were reported (e.g., indehiscence), or monogenic and polygenic (e.g., photoperiod response).

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The genetic control modes of some important traits are presented below (the first phenotype listed is dominant, the second recessive): Branched vs. unbranched, one gene (Nb, nb), a second gene may be involved Normal vs. fasciated stem, single or duplicate genes (F1, f1; F2, f2) Single vs. multiple (usually two or three, but can reach seven) flowers and capsules per leaf axil, one gene (T, t) Fertility vs. complete male and female sterility, one gene (Sc, sc) Fertility vs. male sterility, one gene (Ms, ms) Normal vs. polypetalous (star) flower, one gene Bicarpellate vs. tetracarpellate (quadricarpellate) capsules, one gene (Tc, tc) Dehiscent vs. indehiscent capsules, one gene with pleiotropic effects (Id, id) or two complementary genes Normal vs. paper shell capsule, two complementary genes (P1, p1; P2, p2) Normal vs. seamless capsule (Gs, gs) Normal vs. determinate (Dt, dt) Capsule length, polygenic Photoperiod response, major genes and polygenes Earliness, major genes and polygenes Seed coat color, several genes, one epistatic, white recessive

Many of the traits listed above can serve as morphological markers in studying natural outcrossing rates and in verifying hybridization when the female parents are recessive. The only linkage case reported for sesame so far (Dhillon, cited by Brar and Ahuja, 1979) was in 1974. No other linkage or association of genes with certain chromosomes has been reported except for a molecular marker for closed capsules (Uzun et al., 2003). Both id and dt have side effects. In homozygous idid plants the cotyledons are cupped (spoon shaped), the leaves have enations on the underside, and the styles are bent. In dtdt plants the apical flowers differ from the standard by being bell shaped with no lip, the internodes are telescoped, the capsules are semiclustered, and often the apical capsules are tetracarpellate while the others are bicarpellate. It can be deduced that these genes affect some growth regulators’ controls. 8.4.2

Yield and Yield Components

Sesame’s status as a low-input, high-risk crop stems to a considerable extent from its low yield and low returns (Table 8.1), which motivate growers to shift to other crops. Higher yields are thus most important in assuring sesame’s place in farming areas where it is adapted, so that it will be a crop of choice rather than a crop of default. Higher and more reliable yields have been and are a major objective in most, if not all, breeding programs. Many researchers studied the inheritance of various seed yield components and their relative contribution to the final yield (Weiss, 1983; Brar and Ahuja, 1979; Lee and Chang, 1986; Osman, 1989; Rong and Wu, 1989; Sasikumar and Sardana, 1990; Reddy and Priya, 1991; Tu et al., 1991; Imrie, 1995; Arriel et al., 1999b; Alam et al., 1999; Kavitha et al., 1999; Arulmozhi et al., 2001; Devi et al., 2002; Sankar and Kumar, 2003). The main components are number of branches (especially primary ones) per plant, number of capsules per plant, number of capsules per leaf axil, length of the capsule-bearing zone on the main stem, plant height, duration of the flowering period, capsule length, number of carpels per capsule, number of seeds per capsule, seed weight, harvest index, and also the nature of the root system. Detailed studies were conducted in South Korea to determine the ideal sesame plant type, or ideotype, for the country’s conditions (see Kang, 1994) as well as in Australia (Beech and Imrie, 2001). It is difficult to draw optimal ideotypes because sesame is grown under a wide range of conditions. Many of the factors and their interactions with each other and the environment will vary with the growing conditions, such as cultural practices, stand density, inputs, and water availability. Perforce, studies on yield components in segregating populations were conducted on the basis of single plants, not plots. However, sesame growth habit and development are much affected by the stand density; branching is enhanced in sparse stands and reduced in dense ones. Similarly,

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plant height, which affects the length of the capsule-bearing zone on the stems, is modified by sowing time; plants sown earlier are taller than those planted later. Therefore, the yield components findings noted above, that were derived from single-plant analyses, are lacking. Also, while findings on the yield components obtained by correlations, path coefficients, and heritability values can be useful guides, they vary and are valid only for the genotypes and environments (locations and years) studied. It is difficult to draw generalized conclusions; hence, breeders should refer carefully only to results that are relevant to their genetic materials and conditions. It has been proposed repeatedly to increase the sink of sesame plants by breeding prolific varieties that will have three tetracarpellate capsules per leaf axil. Some breeding programs followed this avenue but did not achieve yield breakthroughs. Van Rheenen (1981a) found that in many traditional growing areas, varieties with one or three capsules per axil were grown side by side, with neither type taking over. Similarly, Van Rheenen showed that in the traditional growing areas there was no preference under the usual growing conditions for long capsules over shorter ones (1981b) or for tetracarpellate varieties over bicarpellate ones (1981c). This conclusion was confirmed in South Korea, where of 1401 local landraces, about 75% had 1 bicarpellate capsule per leaf axil, 20% had 1 tetracarpellate capsule per axil, and only 5% had 3 bicarpellate capsules per axil (Kim and Lee, 1981). In South Korea, where sesame is grown with high inputs, the first few improved varieties had one or two capsules per axil, but the trend has been toward three bicarpellate capsules per axil. Thus, the question of source vs. sink in sesame seed production is still unresolved. According to Beech (1985), low amounts of solar radiation (which is often the case during the monsoon period due to short days and cloudy skies) could be partly responsible for the low yields. Imrie (1995) stressed that yield is the product of intercepted photosynthetically active radiation and conversion efficiency (often expressed in terms of harvest index). Close correlation between photosynthetically active radiation absorption and sesame yield was reported by Yadav et al. (1988). Lee et al. (2002) found in Korea, in comparing two varieties, that the lower-yielding variety had a lower leaf area index (LAI), lower net assimilation rate (NAR), and lower crop growth rate (CGR). The high heritability values for NAR and LAI reported by Singh et al. (2000) indicate that breeding progress can be achieved in source enhancement. 8.4.3

Genotype × Environment Interactions

Stable yield performance of cultivars across seasons and regions is a very important breeding consideration. Genotype ∞ environment interactions were assessed in trials with sets of genotypes (varieties) in several locations or seasons and by studying hybrid populations of various types. It was shown that some genotypes were more stable than others, being less affected by the environmental conditions. The same was true for certain traits, giving them higher heritability values. In Korea, Shim et al. (2003) showed that in trials with seven cultivars in five regions over 3 years, one variety (‘Suwon 171’) stood out with its reliable performance. Such a genotype must have a wide homeostasis, a good buffering response. Similarly, 11 genotypes were tested for yield and other characters in 12 environments in India by Raghuwanshi et al. (2003). They concluded that one variety (‘Uma’) gave good stable yields, while four other genotypes performed consistently well only under favorable conditions. Such phenomena are well known also in other crops. Arriel et al. (1999a) tested 13 genotypes under two soil moisture regimes in a semiarid part of Brazil and found a wide range of phenotypic variation for several reproductive traits. In Sesaco’s experience, stable lines had intermediate expressions in most characters, e.g., medium capsule length and seed size, neither early nor late, neither short nor tall, and not with triple capsules per axil (D.R. Langham, personal communication). 8.4.4

Heritability

Estimates of heritability are very valuable in assessing the share of the genetic components vs. the environmental ones in the total variation of segregating populations. It shows the reliability of

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the phenotype as an indicator of the genotype, and hence the potential for effective phenotypic selection for given traits. Only a few authors have published heritability values. It is encouraging that high heritabilities were reported for several yield-related characters, even though often it is not clear whether the values are narrow- or wide-sense ones. High heritability values for net assimilation rate (NAR), leaf area index (LAI), and crop growth rate (CGR) were reported by Singh et al. (2000) in India. High values were obtained for plant height and seed yield (Arriel et al., 1999b; Govindarasu et al., 1999; Singh et al., 2000), for number of branches per plant (Govindarasu et al., 2000; Krishnaiah et al., 2002), for maturation (Arriel et al., 1999b), and for 1000-seed weight (Zagre et al., 1999). Contrasting values were reported for the heritability of the number of capsules: while high values were calculated in India (Govindarasu et al., 2000; Krishnaiah et al., 2002), in a study in Brazil (Arriel et al., 1999b) heritability was low, 0.30. It should be remembered that these findings are relevant primarily to the genotypes used and the prevailing conditions. Still, the heritability values obtained are encouraging; they show that selection can succeed. Even with the low values, good progress can be made in selection programs, provided a higher number of promising plants are selected and progeny tests are conducted. This has been the case in breeding for various quantitative traits in different crops, where improved performance was achieved despite low heritability values. 8.4.5

Combining Ability

Investigations of hybrid combinations, general and specific combining abilities, genetic control of quantitative traits, and genotype ∞ environment interactions have multiplied in recent years, many of them in India. Some of the studies have general implications, while others are relevant to the particular genotypes and locations involved. The results of a more general nature will be noted here. Conclusions regarding the relative impact of additive vs. nonadditive gene action varied with the traits examined, the genotypes used, the methodologies employed, and the locations. Some researchers considered additive gene action to be more important (Ibrahim et al., 1983; Dharmalingam and Ramanethan, 1993; Imrie, 1995; Das and Gupta, 1999; Ramesh et al., 2000). Other authors reported that nonadditive action was more important (Imrie, 1995; Quijada and Layrisse, 1995; Sumathi and Kalaimani, 2001; Manivannan and Ganesan, 2001). Actually, no general conclusions can be drawn: the role and impact of additive vs. nonadditive gene actions will vary from trait to trait depending on the parental combinations and locations. Thus, for instance, Das and Chaudhury (1999) reported that both additive and nonadditive components were important in the control of oil content and palmitic acid level, while predominately nonadditive ones affected the stearic, oleic, linoleic, and arachidic fatty acids. Both additive and nonadditive genes were involved in the control of net assimilation rate (NAR) and leaf area index (LAI) of plants at full bloom (Singh et al., 2000). The above general conclusion can assist breeders in taking advantage of the large amount of data now available, in making their hybridization plans, in selecting the parents in a more informed way, and in devising selection modes. 8.4.6

Heterosis

Sesame is considered a self-pollinated crop, yet high degrees of heterosis, both over the midparent and over the better parent, were obtained in different countries with diverse parents from various germplasm pools. High yields of F1 hybrids, at times considerably higher than those of the better parent, have been reported from diverse regions (Brar and Ahuja, 1979; Mazzani et al., 1981; Yermanos, 1984; Sharma, 1985; Osman, 1989; Zhan et al., 1990; Khan et al., 1991; Tu, 1993; Quijada and Layrisse, 1995; Pathirana, 1999; Das et al., 2000; Dikshit et al., 2000; Dusane et al., 2002; Saravanah and Nadarajan, 2002). The studies included crosses of many varieties as males with a few testers as females, as well as diallel and half-diallel crosses. In some investigations, as many as 15 quantitative characters were studied, while in others, a few characters were examined,

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focusing on seed yield and yield components. Unfortunately, nearly all the studies analyzed the traits on a plant basis rather than on the basis of plots with the stand density of a commercial field. Higher yields of the F1 hybrids were confirmed on a plot basis in Venezuela by Mazzani et al. (1981). Quijada and Layrisse (1995) grew an F1 hybrid in semicommercial fields of 2 to 5 ha; it proved to be superior in five locations in Venezuela (they produced the large amounts of hybrid seeds needed using genic male sterility). In this connection, it is noteworthy that Kavitha et al. (2000c) obtained yield heterosis in F1 hybrids with the genic-cytoplasmic male-sterile lines that they developed. Farm-level production of hybrid sesame was successful in China (L.C. Tu, personal communication). In view of the need for emasculation and hand pollination to produce F1 hybrid seeds, it was suggested that the heterotic F1 hybrids will be used to produce F2 seeds for commercial growing and even F3. This approach was tested in China by Zhao (1994), who reported that there was residual heterosis in F2 and F3. Kar and Swain (2003) discovered that in certain hybrid combinations there was a significant level of residual heterosis. Mixed results, depending on the hybrid combination, were reported by Karuppaiyan and Ramasamy (2001). Sodani and Bhatnagar (1990) concluded that usually where F1 hybrid vigor was high, F2 inbreeding depression was significant. High inbreeding depression was found where heterosis was due to nonallelic interactions (Ragiba and Reddy, 2000). Generally, high levels of heterosis were obtained when the parents of the hybrids were from very divergent origins. Single-locus heterosis has not been reported in sesame. There were, in some combinations, marked differences between reciprocals in the vigor of the F1 hybrids (Ray and Sen, 1992; Tu, 1993); thus, a cytoplasmic component may be involved (see also Section 8.4.7). At times, accessions or varieties that appear to be from the same genetic germplasm pool produced heterotic F1 hybrids. This can be explained in several ways. First, within the same region selection over the years led to the divergence of two types; such could be the case in India with genotypes adapted to the Kharif vs. Rabi seasons. Bedigian (2003) noted that in Sudan the growers in the Nuba region selected sweet vs. more bitter landraces, which differed also in other attributes. Finally, in some countries, e.g., Ethiopia and Venezuela, the improved local varieties are often introductions or selections from introductions originating in diverse regions. It can be concluded that heterotic F1 hybrid varieties are a most promising way for yield breakthrough in sesame. There is already much data on combining ability (see Section 8.4.5) and on desirable hybrid combinations. This available database could be readily mobilized to produce hybrid varieties once a suitable genic-cytoplasmic male sterility mechanism is established. 8.4.7

Male Sterility

Male sterility in sesame was reviewed by Yermanos and Osman (1981), Weiss (1983), Osman (1985), and Ashri (1998). Earlier accounts described complete sterility (male and female), incomplete male sterility, or male sterility only during the early part of the blooming period. The widely distributed nuclear male sterility allele originated as a spontaneous mutation in Venezuela (Mazzani, personal communication). Sesame lines with the male sterility allele were supplied by B. Mazzani, CENIAP, Maracay, Venezuela, to D.M. Yermanos at the University of California, Riverside, in the mid-1970s; subsequently, both distributed the material widely and freely. The male sterility proved to be monogenic, recessive (ms), and very stable in all environments tested (Yermanos, 1984; Wang et al., 1993; Tu et al., 1995). When pollinated, the male-sterile plants give seed yields that are comparable to male-fertile plants (Yermanos and Osman, 1981), i.e., they are fully female fertile. The anthers of the male-sterile plants are green and translucent, which facilitates identification of the male-sterile plants early in the season, when the flowers are in the bud stage, 3 to 4 days before anthesis. Gao et al. (1992) reported that in the msms plants, the nonvacuolated microspores break down at the tetrad stage.

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The male sterility trait that was discovered in a line adapted to Venezuela has been successfully introgressed into materials adapted to diverse conditions, e.g., California (Yermanos and Osman, 1981), China (Wang et al., 1993; Tu et al., 1995), the Republic of Korea (Kang, personal communication), Israel, and others. However, only in China has this male sterility allele actually been used in pilot production of hybrid seeds for the farmers. Induced mutations for genic male sterility were reported from several sesame research programs. In China, Li et al. (2001) induced six stable mutants in a good commercial variety (‘Yuzhi 4’) using doses of 500 and 700 Grey of gamma rays. Three genic male steriles were induced in Turkey by similar doses of gamma rays (Cagirgan, 2001), and in the Republic of Korea four genic male sterility mutants were induced with sodium azide (NaN3) and used for further studies (Kang, 2001). The mutants noted above are monogenic, with the homozygous recessives being male sterile. So far, it has not been reported whether the induced mutants are in one or more loci and whether some or all of them are allelic to the original Venezuela ms mutant allele. Such findings will lead to better understanding of the inheritance of male sterility in sesame. The use of genic male sterility in producing hybrid varieties is hampered by its maintenance difficulties and by the required roguing of the heterozygous male-fertile (Msms) plants from the intended female rows, which segregate for Msms and msms plants at a 1:1 ratio. This laborious procedure could be eliminated if msms male-sterile plants could be made to produce seeds by the application of some growth regulators. Studies in this direction were conducted by Zheng et al. (2000), who identified some chemical restoration agents that restored fertility when sprayed on the plants and gave a good seed set. Genic-cytoplasmic male sterility (GCMS) was reported in India by Kavitha et al. (2000a, 2000b). They also found several monogenic restorer lines that were homozygous for the dominant restorer allele. Kavitha et al. (1998, 1999, 2000) found that the male sterility in the above lines resulted from degeneration of the tapetum. Male sterility has been obtained in the cross of the S. malabaricum (as female) × S. indicum, and not in the reciprocal (Thangavelu, 1994). Geniccytoplasmic male sterility was reported for the same interspecific cross by Prabakaran (1998a, 1998b) and Prabakaran and Sree Rangaswamy (1995). These findings suggest that an efficient genic-cytoplasmic male sterility mechanism for the production of F1 hybrid cultivars may be obtained in sesame by interspecific hybridization, as in sunflower and other crops. A proposed scheme for identification of restorer alleles and GCMS development using interspecific crosses is presented by Van Zanten (2001). Effective and stable chemical emasculation of sesame could facilitate F1 hybrid production in the absence of male sterility. Recent studies on the subject were not found in the literature. Chemical emasculation of sesame by growth regulators such as dalapon, FW450 (2,3-dichloro-methyl propionic acid sodium salt), and sodium 2,3-dichloropropionate was reviewed by Osman (1985). In China, Zhan et al. (1991) and Zhao (1994) reported that the gametocides caused hypertrophy of the tapetal cells, with FW450 being the most effective. However, since Zhan et al. (1991) found the best time for application to be just after pollen mother cells (PMC) formation, this treatment is not applicable for indeterminate lines. Even for determinate lines that will bloom for only 2 to 3 weeks, more than one application would be required.

8.5 SEED RETENTION 8.5.1

General

Good seed retention is by far the most critical trait that can enhance and expand sesame growing under more intensive conditions. This is the consensus of all concerned — growers, breeders, and traders. Langham and Weimers (2002) predicted that if sesame harvesting is not mechanized by

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2025, its world production will decrease markedly and it will be grown only where other, more suitable crops cannot grow. The manual labor required for weeding and harvesting the crop is becoming scarce and expensive even in developing countries. Seed retention has been a key step in the domestication of most of the crop plants whose yields are seeds, be they grasses or legumes, composites or cruciferous, or others (De Wet, 1989; Gottlieb, 1984). Different seed retention mechanisms evolved in the diverse crops, usually under the control of one or very few major genes (Ashri, 1989a; Gottlieb, 1984). In sesame, although it is a crop of antiquity, the seed dispersal nature of a wild species persists to this day. A seed-retaining mechanism was not known until the indehiscent natural mutant id/id was discovered in Venezuela in 1942 (D.G. Langham, 1946). Ashri and Ladizinski (1964) offered the following possible explanations for this unique situation in sesame: (1) The desired mutation did not occur. (2) It did occur, but was missed by the farmers because the mutants’ seeds were not released in the usual manner of threshing (by inverting the bundled plants and beating them), thus not contributing seeds to the next generation. (3) The mutation did occur, and the growers did notice it, but rejected it because of the difficulty in threshing. In the author’s opinion, the third option is probably the logical one since the traditional early farmers were observant, they did spot seed retaining mutations in other crops, and they would have found them in sesame as well. However, judging from the experience with the id mutation with its many undesirable effects, it is likely that in earlier times the indehiscent mutants were on the whole counterproductive and were rejected. Bedigian (2003) arrived at the same conclusion, that “the cultivators adapted their harvest strategies to the plant’s habits.” Apparently the easy flow of the seeds from the dried plants when inverted and shaken over a hard floor suited the growers. This is all the more important in order to avoid damaging the seeds, which are small and soft with a thin and fragile seed coat, thus preventing rancidity of the oil. 8.5.2

Monogenic Prevention of Seed Shattering

For many years, sesame breeders have looked for the spontaneous major gene mutations that will lead to good seed retention. They also attempted to induce them with mutagenic treatments using radiations and chemicals. An indehiscent spontaneous mutant was discovered in 1942 by D.G. Langham (1946) in Venezuela, which he reported to be monogenically controlled. Later, digenic control was encountered in some crosses (Nafie, 1980) and also modifiers (Ashri and Ladizinski, 1964). In the homozygous recessive idid plants, the capsules are indehiscent due to structural changes in the mesocarp (Ashri and Ladizinski, 1964; Day, 2000a). The idid plants have bent styles, reduced seed set, lower yields, higher susceptibility to diseases, leaf enations on the undersides of the leaves, and other undesirable features, such as bent stems and extra floral growth. Despite intensive breeding efforts by many sesame researchers, the desired indehiscence could not be separated from the undesirable side effects, suggesting pleiotropism. Furthermore, the capsule of the idid indehiscent plants proved too difficult to thresh. The required force and the faster cylinder speeds and additional rasp bars or spikes needed to open the capsules and release the seeds led to high proportions of damaged seeds. In order to ease the threshing, Culp (1960) proposed to combine the capsule paper shell gene and idid. The cross was made by Kinman, and selections from it were used by Sesaco to produce its first cultivar, ‘Sesaco 1’ (SO1); it had the idid and paper shell traits, but the seeds were still badly damaged (Langham and Weimers, 2002). Another trait that could improve seed retention is stronger placentation. Apparently, the trait is controlled by several genes and was incorporated in the Sesaco cultivars SO3 and SO7 (Langham and Weimers, 2002). A recessive mutant (gsgs), termed ‘seamless’, whose capsules do not dehisce, was found by D.G. Langham and D.R. Langham in 1986. Capsules of gsgs plants have very thin shells and appear

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to have only one carpel, although they have two. Unfortunately, in this mutant the stamens often abort and the plants have a lower seed set. In addition, it is difficult to break the capsules open to release the seeds; therefore, breeding efforts with it at Sesaco were terminated (Langham and Weimers, 2002). Mutagenic treatments to obtain good seed retention were undertaken over the years, especially in developing countries that grow sesame, with the help of the Joint FAO/IAEA Division, Vienna, Austria. In Turkey, eight closed-capsule mutants were discovered following seed treatments with 300 to 400 Grey doses of gamma rays (Cagirgan, 2001). Delayed shattering and shatter-resistant capsules’ mutants were obtained in Thailand with gamma rays and ethylmethane sulfonate (EMS) treatments (Wongyai et al., 2001; Maneekao et al., 2001). Some of the induced mutants were sterile or undesirable, but others were promising and were included in cross-breeding programs. Despite progress made by Sesaco in developing nonshattering varieties (see below), the search should still continue for spontaneous and induced effective nonshattering and productive mutants. Treating diverse cultivars with various mutagens using different doses should be a long-term, lowcost effort. Large M2 populations can be grown and screened easily at maturity for seed-retaining mutants, with low labor requirements and budgets. 8.5.3

Nonshattering Achieved by a Combination of Traits

It was clear to the Sesaco Corporation in the U.S. (founded in 1978 by D.G. Langham, family members, and associates) that in order to grow the crop in the U.S., it must be combine harvested. After failing to achieve the desirable levels of seed retention, yield, and quality with the id and gs alleles, Sesaco opted for a multipronged approach, i.e., to combine into successive genotypes characters that promote nonshattering, by intercrossing a wide range of materials and selection. The same conclusion was drawn by Bennett (personal communication), Beech and Imrie (2001), and Wongyai and Chowchong (2003a). Wongyai has developed in Thailand, through a series of crosses with many diverse lines, including determinate (dt45) lines, nonshattering breeding lines, and one of them, known as ‘C plus 1’, is under yield tests in farmers’ fields (Wongyai and Chowchong, 2003b; Wongyai, personal communication, 2005). The ensuing discussion is based on the publications by Langham (2001a) and Langham and Weimers (2002) and on the U.S. Plant Patents awarded to Sesaco Corporation (2000, 2004). The capsule traits that Sesaco combined in order to reach the high levels of shatter resistance required for combine harvesting follow: • Capsule tip opening: Moderate, to allow drying of the seeds. • Capsule constriction: Shrinking of the capsule walls as they dry, holding the seeds in place, but not too tightly. • Membrane completeness and attachment: False membranes (septa) covering the locules (in the inner part of the carpels) from the tip of the carpels to the base, thus holding the seeds in place, yet facilitating their release in the combine. • Stronger placentation: Stronger placenta attachment through assembling genes from many genotypes (probably quantitative). • Capsule split: Splitting of the capsules at both sutures halfway to the base, allowing for drying and for seed release in the combine. Longer splitting would be desirable for seed release in the combine, but could lead to higher seed losses if the membrane attachment is weak. This illustrates well the types of compromises breeders face. • Appressed capsules: The capsules’ tips pointing upwards.

The progressive improvement in seed retention achieved by Sesaco in direct combineharvested varieties is remarkable (Table 8.6). The achievement of Sesaco breeders demonstrates well the need for cumulative contributions. The latest Sesaco varieties have a seed retention rate exceeding 90% in the inverted seed retention test and over 65% in the shaker seed retention test

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(see below). The varieties now available hold their seeds well even under adverse windy and rainy conditions. 8.5.4

Seed Retention Measurements

The many terms used to describe seed retention (or loss), such as indehiscence, nonshattering, shattering resistance, dehiscence, or shattering, demonstrate the need for generally accepted measurement standards to assess progress and performance. In recent years, Sesaco has adopted the shaker shatter resistance (SSR) measurement test (Langham, 2001a; Langham and Weimers, 2002; U.S. Patents, 2000, 2004). In this test two capsules are taken from the middle of the capsule-bearing zone of five plants after they reach physiological maturity. In lines with three capsules per leaf axil, one central and one side capsule are taken from the same axil, from five plants as above. The capsules are dried and placed in a flask, which is placed on a shaker (3.8-cm stroke, 250 strokes/min) for 10 min. The seed that comes out is weighed, the retained seeds are threshed out and weighed, and the SSR rate is calculated as the proportion of seeds that were released over the total of the released and retained (threshed) seeds. This test is suitable for green capsules of shattering and nonshattering types, but if dry capsules are used, it is suitable only for lines that already have some shatter resistance, because the capsules picked for the test must contain all their seeds. Sesaco used two other measurements: upright seed retention (USR) and inverted seed retention (ISR) (Langham, 2001a; Langham and Weimers, 2002; U.S. Patents, 2000, 2004). Ten physiologically mature but green capsules are collected as above, placed upright in a jar, dried with a heat lamp, and lifted out when dry. The seeds that are left in the jar are weighed, and the capsules are then inverted over a pan, twirled, and dropped three times from 15 cm, and the seeds that drop into the pan are weighed. The capsules are then threshed and the seeds weighed. The USR and ISR were calculated from the proportion of the seeds in each lot out of the total of the three. Kang (1997) in South Korea assessed shattering by placing mature (but not dry) plants upside down in bags, allowing them to dry, and weighing the seeds that dropped in the bags; he then threshed the plants and weighed the seeds, thus computing seed shattering percentage out of the total.

8.6 SEED COMPOSITION AND QUALITY 8.6.1

General

Sesame has been grown for millennia for its seeds, prized oil, and oily paste, tahini, obtained by grinding the seeds. It has been primarily an oil crop, but the seeds are used also on baked goods, for condiments, and in making sweets such as halva and candy bars. In view of its major use as a source of oil, seed quality considerations focused for many years mainly on the oil content and fatty acid composition, on the protein content and amino acid composition, and on the antioxidant lignans. These aspects were discussed in an earlier review by the author (Ashri, 1998) and will be presented here too. In addition, seed flavor and sweetness, and findings on sesame seed allergens, will be discussed in view of mounting recent interest in these topics. 8.6.2

Oil Content

Ashri (1998) reviewed findings on oil content in China, India, Israel, South Korea, the U.S., and Venezuela. These studies included hundreds of accessions from very diverse germplasm pools and geographical sources. The mean oil percentages varied from 47.0% in Venezuela to 53.1% in China and California, and they ranged from ca. 40 to 59%. A low one-time value of 34% oil in Israel was caused by seed immaturity due to environmental factors.

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Table 8.7 Ranges of Oil and Protein Contents (%) in Sesame Seeds of Accessions Grown in South Korea and Israel Range (%)

Seeds Grown

No. of Accessions

Oil

Protein

In South Korea In Israel

330 240

45.1–55.2 34.4–54.9

20.0–29.5 19.8

Note: All analyses were made in South Korea using the same equipment and protocols. Source: Ashri, A., in Sesame Biodiversity in Asia: Conservation, Evaluation and Improvement, Arora, R.K. and Riley, K.W., Eds., IPGRI, New Delhi, 1994, pp. 25–39.

Variation in oil content results from both genotypic and environmental factors. Ashri et al. (1991) and Ashri (1994) showed with a large number of identical accessions grown in both Israel and South Korea (and analyzed in the latter) that the oil content of mature seeds in one region could not be used to predict the oil content in the other (Table 8.7 and Figure 8.4). Yermanos et al. (1972) suggested that oil content was governed by quantitative genes. This is generally accepted, but documented reports on the genetic control of oil content have not been found. A successful pure line selection for higher oil content (63%) was reported in Turkey (Baydar et al., 1999), but its field performance is not clear. It was observed that oil content in later maturing cultivars was higher than in earlier ones (Yermanos et al., 1972; Lee et al., 1991b). Oil content is affected by the position of the capsules. In mature seeds, it was highest in basal capsules from the main stem, compared to capsules from apical and side branches (Mosjidis and Yermanos, 1985; Ibrahim and Ragab, 1991). Seed weight varied with capsule position, e.g., central capsules’ seeds on the main stem were the heaviest but had less oil (Mosjidis and Yermanos, 1985). In view of this variation, standardized capsule sampling procedures should be followed in more critical investigations. The question of breeding for higher oil content has been raised from time to time. The author’s opinion is that it should not be attempted. As it is, oil content of about 50% is close to the biological limit; no other commercial annual oil crop exceeds it. Second, the small seeds of sesame are already quite fragile; they might become more so and be badly damaged at harvest if their oil content would be appreciably higher, thus causing poorer seed germination. Finally, higher oil content may lead to lower yields, and thus to lower oil yield per unit area. Breeding and germplasm screening for oil content will be aided by using ultraviolet spectrophotometry (Zhang et al., 2004) to determine it. It has been shown that seed color may be associated with oil content. In Kenyan accessions, those with the highest oil content had white small seeds (Were et al., 2001). Namiki (1995) showed that black seeds had significantly less oil than white or brown seeds (47.8 vs. 55.0 and 54.2%, respectively). Tashiro et al. (1990) and Namiki (1995) attributed the low oil content of their black seeds to their thicker seed coats (14.4 vs. 6.2 and 8.0%, respectively). Also, since black seeds are not used for oil extraction (they color the oil), there was no selection pressure in the black-seeded varieties for higher oil content (D.R. Langham, personal communication). 8.6.3

Fatty Acids

The fatty acid composition of sesame oil is very desirable, with about 80 to 85% unsaturated acids and only 20 to 15% saturated ones. The ranges of fatty acid percentages of the total lipids, as reported by several authors for large samples of diverse genotypes, were palmitic (16:0, number of carbon atoms:number of double bonds), 8.3 to 14.6%; stearic (18:0), 3.4 to 6.8%; oleic (18:1), 32.7 to 53.9%; and linoleic (18:2), 29.9 to 45.6% (Yermanos et al., 1972; Lee et al., 1991a; Namiki, 1995; Kamal-Eldin et al., 1993). The polyunsaturated fatty acids — linolenic, arachidic, eicosenoic,

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Figure 8.4

GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT

The relationship of oil content (%) in Korea and Israel: total of 195 entries (A), with the lowest 40 accessions (B) and highest 37 accessions (C) in Israel, with their oil content (%) in Korea.

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behenic, and lignoceric — occurred in small amounts (Kamal-Eldin et al., 1993). The oils of the wild species S. alatum, S. angustifolium, and S. radiatum contain about the same proportions of fatty acids as the cultivated (Ashri, 1998; Were et al., 2001). As noted above, seed maturity and capsule position affect oil content (Kang et al., 1985; Gupta, 1990) and possibly also the proportions of fatty acids in the oil. Therefore, rare exceptions, as noted in the ranges above, could be due to such causes. Lee et al. (1981) showed that climatic conditions affected the proportions of the fatty acids. In South Korea, seeds produced in the northern cooler areas had a higher proportion of linoleic acid (and lower oleic acid) than seeds from the southern parts. Lee and Kang (1980a) found that accessions that had three capsules per leaf axil and yellow seed coats matured later and had higher proportions of unsaturated fatty acids, but many factors could be involved here. Kang (1994) reported that the proportions of the palmitic, stearic, oleic, and linoleic acids were quite similar in seeds with white, black, brown, or yellow seed coats. However, the stearic acid content of cultivars with white seeds in South Korea was reported to be significantly higher than that of cultivars with black seeds (Kang et al., 2000). Some differences between lines in seed weight, oil and protein content, and fatty acid composition were reported by Brigham and Khan (1989). Mosjidis and Yermanos (1984) suggested that differences between the reciprocal F1 hybrids in the proportions of oleic and linolenic acids resulted from different seed development rates of the parents. In conclusion, there is variability for fatty acid content that may facilitate breeding for modified oil quality if desired, but its genetic control is unknown. The oil composition of sesame could be modified by transgenic manipulation to produce special niche oils, as in other oil crops; initial success in DNA transfer in sesame has been reported (see below). Breeding for fatty acid composition will be helped by the recent findings on the use of nearinfrared (NIR) spectroscopy to estimate fatty acid proportions in whole seeds (Sato et al., 2003). They concluded that with some exceptions, the method is simple, rapid, nondestructive, and reliable, regardless of the color of the seeds. Thus, it is well suited for fast screening of many seed samples in breeding programs. 8.6.4

Proteins and Amino Acids

Sesame oil was prized in ancient times, and the plant continues to be cultivated for its oil. Thus, the oil content was emphasized and, in recent years, also the lignan composition, while little attention was given to the proteins and their amino acids. Ashri (1998) reviewed the reports on protein content in many accessions in China, India, Israel, South Korea, the U.S., and Venezuela. The means were 21% in Venezuela, 24.8% in the U.S., and 26.4% in China. The range varied from 19.4 to 29.5%. The protein content is affected by genetic and environmental factors. The negative relationship of oil and protein content in oilseeds is well known. There are indications that increased nitrogen supply to the plants favors protein synthesis (Beech and Imrie, 2001). Tai et al. (1999) stated that 11S globulin (alpha-globulin) and 2S albumin (beta-globulin) are the two major storage seed proteins of sesame and constitute 80 to 90% of the total seed protein. Lee et al. (1990) found variability in the amino acid composition among 46 accessions. The total amino acid content was higher in seeds with black seed coats, whose oil content is usually lower. 8.6.5

Isozymes and Protein Bands

Studies of isozyme and protein band patterns were quite common in assessing variability, relationships, varietal purity, and origin in various organisms, including plants, before DNA-based approaches were developed. This was the case also with sesame; the few reports published go back to the 1990s. Nava and Gerder (1993) investigated the protein extracts of seeds and seedling leaves of five local Venezuela cultivars. The isozymes identified and the number of phenotypes

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for each were peroxides (2), acid phosphatase (3), esterase A (3), isocitrate lyase (4), malate dehydrogenase (5), esterase B (5), and esterase D (5). Later, Diaz et al. (1999) examined the isozyme variability of 40 accessions from six centers of diversity. They analyzed five loci controlling four isozymes, namely, acid phosphatase, isocitrate dehydrogenase, phosphogluconate dehydrogenase, and shikimate 5-dehydrogenase. They concluded that the total genetic diversity in their sample was fairly low. Diaz and Layrisse (2000) studied two local Venezuelan varieties for seven isozymes and found that several of them were monomorphic. The genetic diversity of 22 progenitors of an indehiscent breeding population in Venezuela was studied using 2 isozyme traits and 14 morphological ones (Diaz et al., 2003). Isshiki and Umezaki (1997) studied seven enzymes in 68 accessions of sesame from divergent sources. They found variation only for isocitrate dehydrogenase, controlled by one gene (Idh) and two alleles. The two alleles were widely distributed in the germplasm collection studied. Using polyacrylamide gel electrophoresis (PAGE), Das et al. (1992) found differences between four cultivars in seed protein band patterns, number, and mobility. They used PAGE also to show protein profile differences between a source cultivar and three derived mutant lines. 8.6.6

Lignans

The lignan antioxidants are unique for sesame and are present in the oil. Their biosynthesis and precursors were described by Kato et al. (1998). The lignans sesamin (2,6-bis-(3,4-methylenedioxy phenyl)-cis-3,7-dioxa-bicyclo (3.3.0)-octane) and sesamolin (2-(3,4-methylenedioxy phenoxy)-6-(3,4-methylenedioxy phenyl)-cis-3,7-dioxabicyclo (3.3.0)-octane) and their derivatives (sesamol, sesaminol) prevent the oxidation of the oil and give it the long shelf-life and stability for which it is renowned. This property of the antioxidant lignans in sesame oil has also been used to prolong the shelf-life of other oils and of peanut butter by mixing small amounts of sesame oil into them. Dachtler et al. (2003) concluded that sesaminol-enriched extract of sesame could increase the antioxidative stability of edible oils high in polyunsaturated fatty acids. Fourier-transform infrared (FTIR) spectroscopy proved efficient and accurate in determining sesamol in edible oils (Mirghani et al., 2003). The antioxidant and health-promoting activities of the lignans (Kato et al., 1998; Morris, 2002; Shirato-Yasumoto et al., 2003) led to enhanced interest in the genetic variability for lignans in the cultivated species and the wild ones. It should be noted that since sesame oil is used as a carrier in some medications, there may also be an effect of the lignans in those cases. Interest in breeding for higher lignan content has increased in several countries. Breeding will be facilitated by the recent development of a rapid, simplified high-performance liquid chromatography (HPLC) method for the quantification of sesamin and sesamolin in sesame seeds (ShiratoYasumoto et al., 2003). It should be noted that sesamin and sesamolin contents in the seeds vary with maturity, being highest in capsules 30 days after flowering (Yasumoto et al., 2005). Therefore, care should be taken to standardize the maturity of the seed samples taken in screening the materials for their lignan content. In another recent development, Suh et al. (2003) obtained expressed sequence tags (ESTs) from immature sesame seeds and identified candidates for genes possibly involved in biosynthesis of sesamin and sesamolin. There are some reports on variation for lignan contents in cultivated sesame and in the wild species, summarized in Table 8.8. In India, wide variation was observed in the total lignan content of 21 seed samples and 9 commercial oils (Ghafoornissa, 2004). The lignan content of white Korean seeds was higher than that of white Chinese seeds (366 vs. 244 mg/100 g); sesamolin content was also higher in the Korean white seeds (242 vs. 125 mg/100 g) (Kang et al., 2003). In Korea, it was also shown that white seeds had significantly more sesamin and sesamolin than black seeds (Kang et al., 2000). Namiki (1995) found that black-seeded accessions had a significantly lower amount of sesamin than white seeds, and there was no significant difference in the sesamolin content between accessions with white, brown, and black seeds. In the Northern Territory, Australia, Bennett (personal communication) examined the contents of lignans and lignan di- and tri-glucosides in

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Table 8.8 Lignans Absence or Presence, and Their Levels, in Seeds of S. indicum, 11 Wild Sesamum Species, and C. sesamoides* Lignans, % in Oil Sesamin Sesamolin

Species 2n = 26 chromosomes S. indicum S. indicum S. indicum a S. alatum S. alatum S. capense S. malabaricum 2n = 32 chromosomes S. angolense S. angustifolium S. angustifolium S. latifolium 2n = 64 chromosomes S. radiatum S. radiatum 2n chromosome number not known S. calycinum S. pedalioides S. rigidum ssp. merenksyanum S. triphyllum Ceratotheca sesamoides

Reference

0.55 ± 0.17 0.07 ± 0.61 P 0.01 A A P

0.50 ± 0.08 0.02 ± 0.48 P 0.01 A T P

Kamal-Eldin, 1993 Namiki, 1995 Bedigian, 2003 Kamal-Eldin, 1993 Bedigian, 2003 Bedigian, 2003 Bedigian, 2003

P 0.32 ± 0.04 P P

P 0.16 ± 0.01 P A

Bedigian, 2003 Kamal-Eldin, 1993 Bedigian, 2003 Bedigian, 2003

2.40 ± 0.10 P

0.20 A

Kamal-Eldin, 1993 Bedigian, 2003

P T A A P

P A A A T

Bedigian, Bedigian, Bedigian, Bedigian, Bedigian,

2003 2003 2003 2003 2003

Note: A = absent; P = present; T = trace. a Called S. orientale.

seeds of ‘Edith’ (selected by Bennett from ‘Yori 77’ of Mexico) and 13 advanced breeding lines. He found a wide range of variation, with some lines containing twice as many lignans as ‘Edith’, which had the lowest level. Variation was noted for sesamin and sesamolin and sesaminol di- and tri-glucosides. The latter were most prevalent, constituting 50 to 90% of the total amount of the lignans in the seeds. On the other hand, Bedigian (2003) found no differences in sesamin and sesamolin content in 50 divergent accessions of sesame sampled from the germplasm collection. Apparently, the sample was not broad enough. Kamal-Eldin (1993) found wide variations in the cultivated species and advocated wider screening efforts to identify accessions with higher lignan contents. It is noteworthy that recently a variety with about twice as much sesamin and sesamolin as in the standard ‘Masekin’ was bred in Japan. Known first as ‘Gomazu’, it was released in Japan as ‘Sesame Norin 1’ in 2002 (Yasumoto et al., 2003). 8.6.7

Allergens

In recent years it has become evident that sesame seeds contain immunoglobulin E (IgE)mediated food allergens, with research reports from France (Fremont et al., 2002; Agne et al., 2003), Israel (Dalal et al., 2002, 2003; Wolff et al., 2003), Italy (Pastorello et al., 2001), and the U.S. (Beyer et al., 2002). Even babies showed allergic reactions, at times very severe. It appears that sesame seed allergy is becoming more prevalent due to the wider and expanding use of sesame seeds in baked goods and fast foods. Several allergens have already been identified. Wolff et al. (2003) identified a 14-kDa protein belonging to the 2S albumin family as the major sesame allergen; they also found some minor sesame allergens of higher molecular weights. Beyer et al. (2002) identified four allergens in the seeds, one of them the 2S albumin. The same 2S albumin was implicated also by Pastorello et al. (2001). Fremont et al. (2002) concluded that white sesame seeds contained at least 10 allergenic proteins, with two of them being major ones. They compared white, brown, and black sesame seed and concluded that the white seed extracts had the highest protein

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concentration. However, since they used only one accession per seed color, no conclusions can be drawn on allergens’ association with seed color. The allergenic reactions to sesame seeds are not exceptional. Food allergens are known in the seed storage proteins of other crops, e.g., peanut, walnut, Brazil nut, and soybean. It is quite possible that there is genetic variation for the allergens in the diverse genetic resources of sesame, but it is not clear if breeding could be effective. So far, reports on such studies have not been found in the literature. It is hoped that as more attention is focused on the seed allergens, more research on their nature will yield rapid identification procedures that will facilitate screening varieties, breeding lines, and germplasm accessions. 8.6.8

Flavor and Taste

Since sesame is used widely in food, as whole seeds or as a paste, its flavor and taste are gaining attention. There are differences between varieties in sweetness (actually degrees of bitterness). In Sudan, there are sweet and standard, somewhat bitter landraces (Bedigian, 2003). In Ethiopia, there are sweet landraces that are highly desired by overseas buyers. Variation was found also among breeding lines in Australia (Malcolm Bennett, personal communication). The bitter taste is imparted to the seeds by an oxalic acid compound in the seed coat. It can be reduced markedly by decortication. In Japan, which is the biggest importer of sesame seed, sweet sesame is sought and much preferred. In taste tests in Japan, the samples are roasted and then judged by panels for several criteria, including sweetness, bitterness, flavor, and aroma (Malcolm Bennett, personal communication). Unfortunately, the seeds must be roasted in order to determine their flavor and taste ratings. This precludes selection based on field tasting of the maturing seeds. It appears to the author that as the markets become more discerning, the demand for sweet sesame will rise and sweeter varieties will be sought, especially if they will draw premium prices. Breeding for sweetness will be complicated, though, by the lack of an objective laboratory analysis method. Obviously, since there are differences in taste between genotypes, selection can be successful. However, at present there is no information on the genetic control of the taste components; most probably several genes are involved. Traditionally, tahini, the sesame paste that is widely used in foods in the Middle East, is prepared by grinding lightly roasted whole or decorticated seeds using stone millstones. The processors prefer sesame seeds that are less bitter and have a nuttier flavor. 8.7 MOLECULAR VARIATION 8.7.1

General

Molecular DNA studies in sesame are only at an initial stage, because as noted above, it is typically a crop of developing countries and it is not a major crop. In a review a few years ago (Ashri, 1998), there were no reports to be cited in this area. Since then, investigations on cloning, DNA transfer, and DNA markers have been undertaken. It is hoped that molecular studies in sesame will be intensified, thus facilitating the efforts to make the breakthrough that will make it a mechanized, more reliable, and remunerative crop. Four protocols for DNA isolation in sesame were compared by Arriel et al. (2002) using young leaves of three cultivars. They concluded that all the protocols they used were adequate, giving sufficient DNA yield for polymerase chain reaction (PCR) applications. 8.7.2

DNA Markers

In the first report on the use of the markers in sesame, 58 accessions were analyzed by random amplified polymorphic DNA (RAPD) for genetic diversity (Bhat et al., 1999). RAPD markers were

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used by other researchers to assess variability in germplasm collections and hybrids (Parani et al., 1996; Davila et al., 2003; Ercan et al., 2004). Genetic relationships among 75 accessions were also studied by inter-simple sequence repeat (ISSR) polymorphism (Kim et al., 2002). Both marker types, RAPD and ISSR, were very instructive in grouping accessions according to their affinities. Linkage of an amplified fragment length polymorphism (AFLP) marker to a closed-capsule mutation was reported recently (Uzun et al., 2003). 8.7.3

DNA Cloning and Transfer

Cloning of sesame DNA was first achieved in 1999 when two full-length cDNA clones were fully sequenced and deduced to code for 11S globulin 2S albumin (Tai et al., 1999); expression was obtained in Escherichia coli for the 11S globulin. In a further development, a chimeric gene from sesame, encoding for a precursor polypeptide of 2S albumin, was expressed in transgenic rice plants (Lee et al., 2003); it increased the protein content and elevated markedly the contents of methionine and cystein in the rice grains. Suh et al. (2003) obtained 3328 expressed sequence tags (ESTs) from a cDNA library of young seeds. They identified a large number of seed-specific genes, particularly EST candidates for genes that might be involved in the biosynthesis of lignans. Several cDNA studies succeeded in identifying DNA sequences that code for certain sesame seed proteins and other components, namely, the protease inhibitor cystatin (Peng et al., 2004; Shyu et al., 2004), 11S albumin and 2S albumin (alpha- and beta-globulin, respectively) (Tai et al., 2001), thiamine binding proteins (Watanabe et al., 2001), myo-inositol 1-phosphate synthase (Chun et al., 2003), omega-6 fatty acid desaturase (Jin et al., 2001), stearoyl-acyl carrier protein desaturase (Yukawa et al., 1996), and oleosin isoforms (Tai et al., 2002). Transformation of sesame was successfully accomplished with Agrobacterium tumefaciens (Taskin et al., 1999). Transformation of S. schinzianum was accomplished by Agrobacteriummediated transfection of a carrot calmodulin gene (Mitsuma et al., 2004). Hopefully, these initial molecular studies in sesame will be followed by more intensive research efforts that will lead to better understanding of its biochemistry and physiology, assist in genetic studies, and contribute to breeding improved cultivars.

8.8 GERMPLASM RESOURCES 8.8.1

Background

The very fact that sesame is a crop of small holders in developing countries, which received limited breeding attention, assured that much genetic variability would be retained in the traditional growing areas. In those areas there have been few outstanding high-yielding improved cultivars that replaced the local landraces on a large scale. Thus, sesame germplasm resources are rich in variability that is enhanced by outcrossing. There are still many heterogeneous local landraces in the diverse sesame-growing areas. On the other hand, the wild species of sesame are very poorly represented in the collections. Despite enhanced collection and studies since the 1980s, much remains to be done with the collection, conservation, and documentation of Sesamum, both the cultivated species and the wild ones. The importance of harmonized, generally agreed criteria for evaluation and documentation cannot be overstated: in the final analysis, an important role of the gene banks is to assist in breeding superior, adapted varieties using the assembled genetic variability. Introductions had key roles in sesame improvement directly or as parents in crosses. A telling example is the breeding program of Sesaco in the U.S. It has 2738 accessions (from 66 countries) in its collection and has studied numerous traits and used them to make and evaluate 33,545 cross combinations in developing commercial cultivars (Langham, 2001b; Langham and Weimers, 2002). The new, revised, and

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Table 8.9 Cultivated Sesame Accessions Evaluated and Multiplied at Rehovot, Israel, Grouped by Source Regions Region

No. of Countries

No. of Accessions

Africa Middle East Far East America, Northa America, Central America, South Europe FAO, Romeb Unknown Total

17 10 14 1 4 3 5 — — 54

278 541 846 952 81 91 141 51 9 2990

a

Some local materials, most from other countries; origins not indicated. Samples from diverse origins. Source: Modified from Ashri, A., in Sesame Biodiversity in Asia: Conservation, Evaluation and Improvement, Arora, R.K. and Riley, K.W., Eds., IPGRI, New Delhi, 1994, pp. 25–39; Ashri, A., Plant Breed. Rev., 16, 179–218, 1998. b

expanded second edition of the sesame descriptors list (IPGRI and NBPGR, 2004) will be of much assistance in the effort. The first germplasm collection of sesame was assembled in 1925 in India (Joshi, 1961). A larger collection was established at about the same period in the Soviet Union (Joshi, 1961; Weiss, 1971), as part of the worldwide collecting efforts of Vavilov and associates. In the 1940s, large collections of sesame accessions from many traditional growing areas were established in Venezuela and the U.S. Some of their improved successful varieties originated from introductions in these collections. There is an urgent need to collect and preserve the genetic variability of sesame, its evolutionary library, which evolved during its long history under diverse conditions, before it is too late. The need was stressed by expert consultations and in conferences sponsored by the FAO, IBPGR (now IPGRI), IDRC (International Development and Research Center, Canada), and FAO/IAEA (Anon., 1981b, 1985a; Ashri, 1987, 1990, 1994; Rana et al., 1994; Zhao, 1994). Ashri (1994) assembled between 1988 and 1992 a collection of 2990 cultivated sesame accessions from diverse geographical and cultural environments (Table 8.9). The seeds were multiplied under open pollination, taking care to retain the heterogeneity of the accessions. Samples of the multiplied seeds have been deposited by the author with the gene banks in South Korea (Rural Development Administration, Suwon) and Kenya (Kenya Agricultural Research Institute, Muguga, near Nairobi). These gene banks were chosen by IPGRI (formerly IBPGR), which has agreements with them to maintain the collections in perpetuity. Duplicate samples were shared by the author with the gene banks of India, China, and Australia, and some were also shared with researchers, e.g., in Turkey and Sri Lanka. In the 1990s, the collection and study of the sesame genetic resources was intensified in China (Zhang et al., 1993) and India. Bisht et al. (1999) reported that in the National Bureau of Plant Genetic Resources (NBPGR) in New Delhi, India, there were 6658 accessions — 4136 local and 2522 introduced (many of these sent there by the author). In China, about 3200 accessions were collected and studied from the 1950s to the late 1980s, while in the 1990s, an additional 4500 accessions were collected in China and 20 countries (Zhang et al., 2000). Other countries and some breeders (e.g., Venezuela, Sesaco) maintain smaller or larger collections. As a result of the efforts in the past two decades, the genetic resources of S. indicum of many areas have been sampled adequately, if not fully, although some traditional sesamegrowing areas are poorly represented. Collection efforts are needed in the more remote regions in Africa, Asia, and Central America. The recent collections made in Nigeria might close some gaps (Falusi and Salako, 2001).

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Table 8.10 Major Features of the Indian and Chinese Core Collections of Sesame and Characters Used For Core Grouping Local Cultivars China Core Element Material Used Accessions in whole collection used for core

India

4100

3129

Data used

Passport and 14 agromorphological characters

Passport and 19 agromorphological characters

Grouping procedure

Ecogeography to produce 8 groups

Combination of ecogeography and cluster analysis to produce 20 groups

Analysis method

Multivariate cluster analysis using Ward’s method

Multivariate cluster analysis using Ward’s method

Within-group selection

Random selection of accessions within identified clusters; number proportional to group size

Random selection of accessions; number proportional to product of log group size and its Shannon diversity index (SDI)

Size of core

Approximately 10%

Approximately 10%

Number of accessions

453

343

Common in both studies

Growing period Seed weight Branching habit Stem hairiness Capsules/axil

Locules/capsule Flower color Seed color

Specific to Chinese or Indian studies

Capsule dehiscence Waterlogging tolerance Fusarium wilt resistance Charcoal rot resistance Oil content Protein content

Days to flower Plant weight Capsules/plant Seeds/capsule Yield/plant Corolla hairiness Internode length Capsule hairiness Capsule length Incidence of phyllody

Characters

Source: Modified from Hodgkin, T. et al., in Core Collections for Today and Tomorrow, Johnson, R.C. and Hodgkin, T., Eds., IPGRI, Rome, 1999, pp. 74–81.

8.8.2

Core Collections

It is clear that in sesame, as in the germplasm collections of other crops, there are many duplicate and triplicate samples. Through seed exchanges, name changes, and mislabeling, similar or identical accessions have been given different identities. Also, when local collections are assembled, neighboring growers or villages may use different names for essentially the same landrace populations. In view of the duplications and the need to study the collections for the ranges of variability for desirable agronomical and other traits, establishment of core collections in sesame became imperative. This was given a high priority by the Asian Regional Workshop on Sesame Evaluation and Improvement in 1993 (Hodgkin, 1994). Subsequently, studies on formation of core collections were undertaken in both China (Zhang et al., 1999, 2000) and India (Bisht et al., 1999). In both countries, core collections development took 2 to 3 years, and although the two research teams used somewhat different characters, grouping strategies, and methods to select the accessions (Table 8.10), they derived core collections that consist of about 10% of the entire collections (Hodgkin et al., 1999). This could probably be true also for collections assembled mainly from introductions from many countries, such as in the U.S. In the above core collection studies, the analyses were based on visual scores and measurement data of morphological and agronomic traits. Molecular means to assess variability, which have been

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Table 8.11 Reported Resistance to Insect Pests and Diseases in Wild Sesamum Species Insect or Disease

Species with Resistance

Reference

Antigastra catalaunalis (shoot webber)

S. malabaricum S. alatum S. laciniatum S. radiatum

Ahuja et al., 2001 Thangavelu, 1994 Manisegaran et al., 2001

Aspondila sesame (capsule borer)

S. alatum

Ahuja et al., 2001

Oidiun spp. (powdery mildew)

S. malabaricum S. alatum

Thangavelu, 1994

Alternaria sesame (leaf blight)

S. alatum S. malabaricum S. radiatum

Lee and Lee, 1991 Shekharappa and Patil, 2001

Phytophthora blight Fusarium wilt Seedling blight

S. alatum S. radiatum

Lee and Lee, 1991

Phyllody-phytoplasma disease

S. malabaricum (as S. mulayanum) S. prostratum

Mehetre et al., 1993

Note: Reports on resistance in S. occidentale were not included, as it is a synonym of S. indicum (IPGRI and NBPGR, 2004).

introduced in recent years to sesame germplasm studies, will certainly facilitate more precise analyses. Several molecular markers have already been used to determine relationships in germplasm collections, as noted in Section 8.2. Such markers can strengthen and validate findings based on morphological and performance traits such as those used in, e.g., Brazil (Arriel et al., 2000) and China (Quenum et al., 2004) (Table 8.10). It is foreseen that intensified marker studies will enhance our knowledge of the genetic resources and aid in pedigree or backcross breeding by facilitating marker-assisted selection (MAS). 8.8.3

Wild Species

The poor level of knowledge on the distribution, cytogenetics, and interrelationships of the wild species Sesamum was noted above. The same is also true for their representation in the collections. Ashri (1998) noted that the wild species collections were very scant. The situation has not been improved since. There are still many gaps in the collections, especially in Africa (Van Rheenen, 1981d; Ashri, 1998), and in the assessment of the wild species. The FAO expert consultations (Anon., 1981b, 1985a) called for intensified collection activities, as did other conferences since then, but the effort is still very limited. Even for widespread wild species there are very few accessions, and they may be duplicates, sent from one collection to another. Much remains to be done to enrich our knowledge base and to benefit breeding. It is important to note that several wild Sesamum species proved resistant in initial studies (Table 8.11) to diseases and pests and to abiotic stresses (Kolte, 1985; Uzo, 1985; Prabakaran and Rangasamy, 1995). 8.8.4

Seed Dormancy and Storage

Generally, there is no seed dormancy in sesame. Dormancy that disappeared 6 months after the harvest was reported by Ashri and Palevitch (1979) in the Mexican cultivar Cola de Burrego. It could be broken by soaking the seeds in gibberellic acid (GA3) solutions. Dormancy was found in the variety ‘Hnanni 25/160’ from Myanmar (Beech and Imrie, 2001), which could be broken by heat treatments (10 min in 60°C or 20 min in 50˚C). Sesame seeds retain their viability and germinability well, probably due to the lignan antioxidants. Sesame breeders customarily store seeds for several years in cool rooms, and the do not lose their viability, provided seed moisture content is low. Sesame seed storage for long periods has received very little research attention. Van Rheenen (1981e) reported that sesame seeds retained their full germination after 10 years of storage at room temperature in Nigeria, in the presence of silica gel,

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while seeds stored in the deep freezer at –18 to –23°C germinated more slowly than seeds that were held at room temperature. In Sesaco’s experience, well-dried seeds that were held in sealed bottles germinated sufficiently well even after storage for 10 or 15 years in warehouses where the temperature exceeded 40°C during the summer (D.R. Langham, personal communication). Lee and Kang (1980b) reported that seed storage at –3°C and 30% relative humidity for 1 year gave the least reduction in germinability. Stanwood (1987) found that sesame seeds with 6% moisture or less survived 7 days storage at –196°C. Long-term seed storage in the gene banks is at –18°C and 6% seed moisture.

8.9 BREEDING OBJECTIVES 8.9.1

General

Sesame breeding objectives are similar to those of other seed-producing crops, especially oil crops, i.e., higher and stable yields, better income for the growers, resistance to diseases and pests, tolerance to abiotic stresses, and improved plant architecture and growth habits. Sesame growing conditions in the developing countries and in the more advanced ones are very different, thus leading to some identical and some divergent objectives. In the developing countries, the traditional small holders use manual labor and rarely apply inputs, if at all. In the U.S. and South Korea, reasonable to high inputs are applied. In the U.S. and Australia, mechanized handling from planting to combine harvesting is a must. This precondition may well be applicable in other producing areas in the near future. Thus, specific objectives vary with the technology, inputs levels, and farming systems — whether sesame is grown as a main crop or a second one, and in a pure stand or a mixed one. If it is to be combine harvested, the stand must be pure and the variety nonshattering; if it is grown traditionally, different stand combinations are possible, and shattering varieties are desired for manual threshing. Market demands and destinations affect sesame breeding objectives, as they would in other crops. Much of the sesame traded in the world is used for baked goods and other confectionery products. In these markets, seed size, shape, flavor, and seed coat color and texture are very critical. On the other hand, for the oil mills, many of the above-mentioned attributes are not too important as long as the oil content and composition (e.g., free fatty acid content) are satisfactory. The tahini mills are somewhere in between — both oil content and seed flavor are important for them. 8.9.2

Objectives

The following list of breeding objectives was derived from Ashri (1998), from recommendations of several conferences (Anon., 1981b, 1985a; Ashri, 1987; Van Zanten, 2001), and from recommendations of a wide array of researchers, especially Joshi (1961), Brar and Ahuja (1979), Mazzani (1983, 1999), Weiss (1983), Anon. (1985b), Sharma (1985, 1994), Thangavelu et al. (1985), Brigham (1985), Lee and Choi (1986), Ashri (1988, 1995, 1998), Kang (1994), Imrie (1995), Ahuja and Kalyan (2001), Beech and Imrie (2001), Langham (2001a), Manisegaran and Patil (2001), Van Zanten (2001), Day et al. (2002), Langham and Weimers (2002), and personal communications from M.R. Bennett, R.D. Brigham, C.W. Kang, D.R. Langham, and W. Wongyai. 1. Seedling traits: Fast, vigorous germination, rapid radicle growth and emergence Ability to germinate and withstand lower temperatures in the early stages of growth in temperate production areas Rapid early growth to overcome weeds and to give good stand establishment 2. Roots: Rapid root growth Deep taproot penetration with a well-distributed secondary root system

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3. Stems and branches: Uniculm or with few appressed basal branches if inputs medium to high Moderately branched at lower nodes, under low-input conditions, to compensate for poor stands Internodes short with corresponding adjustment of capsule angle Height varying with conditions and season, preferably under 1.50 m Lodging resistant 4. Leaves: Medium to broad at the base and narrow-lanceolate toward the apex, to permit maximum sunlight penetration Full leaf drop, abscission early of shaded bottom leaves, and complete at maturity 5. Growth habit: Determinate, with uniform and short flowering period and synchronous capsule ripening, or indeterminate (with prolonged ripening period), according to local needs and farming systems. Under low-input conditions, either may be desirable, depending on the conditions and the growing season. Under medium- or high-input conditions, the first would be preferable. Reduced biomass and improved harvest index. 6. Flowers: To start blooming 20 or 30 cm above the soil surface, under low input or mechanized harvesting, respectively One or three flowers and capsules per axil Genic-cytoplasmic male sterility 7. Capsules: To start forming about 20 to 30 cm above soil surface, as above Bicarpellate (four locules) if larger seed size is desired Long or short, as desired in area One or three per leaf axil; in the latter, rapid transition from one or two capsules per axil in the early stages to three Pointing upward, appressed Full seed set, without aborted ovules Effective seed retention (nonshattering), structure and seed release suitable for machine harvesting Shattering for traditional growing areas with manual threshing 8. Seeds: Uniform, well filled; shape, color, and size to satisfy market demands (large or medium–large for condiments, not too important for the oil and tahini processors) Desirable flavor (e.g., not bitter, “sweet”) Short-term (a few months) dormancy, appropriate for local cropping systems High oil content, about 50% Higher lignan content for longer shelf-life 9. Yield: High and stable seed yields of good quality under a wide range of environmental conditions Potential to respond with higher seed yields to applied inputs (e.g., fertilizers, irrigation) without undesirable effects (e.g., lodging) Show heterosis in hybrid combinations 10. Adaptation: Neutral daylength response and thermoinsensitivity Improved harvest index, more favorable reproductive to vegetative ratio Maturity uniform; early, medium, or late, according to local requirements Rapid dessication at physiological maturity Resistance or tolerance to abiotic stresses, as drought, water logging, salinity 11. Resistance/tolerance to insects, pests, and nematodes: Acherontia styx (sphingid moth) Antigastra catalaunalis (shoot webber, webworm, leaf webber) Bemisia tabaci (white fly); in the Americas, B type, synonym, B. argentifolii Heterodera cajani (nematode)

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12. Resistance/tolerance to diseases: Fungal Alternaria sesami (Kawamura), Mohanty & Behera Cercospora sesami Zimmerman Corynespora cassiicola (Berk & Curt.) Wei Fusarium oxysporum (Schelt.) f. sesami Jacz. Macrophomina phaseolina (Tassi) Goid. ssp. sesamica Oidium sp. & others Phytophthora parasitica (Dastur) var. sesami Prasad

Alternaria leaf spot White spot Corynespora Fusarium Charcoal rot Powdery mildew Phytophthora blight

Bacterial

8.9.3

Pseudomonas syringae Van Hall pv. sesami (Malkoff) Young. Dye & Wilkie Xanthomonas compestris (Pamel) Dowson pv. sesami (Sabet & Dowson) Dye

Bacterial leaf spot

Virus

Leaf curl

Phytoplasma (mycoplasma)

Phyllody

Bacterial blight

Additional Considerations

Most of the breeding objectives are self-evident. Achieving and combining all the desirable attributes in a given genotype by pedigree or backcross breeding would be extremely difficult. Still, the objectives listed above can serve as targets for progressive, cumulative breeding efforts. Hybrid varieties, if and when they become feasible, can have a significant impact (see below). More knowledge on the inheritance mechanisms of the desirable traits, their linkage relations, and association with molecular markers will indeed contribute much to varietal improvement. Increasing oil yield per unit area through higher yields appears to be a better option than raising oil content. As noted elsewhere in this chapter, the oil content at present is close to the biological limit. Furthermore, higher oil content may make the seeds even more damage-prone. Modifications of the fatty acid composition may create additional market niches, but they do not appear necessary at this time. Sesame oil is highly valued as it is. This is also the case with protein content and amino acid modifications. Higher lignan content could expand the use of sesame oil as an antioxidant additive to oil-containing products, such as peanut butter (where it has been used for some time). However, it should be verified that it causes no undesirable flavor or other side effects. In this connection, it is noteworthy that sesame oil and sesame flour improve the flavor and functional properties of many foods (D.R. Langham, personal communication). Much research is needed to study and elucidate the genetic controls of sesame aroma, flavor, and sweetness. In commercial processing (e.g., in Japan), the evaluation of samples for these criteria involves assessment of several components by tasters. The knowledge and the tools that will facilitate screening of large segregating populations are not yet available to the breeders. Breeding for resistance or tolerance to the insect pests and diseases is very important in all cropping systems, especially so in the developing countries, where insecticides or fungicides are rarely applied. There are reports in the literature on genetic variation for resistance or tolerance to all the insects and pests listed above, within the cultivated species or the wild species, or both. Phyllody, which is known in other crops, is a very destructive disease in sesame. It is caused by a phytoplasma (mycoplasma-like organism (MLO)) that is transmitted by leaf hoppers (Orosius albicinctus Distant). In the infected plants, the floral buds revert to vegetative and proliferation is induced. Nakashima et al. (1999) made hybridization assays using a chromosomal DNA segment

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of sesame phytoplasma as a DNA probe. They concluded that the phyllody symptoms may be due to an inhibition of the morphogenesis of the floral organs by metabolites synthesized by the phytoplasma. Still, breeding for tolerance/resistance to phyllody would be difficult. Screening would have to depend on field conditions, vector population buildup, and MLO infestation levels. The development of methods for inoculation and rapid molecular disease identification, as well as screening approaches, is urgently required for breeding for resistance to this disease. Another destructive disease is charcoal rot (Macrophomina phaseolina ssp. sesamica). Since it is not specific and attacks other crops as well, breeding for resistance has been and will continue to be very difficult.

8.10 BREEDING METHODS 8.10.1 General It is generally accepted that especially in developing countries, where sesame is usually grown, breeding improved cultivars is often the most promising avenue to achieve better crop production. Since inputs are limited or rare, better adaptation and improved tolerance to abiotic stress, diseases, and pests can make significant contributions to yield performance and stability. Sesame breeding, though, has lagged behind that of other crops. In many producing areas the crop is still in the early phases of breeding, i.e., relying more on landraces or elections from landraces. This is the situation because research funds in the developing countries where sesame is grown are scarce and sustained long-term programs are lacking. The potential for sesame improvement through breeding is well illustrated by the remarkable achievements of Sesaco Corporation in the U.S. (Langham and Weimers, 2002) and breeding programs in Venezuela (Mazzani, 1983, 1999; ava and Layrisse, 1990), South Korea (Lee and Choi, 1985; Kang, 1994), India (Joshi, 1961; Sharma, 1985, 1994), and China (Zhao, 1994). The breeding approaches used to develop 36 released varieties in China, 99 in India, 30 in South Korea, and 30 in Venezuela were summarized by Ashri (1998). The methods employed in breeding these 195 varieties follow: Introduction (direct) Selection from introductions Selection from local landraces Pedigree Backcross Induced mutations (6 from S. Korea) Methods not known

2 14 69 74 4 11 21

Another illustration of the role of the different breeding methods can be drawn from the progress made in India before and after 1986 (Sharma, 1994), summarized in Table 8.12. Of the 28 varieties released by Sesaco from 1982 until 2005 in the U.S., 7 were selected from introductions, 3 were selected from progenies of outcrosses, and 18 were bred using the pedigree method (D.R. Langham, personal communication). It can be seen that sesame breeding has passed fairly late from the initial phases, typified by selection from local materials, into combination breeding through hybridization. The role of the different methods in sesame breeding is discussed in detail below. 8.10.2 Introduction In the usual progression of breeding, introduction follows soon after initiation of improvement of local landraces. Its objectives are to identify a genotype or genotypes that will be well adapted

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Table 8.12 Number of Released Improved Cultivars Developed in India by Different Breeding Methods, during Two Periods Number of Varieties Method

Up to 1986

1986–1993

Landraces Selection from landraces Hybridization Induced mutation Total

16 26 12 1 55

— 2 6 2 10

to the local conditions and surpass the best local materials, thus facilitating, with or without light selection, the direct release of superior improved varieties in a short time. The second main objective is to identify genotypes containing certain desirable traits (e.g., disease resistance) that will be hybridized with other varieties in order to combine their desirable traits. Introduction in sesame has been effective in achieving both goals. At least four released cultivars in Thailand were pure line selections from introductions: ‘Roi-et 1’ from the Japanese line W-53, ‘Maha Sarakham-60’ from India’s T-85, and ‘Ubon Ratchathani-1’ and ‘Khon Kaen Univ. 3’ from ‘Hnanni 25/160’ of Burma, now Myanmar (Maneekao, 1994; Beech and Imrie, 2001). In Australia, the Mexican cultivar Yori 77 proved adapted in the Northern Territory, and the improved cultivar Edith was selected from it (Bennett, personal communication; Anon., 1996; Beech and Imrie, 2001). The Venezuelan cultivar Morada, selected there from an introduction from Zaire, proved well adapted later on in Tanzania (Mazzani, 1983). Improved varieties were selected in Venezuela from introductions that originated in China, Zaire, Nicaragua, Colombia, and Ethiopia (Nava and Layrisse, 1990). Five of six improved sesame cultivars grown in Ethiopia originated as introductions from India, Uganda, Sudan, and Egypt (Wolde-Mariam, 1985). Two of three outstanding cultivars in Ethiopia were introductions (Wakjira et al., 1993). ‘Early Russian’ introduced from Texas to South Korea was released directly. Its success initiated the high-yielding, intensive, high-input cultivation of sesame there. In the major producing countries, China and India, introductions were not released directly as cultivars. Obviously, these two countries have a tremendous range of growing conditions and genetic variability (see germplasm), which satisfied their needs. Introductions were employed extensively in hybridization projects, as will be described below. The wide range shown above of sources of introduced varieties that were successful demonstrates that it is difficult to recommend promising geographical sources of introductions on a national basis. As noted above, Ashri et al. (1991) and Ashri (1994) showed that oil contents and protein contents in South Korea (Suwon) cannot predict performance for these traits in Israel (Rehovot) and vice versa (Table 8.7 and Figure 8.4). Since introductions can be very useful in breeding programs, it appears advisable to introduce promising and other cultivars and landraces from diverse geographical areas. Introductions from regions with fairly similar photoperiods would be more likely to be adapted to the conditions of the receiving region. However, in searching for sources of specific desirable traits, materials should be obtained from a wide range of environments. In recent years it has become increasingly difficult to expect introductions to yield genetic progress directly. This is partly due to the higher levels of performance required to exceed available cultivars and partly due to restrictions that have been placed in recent years on the free exchange of genetic materials, in sesame, as in other crops. 8.10.3 Selection The small holders in the developing countries who grow sesame usually save some of their seeds for planting, season after season. Over the millennia, conscious and unconscious selection has been practiced by the farmers, as they do to this day. Thus, sesame germplasm collections offer a rich lode of genetic variability for breeding (Table 8.13).

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Table 8.13 Variability for Vegetative and Reproductive Traits of Sesame Lines in Sesaco Nurseries in Uvalde, TX Trait

Range

Plant height, cm Height of first capsule, cm Capsule zone height, cm Branches per plant, number Height of first branch, cm Nodes on main stem, number Internode length at middle of capsule zone, cm Mean internode length for whole capsule zone, cm Leaf size (5th node from base):a Blade length, cm Blade width, cm Petiole length, cm Days to 50% flowers, number Days to 90% flower termination, number Flowering period length, number of days Days to physiological maturity, number Days to first dry capsules, number Capsules per leaf axil, number Locules per capsule, number Capsule length, cm Seed loss (shattering) at dryness, % Seed weight per capsule, g Seeds per capsule

67–245 25–150 18–120 0–20 2–135 4–65 1–11 1.9–8.0 11.7–31.0 2.1–23.6 4.5–20.0 28–98 51–133 10–98 70–167 74–180 1–7 2/4/6 or 8 1.3–7.0 0–100 0.08–0.48 30–120

a

Generally one of the largest leaves on the plant. Source: Modified from Langham, D.R. and Weimers, T., in Trends in New Crops and New Uses, Janick, J. and Whipkey, A., Eds., ASHS, Atlanta, GA, 2002, pp. 157–173.

Both mass- and single-plant selection have been quite important in deriving improved cultivars from local and introduced materials. Mass selection can be very useful in many regions, because the local landraces are often very heterogeneous, containing productive as well as unproductive genotypes. In Myanmar, which has two growing seasons, the author saw fields with mixed populations that were heterogeneous for photoperiod response, with many unproductive plants. This could be the case in India, which has three growing seasons. Mass selection aimed initially at the elimination of the less desirable plants can lead quickly to better adaptation and higher yields. Single-plant selection has also produced successful cultivars. However, there is greater scope for this approach when new variability is created by controlled crosses. 8.10.4 Hybridization Hybridization to create new and desired genetic variation through controlled crosses is usually a more advanced phase in breeding. In sesame it was adopted later than in many other crops, even though several authors (e.g., Kinman and Martinm, 1954; Ashri, 1981) have pointed out that selection within local materials that has been ongoing for a long time, often under low-input conditions, has reached its limits. Thus, much genetic variability for yield within the regions has been exhausted and breakthroughs in productivity will have to come from controlled crosses designed to create new and wider variability, with the potential to respond to higher inputs. At the same time, breeders should also be on the alert for genetic variability resulting from outcrosses. The main hybridization approach in sesame has been the pedigree method; less common are backcross, composite crosses with bulk populations and selection, and population improvement with or without male sterility (Kinman and Martin, 1954; Weiss, 1971; Anon., 1981b, 1985a; Mazzani, 1983, 1999; Lee and Choi, 1985; Ashri, 1988, 1998; Nava and Layrisse, 1990; Kang, 1994; Sharma, 1994; Langham and Weimers, 2002).

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The outstanding achievement of Sesaco Corporation in the U.S., in breeding varieties with progressively better seed retention that can be combinen harvested directly, is the result of a massive crossing and selection effort using mainly the pedigree method. Since 1978, when Sesaco was established, it assembled 2738 introductions from 66 countries and, using them, made 33,545 hybrid combinations to develop varieties (Langham and Weimers, 2002). Generally, the crosses are between two genotypes (cultivars or introductions or germplasm accessions) chosen for their expected contributions to the offspring, e.g., ‘Aussi Gold’ (Anon., 1994; Beech and Imrie, 2001). Sometimes, selected promising plants from advanced generations are hybridized with other genotypes. The segregating offspring are usually managed along the procedures of the pedigree method. More complex crossing schemes are also utilized. Crosses of F1 ∞ F1 and three-way crosses were best for the needs of the Sesaco Corporation and gave more rapid progress when followed by pedigree selection (D.R. Langham, personal communication). Modifications of the classical pedigree method, such as single-seed descent, have been initiated in sesame, but it is premature to make conclusive recommendations. Pathirana (1995) compared breeding progress in five crosses using classical pedigree, bulk, single-seed descent, and modified single-seed descent. He concluded that the bulk method, which is less expensive, was as successful as the other methods. Srinivas et al. (1992) compared the effectiveness of the bulk and singlecapsule descent procedures using six hybrid populations. They found the bulk method to be superior to the single-capsule descent in four crosses, but less successful in two crosses, where the parents were closely related. In this case, however, it could have been less effective because the genetic variability generated by hybridizing two closely related parents was limited. Early-generation testing was studied by Pathirana (1995) and Ranganatha et al. (1994), who concluded that it was ineffective. The composite cross approach was used in sesame, at times with some modifications. D.G. Langham conceived a scheme for crossing indehiscent lines with dehiscent ones, then to cross the F1s among themselves and with additional lines, and repeatedly make more cross combinations to generate sufficiently large populations for selection in several locations (Langham, 2001). From this breeding effort, carried out with Kinman and Martin (1954), the indehiscent varieties ‘Palmetto’ and ‘Rio’ were obtained (Langham, 2001a). Ashri (1981, 1994, 1995) created a highly heterogeneous population by bulking F2 seeds from crosses of a wide range of cultivars with the determinate mutant dt45. Seeds of advanced generations of this population were shared with researchers in many sesame-growing countries. Promising advanced lines were developed by crosses of local varieties with the dt45 determinate plants in various countries. In South Korea, the released variety ‘Pungsan’ has dt45 lines in its parentage (Kang et al., 1993; Kang, 1997). In Thailand, dt45 lines were among the parents of the nonshattering variety ‘C plus 1’ (Wongyai, 1997; Wongyai and Chowchong, 2003a; Wongyai, personal communication). The backcross method has had a limited impact to date. It has been used to transfer desirable traits such as indehiscence and genic male sterility into adapted cultivars. Its use will no doubt expand as better and more productive cultivars are developed and donors of specific useful loci are identified. Sesaco uses a modification of this approach at times. Recurrent selection for high yield gave good progress in Venezuela (Laurentin et al., 2000). Reciprocal recurrent crossing, which exploits both additive and nonadditive gene effects, was recommended by Solanki and Gupta (2001). 8.10.5 Induced Mutations There is a wide range of variability in the germplasm reservoirs of sesame. However, certain highly desirable traits have not been found, or were only rarely encountered, in the collections of sesame, e.g., good seed retention, modified plant architecture, improved harvest index, male sterility, and resistance to some diseases and pests. For this reason, induction of mutations in sesame was adopted already in the 1950s by Kobayashi (1958). Many studies were conducted since then with different mutagens, radiations, and chemicals. Three expert consultations (Anon., 1981b, 1985a;

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Figure 8.5

GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT

Derived induced determinate mutants (Dt45/Dt45): (A) branched; (B) uniculm. Note apical clusters of capsules.

Ashri, 1987) and other bodies recommended that induction of mutations be employed to create the genetic variability needed for breeding improved varieties. The joint FAO/IAEA Division, Vienna, sponsored for several years in the 1990s coordinated research projects (CRPs) on induced mutations in sesame in a dozen sesame-growing countries. The outcome of these projects, which yielded some released varieties and promising genotypes, was summarized by Van Zanten (2001). Ashri (1998) reviewed in detail the achievements of the induced mutations approach in sesame and the procedures employed. Therefore, in the ensuing discussion, some aspects will be highlighted while others will not be. By 1998, 14 sesame varieties derived from induced mutations were released officially in five countries: Egypt, 2; India, 3; Iraq, 3; South Korea, 5; and Sri Lanka, 1 (Ashri, 1998, 2001). Some of these varieties were derived directly from the mutants, multiplying their seeds, while others were selected from progenies of mutants ∞ varieties hybrids. Of those, several were adopted widely in their areas. The induced disease-resistant mutant variety (Lee et al., 1985a) ‘Ahnsangae’ was grown in 1996 on 30% of the South Korean sesame area and is still grown there. In China, ‘Yuzhi 11’, developed from a single mutant of ‘Yuzhi 4’, proved resistant to Fusarium oxysporum f. sp. sesami, Macrophomina phaseolina, and Cercospora sesami (Wei et al., 1999). In the CRPs noted above, 144 mutant lines with agronomically useful characters were obtained in nine countries and were incorporated into breeding programs (Van Zanten, 2001). Diverse mutant lines were obtained, among them the following of special interest: capsule mutations, 76; plant architecture, 60; leaves, 20; and seeds, 10 (Van Zanten, 2001). Especially noteworthy are the first reported mutation for determinate habit, dt45 (Ashri, 1981, 1988, 1998), and mutations for determinate habit and shatter resistance (Wongyai et al., 2001); determinate habit, nonshattering, genic male sterility, and resistance to Phytophthora (Kang, 2001); genic male sterility (Rangaswamy and Rathinam, 1982; Li et al., 2001); closed-capsule, determinate, and wilt-tolerant mutants (Cagirgan, 2001). The monogenic, recessive, determinate dt45 mutation was induced by irradiating with gamma rays (500 Grey) dry seeds of the Israeli cultivar No. 45, shown in Figure 8.2 (Ashri, 1998). The mutation shortens the flowering period markedly and produces shorter unique plants: the internodes are telescoped and five to seven capsules are clustered at the tips of the main stem and branches (Figure 8.5). In these plants there are often also modifications of the apical flowers and capsules. The seeds are oval and large, as in the source variety (Ashri, 1988; Brigham, 1985; Brigham and Khan, 1989). The trait has been bred into various genetic backgrounds, and both branched and unbranched (uniculm) lines are studied (Ashri, 1998).

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The mutagens used have been primarily gamma rays (150 to 800 Grey) to dry seeds and ethylmethane sulfonate (EMS) — soaking the seeds in 0.4 to 1.0% v/v solutions with a phosphate buffer (pH = 7) for 2 to 4 h with occasional shaking, after prior presoaking in water at 4°C for 24 h (Van Zanten, 2001). After soaking in the mutagen solution, the seeds should be washed for 4 h or more in running water and sown immediately. Other mutagens that have been used are sodium azide and fast neutrons (Kang, 2001), diethyl sulfate (Ashri, 1982; Sengupta and Datta, 2003a), N-methyl-N-nitroguanidine (Kar and Swain, 2002), and nitrous acid, hydroxylamine, dimethyl sulfoxide, and hydrogen peroxide (Sengupta and Datta, 2003a). Nearly all researchers applied one mutagen in a treatment, but combinations of mutagens were also used, e.g., gamma irradiation followed by soaking in an EMS solution (Kar and Swain, 2002). As a rule, the treated lines/varieties were homozygous and uniform, in order to ascertain the source of the variation (mutation, outcross, or contamination). Treatments of F1 hybrids were tried and were quite efficient in producing transgressive variation for yield components (Govindarasu et al., 2000). Sesame seeds are less sensitive physiologically to the various mutagenic agents; therefore, higher doses can be used (Ashri, 1982). Genotypic differences in sensitivity to EMS and to gamma rays were reported for diverse genetic materials (Ashri, 1982; Kamala and Sasikala, 1985; Layrisse et al., 1992; Pathirana, 1992; Cagirgan, 2001). In the experience of the participants in the CRPs (Van Zanten, 2001) and in light of other findings (Ashri, 1998) the optimal management for the M1, M2, and M3 generations can be summarized as follows: M1: A large number of plants should be grown, taking care to minimize cross-pollination. The seeds can be harvested individually by taking ca. five capsules from each plant, or in bulk. M2: The well-spaced plants can be grown in progeny rows (30 to 50 progenies per row) or in bulk. They should be examined for mutants repeatedly throughout the season. Promising mutant plants should be selfed when possible. Bulk planting may suffice for recovery of desirable mutants, but better genetic information on the mutations’ nature and frequency can be obtained by planting in progeny rows. M3: Grow progeny rows from promising mutants identified in M2, and also grow bulk populations to screen for quantitative trait mutants. In all stages,sufficiently large populations are required. Promising mutants can be tested further and released as new, improved varieties. Also, they can be hybridized with other materials, thus deriving superior varieties by combining the desirable traits of the parents by the pedigree or backcross methods. In some cases, F1 heterosis was noted in mutant ∞ mutant F1 hybrids (Cagirgan, 2001). The mutations can be expected to cover a wide range of desired traits, as in other crops. Especially worthy for sesame are high-yielding mutants with full seed retention and mutants for altered growth habit, such as dt45 (Ashri, 1998), higher harvest index, genic-cytoplasmic male sterility, and resistance to the mycoplasma (phytoplasma) causing phyllody and to other diseases. Some successes have been achieved, but many are still needed. Mutations for oil contents or fatty acid composition, or for other seed quality components (e.g., flavor or lignans), can be expected, but their identification would depend on the availability of efficient screening procedures. Hopefully, before too long, sesame will receive more attention in molecular manipulations that will facilitate more effective, targeted mutation induction. 8.10.6 Hybrid Cultivars As noted above, heterosis — high-yield increments (50% and more) — was obtained in some F1 hybrid combinations. The prospect of hybrid cultivars for sesame is therefore very attractive: it could produce the yield breakthrough that is so needed, since progress in increasing the yields of the improved pure line cultivars has been slow.

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A genic-cytoplasmic male sterility discovered in India (see above) may usher in the hybrid approach, once it is tested and verified and good parental hybrid combinations are identified. High levels of cross-pollination of male-sterile flowers by honey bees (Sarker, 2004) and other insects (Yermanos, 1980) and Apis spp. (Sachdeva et al., 2003) can be achieved easily. Even a cross-pollination rate of 60 to 70% in the seed-producing blocks would suffice (as was the case in sunflower). As reviewed by Ashri (1998), there were attempts in China and India to produce F1 hybrid seeds by hand pollination of genic male-sterile plants, or even with hand emasculation of the intended female parent, but these approaches were not adopted. The labor costs of hand emasculation in India were too high (Karuppaiyan and Ramasamy, 2000). Another suggestion was to produce heterotic F1 hybrids by hand pollination, obtain the F2 seeds, and sow them in the farmers’ fields. This approach would have led to reduced vigor and heterogeneous populations, and therefore was not adopted. The use of the split-corolla mutant (which reduces self-pollination) as the maternal line is an alternate approach for the production of hybrid seeds. If the crossing blocks are in isolated areas without other flowering plants around them, it could be expected that insect pollinators could produce a high percentage of outcrossed progenies. As noted earlier, even if the cross-pollination rate is 60 to 70%, the yield increment can be considerable, provided parents with high combining abilities are used. An advantage of this system is that the split-corolla female parent can be maintained easily by being grown in isolated self-pollination plots. Unfortunately, bees do not visit the split-corolla flowers because they cannot land on them (D.R. Langham, personal communication).

8.11 IN VITRO TECHNIQUES 8.11.1 Tissue Culture Very limited research on sesame tissue culture was reported in the last few years. Micropropagation in vitro, the induction of sprouting in cotyledon shoots of three varieties, was investigated in Brazil (Batista et al., 2001). They found that solid Murashige and Skoog (MS) media with low NAA concentrations gave the best results. Previously, micropropagation from shoot tip cultures had been reported by George et al. (1987), Lee et al. (1985b), and Ram et al. (1990). Only root formation was obtained from hypocotyl and cotyledon explants (Batra et al., 1991). Kim (2001) studied callus induction and shoot regeneration from sesame hypocotyls and cotyledons. He found that shoot regeneration from them was highest in MS medium containing 3.0 mg benzyladenine (BA)/l, 0.5 mg NAA/l, 30 g sucrose/l, and 8 g agar/l with AgNO3 at 5, 10, and 15 μM, while 2,4-D treatments were not effective for callus induction and shoot regeneration. Ram et al. (1990) regenerated plants from shoot apical meristems and hypocotyl segments. Somatic embryos were produced directly from zygotic embryos in culture and from callus cultures (at a low frequency) originating from cotyledon and hypocotyl segments. Embryo-like structures were obtained by George et al. (1987) from callus tissue. Somatic embryos were induced from hypocotyl-derived calli; of the seven auxins and four cytokinins tested, 2,4-D was the most effective (Jeya Mary and Jayabalan, 1997). There are no reports assessing somaclonal variation in sesame, although Ram et al. (1990) noted that some regenerants deviated from the normal source phenotype. 8.11.2 Embryo Culture Embryo culture procedures were suggested and developed primarily in order to rescue interspecific F1 hybrids (Lee et al., 1991b; Shi, 1993; Tarihal et al., 2003), thereby facilitating genetic exchange between the more distant wild species and the cultivated ones. Qu et al. (1994) have described a successful three-stage embryo culture. The embryos of S. schinzianum × S. indicum

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were cultured 9 to 13 days after pollination on MS medium containing Nitsch organic salts, 0.2 mg BA, and 30 mg GA/l and 5% sucrose. The embryos that germinated were transferred to MS medium supplemented with 0.2 mg BA/l for 2 weeks, and then to MS medium supplemented with 0.5 mg NAA/l. Ram et al. (1990) developed an embryo culture protocol for the cultivated species. Recent reports have not been found. 8.11.3 Protoplast Culture Very few reports were published on protoplast isolation culture and fusion in sesame (Shoji et al., 1988; Bapat et al., 1989; Dhingra and Batra, 1990). Also, selective media or suitable experimental lines to isolate the fused cells are lacking. Shoji et al. (1988) determined the proportion of fused protoplasts by measuring the relative amounts of DNA. After 5 weeks of culturing, 7.4% of the protoplasts were tetraploid, 4.2% aneuploid, and 84.4% diploid. More recent reports have not been found. 8.11.4 Anther Culture Studies on anther culture in order to obtain haploid plants were reported by Ranaweera and Pathirana (1992) and Kang (1994). However, haploid plants have not been produced so far. As above, more recent reports have not been found.

8.12 LOOKING AHEAD Sesame, a very important crop for small holders, is grown in many countries, mainly in Africa, Asia, and Latin America. Due to low yields, seed shattering, and high manual labor requirements, its cultivated areas may be markedly reduced in the next decades. It is not only that the growers will have fewer crop options then, but that in some areas other options are very limited. Sesame research has suffered for a long time from lack of funds, lack of continuity, and lack of involvement of major international research organizations. A collaborative program to invigorate sesame research should be undertaken by the countries where it is grown and by relevant international organizations. It would be most appropriate for the major producers (e.g., China, India, Sudan) and consumers (e.g., Japan) to develop research programs that would also include coordination with a major international research center such as ICRISAT in India. These programs should lead to the technological advancement that will make sesame a more successful crop, more remunerative, higher yielding, less risky, and a better competitor with other crops. Over the years, sesame research and breeding with public funds has been successful at times, but it has also suffered from lack of continuity and funding difficulties. Therefore, it is to be hoped that commercial companies will follow the example of Sesaco Corporation and go into enhanced long-term breeding efforts in the major growing areas. This would help ascertain that sesame will remain an important oil crop in the next decades. Genetic and breeding research can make major contributions toward the above goals, as has been the case in other crops. Many breeding objectives were outlined above (Section 8.9.2). Very important high-priority breeding objectives are full seed retention combined with easy release of the seeds in mechanized threshing, improved harvest index, modified plant architecture, and development of genic-cytoplasmic male sterility to facilitate production of hybrid varieties. More emphasis should be given in the future to the quality of the seeds, including flavor and taste and lignan content. Significant advances can be obtained by intensified studies of the germplasm resources of sesame, emphasizing also the Sesamum wild species. The latter may furnish the much needed genes for resistance to biotic and abiotic stresses, especially to the very destructive phyllody disease caused by phytoplasma.

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Acceleration of the achievement of the desired objectives will require intensified inheritance studies of various traits, linkage and chromosome studies, and molecular investigations that will make DNA markers available to the breeders. Breeding successful varieties, accompanied by suitable agronomic research, will ensure that sesame retains its place in the relevant cropping systems, to the benefit of the small holders in the less developed countries, and potentially also contribute to its adoption in areas with higher inputs. ACKNOWLEDGMENTS I thank first and foremost the many sesame researchers who shared with me their findings and discussed with me their ideas. I am very grateful to D.F. Beech and M.R. Bennett for reviewing the manuscript and making helpful comments, and especially to D.R. Langham, who made many valuable comments. My deep appreciation is extended to Mrs. Nili Ben-Yehezkel for her patience and expert help with the manuscript. Last, but not least, I am very grateful to my wife, Lydia, for her editorial help and encouragement. REFERENCES Agne, P.S.E., F. Rance, and E. Bidat. 2003. Sesame seed allergy. Rev. Franc. Allergol. Immunol. Clin. 43: 507–516. Ahuja, D.B. and R.K. Kalyan. 2001. Field screening of genotypes of sesame against leaf webber/capsule borer, Antigastra catalaunalis Dup., gallfly, Asphondylia sesami Felt., and mite, Polyphagotarsonemus latus (Banks). Pest Manage. Econ. Zool. 9: 5–9. Alam, S., A.K. Biswas, and A.B. Mandal. 1999. Character application and path coefficient analysis in sesame (Sesamum indicum L.). Environ. Ecol. 17: 283–287. Anon. 1981a. Descriptors for Sesame, AGP:IBPGR/80/71. IBPGR Secretariat, Rome. Anon. 1981b. Conclusions and recommendations. In Sesame: Status and Improvement, Paper 29, Ashri, A., Ed. FAO Plant Production and Protection, Rome, pp. 192–195. Anon. 1985a. Conclusions and recommendations. In Sesame and Safflower: Status and Potential, Paper 66, Ashri, A. Ed. FAO Plant Production and Protection, Rome, pp. 218–220. Anon. 1985b. Summary of recommendations of the oil crops network. In Oil Crops: Sesame and Safflower, IDRC-MR105e, Omran, A., Ed. IDRC, Ottawa, pp. 249–251. Anon. 1994. Aussi Gold. Plant Var. J. (Australia), 7: 14–15. Anon. 1996. Edith. Plant Var. J. (Australia) 9: 64. Anon. 2005. Notices to readers. Sesame Safflower Newsl. 20: VII. Arriel, N.H.C. et al. 1999a. Genotype evaluation and genetic parameter estimates of Sesamum in the semiarid area of Northeast of Brazil. Revista Oleaginosas Fibrosas 3: 165–173 (in Portuguese). Arriel, N.H.C. et al. 1999b. Genetic and phenotypic correlations and heritability in sesamum (Sesamum indicum L.) genotypes. Revista Oleaginosas Fibrosas 3: 175–180 (in Portuguese). Arriel, N.H.C. et al. 2000. Evaluation of quantitative descriptors in preliminary characterization of sesame (Sesamum indicum L.) germplasm. Revista Oleaginosas Fibrosas 4: 45–54 (in Portuguese). Arriel, N.H.C. et al. 2002. Comparative analysis of four genomic DNA extraction protocols in sesame. Revista Oleaginosas Fibrosas 6: 525–535 (in Portuguese). Arulmozhi, N., S. Santha, and S.E.N. Mohammed. 2001. Correlation and path co-efficient analysis in sesame. J. Ecobiol. 13: 229–232. Ashri, A. 1981. Increased genetic variability for sesame improvement by hybridization and induced mutations. In Sesame: Status and Improvement, Paper 29, Ashri, A., Ed. FAO Plant Production and Protection, Rome, pp. 141–145. Ashri, A. 1982. Status of breeding and prospects for mutation breeding in peanuts, sesame and castor beans. In Improvement of Oil-Seed and Industrial Crops by Induced Mutations. IAEA, Vienna, pp. 65–80. Ashri, A. 1985. Sesame (Sesamum indicum L.). In Handbook of Flowering, Vol. IV, Halevy, A.H., Ed. CRC Press, Boca Raton, FL, pp. 309–312.

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Index A Afghanistan, cottonseed production in, 92 Aflatoxin contamination, 5, 10, 73–75 Africa cottonseed production in, 91 sesame production in, 233, 264 sesame seed trade, 235–236 Agelaius phoeniceus, 145 Agrobacterium, 41–42, 68 Agrobacterium-mediated transfection, 263 Agrobacterium tumefaciens, 68, 187, 218, 263 Albugo, 142 Albugo candida, 214 Allotetraploids, 94 Alternaria, 178, 183, 187 Alternaria brassicae, 214 Alternaria carthami, 183 Alternaria helianthi, 117, 143 Ambinervosae, 56, 62 Angustifolius, 134 Antigastra catalaunalis, 266 Antinutritional elements breeding oilseeds for, 9–10 Apis, 276 Apomixis, Carthamus tinctorius, 188 Arabidopsis, 8, 208–210, 221 Arabidopsis thaliana, 8, 68, 208–210 Arachis, 51–88 aflatoxin contamination, resistance to, 73–75 bacterial wilt, resistance to, 72 breeding progress for important traits, 69–76 China, mulch conditions, 52 Consultative Group on International Agricultural Research (CGIAR), 59 Convention on Biological Diversity, 59 core collections, 59–61 crop improvement, 63–76 cytogenetics, 62–63 dissemination, 54–55 distribution, 54 drought tolerance, 75 evaluation, 59–61 foliar diseases, resistance to, 69–71 future developments, 76–77 genetic resources, 54–61 genetic transformation, 68–69 genomes, 62–63 germplasm

collection, 57–59 conservation, 57–59 ICRISAT, 59 improved oil quality, 75–76 International Board for Plant Genetic Resources, 59 International Crops Research Institute for Semiarid Tropics, 53, 58 interspecific hybridization, 64–66 marker-assisted selection, 66–68 mutation breeding, 66 National Center of Genetic Resources, 58 nematodes, resistance to, 73 oil content, 75–76 Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, 58 origin, 54 soilborne fungi diseases, resistance to, 71–72 taxonomy, 55–56 testa color, groundnut cultivars, diversity, 57 traditional breeding methods, 63–64 wild relatives, 57–59 wild species, 59 Arachis batizocoi, 62, 66–67, 70, 73 Arachis benthamii, 56 Arachis cardenasii, 63, 65–67, 73 Arachis correntina, 65–66 Arachis cruziana, 62 Arachis dardani, 56 Arachis decora, 62 Arachis diogoi, 66–67, 73 Arachis doiogoi, 73 Arachis duranensis, 67, 70 Arachis glabrata, 56, 58–59, 64 plants, flowers of, 59 wild species, plants, flowers of, 59 Arachis glandulifera, 62 Arachis guaranitica, 56, 58 Arachis hoehnei, 62 Arachis hypogaea, 51, 54, 56–58, 61–69, 71–74, 77 Arachis ipaensis, 63, 67–68 Arachis ipnaensis, 62 Arachis magna, 62 Arachis marginata, 58 Arachis monticola, 56, 62–64, 68 Arachis palustris, 62 Arachis pintoi, 58 Arachis praecox, 62 Arachis prostrata, 56 Arachis repens, 56, 58 291

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Arachis rigonii, 56 Arachis stenosperma, 58, 67 Arachis triseminata, 56 Arachis tuberosa, 58 Arachis villosa, 63, 65, 67–68 Arachis villosulicarpa, 58 Arachis williamsii, 62 Archer Daniels Midland Company, Decatur, IL, 6 Argentina cottonseed production, 91 soybean yield, 14 Arthritis, prevention, 5 Asia cottonseed production, 91–92 sesame production, 233 sesame seed trade, 235–236 Asian Vegetable Research and Development Center, 6 Aspergillus, 53, 74 groundnut cultivars resistant to, 10 Aspergillus flavus, 74, 76, 97 Aspergillus infection, 5 Aspis mellifera, 105 Aspondila sesame, 266 Atmospheric nitrogen, 2 Atractylis, 176 Atrorubens, 111 Australia cottonseed production, 92 sesame seed trade, 236 AVRDC. See Asian Vegetable Research and Development Center Azerbaijan, cottonseed production, 92

B B carinata, 202 Babassu nut oil, 3 Bacillus thuringensis, 69 Bacterial wilt, 72 Bangladesh, sesame production, 233 Beef, fatty acid composition, 4 Benin, cottonseed production, 91 Bison bison, 110 Bradyrhizobium, 37 Bradyrhizobium japonicum, 17 Brassica, 2–3, 8–11, 195–230 abiotic stress, 214–216 biotic constraints, resistance to, 214–216 botany, 199 breeding for end use, 212–213 breeding methods, 210–212 classical cytogenetics, 202–203 crop use, 197–199 cultivar development, 210–211 cytogenetic maps, 204–205 cytogenetics, 202–204 description, 197–199 diploid species, genomic relationships among, 202 doubled haploids, generation of, 217–218 fungal disease, 214–215 genetic mapping, 204–210 genetic maps, 205–207

genetic modification, 218–219 genetic transformation, 217–219 germplasm enhancement, conventional breeding, 210–216 germplasm resources, 199–202 hybrid breeding, 211–212 intergeneric hybridization, 219–221 interspecific hybridization, 219–221 meal quality, 212 molecular cytogenetics, 203–204 molecular genetic variation, 216–217 oil content, 213 pests, 215 physical maps, 207–209 primary gene pool, 201 protein content, 212 quality of oil, 213 reproductive system, 199 secondary gene pool, 201 sexual hybridization, 218 somatic hybridization, 218 stability, 214 tertiary gene pool, 201–202 tissue culture, 217–219 utilization, worldwide, 197–199 viral disease, 214–215 whole genome sequencing, 209–210 world production area, 197–199 yield potential, 214 Brassica alboglabra, 7, 220 Brassica bourgeaui, 7 Brassica carinata, 7, 196, 200–201, 206, 212–213, 220 Brassica chinensis, 220 Brassica Collections for Broadening Agricultural Use, 200 Brassica Consortium, 207 Brassica cretica, 7 Brassica fruticulosa, 201 Brassica Genome Project, 208 Brassica gravinae, 201 Brassica hilarionis, 7 Brassica incana, 7 Brassica insularis, 7 Brassica juncea, 7, 196–197, 201–202, 206, 209–210, 212–214, 216–217, 220–221 Brassica macrocarpa, 7 Brassica mauronum, 201 Brassica montana, 7 Brassica napus, 7, 9, 196, 199–217, 219–221 Brassica nigra, 7, 196, 201–202, 206, 221 Brassica oleracea, 7–8, 196, 199–203, 205–207, 209–210, 213, 219–221 Brassica oxyrrhina, 201 Brassica rapa, 7–8, 196, 199–203, 205–210, 212–215, 217, 219–221 Brassica repanda, 201 Brassica rupestris, 7 Brassica souliei, 201 Brassica tournefortii, 201, 212 Brassica triangle, 197 Brassica villosa, 7

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INDEX

Brazil cottonseed production, 91 sesame production, 233 soybean yield, 14 Breeding for high oil, protein, 10 Breeding for high-quality fatty acids, 10 Breeding for high yield, 9 Breeding method development, 11 Burkina, cottonseed production, 91 Burkina Faso sesame production, 233 sesame seed trade, 235 Burma, cottonseed production, 92 Butter, fatty acid composition, 4

C CAAS. See Oil Crops Research Institute of Chinese Academy of Agricultural Sciences Cameroon, cottonseed production, 91 Canada, 181 Canada, sesame seed trade, 236 Cancer, prevention of, 3 Candies, oils used in, 3 Canning industry, oils used in, 3 Canola oil, 3–4, 10. See also Rapeseed oil erucic acid content, 5 Capsule borer, 266 Carcinogenic chemicals, vegetable oils heated at high temperature, 5 Cardiovascular diseases, prevention of, 5 Carduncellus, 175–176 Caribbean, sesame seed trade, 235–236 Carthamus, 168, 174–177 Carthamus alexandrinus, 174 Carthamus ambiguus, 174 Carthamus arborescens, 175 Carthamus baeticus, 175–176 Carthamus boissier, 174, 176 Carthamus caeruleus, 175 Carthamus creticus, 176 Carthamus curdicus, 176 Carthamus dentatus, 174, 176 Carthamus divaricatus, 175–176 Carthamus flavescens, 173, 185 Carthamus glaucus, 174–176 Carthamus gypsicola, 176 Carthamus lanatus, 175–178, 185 Carthamus leucocaulos, 174, 176 Carthamus monspelienium, 175 Carthamus nitidus, 175–177 Carthamus oxyacantha, 174, 177–178 Carthamus oxyacanthus, 176 Carthamus palaestinus, 173–174, 176–177 Carthamus persicus, 176 Carthamus rechingeri, 175 Carthamus rhiphaeus, 175 Carthamus ruber, 175 Carthamus sartori, 175 Carthamus syriacus, 174 Carthamus tenuis, 174, 176

293

Carthamus tinctorius, 2, 167–194 abiotic stresses, 187 resistance to, 185 apomixis, 188 backcross method, segregating populations, 181 biotic stresses, 187 botany, 170–177 breeding for end use, 183–186 breeding methods, 178–182 bulk population method, segregating populations, 181 centers of origin, 173–174 classical cytogenetics, 176–177 crop use, 169–173 cytogenetics, 174–177 cytoplasmic-genetic male sterility, 183 disease resistance, 183–184 distinguishing characteristics, 174 dominant genetic male sterility, 182 florets, 173 flower color variability, 172 genetic modification, 187–188 genetic transformation, 186–188 genomic relationships, 174–176 germplasm enhancement, conventional breeding, 178–185 germplasm resources, 177–178 hybrid breeding, 182–183 hybridization, 179–182 insect resistance, 185 molecular classification, 176 molecular cytogenetics, 177 molecular genetic variation, 185–186 oil content, quality, 184–185 pedigree method, segregating populations, 181 polyembryony, 188 production, worldwide, 169 pure line selection, 179 reclassification of Carthamus, 175–176 reproductive system, 171–173 segregating populations, 181 single recessive genetic male sterility, 182 single-seed descent method, segregating populations, 181 somaclonal variation, 187 somatic embryogenesis, 186–187 species classification, 174–175 spineless safflower, 185 tissue culture, 186–188 utilization, 169–170 world distribution, 169 Carthamus turkestanicus, 175–176 Castor oil, 3 Caulks, safflower oil in, 4 Caulorhizae, 62 CENARGEN. See National Center of Genetic Resources Central African Republic, sesame production, 233 Central America sesame production, 233, 264 sesame seed trade, 235–236 Ceratotheca, 239, 244 Ceratotheca sesamoides, 247, 261 Ceratotheca triloba, 247

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Cercospora arachidicola, 53, 60, 67, 69 Cercospora carthami, 183 Cercospora sesami, 269, 274 Cercosporidium personatum, 60, 69 Ceutorhynchus picitarsis, 215 Ceutorhynchus quadridens, 215 CGIAR. See Consultative Group on International Agricultural Research Chad, cottonseed production, 91 Chemicals, oils used in, 3 China cottonseed production, 91 sesame production, 233 sesame seed trade, 235–236 soybean yield, 14 Classification of oils, based on utilization, 3 CMS system. See Cytoplasmic male sterility system Coating, vegetable oils for, 3 Coatings, oils used in, 3 Cochylis hospes, 145 Coconut oil, 3–4 fatty acid composition, 4 Coincya monensis, 221 Colombia, cottonseed production, 91 Confectionery industry fats, 3 Consultative Group on International Agricultural Research (CGIAR), 59 Contarinia schulzi, 145 Convention on Biological Diversity, 59 Conventional breeding methods, 11 Cooking oils, 3 Core collection approach, germplasm evaluation, 9 Corn oil, 3–4 Corynespora cassilcola, 269 Cosmetics, oils used in, 3 Cote d'lvoire, cottonseed production, 91 Cotton, earliness as desired trait, 9 Cottonseed (Gossypium), 2–3, 89–102. See also Gossypium allotetraploids, 94 diploids, 93 distribution, 90 evolutionary relations, 95 genetic improvement, 92–98 genetic resources, 90–92 genomic symbols, 93–94 history, 95–96 importance of, 90 methods, 96 mode of reproduction, 92–95 nutritional value, 5 objectives, 96–97 origin, 90–92 pests, 97 pigment glands, 10 product utilization, 99–100 production, 2, 91–92 quality of cottonseed, 97–98 species origins, 93–94 yield, 91–92 Crambe abyssinica, 201, 203–204, 221 Cylindrocopturus adspersus, 117

Cylindrosporium concentricum, 214 Cytoplasmic male sterility system, 9, 11

D Deroceras agreste, 215 Deroceras reticulatum, 215 Descriptors for Sesame, 239 Detergents, sunflower oil in, 5 Diabetes, prevention of, 3 Diabrotica undecimpuncata howardi, 65 Didymella arachidicola, 53 Diplotaxis acris, 201 Diplotaxis assurgens, 201 Diplotaxis berthautii, 201 Diplotaxis catholica, 201 Diplotaxis cossoniana, 201 Diplotaxis erucoides, 201–202, 221 Diplotaxis harra, 201 Diplotaxis muralis, 201 Diplotaxis siettiana, 201 Diplotaxis siifolia, 201 Diplotaxis tenuifolia, 201, 221 Diplotaxis tenuisiliqua, 201 Diplotaxis viminea, 201 Diplotaxis virgata, 201 Directorate of Oilseeds Research, Hyderabad, 9 Divaricati, 111 Drought tolerance, groundnut, 75

E Eggert's sunflower, 113–114, 117 Egypt cottonseed production, 91 sesame production, 233 sesame seed trade, 236 Elasmopalpus lignosellus, 69 Empoasca fabae, 66 Erasers, vegetable oils for, 3 Erectoides, 55, 62, 64, 67 Eruca sativa, 202 Erucastrum abyssinicum, 201 Erucastrum canariense, 201 Erucastrum elatum, 201 Erucastrum gallicum, 201 Erucastrum nasturtiifolium, 201 Erucastrum strigosum, 201 Erucastrum varium, 201 Erucastrum virgatum, 201 Erucic acid, 5 Erysiphe cichoracearum, 134 Erysiphecichoracearum, 117 Escherichia oil, 263 Ethiopia sesame production, 233 sesame seed trade, 235 Ethiopian mustard. See Brassica carinata Europe cottonseed production, 91 sesame production, 264 sesame seed trade, 235–236 Extranervosae, 55, 62

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F Far East, sesame production, 264 Fats for bakery, 3 Fatty acid compositions, oilseed crops, 4 Feed for animals, oilseed crops, 3 Folate, 3. See also Vitamin B Foliar diseases, 69–71 Franc-Zone Africa, cottonseed production, 91 France, sesame seed trade, 236 Fusarium oxysporum, 183, 187, 269, 274 Fusarium solani, 37 Fusarium wilt, 266

G Gene pools, oilseed crops, 6–7 Genetic transformation, 11 Genomes, 62–63 Germany, sesame seed trade, 236 Germplasm enhancement, 8–11 Germplasm resources, oilseed crops, 7–8 Glycine, 7, 16, 21, 25, 27–32, 37–38 Glycine albicans, 23, 30 Glycine aphyonota, 23, 30 Glycine arenaria, 23, 30 Glycine argyrea, 21, 23, 29, 31 Glycine canescens, 21, 23, 29, 31 Glycine clandestina, 23, 27, 29, 31 Glycine crytoloba, 29–30 Glycine curvata, 23, 28–30 Glycine cyrtoloba, 23, 27–28 Glycine dolichocarpa, 23, 31 Glycine falcata, 23, 27, 29 Glycine formosana, 15 Glycine gracei, 23 Glycine gracilis, 19 Glycine hirticaulis, 23, 30–31 Glycine lactovirens, 23, 30 Glycine latifolia, 23, 28–29, 31 Glycine latrobeana, 23 Glycine max, 2, 7, 13–50 aneuploidy, 33–34 monosomics, 34 primary trisomics, 33–34 tetrasomics, 34 autopolyploidy, 32–33 average annual soybean production, 17 biotechnology, 40–42 botany, 17–19 callus, cell suspension cultures, plant regeneration from, 41 chromosomal aberrations numerical changes, 32–34 structural changes, 32 cytogenetics, 21–36 diploid species, genomic relationships, 27–31 chromosome pairing, 28–29 classical taxonomy, 27–28 crossing affinity, 28 molecular methods, 29–31genome designation, 27 dissemination, 15–17 domestication, 15–17

295

epigeal germination, 17 gene pools, 19–22 genetic transformation, 41–42 genome evolution, 25–27 geographical map showing, 22 germplasm enhancement, 36–42 conventional breeding, 36–37 interspecific hybridization, 37 intersubgeneric hybridization, 37–38 Kunitz trypsin inhibitor protein, 36–37 mutation breeding, 38–40 hybrid soybeans, potential to produce, 42 linkage mapping, 34–36 chromosome map, 35 classical genetic linkage map, 35 molecular linkage map, 35–36 mature pods, 20 maturity groups, soybean cultivars, 21 mitotic metaphase cell, 25 morphology, 17–19 mutagensis, soybean produced through, fatty acid content changes, 39 protoplasts culture, 41 soybean GP-1, 19–20 soybean GP-2, 20–21 soybean GP-3, 21 taxonomy, 17, 23–24 world soybean production, 16 Glycine microphylla, 23, 28–29, 31 Glycine montis-douglas, 23 Glycine peratosa, 23, 30 Glycine pescadrensis, 23 Glycine pindanica, 23, 30 Glycine pullenii, 23, 30 Glycine rubiginosa, 23 Glycine soja, 7, 15, 19–21, 24–25, 28–30, 32, 37, 42 Glycine stenophita, 23, 30 Glycine syndetika, 23, 31 Glycine tabacina, 23, 25, 28–29, 31–32 polyploid complexes, 31–32 Glycine tomentella, 21, 24–25, 27, 29–32, 38 Glycine ussuriensis, 15 Glycosinolate, 5 Glyphosate-tolerant soybean, 11 Gossypium, 2, 7, 89–102 allotetraploids, 94 diploids, 93 distribution, 90 evolutionary relations, 95 genetic improvement, 92–98 genetic resources, 90–92 genomic symbols, 93–94 history, 95–96 importance of, 90 methods, 96 mode of reproduction, 92–95 objectives, 96–97 origin, 90–92 pests, 97 product utilization, 99–100 production, 91–92

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quality of cottonseed, 97–98 species origins, 93–94 yield, 91–92 Gossypium aboreum, 90 Gossypium amourianum, 93 Gossypium anapoides, 93 Gossypium anomalum, 93 Gossypium arboreum, 93 Gossypium areysianum, 94 Gossypium aridum, 93 Gossypium austale, 93 Gossypium barbadense, 7, 90, 94, 96–97 Gossypium benadirense, 94 Gossypium bickii, 93 Gossypium bricchettii, 94 Gossypium capitis-viridis, 94 Gossypium costulatum, 93 Gossypium cunninghamil, 93 Gossypium darwinii, 94 Gossypium davidsonii, 93 Gossypium enthyle, 93 Gossypium exiguum, 93 Gossypium gossypioides, 93 Gossypium harknessi, 93 Gossypium herbaceum, 90, 93 Gossypium hirsutum, 7, 90, 94–95, 98 Gossypium incarum, 94 Gossypium klotzschianum, 93 Gossypium laxum, 93 Gossypium lobatum, 93 Gossypium londonderriense, 93 Gossypium longicalyx, 94 Gossypium marchantii, 93 Gossypium mustelinum, 94 Gossypium nelsonii, 93 Gossypium nobile, 93 Gossypium pilosum, 93 Gossypium populifolium, 93 Gossypium pulchellum, 93 Gossypium raimondii, 93 Gossypium robinsonii, 93 Gossypium rotundifolium, 93 Gossypium schwendimanii, 93 Gossypium somalense, 94 Gossypium stocksii, 94 Gossypium sturtianum, 93 Gossypium thurberi, 93 Gossypium tomentosum, 94 Gossypium trilobium, 93 Gossypium triphyllum, 94 Gossypium turneri, 93 Gossypium vollesenii, 94 Gossypol-free cotton, 10 Gossypol precursors, 10 Greases, vegetable oils for, 3 Greece cottonseed production, 91 sesame seed trade, 236 Groundnut (Arachis), 2, 5, 51–88. See also Arachis aflatoxin contamination, resistance to, 73–75

allergic reaction, 5 in archaeological evidence of Peru, 1 bacterial wilt, resistance to, 72 breeding progress for important traits, 69–76 China, mulch conditions, 52 Consultative Group on International Agricultural Research (CGIAR), 59 Convention on Biological Diversity, 59 core collections, 59–61 crop improvement, 63–76 cytogenetics, 62–63 dissemination, 54–55 distribution, 54 drought tolerance, 75 evaluation, 59–61 foliar diseases, resistance to, 69–71 food quality of, 5 future developments, 76–77 genetic resources, 54–61 genetic transformation, 68–69 genomes, 62–63 germplasm collection, 57–59 conservation, 57–59 ICRISAT, 59 improved oil quality, 75–76 International Board for Plant Genetic Resources, 59 International Crops Research Institute for Semiarid Tropics, 53, 58 interspecific hybridization, 64–66 marker-assisted selection, 66–68 mutation breeding, 66 National Center of Genetic Resources, 58 nematodes, resistance to, 73 oil, fatty acid composition, 4 oil content, 75–76 Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, 58 origin, 54 production, 2 soilborne fungi diseases, resistance to, 71–72 in South America of antiquity, 1 taxonomy, 55–56 testa color, groundnut cultivars, diversity, 57 traditional breeding methods, 63–64 wild relatives, 57–59 Guatemala sesame production, 233 sesame seed trade, 235

H Haploidy technique, 11 Heart disease, prevention of, 3 Helianthopsis, 111 Helianthus, 2, 103–166 alteration of fatty acid composition, 136 aneuploidy, 118–119 BAC library, 150 bacteria, 144 bird depredation, 145–146

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botanical traits, 105–108 breeding for adaptation, 139–140 breeding for end use, 137–139 center of diversity, 110–111 chiasma frequency per cell, 123 chromosome doubling, 136 CMS sources, 126–127 core collection, 116 crop use, 105–109 cytogenetic stocks, 128–129 cytogenetics, 118–136 description, 105–109 diseases, 141–144 dispersion, 109–110 DNA content, 119–121 domestication, 109–110 drought tolerance, 146 euploidy, 118–119 fatty acid composition, 138 gene mapping, 149–150 genetic diversity, 147–148 genetic stocks, 116 genomes, 118–122 germplasm characterization, molecular applications, 147–150 germplasm enhancement, conventional breeding, 137–147 germplasm evaluation, use, 116–118 germplasm resources, 110–118 grain, 137 growth habit, 106–108 herbicide tolerance, 146 hybridization techniques, 129–131 infrageneric classification annual species, 112 perennial species, 113 insects, 117, 144–145 interspecific hybridization, 129–135 interspecific hybrids, 131–132 between wild, cultivated lines, 132–135 karyotype, 121–122 karyotype comparisons, 122 male sterility, 124–128 male sterility induction, 135–136 microsporogenesis, 122–124 molecular mapping, 148–149 morphological traits, 105–108 mutagenesis, 135 origin, 109–110 pathogens, 116–117 pests, 144–146 phenology, 140 plant structures, 106–108 plant type, 139–140 protein, 117–118, 137–138 quality of oil, 117, 138–139 reproductive system, 105–106 resistance to abiotic constraints, 146–147 resistance to biotic constraints, 141–148 salt tolerance, 146

297

soil nutrition, 147 stability, 141 taxonomy, 110–118 tocopherol composition, 138–139 utilization, 108–109 viruses, 144 wild, weedy relatives, 114–116 world production area, 108–109 yield potential, 141 Helianthus agrestis, 112, 117, 119, 130, 145 Helianthus angustifolius, 113, 119, 122, 129, 134 Helianthus annuus, 104, 109–112, 114–119, 121–124, 126, 128, 130–136, 142–144, 146, 149 Helianthus annuus lenticularis, 126 Helianthus annuus texanus, 126 Helianthus annuus wild, 126 Helianthus anomalous, 112, 114, 117, 126, 144, 149 Helianthus argophyllus, 112, 116, 122–124, 126, 131, 142–144, 146 Helianthus arizonensis, 113 Helianthus atrorubens, 113, 123 Helianthus bolanderi, 111–112, 117, 126, 129, 131, 143 Helianthus californicus, 113, 121, 129, 133 Helianthus carnosus, 113–114 Helianthus ciliaris, 113, 117–118, 145 Helianthus cusickii, 113, 129 Helianthus debilis, 112, 114, 117, 121–122, 126, 131, 134, 143 Helianthus decapetalus, 113, 117–118, 121–123, 128, 130–131, 133, 136 Helianthus deserticola, 112, 114, 117 Helianthus divaricatus, 113, 117, 119, 122–123, 132–134, 143 Helianthus eggertii, 113–114, 117 Helianthus exilis, 112, 114, 126, 144 Helianthus floridanus, 113 Helianthus giganteus, 113, 117, 123, 126, 128–130, 132–136 Helianthus glaucophyllus, 113, 132 Helianthus gracilentus, 113, 129, 134 Helianthus grosseserratus, 113, 117, 123–124, 129, 132–134, 143, 145 Helianthus heterophyllus, 113 Helianthus hirsutus, 113, 117, 119, 121–122, 129–130, 133–135, 143 Helianthus laciniatus, 113, 130, 133 Helianthus laevigatus, 113–114, 133 Helianthus lenticularis, 122, 126 Helianthus longifolius, 113 Helianthus maximiliani, 113, 117, 119, 122, 126, 128–130, 132–134, 136, 142–145 Helianthus microcephalus, 113, 128, 132, 136 Helianthus mollis, 113, 117, 121–123, 126, 129–134, 142, 144 Helianthus neglectus, 112, 118–119, 126, 131 Helianthus niveus, 111–112, 114, 117, 146 Helianthus niveus canescens, 126 Helianthus nuttallii, 113–114, 117–118, 129–130, 133–134, 142 Helianthus orgyalis, 133 Helianthus paradoxus, 112, 114, 146

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298

GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT

Helianthus pauciflorus, 113, 117, 123, 130, 132–133, 143–145 Helianthus petiolaris, 111–112, 115–117, 119, 124, 126–127, 131–133, 136, 142–143, 145–147, 149 Helianthus petiolaris fallax, 126 Helianthus petiolaris Nutt, 126–127 Helianthus porteri, 111–112, 118 Helianthus praecox, 112, 114, 116–117, 127, 131, 142–143, 145 Helianthus praecox hirtus, 127 Helianthus pumilus, 113, 122, 131, 134 Helianthus radula, 113 Helianthus resinosus, 113, 117, 127, 132–133, 142–144 Helianthus rigidus, 111, 113, 117, 123, 127, 130, 132–133, 143–145 Helianthus salicifolius, 113, 117, 121–123, 131–133, 145 Helianthus scaberimus, 133 Helianthus schweinitzii, 113–114 Helianthus silphioides, 113 Helianthus simulans, 113, 133 Helianthus smithii, 113–114 Helianthus strumosus, 113, 117–118, 127, 129–130, 132, 134, 145 Helianthus trachaelifolius, 122 Helianthus tuberosus, 113, 117–118, 122, 130, 132–134, 142–145 Helianthus verticillatus, 111, 113–114 Heterodera glycines, 37 Heterodera schachtii, 221 Homoeosoma electellum, 117 Hordeum vulgare, 121 Hypertension, prevention of, 5

I IBPGR. See International Board for Plant Genetic Resources ICRISAT. See International Crops Research Institute for Semiarid Tropics Immunoglobulin E-mediated food allergens, 10 India cottonseed production, 91 sesame production, 233, 271 sesame seed trade, 235 soybean yield, 14 Indian mustard. See Brassica juncea Industrial lubricants, soybean oil, 4 Industrial oil uses, 3 Industrial suitability, oilseed crops, 9 Ink, printing soybean oil, 4 vegetable oils, 3 International Board for Plant Genetic Resources, 59 International Crops Research Institute for Semiarid Tropics, 5, 53, 58 International Plant Genetics Resources Institute, 6 International programs, establishment of, 5–6 International Soybean Program, 6 INTSOY. See International Soybean Program IPGRI. See International Plant Genetics Resources Institute Iran cottonseed production, 92 sesame production, 233

sesame seed trade, 236 Iraq, sesame in antiquity, 1 Israel cottonseed production, 92 sesame production, 257, 264 sesame seed trade, 236

J Japan, sesame seed trade, 236 Jojoba oil, 3 Jordan, sesame seed trade, 236

K Kazakhstan, cottonseed production, 91 Kentrophyllum, 176 Kenya, sesame production, 233 Kidney disease, prevention of, 3 Korea, Republic of sesame production, 233 sesame seed trade, 236 Kunitz trypsin inhibitor protein, 10, 36–37 Kygyzstan, cottonseed production, 92

L Lamottea, 175 Landmark research, oilseed crops, 1–12 Lard, fatty acid composition, 4 Leaf blight, 266 Lebanon, sesame seed trade, 236 Lecithin, 3 Lepidopappus, 176 Leptosphaeria maculans, 214, 221 Leptosphaerulina crassiasca, 60 Lignans, 4 Linolenic acid, oils high in, yellowing problems, 5 Linoleum oils used in, 3 safflower oil in, 4 Linseed oil, 2–3 production, 2 Lubrication, oils used in, 3 Lycopersicon esculentum, 121 Lygus, 145

M Macrophomina phaseolina, 144, 269–270, 274 Maize, 4. See also Corn fatty acid composition of oil, 4 oil produced from, 3 Malaysia, sesame seed trade, 236 Mali, cottonseed production, 91 Margarine, 3 Martynia annua, 247 Mayonnaise, 3 MBGP. See Multinational Brassica Genome Project Meloidogyne, 73 Meloidogyne arenaria, 60, 66–67, 73 Mexico cottonseed production, 91 safflower production, 181 sesame production, 233

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sesame seed trade, 235–236 Microsphaera diffusa, 37 Middle East, sesame production, 264 Molds, aflatoxin from, 5, 10, 73–75 Molecular linkage groups, soybean, 8 Monounsaturated fat, fatty acid composition, 4 Mozambique cottonseed production, 91 sesame seed trade, 235 Multinational Brassica Genome Project, 8 Mustard production, 2 Myanmar sesame production, 233 sesame seed trade, 235 Sesamum indicum, drying, threshing in, 237

N N-nitroso-N-methylurea, 38 National Bureau of Plant Genetic Resources, 9 National Center of Genetic Resources, 58 National programs, establishment of, 5–6 National Soybean Research Laboratory at University of Illinois, Urbana, 6 NBPGR. See National Bureau of Plant Genetic Resources Nematodes, 73 Nicaragua, sesame seed trade, 235 Nigeria cottonseed production, 91 sesame production, 233 sesame seed trade, 235 North America sesame production, 233, 264 sesame seed trade, 235–236 NuSun, 3 Nutritional value, oilseed crops, 9

O Obesity, prevention of, 3 Oceania, cottonseed production, 91–92 Odontagnathius, 176 Oidiun, 266 Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, 58 Oil palms, 3 Oilseeds, 195–230 abiotic constraints, resistance to, 214–216 abiotic stress, 215–216 biotic constraints, resistance to, 214–216 botany, 199 breeding for end use, 212–213 breeding methods, 210–212 classical cytogenetics, 202–203 crop use, 197–199 cultivar development, 210–211 cytogenetic maps, 204–205 cytogenetics, 202–204 description, 197–199 diploid species, genomic relationships among, 202 doubled haploids, generation of, 217–218 fungal disease, 214–215 genetic mapping, 204–210

299

genetic maps, 205–207 genetic modification, 218–219 genetic transformation, 217–219 germplasm enhancement, conventional breeding, 210–216 germplasm resources, 199–202 hybrid breeding, 211–212 intergeneric hybridization, 219–221 interspecific hybridization, 219–221 meal quality, 212 molecular cytogenetics, 203–204 molecular genetic variation, 216–217 oil content, 213 pests, 215 physical maps, 207–209 primary gene pool, 201 protein content, 212 quality of oil, 213 reproductive system, 199 secondary gene pool, 201 sexual hybridization, 218 somatic hybridization, 218 stability, 214 tertiary gene pool, 201–202 tissue culture, 217–219 utilization, worldwide, 197–199 viral disease, 214–215 whole genome sequencing, 209–210 world production area, 197–199 yield potential, 214 Oleosin, 5 Olive oil, 3 fatty acid composition, 4 Onchophragmus violaceus, 201 Orobanche cernua, 117 Orobanche cumana, 135, 144, 147 Orosius albicinctus, 269 Oryza sativa, 15, 121 Osteoporosis, prevention of, 3

P Paints oils used in, 3 sunflower oil in, 5 vegetable oils for, 3 Pakistan cottonseed production, 91 sesame production, 233 sesame seeds in archaeological excavations, 1sesame seed trade, 235 Palm kernel oil, 4 Palm oil, fatty acid composition, 4 Paraguay cottonseed production, 91 sesame seed trade, 235 Peanut, 2. See also Arachis Pedalium, 244 Perenospora parasitica, 214 Peru, cottonseed production, 91 Phaeoisariopsis personata, 53 Phakopsora pachyrhizi, 37

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300

GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT

Pharmaceutical products oils used in, 3 vegetable oils for, 3 Phoma lingam, 214 Phoma macdonaldii, 117 Phomopsis helianthi, 117 Phonus, 175 Phytophthora blight, 266 Phytophthora drechsleri, 181, 184 Phytophthora parasitica, 269 Phytophthora sojae, 37 Phytophtora, 274 Plant Introduction Station, USDA, Griffin, GA, 59 Plasmodiophora brassicae, 200, 214 Plasmopara halstedii, 116, 118, 148 Plastic coatings, oils used in, 3 Plastics sunflower oil in, 5 vegetable oils for, 3 Poland, sesame seed trade, 236 Polyembryony, Carthamus tinctorius, 188 Polyploid complexes, Glycine tomentella, 31–32 Polyunsaturated fat, fatty acid composition, 4 Powdery mildew, 266 Primary gene pool, oilseed crops, 6–7 Printing ink soybean oil, 4 vegetable oils, 3 Processing industries, development of, 6 Prorhizomatosae, 56 Protein energy malnutrition, 3 Pseudomonas syringae, 144, 269 Psylliodes chrysocephala, 215 Puccinia arachidis, 53, 69 Puccinia helianthi, 116, 134, 143–144 Putties safflower oil, 4 vegetable oils, 3 Pyrenopeziza brassicae, 214 Pyrethrum insecticides, 4

R Ralstonia solanacearum, 53, 55, 72 Ramularia carthami, 183 Rapeseed, in Indian Sanskrit writings, 1 Rapeseed oil, 2, 4–5, 10 fatty acid composition, 4 Raphanus sativus, 203, 206, 221 Rehovot, Israel, sesame production, 264 Resins, coatings, oils used in, 3 Rhizobium, 2, 11 Rhizoctonia bataticola, 183 Rhizoctonia solani, 60, 183 Rhizomatosae, 55–56, 58, 62, 64 Rhizopus arrhizus, 117, 143 Rhizopus oryzae, 143 Rome, sesame production, 264 Roundup Ready soybean, 11

S Safflower (Carthamus tinctorius), 2, 167–194. See also Carthamus tinctorius abiotic stresses, 187 resistance to, 185 apomixis, 188 backcross method, segregating populations, 181 biotic stresses, 187 botany, 170–177 breeding for end use, 183–186 breeding methods, 178–182 bulk population method, segregating populations, 181 centers of origin, 173–174 classical cytogenetics, 176–177 crop use, 169–173 cytogenetics, 174–177 cytoplasmic-genetic male sterility, 183 disease resistance, 183–184 distinguishing characteristics, 174 dominant genetic male sterility, 182 florets, 173 flower color variability, 172 genetic modification, 187–188 genetic transformation, 186–188 genomic relationships, 174–176 germplasm enhancement, conventional breeding, 178–185 germplasm resources, 177–178 hybrid breeding, 182–183 hybridization, 179–182 insect resistance, 185 medicinal properties, 5 molecular classification, 176 molecular cytogenetics, 177 molecular genetic variation, 185–186 oil, fatty acid composition, 4 oil content, quality, 184–185 orange-red dye, 5 pedigree method, segregating populations, 181 polyembryony, 188 production, 2 worldwide, 169 pure line selection, 179 reclassification of Carthamus, 175–176 reproductive system, 171–173 segregating populations, 181 single recessive genetic male sterility, 182 single-seed descent method, segregating populations, 181 somaclonal variation, 187 somatic embryogenesis, 186–187 species classification, 174–175 spineless safflower, 185 spineless varieties, 10 tissue culture, 186–188 utilization, 169–170 world distribution, 169 Salad oil, 3 Saturated fatty acids, 4 Saudi Arabia, sesame seed trade, 236 Schlerotinia sclerotiorum, 37 Schweinitz's sunflower, 114

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INDEX

Sclerotinia infection, 148–149 Sclerotinia minor, 71 Sclerotinia sclerotiorum, 117, 142, 148, 184, 214 Sclerotium rolfsii, 71 Secale cereale, 121 Secondary gene pool, oilseed crops, 7 Septoria glycines, 37 Septoria helianthi, 143 Sesame and Safflower Newsletter Descriptors for Sesame, 238 Sesame (Sesamum indicum), 2, 231–290. See also Sesamum indicum allergens, 261–262 alternaria leaf spot, 269 amino acids, 259 in antiquity, 1 bacterial blight, 269 bacterial leaf spot, 269 bacterial resistance, 269 botany, 238–244 breeding methods, 270–276 breeding objectives, 267–270 breeding research, 237–238 capsules, 242–244 charcoal rot, 269 chromosomes, description, numbers, 244 combining ability, 251 core collections, 265–266 corynespora, 269 cross compatibilities, 245 cytogenetics, 244–248 dehiscence, 256 disease resistance, 266 DNA cloning, transfer, 263 DNA markers, 262–263 domestication, 247–248 drying, threshing in Myanmar, 237 exporting countries, 235 fatty acids, 257–259 flavor, 262 floral biology, 241–242 flowering, 241–242 fungal resistance, 269 fusarium, 269 genetic research, 237–238 genetic studies, 248–249 genetics, 248–253 genotype, environment interactions, 250 germplasm resources, 263–267 growth habit, 240 in Hellenic era, 1 heritability, 250–251 heterosis, 251–252 hybrid cultivars, 275–276 hybridization, 272–273 importing countries, 236 indehiscence, 256 indeterminate growth habit, 240 India, released improved cultivars developed in, 271 induced mutations, 273–275 insects, disease, 266

301

interspecific relationships, 244–247 isozymes, 259–260 Israel, ranges of oil, protein contents, 257 leaf curl, 269 lignans, 260–261 male sterility, 252–253 molecular variation, 262–263 monogenic prevention, seed shattering, 254–255 nonshattering, by combination of traits, 255–256 nonshattering sesame, Sesaco Corporation development, 248 objectives, 267 oil, pyrethrum insecticide synergist, 4 oil antioxidants, 4 oil content, 256–257 oil for lighting, 4 origin, 247–248 phyllody, 269 phyllody-phytoplasma disease, 266 phytophthora blight, 269 phytoplasma, 269 plant structure, 240 powdery mildew, 269 protein bands, 259–260 proteins, 259 Rehovot, Israel, cultivated sesame accessions evaluated, multiplied at, 264 resistance to diseases, 269 resistance to insect pests, diseases, 266 in Roman era, 1 seed, 10, 244 fatty acid composition, 4 seed composition, quality, 256–262 seed dormancy, storage, 266–267 seed in archaeological excavations, 1 seed retention, 253–256 measurements, 256 seed trade, 236 seedling blight, 266 selection, 271–272 shattering resistance, 256 South Korea, ranges of oil, protein contents, 257 taste, 262 taxonomy, 238–240 trade, 235 in vitro techniques, 276–277 anther culture, 277 embryo culture, 276–277 protoplast culture, 277 tissue culture, 276 white spot, 269 wild species, 266 world production, 232–237 yield, 249–250 Sesame stover, 4 Sesamum, 238–239, 244, 247–248, 266 wild species, 277 Sesamum abbreviatum, 239 Sesamum alatum, 239, 244–247, 259, 261, 266 Sesamum angolense, 239, 244–246, 248, 261 Sesamum angustifolium, 239, 244, 248, 259, 261

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GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT

Sesamum calycinum, 239, 248, 261 Sesamum capense, 239, 244–246, 261 Sesamum grandiflorum, 246 Sesamum indicatum, 247 Sesamum indicum, 2, 231–290 allergens, 261–262 alternaria leaf spot, 269 amino acids, 259 bacterial blight, 269 bacterial leaf spot, 269 bacterial resistance, 269 botany, 238–244 breeding methods, 270–276 breeding objectives, 267–270 breeding research, 237–238 capsules, 242–244 charcoal rot, 269 chromosomes, description, numbers, 244 combining ability, 251 core collections, 265–266 corynespora, 269 cross compatibilities, 245 cytogenetics, 244–248 dehiscence, 256 disease resistance, 266 DNA cloning, transfer, 263 DNA markers, 262–263 domestication, 247–248 drying, threshing in Myanmar, 237 exporting countries, 235 fatty acids, 257–259 flavor, 262 floral biology, 241–242 flowering, 241–242 fungal resistance, 269 fusarium, 269 genetic research, 237–238 genetic studies, 248–249 genetics, 248–253 genotype, environment interactions, 250 germplasm resources, 263–267 growth habit, 240 heritability, 250–251 heterosis, 251–252 hybrid cultivars, 275–276 hybridization, 272–273 importing countries, 236 indehiscence, 256 indeterminate growth habit, 240 India, released improved cultivars developed in, 271 induced mutations, 273–275 insects, disease, 266 interspecific relationships, 244–247 isozymes, 259–260 Israel, ranges of oil, protein contents, 257 leaf curl, 269 lignans, 260–261 male sterility, 252–253 molecular variation, 262–263 monogenic prevention, seed shattering, 254–255 nonshattering, by combination of traits, 255–256

nonshattering sesame, Sesaco Corporation development, 248 objectives, 267 oil content, 256–257 origin, 247–248 phyllody, 269 phyllody-phytoplasma disease, 266 phytophthora blight, 269 phytoplasma, 269 plant structure, 240 powdery mildew, 269 protein bands, 259–260 proteins, 259 Rehovot, Israel, cultivated sesame accessions evaluated, multiplied at, 264 resistance to diseases, 269 resistance to insect pests, diseases, 266 seed composition, quality, 256–262 seed dormancy, storage, 266–267 seed retention, 253–256 measurements, 256 seed trade, 236 seedling blight, 266 seeds, 244 selection, 271–272 shattering resistance, 256 South Korea, ranges of oil, protein contents, 257 taste, 262 taxonomy, 238–240 trade, 235 in vitro techniques, 276–277 anther culture, 277 embryo culture, 276–277 protoplast culture, 277 tissue culture, 276 white spot, 269 wild species, 266 world production, 232–237 yield, 249–250 Sesamum laciniatum, 239, 244–247, 266 Sesamum latifolium, 239, 244, 261 Sesamum malabaricum, 239, 244–248, 253, 261, 266 Sesamum marlothii, 239 Sesamum mulayanum, 239, 246 Sesamum occidentale, 113, 123, 134, 244, 246–247 Sesamum orientale, 239, 244, 247 Sesamum parviflorum, 239 Sesamum pedalioides, 239, 261 Sesamum prostratum, 239, 244–247, 266 Sesamum radiatum, 113, 123, 134, 239, 244–247, 259, 261, 266 Sesamum rigidum, 239, 261 Sesamum schenckii, 244 Sesamum schinzianum, 239, 244–246, 263, 276 Sesamum triphyllum, 239, 261 Shoot webber, 266 Shortenings, 3 Sinapis alba, 201, 221 Sinapis arvensis, 201–203, 219, 221 Sinapis aucheri, 201 Sinapis flexuosa, 201

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Sinapis pubescens, 201 Smicronyx fulvus, 145 Soap oils used in, 3 sunflower oil in, 5 vegetable oils for, 3 Soilborne fungi diseases, 71–72 Soja, 19, 28, 30 Somatic hybridization, 11 South Africa, cottonseed production, 91 South America sesame production, 233, 264 sesame seed trade, 235–236 South Korea, sesame production, 257 Soy meal, as source of protein, 3 Soy milk, 3 as source of protein, 3 Soybean (Glycine max), 2–4, 13–50. See also Glycine max amino acids, 3 aneuploidy, 33–34 monosomics, 34 primary trisomics, 33–34 tetrasomics, 34 annual soybean production, 17 autopolyploidy, 32–33 biotechnology, 40–42 botany, 17–19 callus, cell suspension cultures, plant regeneration from, 41 chromosomal aberrations numerical changes, 32–34 structural changes, 32 cytogenetics, 21–36 diploid species, genomic relationships, 27–31 chromosome pairing, 28–29 classical taxonomy, 27–28 crossing affinity, 28 genome designation, 27 molecular methods, 29–31 dissemination, 15–17 domestication, 15–17 epigeal germination, 17 gene pools, 19–22 genetic transformation, 41–42 genome evolution, 25–27 geographical map showing, 22 germplasm enhancement, 36–42 conventional breeding, 36–37 interspecific hybridization, 37 intersubgeneric hybridization, 37–38 Kunitz trypsin inhibitor protein, 36–37 mutation breeding, 38–40 glyphosate-tolerant, 11 hybrid soybeans, potential to produce, 42 linkage mapping, 34–36 chromosome map, 35 classical genetic linkage map, 35 molecular linkage map, 35–36 mature pods, 20 maturity groups, soybean cultivars, 21 mitotic metaphase cell, 25

303

morphology, 17–19 mutagensis, soybean produced through, fatty acid content changes, 39 oil fatty acid composition, 4 oil for disease prevention, 3 polyploid complexes, 31–32 production, 2 protoplasts culture, 41 soybean GP-1, 19–20 soybean GP-2, 20–21 soybean GP-3, 21 taxonomy, 17, 23–24 trypsin inhibitor, 5 vitamins, 3 world soybean production, 16 Spain, cottonseed production, 91 Spineless safflower, 185 Spodoptera, 53 Spondylosis, prevention, 5 Sterility, prevention, 5 Striga damages, 238 Sudan cottonseed production, 91 sesame production, 233 sesame seed trade, 235 Sunflower (Helianthus), 2, 103–166. See also Helianthus alteration of fatty acid composition, 136 aneuploidy, 118–119 BAC library, 150 bacteria, 144 bird depredation, 145–146 botanical traits, 105–108 breeding for adaptation, 139–140 breeding for end use, 137–139 center of diversity, 110–111 chiasma frequency per cell, 123 chromosome doubling, 136 CMS sources, 126–127 core collection, 116 crop use, 105–109 cytogenetic stocks, 128–129 cytogenetics, 118–136 description, 105–109 diseases, 141–144 dispersion, 109–110 DNA content, 119–121 domestication, 109–110 drought tolerance, 146 euploidy, 118–119 fatty acid composition, 138 gene mapping, 149–150 genetic diversity, 147–148 genetic stocks, 116 genomes, 118–122 germplasm characterization, molecular applications, 147–150 germplasm enhancement, conventional breeding, 137–147 germplasm evaluation, use, 116–118 germplasm resources, 110–118 grain, 137

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304

GENETIC RESOURCES, CHROMOSOME ENGINEERING, AND CROP IMPROVEMENT

growth habit, 106–108 herbicide tolerance, 146 hybridization techniques, 129–131 infrageneric classification annual species, 112 perennial species, 113 insects, 117, 144–145 interspecific hybridization, 129–135 interspecific hybrids, 131–132 between wild, cultivated lines, 132–135 karyotype, 121–122 comparisons, 122 male sterility, 124–128, 135–136 microsporogenesis, 122–124 molecular mapping, 148–149 morphological traits, 105–108 oil for industrial purposes, 4mutagenesis, 135 origin, 109–110 pathogens, 116–117 pests, 144–146 phenology, 140 plant structures, 106–108 plant type, 139–140 production, 2 protein, 117–118, 137–138 protein chlorogenic acid, 10 quality of oil, 117, 138–139 reproductive system, 105–106 resistance to abiotic constraints, 146–147 resistance to biotic constraints, 141–148 salt tolerance, 146 seed, fatty acid composition, 4 soil nutrition, 147 stability, 141 taxonomy, 110–118 tocopherol composition, 138–139 utilization, 108–109 viruses, 144 wild, weedy relatives, 114–116 world production area, 108–109 yield potential, 141 Syria, cottonseed production, 92

T Tajikistan, cottonseed production, 91 Tanzania cottonseed production, 91 sesame production, 233 sesame seed trade, 235 Technical products, oils used in, 3 Tertiary gene pool, oilseed crops, 7 Testa color, groundnut cultivars, diversity, 57 Thailand sesame production, 233 sesame seed trade, 235 Thlaspi arvense, 219 Tofu, 3 as source of protein, 3 Togo, cottonseed production, 91 Trachystoma labasil, 202 Triticum aestivum, 142

Triticum turgidum, 148 Tunisia, sesame seed trade, 236 Turkey cottonseed production, 92 sesame in antiquity, 1 sesame production, 233 sesame seed trade, 235–236 Turkmenistan, cottonseed production, 91

U Uganda cottonseed production, 91 sesame production, 233 U.K., sesame seed trade, 236 United States cottonseed production, 91 sesame seed trade, 236 soybean yield, 14 Unsaturated fatty acids, 4 Urethane resins, safflower oil in, 4 USDA, 59 Uzbekistan cottonseed production, 91 sesame production, 233

V Vanaspati, 3 Varnishes sunflower oil in, 5 vegetable oils for, 3 Vegetable oils, for pharmaceutical products, 3 Vegetable proteins, 3 Venezuela sesame production, 233 sesame seed trade, 235 Verticillium dahliae, 116, 214 Verticillium longisporum, 214 Vietnam sesame production, 233 sesame seed trade, 235 Viguiera porteri, 111 Vitamin B, 3

W Western hemisphere, cottonseed production, 91 World production, oilseed crops, 2 World War II, soybean production, 2

X Xanthomonas compestris, 269

Y Yellowing problems, oils high in linolenic acid, 5 Yemen sesame production, 233 sesame seed trade, 236

Z Zambia, cottonseed production, 91 Zea mays, 56, 121 Zimbabwe, cottonseed production, 91 Zygogramma exclamationis, 117

3639_color_insert.fm Page 2 Friday, September 15, 2006 10:58 AM

Color Figure 2.4

Color Figure 2.12

Color Figure 3.2

3639_color_insert.fm Page 3 Friday, September 15, 2006 10:58 AM

Color Figure 7.1

Color Figure 7.2

Color Figure 7.3

Color Figure 8.3