Transgenic Cotton

转基因棉花

Editor in Chief

JIA Shirong Editors GUO Sandui AN Daochang XIA Guixian

Science Press

Beijing, New York

Responsible Editor: PANG Zaitang

http://www.lifescience.com.cn

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means,electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the copyright owner.

ISBN 7-03-014470-8 ISBN 1-933100-02-8

Editor in Chief

JIA Shirong Editors GUO Sandui AN Daochang XIA Guixian

Contributors for Chinese Version

CHANG Tuanjie CHEN Lei CHEN Mingnan CHEN Xiaoya

CHENG Hongmei CUI Hongzhi DING Zhiyong FENG Dejiang

GUO Sandui JIA Shirong JIAN Guiliang LI Huifen

LI Xugang LI Yan’e LIN Zhongping LIU Xiang

LIU Yuhui LU Meiguang LU Zixian NI Wanchao

RUI Changhui TANG Canming TIAN Yingchuan WANG Wugang

WANG Zhixing WU Kongming WU Qian XIA Guixian

XU Chongren XU Honglin XU Junwang YUAN Zhengqiang

ZHANG Ning ZHANG Tianzhen ZHAO Guozhong ZHAO Jianzhou

ZHOU Xiangjun ZHU Zhen

Contributors for English Version

CHEN Xiaoya CUI Hongzhi FAN Cunhui GONG Wankui

HU Ruifa HUANG Jikun HUANG Jizhang JIA Shirong

JIN Wujun LI Jigang LI Weimin LIN Zhongping

PRAY Carl ROZELLE Scott TIAN Yingchuan WANG Qinfang

WANG Xujing WU Kongming XIA Guixian XU Chongren

ZHANG Yongqiang ZHAO Jianzhou ZHU Zhen

Preface

In March 1996, a National High-Tech R & D Program (also referred as ‘863 Program’) was initiated under the leadership and supervision of the Ministry of Science and Technology, China. Biotechnology applicable to agriculture and medicine was listed as the key priority in this program, in which the ‘R & D of transgenic insect-resistant cotton’ was incorporated as one of the key projects. The initial phase of 863 Program was terminated in the year 2000, and the second phase of the program is continued since 2001. In summarizing the scientific achievements obtained during the first phase, ‘863 serial books’ were compiled and published in 2001, among which a Chinese version of ‘Transgenic Cotton’ was included. Transgenic Bt cotton is the first biotechnology product applied in Chinese agriculture, by which China has become the second country in the world in developing Bt cotton with own IPR and the production area of Bt cotton is now accounted for more than 60% of the total acreage of cotton in this country. In August 2000, a China-ASEAN Workshop on Transgenic Plants was held in Beijing, during which Chinese scientists presented data on the R & D of transgenic cotton and scientists from ASEAN member countries had a field trip to see the performance of Bt cotton. All the participants showed great interests in obtaining detailed information on Bt cotton development in China. Unfortunately, the book was published in Chinese so that most of scientists are inaccessible to the information. In accordance with the recommendation raised in the workshop, the Secretary of ASEAN Committee collaborated with the Chinese Government have decided to translate Chinese version of the book into English. Since biotechnology is a fast growing technology, the content in the Chinese version published in 2001 may be out of date to some extent, although the major parts of the book remain unchanged. To cope up with the rapid development in international scientific community, we have tried our best in updating the information in this English edition accordingly. To the best of our knowledge, neither Chinese nor English monograph of ‘Transgenic cotton’ has been published to date. The editors and authors would be highly appreciated if this book would be valuable to the readers. The research and development of transgenic cotton is organized multidisciplinary that include molecular cloning and characterization, transformation, genetics and breeding, cultivation, entomology, pathology, and biosafety assessment, etc. More than 30 institutions nationwide have been involved in the project. The editors would like to

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express our sincere thanks to the financial support of the 863 Program from the Ministry of Science and Technology, eminence respect and acknowledgement to those people who have devoted their intelligence to the project, and all the contributors and translators who made it possible for the book to be published both in Chinese and English.

Editor in chief: JIA Shirong Biotechnology Research Institute Chinese Academy of Agricultural Sciences Chief scientist for the project of ‘R & D of transgenic insect-resistant cotton’ in 863 Program

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Content

Preface Introduction············································································································1 1 Field performance of Bt cotton··············································································2 2 Variety registrations·······························································································4 3 Inheritance of Bt gene ···························································································4 4 Insect resistance management for Bt cotton ··························································4 5 Gene flow from transgenic cotton ·········································································6 6 Safety assessments of Bt cotton byproducts··························································6 7 Development of transgenic cotton resistant to Fusarium and Verticillium wilt ····6 8 Genetic engineering for improvement of cotton fiber quality·······························7

Chapter 1 General Situation of Cotton Production and Breeding in China···························································································8 1 General situation of cotton production in China ···················································8 1.1 Role of cotton production in China’s national economy ································9 1.2 Development of cotton production in China ················································10 1.3 Cotton production regions in China ·····························································12 1.4 Current situation of cotton production ·························································14 2 Achievements and present status of cotton breeding in China····························15 2.1 Cotton breeding achievements in China·······················································15 2.2 Two key issues confronting cotton breeding in China··································17

Chapter 2 Bt Insecticidal Crystal Proteins and Their Genes····20 1 The structures and functions of Bt ICPs ····························································21 1.1 Bt Cry genes ·······························································································21 1.2 Features of Bt ICPs ·····················································································21 1.3 The structure and function of Bt ICPs ························································22 2 Mode of action of Bt insecticidal proteins ·························································25 2.1 The processing and action mechanism of Bt ICPs ·····································25 2.2 Toxicity of Cyt insecticidal proteins ···························································27 3 Application and limitation of Bt bio-insecticides ··············································27 3.1 Application of Bt products and its limitations ············································27 3.2 Bt insect-resistant transgenic plants ····························································28

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3.3 Bt ICPs resistance developed by insects ·····················································29 4 Classification of Bt toxin genes ·········································································30

Chapter 3 Protease Inhibitors, Agglutinins and Other Pest- control Genes ·····························································································43 1 Classification of protease inhibitors ··································································43 1.1 Serpins ········································································································43 1.2 Cystatins ·····································································································47 1.3 Metallocarboxypeptidase and aspartyl protease inhibitors ·························48 2 Structure and function of PIs ·············································································48 2.1 Structure of PIs ···························································································49 2.2 Physiological function of PIs ·····································································52 3 The insecticidal mechanism of PIs ····································································54 3.1 Digestive enzymes of insects ·····································································54 3.2 Formation of complex between proteases and their inhibitors ···················55 3.3 Mechanism of insecticidal action of PIs ·····················································56 4 Lectins and their coding genes ··········································································57 4.1 Categories of phyto-lectins ·········································································57 4.2 Structure and biological function of phyto-lectins ·····································61 4.3 Homology, expression and posttranslational processing of lectin genes ····63 4.4 Application of phyto-lectins in pest control genetic engineering ···············64 5 Other pest control genes ····················································································65 5.1 Other pest control genes of plant-origin ·····················································65 5.2 Other pest control gene related to insect development ·······························67 5.3 Second-generation of insecticidal genes ····················································68

Chapter 4 Insecticidal Genes and Elements Regulating Their Expression·······································································································74 1 Design and synthesis of Bt insecticidal gene·······················································74 1.1 Design of Bt GFM Cry1A insecticidal gene·················································74 1.2 In vitro synthesis of Bt GFM Cry1A insecticidal gene ·································76 2 Construction of plant expression vector harboring Bt GFM insecticidal gene····77 2.1 Preparation and addition of 5′regulatory elements·······································77 2.2 Preparation of 3′ regulatory elements···························································78 2.3 Construction of plant expression vector containing Bt insecticidal gene ·····80 3 Proteinase inhibitor genes ···················································································82 3.1 Cowpea trypsin inhibitor (CpTI) genes ························································82 3.2 Potato proteinase inhibitor II gene ·······························································83 3.3 Oryzacystatin (Oc) gene···············································································83 · iv·

4 Construction of plant expression vector harboring protease inhibitor genes·······83

4.1 Construction of dicots expression vector pRCL27 ········································84 4.2 Construction of monocots expression vector pBCA-hpt······························85 4.3 Improving the expression of pest-control genes by protein retention ··········86 4.4 Construction of plasmid pBinΩSCK····························································87 4.5 Construction of plasmid pUSCK-Hyg··························································87 5 Gene expression regulation elements ··································································88 5.1 Promoters ·····································································································88 5.2 Enhancers, exons and introns ·······································································96 5.3 Terminators···································································································98 5.4 Modification sequences················································································99 5.5 Sequences for localized-expression····························································101

Chapter 5 Genetic Transformation of Cotton····························113 1 Agrobacterium-mediated transformation ··························································113 1.1 Progress in Agrobacterium-mediated gene transfer in cotton ····················114 1.2 Protocol for Agrobacterium-mediated transformation in cotton ··············114 2 Pollen-tube pathway (PTP) transformation·······················································119 2.1 Biology of cotton flowering and fertilization·············································120 2.2 Mechanism of cotton germ line transformation ·········································122 2.3 Cotton transformation by PTP····································································123 2.4 Advantages and disadvantages of PTP·······················································124 2.5 Manipulation procedures and influencing factors ······································125

Chapter 6 Development of Transgenic Cotton ·····························128 1 Molecular characterization of transgenic Bt cotton···········································128 1.1 Integration of insecticidal gene in transgenic cotton at DNA level ············128 1.2 Detection of transcription of insecticidal gene at RNA level ·····················132 1.3 Western blotting analysis············································································133 2 Detection of Bt protein in transgenic cotton······················································134 2.1 Establishment of detection method ····························································135 2.2 Results of detection ····················································································137 2.3 Discussion ··································································································141 3 Detection of pest-resistant cotton with trypsin inhibitor gene at molecular level ···············································································································142 3.1 General method for detection of exogenous gene in transgenic plants ······142 3.2 ELISA test of cowpea trypsin inhibitor······················································143 3.3 Detection of inhibition activity of cowpea trypsin inhibitor ······················144

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4 Detection of insecticidal activity of transgenic cotton ······································149 4.1 Method for evaluation of pest-resistance of transgenic cotton···················149 4.2 Evaluation of pest-resistance of transgenic cotton in field·························153 4.3 Pest-resistance of transgenic cotton lines ···················································153 5 The heredity of Bt gene in transgenic cotton·····················································157 5.1 Inheritance of Bt gene in transgenic cotton ················································157 5.2 Genetic stability of pest-resistance in transgenic cotton·····························161 5.3 Pest-resistance of Bt cotton ········································································162 6 Deletion of insect-resistant gene in the filial generations of transgenic cotton by site-specific recombination system·····························································165 6.1 Mechanism of site-specific recombination system·····································166 6.2 Using site-specific recombination system to delete insect-resistant gene in the offspring of transgenic cotton ·······················································168

Chapter 7 Bt/CpTI Insect-Resistant Cotton·································172 1 Construction of plant expression vector harboring two insecticidal genes ·······172 1.1 The ideology of constructing Bt/CpTI genes··············································172 1.2 Construction of expression vector with Bt/CpTI genes ······························174 1.3 Expression of Bt/CpTI genes in tabacco·····················································177 2 Molecular characterization of Bt/CpTI transgenic cotton··································178 2.1 PCR and PCR-Southern detection of Bt/CpTI cotton·································178 2.2 Other molecular analysis method ·······························································178 3 Insect resistance of Bt/CpTI cotton····································································178 3.1 Test of insect resistance of transgenic cotton lines in green house·············179 3.2 Insect-resistance bioassay in laboratory ·····················································179 4 Breeding of the Bt/CpTI cotton ·········································································181 4.1 Insect resistance of sGK321·······································································181 4.2 Yield of sGK321·························································································181 4.3 Agronomic traits of sGK321 ······································································182 4.4 Experiences in the breeding of sGK321·····················································182

Chapter 8 Insect Resistance Management (IRM) for Transgenic Bt Cotton ···············································································184 1 Detection and monitoring techniques for insect resistance ·······························185 1.1 Review of research progress abroad···························································185 1.2 Research progress in China ········································································187 1.3 Discussion ··································································································189 2 Resistance risk assessment ················································································189

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2.1 Review of research progress abroad···························································189 2.2 Research progress in China ········································································190 2.3 Discussion ··································································································194 3 Resistance management ····················································································195 3.1 Review on IRM experience abroad ····························································195 3.2 Research progress in China ········································································198

Chapter 9 Integrated Control of Insect Pests in Bt Cotton ·································································································································205 1 Population dynamics of insect pests and natural enemies·································205 1.1 Target insect pests·······················································································205 1.2 Non-target insect pests ···············································································208 1.3 Natural enemies··························································································211 1.4 Impacts on community structure and diversity of arthropods ····················211 2 Strategies for integrated insect pest control·······················································212 2.1 Economic threshold and control strategy for H. armigera ·························213 2.2 Utilization of natural enemy·······································································213 2.3 Chemical control ························································································214 2.4 Agricultural control ····················································································215

Chapter 10 Safety Assessment of Insect Resistant Transgenic Cotton ··············································································································218 1 Safety assessment techniques and animal models·············································218 1.1 Safety assessment techniques for transgenic cotton ···································218 1.2 Animal model for safety assessment ··························································224 2 Biosafety of Bt cotton························································································230 2.1 Study on gene flow from transgenic Bt cotton ···········································230 2.2 Assessment of toxicity and genetic toxicity of Bt cotton byproducts·········232 2.3 Conclusions ································································································238 3 Safety assessment of transgenic cotton with proteinase inhibitor gene·············239 3.1 The effect on natural environment······························································239 3.2 Effects on non-target organisms ·································································240 3.3 Effect on human and domestic animals······················································240

Chapter 11 Economic Impacts of Bt Cotton in China ················245 1 Introduction·······································································································245 2 Bt cotton development and adoption in China ··················································246 3 Data and surveys ·······························································································248

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4 Performance of Bt cotton in farm fields ····························································249 4.1 Yield impacts······························································································249 4.2 Cost of production impacts·········································································250 4.3 Farmer income impacts ··············································································251 5 Farmer health and environmental impacts ························································252 5.1 Production and price impacts ·····································································253 5.2 Model and estimation results······································································254 6 China and other developing countries ·······························································261 7 Conclusions·······································································································262

Chapter 12 Development of Transgenic Disease-Resistant Cotton················································································································265 1 Chitinase············································································································266 1.1 Primary structure, classification and subcellular localization of chitinase ···············································································································266 1.2 Genes encoding for chitinases····································································267 2 β-1, 3-glucanases and its coding genes ·····························································268 2.1 Basic structure, classification and subcellular localization of β-1, 3-glucanases ································································································268 2.2 Genes encoding the β-1, 3-glucanases ·······················································268 2.3 Biological function of chitinase and β-1, 3-glucanase ·······························269 3 Glucose oxidase and its coding gene·································································271 3.1 Primary structure of glucose oxidase ·························································271 3.2 Function of glucose oxidase·······································································271 4 Other anti-fungal proteins and their genes ························································272 4.1 Plant defensins····························································································273 4.2 Thionin ·······································································································274 4.3 Ribsome-inactivating proteins (RIPs) ························································275 5 Signaling components in plant defense response and their genes ·····················276 5.1 Primary structure and function of signaling components in plant defense response··························································································276 5.2 Genetic engineering of plant broad-spectrum disease resistance ···············278 6 Development of transgenic disease-resistant cotton··········································279 6.1 Development of disease-resistant cotton····················································280 6.2 Evaluation of disease resistance in transgenic cotton·································280 7 Development of disease/insect-resistant transgenic cotton ·······························284 7.1 Disease resistance of transgenic disease/insect resistant cotton ·················284 7.2 Insect-resistance of transgenic disease/insect resistant cotton····················285

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Chapter 13 Genetic Engineering for Cotton Fiber Quality Improvement ·········································································291 1 Introduction·······································································································291 2 Development of cotton fiber··············································································291 3 Strategies and schemes for genetic engineering of cotton fiber ························292 3.1 Identification of fiber quality-related genes ···············································292 3.2 Isolation of fiber-specific promoters ··························································294 3.3 Establishment of experimental systems for rapid functional characterization of selected genes and promoters······································294 3.4 Transformation of gene into cotton ····························································296 3.5 Molecular characterization and phenotypic analysis of transgenic cotton ·296 4 Progress and status of research on fiber modification in China ························296 5 Summary and perspectives················································································297

Chapter 14 Prospect of Cotton Genetic Engineering·········301 1 Cotton functional genomics···············································································301 2 Marker-assisted breeding and gene stacking·····················································302 3 Isolation and utilization of new genes·······························································303 3.1 Insect resistance··························································································304 3.2 Disease resistance·······················································································304 3.3 Herbicide tolerance·····················································································306 3.4 Stress tolerance···························································································307 3.5 Plant nutrition·····························································································308 3.6 Increasing production·················································································309

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Introduction

This introduction aims at briefly describing the essence of the book and summarizing the major research achievements obtained in transgenic cotton research and development under the 863 Program, so that the readers may have a better understanding of the whole book from the very beginning. The development of molecular biology is the greatest event in the 20th century. Plant genetic engineering was a result of perfect combination of recombinant DNA technology and plant tissue culture, by which a first transgenic plant was generated in 1983 that displayed significant academic importance, although the question ‘when it could be used practically’ was unclear at that time. Being contrary to people’s expectation, the growing area of transgenic crops in the world had increased from 1.7 million ha in 1996 to 67.7 million ha in 2003 (40 fold increase, James, 2003, ISAAA Briefs No. 30), demonstrating that the transgenic technology is the most fast growing technology than ever before in the agriculture technology development. Cotton is the most important cash crop in China. The maximum cultivated area had reached 6.66 million ha in the history. However, due to continued breakout of cotton bollworm in Northern China in the early 1990s, the country had suffered heavy economic losses. For example, in the year of 1992 alone, the lint yield reduced 90 million kg, which caused direct economic loss of RMB 5 billion Yuan (1USD≈8.2 Yuan). The disaster caused by cotton bollworm became one of the major factors for the sharp drop down of cotton production in China. Under this severe situation, aiming at solving the key problems faced in the national economy development, the project of “Development of Transgenic Cotton” was launched under the support of National High-Tech R & D Program in 1992. Another reason for selecting cotton as a target crop for genetic engineering of insect resistance is based on considerations of environment and food safety issues. As it is known to all, Bacillus thuringiensis (Bt) has been safely used as a bio-insecticide in crop production for several decades. The crystal insecticidal protein produced in the process of spore formation has a specific spectrum of insect toxicity. So far there is no evidence to show that Bt is toxic to human and livestock, because: (1) The non-toxic Bt preprotein is converted into mature active insecticidal protein only under the basic conditions in the insect mid-gut. The pH in gastrointestinal system of human and livestock is acidic, so it is not optimal for the conversion of Bt protein precursor into toxic protein; (2) It is known that there is no Bt protein receptor on the surface of intestine cells of mammals to which Bt protein may bind. Regarding to cotton, although cottonseed meal and cake are used as animal feed and cottonseed oil is used for human consumption in some areas, cotton fiber is the major target product used as raw material for textile industry. Under the situation of no detailed information available for risk assessment of transgenic plants in the early 1990s, the first choice is

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to use cotton as the first target crop for genetic engineering. The disadvantage is that the cotton transformation technique is not well developed at that time, which is the technical barrier or “bottle neck” for obtaining transgenic cotton plants. At the beginning, the key point was to obtain insecticidal genes. Three research groups from the Biotechnology Research Institute of Chinese Academy of Agricultural Sciences (BRI, CAAS), the Institute of Microbiology and the Institute of Genetics of Chinese Academy of Sciences (CAS) were involved in the synthesis and cloning of Bt insecticidal gene and cowpea trypsin inhibitor (CpTI) gene. According to the plant preferred codon, the Cry1A crystal protein gene together with a series of regulatory elements (for enhancing gene transcription, translation and stabilization of gene expression) were synthesized and plant expression vectors constructed in 1992 by Prof. Guo’s group in the BRI, CAAS. Thereafter, the active Cry1Ac and CPTI gene were synthesized by other two groups, which formed the basis for the development of transgenic insect-resistant cotton. For the establishment of cotton transformation system, the 863 Expert Committee in 1993 decided to organize a project in which several research groups with expertise were involved in order to make a breakthrough to overcome the “bottle neck” problem. A great achievement has been made on cotton transformation by either Agrobacterium-mediated gene transfer or pollen tube pathway (PTP) approach, a unique method developed by Chinese scientist. With the improvement and further optimization of protocol thereafter, the PTP transformation has become a routine procedure for large-scale transformation of cotton with a frequency as high as 1%. The major advantage of this approach is without genotype-dependency; target genes can be transferred into any cotton cultivars in principle. Up to now, different genes have been transferred into more than 30 major cotton cultivars. Compared with the other transformation methods, the PTP is simple, easy to handle, with a relatively short duration for obtaining transgenic plants and no specific facility is required. The histochemical assays, antibiotic resistance, various molecular analyses and bio-functional characterization, etc. have confirmed the integration and expression of the target genes, selectable marker or reporter genes. Through selections and characterizations of the transgenic cotton plants and multiplication in Hainan Island in winter season, the breeding process of homozygous lines or superior cultivars has been accelerated. Since 1995, the Institute of Plant Protection, CAAS, has been authorized by the 863 Expert Committee to do the evaluation of insect resistance in order to confirm or guarantee the results reported by different laboratories. At the same time, breeding programs of insect-resistant cotton have been conducted in different institutions nationwide. Through multidisciplinary research, a great progress has been made as following. 1 Field performance of Bt cotton

The field test and risk assessment of Bt cotton has been initiated since 1996. Generated data was submitted to the National Biosafety Committee of Agricultural GMOs for evaluation. In 1997 the Ministry of Agriculture approved commercialization

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of Bt cotton developed by CAAS. Laboratory and field test of transgenic Bt cotton have shown it has high resistance against cotton bollworm (Helicoverpa armigera). The newly hatched larvae fed on artificial diet with Cry1Ac protein or leaves of transgenic cotton died 3 days after feeding. Under field conditions, the percentage of apical bud and square damage on Bt cotton is less than 1%, while it is generally 40%~90% for non-Bt cotton depending on the year and location. Field evaluation of Bt cotton cultivars GK12 and GK2 in Northern China showed that their efficacy against H. armigera remained over 90% in whole growing season. The number of pesticide spray needed for Bt cotton is only 2~3 times, while it needs 10~15 times or even more for non-Bt cotton. It is absolutely unnecessary to spray pesticide on Bt cotton against the 2nd generation of H. armigera. Therefore the total use of pesticides is reduced by 70%~80%. The reduction of pesticide use is not only environment friendly but also dramatically reduced the accident injury of farmers by pesticides. It has been observed that there is a variation in Bt gene expression in different developmental stages and organs of Bt cotton plants. The insecticidal activity is reduced in the late growing season when plants enter reproductive stage. The order of efficacy of different organs against bollworm is: leaf > square or boll > flower based on the mortality of first and second instars. A certain percentage of neonates may survive after feeding on flowers of Bt cotton that may further feed on bolls until pupation and emergence during the 3rd and 4th generation (July to August). Therefore, it is needed to spray pesticides in late growing season. When it is necessary to spray pesticide against the 3rd or 4th generation of H. armigera depends on the insect density in a particular year and region. Considering the insecticidal activity of Bt cotton against bollworm, the larval number per hundred plants, rather than the egg number per hundred plants, should be used as an indicator for pesticide spray. Assuming the threshold of yield loss is set at 3%, the 13 newborn larvae per hundred plants is proposed as an indicator for spraying pesticide in Northern China. Along with the commercialization of Bt cotton, the cultivation techniques have also been systematically studied. One key point is that because the square and boll dropping rate on Bt cotton is dramatically reduced and the number of bolls per plant increased significantly, it is necessary in supplementing more NPK fertilizers, particularly for potassium since cotton is sensitive to potassium deficiency, which will lead to a physiological disease-red leaf/stem syndrome, resulting in leaf falling, early senescence, and heavy reduction of yield. The commercialization of Bt cotton continuously succeeded and is expanded to a large acreage as cotton growers warmly welcome it. Even in the case of 1999, when cotton growing area was decreased due to the opening up of a cotton free market and drop down of lint price, the Bt cotton was still significantly expanded. In recently years, nearly all farmers would like to grow Bt cotton rather than non-Bt cotton. The total growing area of Bt cotton in 2003 is estimated to be approximately 60% of the total cotton acreage in this country, which provided a huge social, economic and environmental benefit. A survey conducted in Liangshan County, Shandong province showed that the income increased 7,650 Yuan per ha of Bt cotton that includes：4,500 ·3·

Yuan derived from lint increase, 750 Yuan from cottonseed increase, 900 Yuan from reduction of insecticide use, and 1500 Yuan from labor saving. Based on above calculation, the annual economic benefit in whole nation is significant. 2 Variety registrations

To date, 14 cultivars and 3 hybrid cotton varieties containing single Bt gene, and 4 cultivars with Bt/CpTI double genes have been registered. The efficacy of Bt/CpTI cotton against cotton bollworm is superior to the single Bt cotton, particularly in the late growing stage. In addition, transgenic cotton with Bt+API (arrowhead proteinase inhibitor) gene and SKTI (soybean Kunitz trypsin inhibitor) + GNA (snowdrop lectin) gene have been developed. The expression level and insecticidal activity of the modified CpTI gene (targeting signal sequence of KDEL added at C-terminus) are 2~3 times higher than that of the non-modified ones. 3 Inheritance of Bt gene

Genetic studies have shown that both insecticidal activity and agronomic traits of transgenic cotton lines Zhongxin94 and Shanxi94-24 are homozygous. The insecticidal activity of F2 progeny showed 3:1 segregation，selfing showed 1:1 segregation, and intercross of these two lines showed 15:1 segregation, indicating the Bt gene was integrated at two different loci and was non-allelic. No gene silencing was observed when the two loci were heterozygous. After crossing and backcrossing of Shanxi 94~24 with another Bt transgenic line R19, all BC1 were insect resistant, indicating Bt gene was inserted in the same locus in these two lines. Study of cytogenetics showed that the Bt gene is located at the short arm of the chromosome 22 in Zhongxin94, the distance to centromere is 27 centimorgan. 4 Insect resistance management for Bt cotton

rd (1) Based on the assays of ID50 (50% inhibition of new-born larvae to 3 instar) of Cry1Ac protein on different H. armigera populations sampled from 23 sites of 5 major cotton production regions, it was demonstrated that the extensive use of Bt insecticides in early 1990s and widespread use of Bt cotton since late 1990s did not cause detectable increases in resistance of H. armigera to Cry1Ac protein and Bt cotton in Northern China from 1996 to 2003. This baseline information is critical for monitoring of resistance development in bollworm population and adoption of proper measures for resistance management. (2) A high dose/refuge strategy has been adopted in the US and Australia for resistance management of cotton bollworm. The underline scientific basis of ‘refuge strategy’ is that the resistance of bollworm to Bt protein is controlled by a single autosomal locus (G) with two alleles (GR and GS), thus there are three genotypes (GSGS, homozygous susceptable; GRGS, heterozygous; and GRGR, homozygous

·4·

resistant) existed in insect population. If homozygous resistant individuals produced in Bt cotton field breed with homozygous susceptible ones in non-Bt cotton (refuge), the resulted heterozygous progeny will still be susceptible to Bt. However, in practice, the ‘refuge strategy’ by parallel planting of Bt cotton and non-Bt cotton is not feasible for cotton production in China (except Xinjiang autonomous region), where the multiple cropping systems are adopted and the farmers only own a small piece of land. Entomologists have evaluated the function of ‘natural refuge’ provided by the multiple cropping systems for insect resistance management. It has been found that 70% of the 4th generation of cotton bollworm larvae is raised in the cornfield in northern China, which provides a huge ‘natural refuge’. Entomologists have also found that the long distance migration behavior of bollworm that migrates from central part of China to northern (where cotton is not planted) in spring and returns back to central in autumn also played a role in minimizing insect resistance development. (3) Research has indicated that the bollworm strain resistant to Cry1Ac is not cross-resistant to Cry1C and Cry2Aa. However, the efficacy of Bt corn expressing Cry1Ab to a Cry1Ac-resistant strain of bollworm is significantly lower than that to non-selected control strain, indicating there is a cross resistance in H. armigera between Bt cotton and Bt corn. Therefore, to reduce the selection pressure and delay the resistance development in H. armigera and Asian corn borer, it is recommended that simultaneously commercialize of Bt cotton and Bt corn in the same region is inadvisable. (4) According to a laboratory simulation model for analysis of resistance development in H. armigera to Bt protein, it has been predicted that the single Bt cotton can effectively control bollworm for at least ten years. Due to the complexity under the field condition, this prediction should be further evaluated. (5) Gene stacking study has shown that the resistance ratio (RR) of transgenic Bt and Bt/CpTI tobacco to Cry1Ac were 13.1 and 3.0, respectively, after 18 generation of selection in the laboratory. This is the first laboratory data to demonstrate that double-gene can significantly reduce the resistance development in H. armigera. Similar to the single Bt cotton, the insecticidal activity of Bt/CpTI cotton is higher in early growing stage than that in late stage. However, the overall insecticidal activity of squares and flowers on Bt/CpTI cotton is higher than that of single Bt cotton, and the insecticidal activity to the 2nd to 4th instars of a selected resistant strain of H. armigera is significantly enhanced. The first to fourth instars of resistant strain of H. armigera lost ability of pupation after continuous feeding with leaves of Bt/CpTI cotton; only a few 5th instar larvae could develop into pupae. While for the single Bt cotton, some percentage of 2nd instar larvae of resistant strain was able to develop into pupae. (6) Studies of population dynamics of non-target pests in Bt cotton field showed that due to no spray of pesticides in the 2nd generation of bollworm, the number of natural predator enemies such as ladybirds, lacewings and spiders in Bt cotton field increased significantly, which effectively controlled the size of aphid population in summer. The number of aphids on top three leaves of non-Bt cotton sprayed four times with pyrethroid or organophosphorus was 443~1,546 fold higher than that of Bt cotton. At the same time, due to the less pesticide used, the mirids and spider mites might become major pests in Bt cotton field. Attention should be paid for control of such pests. ·5·

The general conclusion is that the integrated pest management (IPM) for Bt cotton should be mandatory carried out and further strengthened, and the development of insect resistance to Bt cotton should be continuously monitored. 5 Gene flow from transgenic cotton

It is known that cotton pollen dispersal is mediated by insects. Experimental data of gene flow from transgenic 2,4-D-resistant upland cotton containing tfdA gene to non-transgenic upland cotton showed that at 1 meter apart the gene flow frequency could be as high as 11.2%, while it was falling down to 0.03% at 50 m; no outcrossing was found at 100 m or farther. Thus a distance of 100 m isolation buffer is a proper measure for small-scale field test of transgenic cotton, which is in consistent with the experiences in cotton breeding and seed production. It should be noted that the frequency and maximum distance of gene flow may differ from place to place depending on the kinds and population size of pollinators existed in different ecological zones. Since there is no wild relatives existed in the cotton growing area in China, the environment consequences of transgene flow to wild relatives is of little concern. 6 Safety assessments of Bt cotton byproducts

Toxicity and mutagenicity test of Bt cottonseed meal and oil on mammal (mouse as a model) and fish (zebrafish, Branchynanio rerio) have proved that neither Bt cotton nor non-Bt cotton is harmful to experimental animals in 30 d and 90 d feeding test. (1) By using cyclophosphamide or sodium fluoride as revulsant, conventional cottonseed meal and oil as negative control, the micronuclei frequency (MN%) in polychromatic erythrocytes (PCE) and the sperm malformation rate in mice fed with Bt cottonseed meal or oil for 30 d were assayed. No mutagenicity and dose effect to mouse somatic and sexual cells were observed. (2) No toxicity effect of Bt and non-Bt cotton byproduct was observed as indicated by MN% and sperm malformation rate in zebrafish fed with Bt and non-Bt cottonseed meal or oil for 90 d. No abnormal symptoms were found in anatomic and pathological assays. Meanwhile, polyclonal, monoclonal antibodies and detection kits have been made available for identifying Bt protein expression in transgenic cotton. No false positive result was obtained on non-Bt cotton. 7 Development of transgenic cotton resistant to Fusarium and Verticillium wilt Fusarium and Verticillium wilt are two major diseases in cotton production in China. Although Fusarium wilt is no longer a problem as resistant varieties have been developed through conventional breeding, there are no effective measures yet to control Verticillium wilt and no resistant germplasm or cultivars of upland cotton (Gossypium hirsutum) available. The current major cotton cultivars are only tolerant to

·6·

this disease (relative disease index <10, highly resistant; 10∼20, resistant; 20∼35, tolerant; >35, susceptible). Although the Sea Island cotton, G. barbadense, closely related species of G. hirsutum, has higher resistance to Verticillium wilt, it is not yet succeed to transfer their resistant genes into upland cotton by conventional breeding. The disease resistant genes are usually lost during continuous backcrossing. Therefore, Verticillium wilt is still a big headache problem in cotton production, which has become more and more serious and occurred in all cotton production regions in China since 1990s. According to the incompleted statistics, the infected area by Verticillium is over 3 million ha. To solve this problem, a project on the development of transgenic cotton resistant to Fusarium and Verticillium wilt was initiated under the support of 863 Program. Several fungal resistant genes including chitinase, β-1,3-glucanase, antifungal protein 1 (R-afp1) from Raphanus sativus, glucose oxidase, NPR1, Sgt1 and Rar1, etc. have been transferred into cotton by pollen tube pathway. Several transgenic cotton lines with enhanced disease resistance have been developed by continuous selection and characterization in a period of severe years. Meanwhile, the fungal resistant genes have been successfully introduced into insect-resistant Bt cotton. 8 Genetic engineering for improvement of cotton fiber quality A number of cotton varieties with higher adaptability, yield, fiber quality and resistant to Fusarium wilt have been bred and cultivated since the establishment of People’s Republic of China, which terminated the history of dependence on US cotton varieties for cotton production in China. In general, the fiber quality of domestically developed varieties meets the requirement of textile industry, but it is still necessary to develop special cotton varieties for particular uses. On the other hand, the focus of international research on crop genetic engineering has been shifted from the first generation of genetically modified (GM) crops with herbicide-, insect- and disease- resistance to the second generation of GM crops with higher quality and added value, etc. Based on the rapid development of recombinant DNA technology, especially the studies of genomics, the 863 Program has initiated a project on the genetic engineering of cotton for fiber quality improvement since March 1999, by which preliminary results and progress have been made.

In conclusion, our practice has demonstrated that it is a right decision for 863 Program to not only emphasize the innovation required for high technology development, but also combine the biotechnology with conventional breeding and multidisciplinary technologies for target product development. As a national coordinator for this project, I would highly appreciate and sincerely thank all the people who have devoted their efforts to the project.

(Written by JIA Shirong)

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Chapter 1 General Situation of Cotton Production and Breeding in China

There are 75 cotton-producing countries all over the world, located between 32° south and 47° north latitude. Over 70% of the cotton production is concentrated in 5 countries, including China, United States, India, Pakistan and Uzbekistan where each produced lint cotton over 1 million tons. Since the 1950’s, the total cotton growing area has been relatively stabled at 31.8~33.1 million ha, which occupying about 5% of the harvesting area of field crops all over the world (Liu, 1995). Cotton is a fine natural fiber with low production cost and high yield. Unlike wool and silk, its price is not so dear and the consumption is not limited. It possesses characteristics of moisture absorption, air ventilation, warm keeping, soft feeling and electrostatic resistance, etc. that artificial fiber could not imitate and replace. Since the 1990’s, along with the improvement of people’s living standards, wearing clothe made of natural fiber has become an irreversible international trend. Besides, cottonseed oil and protein are important sources of vegetable oil and protein. They accounted for 10% and 6%, respectively, of the total supplying quantity of edible vegetable oil and proteins in the world. Cotton staple (short fiber), gossypol, straw and shell of cottonseed is row material with industrial usage. There are four cultivated cotton species in Gossypium genus. Two of them are diploid species: African cotton [Gossypium herbaceum L. (A1)] and Asian cotton [G. arboreum L. (A2)]. The other two are tetraploid species: upland cotton [G. hirsutum L. (AD)1] and Sea Island cotton [G. barbadense L. (AD)2]. In the world’s cotton production, upland cotton occupies the most growing areas that produces 90% of the total cotton lint, followed by Sea Island cotton, which accounts for 5%~8% of the total (Huang, 1996). 1 General situation of cotton production in China

At the initiate stage, cotton was introduced into China and its first settlements are in South China and Xinjiang. In the 3rd century B.C., cultivation and processing of annual cotton in Hainan Island already existed. In the 13th century, cotton was planted in the Yangtze River Valley and the Yellow River Valley. Asian cotton and a portion of African cotton (mainly in Xinjiang) were planted for a long period of time. The fibers of Asian cotton and African cotton are thick and short, which could not meet the requirement of textile machines. Along with the upsurge of textile industry, upland

·8·

cotton was introduced from the United States since 1870’s, which is suitable for machine woven and with better fiber quality and high yield. At present, except Xinjiang, where a proportion of Sea Island cotton is planted, the rest parts of China are grown with upland cotton.

1.1 Role of cotton production in China’s national economy

China is a big cotton producing and raw cotton consuming country. In other words, it is either a big importer of raw cotton and a big exporter of textile products. Cotton cultivation has approximately 2,000 years of history in our country (Huang, 1996). During recent 20 years since reform and open-door policy has been implemented, China has been ranked as one of the largest cotton producer in the world. Therefore, up and down of cotton production will have a decisive influence to the economic development. In the 1980’s, the normal cotton growing area was 6~6.3 million ha, accounting for 1/5~1/6 of the world’s total and was ranked the second in the world. The total cotton lint yield for an average year was about 4.5 million tons, taking 1/4 of the world’s total and was ranked first in the world. The average annual raw cotton consumption is 4.5 million tons, also taking 1/4 of the world’s total consumption and ranked first in the world. The total production value of the cotton takes 7%~10% of that of the entire plant industry in China. In recent years, the cotton growing area has been reduced for various reasons. In 1998, it was only 4.46 million ha. There are over 300 million populations in the cotton producing areas and about 200 million farmers make a living on cotton growing. Not any other industry could support such a big rural population, especially in the less developed and developing Mid-West part of China. Cotton production is a labor-intensive industry, for example, the average labor expense was 585 labor/ha in 1990. Even if in 2010, it will still be about 450 labor/ha (Mao, 1998). Just like the Chinese people solved the grain provision, they have to solve clothing problem by themselves. There exists a huge domestic garment consuming market. In 1995, the average fiber consumption per Chinese was only 4.6 kg, which was far lagged behind the developed countries (that for per American was 30.9 kg, 23.4 kg for Japanese and 19.5 kg for EU resident). Along with the improvement of people’s living standards, garment market will be further expanded. It is forecasted that by 2010 population in China will be 1.41 billion and the average fiber consumption per capita will reach 6.2~6.4 kg, increasing by 2.5% annually, i.e. a net of 1.5 million tons of raw cotton need to be increased compared with that in 1990 (Mao, 1998). Cotton and textile products are two supporting industries for exporting in China. The total annual value earned from its exportation takes nearly 1/4 of the country’s export value. There is a tendency of increasing export year after year, for example, in 1970, the total export value was 2.932 billion US dollars; in 1980, the figure increased to 6.666 billion US dollars. It became 8.0 billion in 1987, 13.8 billion in 1989 and 27.48 billion US dollars in 1995. In 1998 the export of textile products and garments amounted to as high as 42.85 billion US dollars, which takes 23.3% of the country’s export value. This indicates that textile products and garments mainly made of cotton ·9·

are one of the major exporting commodities in China (Mao, 1998).

1.2 Development of cotton production in China

During the 30 years before P. R. China was founded, the average annual lint yield was 9.16 million Dans (1 Dan = 50 kg). In 1949, cotton growing area was 2.77 million ha, unit lint yield was 160.5 kg/ha and total lint production was 444,000 tons, which took 6.2% of the total world production and was ranked 4th place. After the founding of P. R. China, cotton production was increased rapidly. In the 1980’s, average annual cotton growing area was 5.396 million ha and annual cotton lint production was 4 million tons. Cotton growing area was doubled; unit yield and total production increased 4.5 and 9 times, respectively. China has joined the rank of countries with high-yield of cotton and become the number one big cotton producing and consuming country in the world (Huang, 1996). Cotton growing area and production during 1949~1994 in China are shown in Fig. 1-1, 1-2 and 1-3 (Agriculture Bureau of Ministry of Agriculture & Jiangsu Bureau of Agriculture and Forestry, 1998). Owing to the constant attention paid by the government, cotton production has gained remarkable achievements. Especially during 1980~1984, which was a golden period for cotton production in China, there were 5 consecutive years of bumper harvests. A great achievement in cotton production has attracted worldwide attention: (1) In 1982, China for the first time realized self-sufficient on raw cotton and completely ended the long history of importation. (2) In 1983, China became a country with unit lint yield surpassing 750 kg/ha. The ration system of cotton products, which was implemented for nearly 30 years, was all-out abandoned. (3) Meanwhile in 1983, China became a net cotton exporting country. However, during the past 40 years, the development of cotton production in China was extremely uneven. There existed 5 big ups and downs. Among these over 40 years, there were only 4 years (1982~1984 and 1991), when demand and supply were in balance or supply surpassed demand, which was less than 1/10. In most of those years, demand surpassed supply, thus we had to spend huge amount of foreign currency to import large quantities of raw cotton at high prices. Reasons for ups and downs of cotton production are as follows: the low yield in 1962 and 1972 was mainly because of the sharp conflict between grain crops and cotton, which competed each other for land, labor, fertilizer and water. In addition, infrastructure at that time was incomplete; technology of cotton breeding and cultivation was not advanced, which greatly limited the development of cotton production. The later 3 times of low yield was mainly due to the high cost/low output, which significantly affected the enthusiasm of farmers to grow cotton. Recently, cotton production has dropped down again, since there is a relatively high amount of cotton in the storage and many textile enterprises directly use lint fibers from abroad because of higher price of domestic one. Technically, another important issue was the breakout of cotton bollworm in Northern China starting from 1990’s, which severely damaged cotton plants and caused a great deal of economic loss.

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Fig. 1-1 Cotton growing areas from 1935 to 1995 in China

Recalling the previous cotton production, it is found that cotton production in China is lack of an atmosphere for a planned and stable development way, in which it is unable to adjust between year of bumper harvest and poor harvest. Cotton production in our country has been in a passive situation for many years, sometimes even out of control.

Fig. 1-2 Total cotton lint production from 1935 to 1995 in China

Since China is a large cotton producer, the yield of cotton produced in China directly influences the price of raw cotton in international market. In the late 1970’s, cotton production in China had poor harvest for consecutive years. In 1980, 900,000 tons of raw cotton was imported, which took 21% of the total cotton trade of that year in the world resulting in lint price in international market soaring to 82~87 cents/lb. In ·11·

the early 1980’s, cotton production in China had bumper harvest for consecutive 5 years. In 1987, 755,000 tons of lint cotton was exported, which took 18% of the total international cotton trade and therefore, price of lint cotton dropped down to the lowest point over past 10 years, only 32~36.5 cents/lb. Cotton production was dropped again in 1993. As a result, in the following year about 500,000 tons of raw cotton was imported, which once again enabled international price rising to a new height, almost 1 dollar/lb. In 1995, price of raw cotton broke 1 dollar/lb and reached 106.5 cents/lb (Ma, 1999).

Fig. 1-3 Average national cotton lint yield

1.3 Cotton production regions in China

Cotton growing covers a vast area in our country including the Liao River Valley, the Yangtze River delta in the east, west to Talimu Basin, south to Ai County of Hainan Island and north to the Manarshi River Basin. In these broad areas, there are big differences in terms of climate and cultivars suitable to grow. In the early 1950’s, 5 big cotton-growing regions were divided. They are south China, the Yangtze River Valley, the Yellow River Valley, the Liao River Basin (it is now referred to as north earliest-maturity region) and north-west inland (Table 1-1). After over 40 years’ change, there are only a few cotton growing areas in the Liao River Basin and south China region. At present cotton growing mainly concentrated in the following 3 regions (Huang, 1996). 1.3.1 North-west inland cotton region It mainly refers to Xinjiang including a few cotton fields in Hexi Corridor of Gansu. Weather in this region is typical continental, dry and arid, small amount of rainfall, abundant sunlight and big temperature differences between day and night. These conditions are very favorable for photosynthesis of cotton plants to accumulate higher amount carbohydrates and other organic compounds. By adopting appropriate cultivation measures it is possible to increase plant density, reduce dropping of squares

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Table 1-1 Natural conditions for the five major cotton growing regions

Main border of the Non-frost Average annual Annual precipitation region Major soil Suitable cotton varieties region (d) temperature(ć) (mm) Yellow River Valley South of Great Wall, 180 ~ 230 ˚11 400 ~ 700 Lime alluvial soil Medium (early) mature upland Qin Mountain, Funiu cotton Mountain, north of Huai River

Yangtze River Valley Qin Mountain, Funiu 230 ~ 280 ˚15 750 ~ 1500 Lime-null alluvial Medium mature upland cotton Mountain, south of soil Huai River and north of Nan Mountain

North (earliest North of Great Wall, 150 ~ 170 8 ~ 10 500 ~ 700 Lime alluvial soil Earliest maturity of upland maturity) and central Shanxi, cotton south Gansu

North-west North, south and east of 180 ~ 230 8 ~ 12 ˘200 Saline-alkali soil Medium (early) mature upland Inland Xinjiang, Lime soil cotton, early mature Sea Island Gansu Hexi corridor 130 ~ 160 8 ~ 10 200 cotton, earliest maturity of upland cotton

South China Guizhou, south of ˚320 ~ ˚20 1200 ~1800 Yellow soil, red soil Upland cotton and Sea Island Fujian, Yunnan, All year cotton Guangxi and Taiwan non-frost

and bolls, so as to achieve high and stable yield. In addition, cotton fields in this region are mainly vast and plane, which is favorable for large-scale machinery cotton production to save labor and reduce cost. Land per capita in Xinjiang is larger and conflict between grain crops and cotton for competing land is smaller. In 1988, the State Council decided to list Xinjiang as a national key base for cotton production. The acreage of cotton in Xinjiang has extended from 267,000 ha in the 1980’s to 435,000 ha in 1990 with yield of 468,800 tons. In 1998, the cotton-cultivated area increased to nearly 1 million ha and the total output was 1.4 million tons. It accounted for a 22.4% of the total cotton acreage and 31.1% of the total lint production that year of the nation. 1.3.2 The Yangtze River Valley cotton region Cotton is mainly cultivated in the riverbanks of the middle and lower valley of the Yangtze River, around lakes, plains near seacoast and partly in hilly areas. Weather conditions in this region are rich in heat resources allowing cotton plants to grow for a longer period of time, but sunlight is insufficient. In most areas there is plenty of rainfall in spring and early summer and frequent drizzles in autumn. Grain or oil crops are commonly inter-planted with cotton in this region or a multiple cropping system is adopted with two crops per year. In the 1970’s, this region with Jiangsu and Hubei as its representative, took 46.0% of the cotton growing area and approximately 60.0% of the total lint production. In the 1980’s, cotton-growing area was reduced owing to the low comparative efficiency of cotton and the well-developed economy in this region. It was stabled at about 2 million ha in the 1990’s. 1.3.3 The Yellow River Valley cotton region It is the largest cotton growing area in China located at the sub-moisture east monsoon climate region of the South Temperate Zone. Hebei, Shandong and Henan are its representatives. In the early 1980’s, the cotton growing area and yield took 50% and 46% of the total respectively. Since 1990’s, cotton planting has been reduced greatly and yield dropped, due to severe damage caused by insect pests and diseases, decrease of cotton fields’ quality, low cotton yield per unit and poor comparative efficiency. In 1993, there were 2.44 million ha of cotton planted and 1.386 million tons of lint produced, which was 48% and 37% of the whole nation’s figure respectively.

1.4 Current situation of cotton production

Along with the reformation of cotton marketing system as well as the gradual formation of internationalized market, cotton production in China is confronting new challenges. Recent situation of cotton production, importation, consumption and storage are shown in Table 1-2. It was clear from this Table that the total cotton production remained between 4.2~4.5 million tons. Since 1994, China has owned over 41 million spindles, which will consume 4.6~4.9 million tons of cotton. Therefore, cotton supply and consumption in China is basically balanced with a small deficiency. But since 1995, serious problems have occurred including the stagnant selling of ·14·

domestically produced cotton, the sharply increasing of cotton storage and the deficit of cotton and flax companies. The reason for that is China has become the largest cotton importing country with the accumulated import volume of 2.025 million tons during 3 years (Xiang et al., 1999). The sharp increase of import is due to the price of imported cotton Power than that of domestic one, yet with guaranteed quality. Facing this severe situation, it is necessary to adjust allocation of cotton production in different regions, e. g. to slow down the development speed in North-west inland, to adjust structure of cropping system in the Yangtze River Valley, to reduce cotton acreage in the Yellow River Valley etc.; at the same time to adopt other necessary measures in enhancing competitiveness and economic efficiency of cotton plantation (Ma, 1995).

Table 1-2 Cotton production, importation and consumption in China(10 thousand tons)

1995/1996 1996/1997 1997/1998 Production 450 420 430 Net import 85 78.5 39 Availability 821.4 870.2 884.2 Consumption 450 455.0 470 Closing Inventory 371.4 415.2 414.2

2 Achievements and present status of cotton breeding in China Historically all cotton grown in China was Asian cotton. In the 1920’s and 1930’s, some varieties of Asian cotton were developed, e. g. chicken-paw cotton (leaf shape like chicken-paw). They were later replaced by the introduced upland cotton cultivars such as Stoneville and Deltapine varieties. Since the founding of the People’s Republic of China, attention has been paid to the introduction and characterization of foreign varieties (Huang, 1996). Along with the establishment of institutions, cotton breeding system has gradually been developed aiming at selection of cotton varieties adaptable to different regions. Especially in the early 1980’s when a task force project was set up, cotton breeding was further strengthened. The breeding methods were shifted from systematic selection to hybridization between different cultivars, followed by mutagenesis breeding by radiation and gene transfer in succession.

2.1 Cotton breeding achievements in China

Since the founding of the People’s Republic of China, 6 times for large-scale variety replacement have been carried out. Each replacement brought about big yield increase and fiber quality improvement. In 1950, Deltapine and Stoneville cotton varieties were introduced from the

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United States that replaced Asian cotton and deteriorated foreign varieties that had been grown for a quite long time in China. As a result, the improvement of cotton cultivars was realized. Firstly, Stoneville 2B and 5A were extended in the Yellow River Valley and Deltapine 15 was extended in the Yangtze River Valley as well as Yellow River Valley. The largest yearly growing area reached 3.5 million ha. Apart from these, 108 Fu and KK1534 were also introduced from the former Soviet Union to Xinjiang. In the 1960’s and 1970’s, by focusing on the varietal improvement, the domestically developed cultivars reached international level with increased yield and fiber length. By the end of 1970’s, the growing area of domestically developed varieties occupied over 80% of the total, hence the situation that alien varieties took the leading place in cotton production was ended. For example, there were Xuzhou 1818, Xuzhou 209, Shiduan 5, and Jihang 3 in the Yellow River Valley, Dongtinghu 1, Penze 4, Daihongdai, and Shimian 1 in the Yangtze River Valley. Lumian 1 and Shimian 2 selected and released in the early 1980’s have brought about another big yield increase. Since 1980’s, the rapid spread of cotton Fusarium and Verticillium wilt diseases have led to a large area of cotton plants death, even no harvest in some severe occasions. Requirement for cotton with superior quality has also been raised. Therefore, the aim of breeding projects has been shifted to and focused on the selections of varieties with high yield, superior fiber quality, resistant to diseases, and with different maturity time suitable for different regions. During this period, the disease susceptible varieties or varieties with poor quality were replaced by relatively resistant varieties or varieties with medium class of fiber quality, respectively. At the same time, cotton cultivars with short growing season were expanded in order to meet the requirement for growing wheat and cotton per year (two-crop system) in some areas. For example, disease resistant varieties are bred including Zhongmiansuo 12, Jimian 14, Yumian 4, and Yanmian 48. Zhongmiansuo 12 is an upland cotton variety domestically developed by our scientists with high and stable yield, resistant to Fusarium wilt and tolerant to Verticillium. In 1991, its growing area reached 1.7 million ha. Summer sowing varieties suitable for two-crop system (wheat followed by cotton) and short season varieties have also been bred including Zhongmiansuo 16, Lumian 11, Emian 13 and Xinluzhao 2, etc. (Huang, 1996). At present, growing areas of hybrid cotton are continuously expanding. In 1998, it was extended to 270,000 ha, taking about 6% of the total in China. Along with the development of plant biotechnology, two kinds of transgenic insect-resistant Bt cotton, Bollguard 33B developed by Monsanto and a series of Guokang varieties developed by Chinese scientists were grown around 150,000 ha in 1998. In 2003 it is estimated that the area of Bt cotton is about 2,797,000 ha occupying 57.8% of the total in China, out of which Monsanto’s Bt cotton accounted for 45% while the domestically developed Bt cotton accounted for 55% (CCAP data base, 2003). Germplasm resources are important bases for cotton breeding. Early in the 1920’s, China started to collect cotton germplasm resources. Since 1950’s, cotton research institutions all over China have carried out study tours and collections. Through international exchange, cotton germplasm resources in China have been expanded and enriched, which provided abundant materials for breeding and basic research. At

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present, 6,224 accessions are preserved, including 5,020 upland cotton, 492 Sea Island cotton, 348 Asian cotton, 14 African cotton and 350 semi-wild accessions of upland cotton. Seventy characters of these collections, in terms of biology, agronomy and economic traits, have been evaluated. For over 40 years, cotton-breeding methods changed a great deal. From the 1940’s to 1960’s, varieties developed by systematic selection accounted for about 50%, while about 25% were derived from hybridization. In 1970’s and 1980’s, varieties bred by hybridization method increased to 56% and 84% respectively. Hybridization has been shifted from mainly single cross to complex crosses in which multiple parents are involved. Varieties of Zhongmiansuo 12, Shimian 2 and 3, which have large extension areas, are all developed by hybridization method. In addition, modified backcrossing, recurrent selection and mixed crossing and selection, etc. is also performed (Pan, 1998). In the area of basic research, China has first reported the regeneration of plants from protoplast culture of upland cotton and created a genetic transformation system via pollen-tube pathway that promoted cotton genetic engineering. Besides, China is also leading in the utilization of cotton male-sterility and heterosis, the comprehensive utilization of cotton-seed protein, and the techniques for propagation and refinement of the elite varieties, etc. (Pan, 1998).

2.2 Two key issues confronting cotton breeding in China 2.2.1 Fiber quality improvement At present, the fiber quality of varieties largely popularized in cotton production is of medium grade, although it can basically meet the needs of textile industry, the quality is too unitary. In 1996 and 1997, The Center of Cotton Fiber Quality Supervision and Testing, Ministry of Agriculture, sampled and tested 39 major cotton varieties from 11 provinces (regions). Results indicated that varieties with average fiber length exceed 29 mm accounted for 89.1%. The average fiber strength was 19.79 cN/tex (1 cN/tex = 0.98 g/tex) in 1996 and 20.97 cN/tex in 1997. Varieties with strength >19.6 cN/tex was 64.5% and 80% in 1996 and 1997 respectively. Fiber fineness of varieties within the normal range (3.5~4.9) of Macronaire value was 90.3% in 1996 and 84% in 1997. In conclusion, most (over 70%) of the quality of raw cotton produced in China is ranked at a level of medium or upper medium internationally. They can meet the needs of Chinese textile industry at present. The fiber length is sufficient to meet the requirements for producing fabrics with medium or medium to high yarn sizes (Table 1-3, 1-4). Besides, the percentage of short fibers in raw cotton in China is low and there are few leaf debris and other substances inside. Therefore, the raw cotton produced in China is competitive in the international market (Xiang et al., 1999). The major problem of Xinjiang cotton is cotton fiber attached by external sugars that brings stickiness to textile processing. Cotton varieties grown in Xinjiang, no matter if it is introduced from inland or self-bred in Xinjiang, all contain different degrees of external sugars, while all varieties grown in inland do not contain external

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sugars. The main reason for this is aphid infection, especially autumn aphid that contaminated cotton fibers with external sugar waste. Therefore, it is necessary to develop cotton varieties resistant to aphids by genetic manipulation or by insecticide control.

Table 1-3 Quality index of cotton fibers needed for three types of machine woven 12 (yarn number, British system) 36 (yarn number, British system) Fiber Quality Round spindle Airflow Air-jet Round spindle Airflow Air-jet Fiber strength (cN/tex) 16.75 18.22 / 18.22 22.05 20.48 Micronaire value 5.0 4.5 / 4.5 3.7 3.8 Fiber length (mm) 25.4 22.9 / 29.2 27.9 31.8

Table 1-4 Quality comparison between domestic cotton and imported cotton No. of Fiber strength Micronaire Sample source Length(mm) Uniformity (%) samples (cN/tex) value Xinjiang (China) 14 29.1 47.7 22.1 (20.4 ~ 23.9) 4.5 United States 12 28.3 46.1 22.7 (20.4 ~ 25.8) 4.4 Russian and its associated 6 28.7 46.1 22.1 (20.2 ~ 23.9) 4.5 independent countries Australia 3 29.6 42.0 21.0(21.1 ~ 22.6) 3.8

At present, China still uses round spindle as major textile processing. Airflow only takes 7.5% of the total, mainly for weaving fabrics with low yarn numbers so as to increase the rate of raw material utility, reduce production cost and improve production efficiency. In round spindle weaving, the fiber length is a basic index that determines different numbers of yarns to be woven. If fine and high yarns above 40 and 60 are woven, the raw cotton must possess fiber length > 29 mm and 31 mm, with Macronaire value around 4.0. For airflow weaving (Table 1-5), the domestically produced cotton is still not applicable due to its lower fiber strength and higher Macronaire value (Xiang et al., 1999).

Table 1-5 Percentage of various numbers of yarns and requirement for fiber length Number of Yarn (Ne) Number (tex) Percentage (%) Fiber length (mm) British system 5 ~ 6 120 ~ 80 1.56 Long fiber cotton 10 ~ 14 60 ~ 50 48 31 14 ~ 20 42 ~ 30 48 29 22 ~ 30 26 ~ 20 31.88 27 32 and above 18 and under 18.55 25

2.2.2 Stress-tolerance, in particular, Verticillium wilt-resistance According to the statistics made by Ma et al. (1997), there were 44 varieties in Northern China registered provincially. As a matter of fact, none of them is indeed

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