棉花规模化转基因技术体系构建及其应用

doi:10.3864/j.issn.0578-1752.2014.21.005

摘要/Abstract

摘要： 文中综述了国内外转基因技术在棉花中的应用概况，主要介绍了近年来中国棉花规模化转基因技术体系的建立及应用研究进展，并对棉花转基因技术中存在的主要问题和未来发展趋势做了相关陈述。对棉花科研工作者了解棉花转基因研究进展并有效利用转基因技术开展工作具有重要意义。转基因技术在克服棉铃虫危害上取得了巨大成功，并将逐步在棉花抗病、抗逆等方面取得重要进展。世界上，棉花转基因初期主要建立了以珂字棉为受体的转基因体系，随着雷蒙德氏棉、亚洲棉、海岛棉、陆地棉等其他棉种组织培养体系的建立，农杆菌介导法、基因枪轰击法、花粉管通道法及其他转基因方法的应用，使得棉花转基因技术研究取得长足的进步。中国棉花规模化转基因技术体系主要是依托中国农业科学院棉花研究所及其他科研单位建立起来的，通过高效转化载体的筛选、主要棉花品种（系）的转基因技术体系的建立、组织培养条件的优化等措施，重点对农杆菌介导法转化棉花技术进行了改良，同时优化了基因枪轰击法及花粉管通道法转化技术，形成了三位一体的棉花规模化转基因技术体系。该体系建立了以中棉所24等材料为转基因受体的农杆菌介导体系，并利用叶柄组织培养筛选获得了组织培养分化率达100%的新材料W12等，使转化率提高到原有效率的2.88倍，同时建立了基因枪胚性愈伤组织轰击转化体系，并提高了花粉管通道的转化效率。该体系为棉花育种提供了大量材料，中国农业科学院棉花研究所已培育多个棉花抗虫新品种，并为国内41家科研单位转化基因200多个，验证了多个功能基因作用获得大量育种价值新材料。笔者认为基因型依赖性仍然是限制棉花规模化转基因技术发展的瓶颈，扩大棉花转基因受体材料基因型范围、提高转化效率、扩大转化规模是棉花转基因技术体系的长久主题。同时，为提高效率和降低安全性的公众焦虑，探索和发现新的更为安全和有效的转化体系，如多基因共转化、质体遗传转化、定点转化或基因叠加、开发安全型转化技术等研究引起人们的普遍关注，是未来棉花转基因技术的发展趋势。大量新基因处于材料阶段，同时安全性评价要求提高也需要转基因材料深入的研究。随着棉花基因组序列的公布，棉花自身基因的克隆将会成为棉花基础研究和应用研究的新方向，对于促进转基因棉花新品种培育和功能基因转化研究具有重要参考价值。

关键词: 棉花, 转基因技术, 规模化, 基因型依赖, 安全转化体系

Abstract: This assay is a summary of the application of transgenic technology in cotton all over the world, including the establishment and research progress of cotton transgenic technology for large-scale performance in China in recent years, the main problems and development trend of transgenic technology in cotton, which gives an insight into the transgenic cotton for researchers and makes a great significance to help them working efficiently. Transgenic technology has achieved great progress in cotton bollworm resistance by GMOs, and will gradually get important progresses in disease and adverse resistance in cotton, etc. Early transformation events are transformed by Coker cultivars, and nowadays rapid progresses have been made in transgenic technology for the success of different cotton species of tissue culture in Gossypium raimondii, G. arboretum, G. barbadense, G. hirsutum, etc. and three major transformation methods containing biolistic particle, pollen tube pathway and Agrobacterium-mediated transformation. Cotton transgenic technology for large-scale performance in China is established mainly by Cotton Research Institute of Chinese Academy of Agricultural Sciences and other institutes in China. It forms a trinity system for cotton large-scale transformation by focusing on the optimization of Agrobacterium-mediated method through the selection of efficient transformation vectors, transformation system establishment of major cotton varieties or lines, optimization of tissue culture conditions, in addition to the improvement of biolistic particle and pollen tube pathway methods. This transformation system involves in establishment of Agrobacterium mediated transformation system selecting CRI24 as the transgenic receptor, getting new materials such as W12 whose differentiation rate is up to 100% by petiole tissue culture screening and transformation efficiency increased by 2.88 times compared with before, development of embryonic callus transformation system by biolistic particle method, improvement of the transformation efficiency by pollen tube pathway. Using the transform system, many transgenic materials or lines are obtained and some of them are bred to insect-resistant cotton varieties, more than 200 genes for 41 scientific research institutes in China have been identified their gene functions, and provides a large number of new materials for cotton breeding. It is concluded that strong genotype-dependence is the bottleneck for cotton transformation, expanding the genotype of transgenic receptor, improving the transformation efficiency, and scaling up the system are the subjects of cotton transformation for a long time. At the same time, concerning the efficiency and the public anxiety, further studies in this field should be focused on exploring and discovering safer and more effective transformation system such as multi-gene transformation, plastid transformation, fixed-point conversion or gene stacking, developing safe or maker-free transgenic technique. At last, the strict requirement for safety evaluation needs to be further researched on the genetically modified materials. With the publication of cotton genome sequences, it will be a new direction for cotton basic and applied research to clone cotton genes, which will provide valuable information for the transformation of candidate genes and breeding of transgenic cotton varieties.

Key words: cotton, transgenic system, largescale, genotype dependence, safe transformation system

刘传亮，田瑞平，孔德培，李凤莲，商海红，陈秀军. 棉花规模化转基因技术体系构建及其应用[J]. 中国农业科学, 2014, 47(21): 4183-4197.

LIU Chuan-liang, TIAN Rui-ping, KONG De-pei, LI Feng-lian, SHANG Hai-hong, CHEN Xiu-jun. Establishment and Application of Efficient Transformation System for Cotton[J]. Scientia Agricultura Sinica, 2014, 47(21): 4183-4197.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.21.005

https://www.chinaagrisci.com/CN/Y2014/V47/I21/4183

参考文献

[1] Wilkins T A, Rajasekaran K, Anderson D M. Cotton biotechnology. Critical Reviews in Plant Sciences, 2000, 19(6): 511-550.

[2] Perlak F J, Deaton R W, Armstrong T A, Fuchs R L, Sims S R, Greenplate J T, Fischhoff D A. Insect resistant cotton plants. Biotechnology, 1990, 8: 939-943.

[3] Jenkins J N, Parrott W L, McCarty J C, Callahan F E, Berberich S A, Deaton W R. Growth and survival of Heliothis virescens (Lepidoptera, Noctuidae) on transgenic cotton containing a truncated form of the delta endotoxin gene from Bacillus thuringiensis. Journal of Economic Entomology, 1993, 86: 181-185.

[4] Nida D L, Kolacz K H, Buehler R E, Deaton W R, Schuler W R, Armstrong T. Glyphosate tolerant cotton: Characterization and protein expression. Journal of Agricultural and Food Chemistry, 1996, 44: 1960-1966.

[5] 李付广, 刘传亮. 生物技术在棉花育种中的应用. 棉花学报, 2007, 19(5): 362-368.

Li F G, Liu C L. Applications of biotechnology on the cotton breeding. Cotton Science, 2007, 19(5): 362-368. (in Chinese)

[6] Umbeck P, Johnson G, Barton K, Swain W. Genetically transformed cotton (Gossypium hirsutum L.) plants. Nature Biotechnology, 1987, 5: 263-266.

[7] Firoozabady E, DeBoer D L, Merlo D J, Halk E L, Amerson L N, Rashka K E, Murray E E. Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants. Plant Molecular Biology, 1987, 10: 105-116.

[8] Cousins Y L, Lyon B R, Llewellyn D J. Transformation of an Australian cotton cultivar: Prospects for cotton improvement through genetic engineering. Australian Journal of Plant Physiology, 1991, 18(5): 481-494.

[9] Bayley C, Trolinder N, Ray C, Morgan M, Quisenberry J E, Ow D W. Engineering 2,4-D resistance into cotton. Theoretical and Applied Genetics, 1992, 83(5): 645-649.

[10] Thomas J C, Adams D G, Keppenne V D, Wasmann C C, Brown J K, Kanost M R, Bohnert H J. Protease inhibitors of Manduca sexta expressed in transgenic cotton. Plant Cell Reports, 1995, 14(12): 758-762.

[11] Chlan C A, Lin J, Cary J W, Cleveland T E. A procedure for biolistic transformation and regeneration of transgenic cotton from meristematic tissue. Plant Molecular Biology Reporter, 1995, 13(1): 31-37.

[12] Keller G, Spatola L, Mccabe D, Martinell, B, Swain, W, John, M E. Transgenic cotton resistant to herbicide bialaphos. Transgenic Research, 1997, 6(6): 385-392.

[13] 朱卫民, 吴敬音, 佘建明, 蔡小宁, 朱祯, 李向辉. 棉花茎尖分生组织在微粒轰击法基因转化中的应用. 江苏农业学报, 1998, 14(2): 74-79.

Zhu W M, Wu J Y, She J M , Cai X N, Zhu Z, Li X H. Application of the shoot apical meristem of cotton in gene transformation by particle bombardment. Jiangsu Journal of Agricultural Sciences, 1998, 14(2): 74-79. (in Chinese)

[14] Huang G C, Dong Y M, Sun J S. Introduction of exogenous DNA into cotton via the pollen-tube pathway with GFP as a reporter. Chinese Science Bulletin, 1999, 44(8): 698-701.

[15] Zhang Z L, Ni W C, Zhang B L, Xu Y J, Guo S D, Cui H Z, Shu C E. Insect-resistant transgenic cotton plants transformed with pollen tube pathway. Acta Agriculturae Boreali-Occidentalis Sinica, 1999, 9(2): 1-4.

[16] Rajasekaran K, Hudspeth R L, Cary J W, Anderson D M, Cleveland T E. High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures. Plant Cell Reports, 2000, 19(6): 539-545.

[17] Chen W X, Xiao G F, Zhu Z. Obtaining high pest-resistant transgenic upland cotton cultivars carrying cry1Ac3 gene driven by chimeric OM promoter. Acta Botanica Sinica, 2002, 44(8): 963-970.

[18] Wu X, Wang J J, Zhu Z, Shangguan X X, Zhang L S, Li B, Du C F, Li Y E. Study of transgenic cotton carrying Bt-CpTI-GNA genes. Cotton Science, 2005, 17: 353-359.

[19] 刘锡娟, 刘昱辉, 王志兴, 王旭静, 张永强. 转 5-烯醇式丙酮酰莽草酸-3-磷酸合酶 (EPSPS) 基因抗草甘膦烟草和棉花的获得. 农业生物技术学报, 2007, 15(6): 958-963.

Liu X J, Liu Y H, Wang Z X, Wang X J, Zhang Y Q. Generation of glyphosate-tolerant transgenic tobacco and cotton by transformation with a 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) gene. Journal of Agricultural Biotechnology, 2007, 15(6): 958-963. (in Chinese)

[20] 范小平, 范博红, 李学宝, 杨维才, 徐子勤. 肌动蛋白基因在棉花纤维中的作用研究. 华北农学报, 2008, 23(5): 73-75.

Fan X P, Fan B H, Li X B, Yang W C, Xu Z Q. Transformation of GhACT1 RNAi and its effect on fiber length of cotton (Gossypium hirsutum). Acta Agriculturae Boreali-Sinica, 2008, 23(5): 73-75. (in Chinese)

[21] 李付广, 侯玉霞, 方鑫, 龚玉梅, 张雪妍. 来源于耐旱荒漠植物蛋白 CkND 对棉花黄姜病的抑制作用及其抗旱性研究. 棉花学报, 2009, 21(2): 89-93.

Li F G, Hou Y X, Fang X, Gong Y M, Zhang X Y. Studies on the inhibition of plant derived protein CkND against Verticillium dahlia and its drought resistance. Cotton Science, 2009, 21(2): 89-93. (in Chinese)

[22] Hashmi J A, Zafar Y, Arshad M, Mansoor S, Asad S. Engineering cotton (Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences. Virus Genes, 2011, 42(2): 286-296.

[23] Liu J F, Zhao C Y, Ma J, Zhang G Y, Li M G, Yan G J, Ma Z Y. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) with a fungal phytase gene improves phosphorus acquisition. Euphytica, 2011, 181(1): 31-40.

[24] Liu Y D, Yin Z J, Yu J W, Li J, Wei H L, Han X L, Shen F F. Improved salt tolerance and delayed leaf senescence in transgenic cotton expressing the Agrobacterium IPT gene. Biologia Plantarum, 2012, 56(2): 237-246.

[25] Liu G Z, Jin S X, Liu X Y, Tan J F, Yang X Y, Zhang X L. Overexpression of Arabidopsis cyclin D2;1 in cotton results in leaf curling and other plant architectural modifications. Plant Cell Tissue and Organ Culture, 2012, 110(2): 261-273.

[26] Xu W L, Zhang D J, Wu Y F, Qin L X, Huang G Q, Li J, Li X B. Cotton PRP5 gene encoding a proline-rich protein is involved in fiber development. Plant Molecular Biology, 2013, 82(4/5): 353-365.

[27] Rao Q, Bajwa A, Puspito K S, Khan A N, Abbas M, Bakhsh M A, Husnain T. Variation in expression of phytochrome B gene in cotton (Gossypium hirsutum L.). Journal of Agricultural. Science and Technology, 2013, 15(5): 1033-1042.

[28] Liu, Z, Zhu, Z, Zhang, T Z. Development of transgenic CryIA (c) + GNA cotton plants via pollen tube pathway method confers resistance to Helicoverpa armigera and Aphis gossypii Glover//Zhang B H. Transgenic Cotton, Methods and Protocols. New York: Humana Press, 2013: 199-210.

[29] Zhang B H, Wang M, Zhang X, Li C Q, Wang Q L. Overexpression of miR156 in cotton via Agrobacterium-mediated transformation// Zhang B H. Transgenic Cotton, Methods and Protocols. New York: Humana Press, 2013: 189-197.

[30] Kumar V, Joshi S G, Bell A A, Rathore K S. Enhanced resistance against Thielaviopsis basicola in transgenic cotton plants expressing Arabidopsis NPR1 gene. Transgenic Research, 2013, 22(2): 359-368.

[31] 欧婷, 何秋伶, 陈进红, 祝水金. 基于抗草甘膦基因的棉花茎尖农杆菌介导转化方法的研究. 棉花学报, 2013, 25(5): 410-416.

Ou T, He Q L, Chen J H, Zhu S J. Studies on the genetic transformation method by cotton shoot tip-Agrobacterium medium using the glyphosate resistant gene. Cotton Science, 2013, 25(5): 410-416. (in Chinese)

[32] Beasley C A. In vitro culture of fertilized cotton ovules. Bioscience, 1971, 21(17): 906-907.

[33] Shoemaker R C, Couche L J, Galbraith D W. Characterization of somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.). Plant Cell Reports, 1986, 5(3): 178-181.

[34] 刘传亮, 武芝霞, 张朝军, 李凤莲, 王玉芬, 李付广. 农杆菌介导棉花大规模高效转化体系的研究. 西北植物学报, 2004, 5(24): 768-775.

Liu C L, Wu Z X, Zhang C J, Li F L, Wang Y F, Li F G. Study on large-scale and high efficient transformation system mediated by Agrobacterium tumefaciens of cotton. Acta Botanica Boreali- occidentalia Sinica, 2004, 24(5): 768-775. (in Chinese)

[35] Wu J H, Zhang X L, Nie Y C, Jin S X, Liang S G. Factors affecting somatic embryogenesis and plant regeneration from a range of recalcitrant genotypes of chinese cottons. In vitro Cellular and Developmental Biology-Plant, 2004, 40(4): 371-375.

[36] Sakhanokho H F, Ozias-Akins P O, Lloyd M, Chee P W. Induction of somatic embryogenesis and plant regeneration in select georgia and Pee Dee cotton Lines. Crop Science, 2004, 44(6): 2199-2205.

[37] Chaudhary B, Kumar S, Prasad K V S K, Oinam G S, Burma P K, Pental D. Slow desiccation leads to high-frequency shoot recovery from transformed somatic embryos of cotton (Gossypium hirsutum L. cv. Coker 310 FR). Plant Cell Reports, 2003, 21(10): 955-960.

[38] Wu J H, Zhang X L, Nie Y C, Luo X L. High-efficiency transformation of Gossypium hirsutum embryogenic calli mediated by Agrobacterium tumefaciens and regeneration of insect-resistant plants. Plant Breeding, 2005, 124(2): 142-146.

[39] Jin S X, Liang S G, Zhang X L, Nie Y C, Guo X P. An efficient grafting system for transgenic plant recovery in cotton (Gossypium hirsutum L.). Plant Cell, Tissue and Organ Culture, 2006, 85(2): 181-185.

[40] Wang Y X, Wang X F, Ma Z Y, Zhang G Y, Han G Y. Somatic embryogenesis and plant regeneration from two recalcitrant genotypes of Gossypium hirsutum L.. Agricultural Sciences in China, 2006, 5(5): 323-329.

[41] Li X B, Fan X P, Wang X L, Cai L, Yang W C. The Cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation. The Plant Cell, 2005, 17(3): 859-875.

[42] Liu Q, Singh S P, Green A G. High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiology, 2002, 129(4): 1732-1743.

[43] Ganesan M, Jayabalan N. Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR 2). Plant Cell Reports, 2004, 23(4): 181-187.

[44] 刘春明, 姚敦义. 陆地棉体细胞胚发生及其细胞组织学研究. 植物学报, 1991, 33(5): 378-384.

Liu C M, Yao D Y. Studies on somatic embryogenesis and histological observation in GossypIum hirsutum L. CV. Coker 312. Journal of Integrative Plant Biology, 1991, 33(5): 378-384. (in Chinese)

[45] 夏启中, 张献龙, 聂以春, 郭小平. 撤除外源生长素诱发棉花胚性悬浮细胞程序性死亡. 植物生理与分子生物学学报, 2005, 31(1): 78-84.

Xia Q Z, Zhang X L, Nie Y C, Guo X P. With drawal of exogenous auxin induces programmed cell death of cotton embryogenic suspension cultures. Journal of Plant Physiology and Molecular Biology, 2005, 31(1): 78-84. (in Chinese)

[46] 张朝军, 李付广, 喻树讯, 范术丽, 王玉芬, 武芝侠, 李凤莲, 刘传亮. 棉花叶柄组织培养与高分化率材料选育方法: 中国, 200610089439.1[P] 2006-11-22.

Zhang C J, Li F G, Yu S X, Fan S L, Wang Y F, Wu Z X, Li F L, Liu C L. Cotton leafstalk tissue cultivation and high-differentiation cotton material selective breeding method: China, 200610089439.1[P]. 2006-11-22.

[47] Klein T M, Wolf E D, Wu R, Sanford J C. High-velocity microprojectiles for delivering nucleic acids into living cells. Nature, 1987, 327(6117): 70-73.

[48] Zhou G Y, Weng J, Zheng Y S, Huang J G, Qian S Y, Liu G L. Introduction of exogenous DNA into cotton embryos. Methods in Enzymol, 1983, 101: 433-481.

[49] Trolinder N L, Linda K. In-planta method for the production of transgenic plants: US, US5994624 A[P]. 1999-11-30.

[50] 张家明, 孙雪飘, 郑学勤, 张献龙, 赵燕, 刘金兰, 孙济中. 陆地棉愈伤诱导及胚胎发生能力的遗传分析. 中国农业科学, 1997, 30(3): 36-43.

Zhang J M, Sun X P, Zheng X Q, Zhang X L, Zhao Y, Liu J L, Sun J Z. Genetic analysis of callus induction and somatic embryogenesis in upland cotton. Scientia Agricultura Sinica, 1997, 30(3): 36-43. (in Chinese)

[51] 张朝军, 王晔, 王玉芬, 李凤莲, 李付广. 棉花叶柄分化率主基因+多基因混合遗传分析. 棉花学报, 2012, 24(1): 3-9.

Zhang C J, Wang Y, Wang Y F, Li F L, Li F G. Mixed major gene and polygene inheritance analysis of embryogenesis callus induction ratio in upland cotton mature leaf petioles as explants. Cotton Science, 2012, 24(1): 3-9. (in Chinese)

[52] 商海红. 陆地棉体细胞胚胎发生的细胞学研究[D]. 北京: 中国农业科学院, 2009.

Shang H H. Cytological studies on the somatic embryogenesis of Gossypium hirsutum L. [D]. Beijing: Chinese Academy of Agricultural Sciences, 2009. (in Chinese)

[53] Gawel N J, Robacker C D. Somatic embryogenesis in two Gossypium hirsutum L. genotypes on semi-solid versus liquid proliferation media. Plant Cell, Tissue and Organ Culture, 1990, 23(3): 201-204.

[54] Xu Z Z, Zhang C J, Zhang X Y, Liu C L, Wu Z X, Yang Z R, Zhou K H, Yang X J, Li F G. Transcriptome profiling reveals auxin and cytokinin regulating somatic embryogenesis in different sister lines of cotton cultivar CCRI24. Journal of Integrative Plant Biology, 2013, 55(7): 631-642.

[55] 李静, 韩秀兰, 沈法富, 刘莲. 提高棉花花粉管通道技术转化率的研究. 棉花学报, 1999, 17(2): 67-71.

Li J, Han X L, Shen F F, Liu L. Study on promoting the pollen-tube pathway transformation in cotton. Cotton Science, 2005, 17(2): 67-71. (in Chinese)

[56] 马盾, 黄乐平, 黄全生, 孟庆玉, 危晓薇, 陈勋基, 美丽古丽. 提高棉花花粉管通道法转化率的研究. 西北农业学报, 2005, 14(1): 10-12.

Ma D, Huang L P, Huang Q S, Meng Q Y, Wei X W, Chen X J, Meili G L. Study on improvement of pollen tube pathway transformation efficiency through field concrete operation. Acta Agriculturae Boreali-Occidentalis Sinica, 2005, 14(1): 10-12. (in Chinese)

[57] 王志才, 艾尔肯木合热皮亚, 廖茂森, 张富春. 影响棉花体细胞胚胎发生和植株再生的关键因素分析. 新疆农业科学, 2011, 48(1): 39-44.

Wang Z C, Aierken M, Liao M S, Zhang F C. Analysis on key factors affecting somatic embryogenesis and plant regeneration in cotton. Xinjiang Agricultural Science, 2011, 48(1): 39-44. (in Chinese)

[58] Jin S X, Zhang X L, Liang S G, Nie Y C, Guo X P, Huang C. Factors affecting transformation efficiency of embryogenic callus of upland cotton (Gossypium hirsutum) with Agrobacterium tumefaciens. Plant Cell, Tissue and Organ Culture, 2005, 81(2): 229-237.

[59] Salm T V D, Bosch D, Honee G, Feng L X, Munsterman E, Bakker P, Stiekema W J, Visser B. Insect resistance of transgenic plants that express modified Bacillus thuringiensis cryIA(b) and cryIC genes: A resistance management strategy. Plant Molecular Biology, 1994, 26(1): 51-59.

[60] Mullen J, Adam G, Blowers A, Earle E. Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation. Molecular Breeding, 1998, 4(5): 449-457.

[61] Hamilton C M. A binary-BAC system for plant transformation with high-molecular-weight DNA. Gene, 1997, 200(1): 107-116.

[62] Paszkowski J, Baur M, Bogucki A, Potrykus I. Gene targeting in plants. The EMBO Journal, 1988, 7(13): 4021-4026.

[63] Kempin S A, Liljegren S J, Block L M, Rounsley S D, Yanofsky M F, Lam E. Targeted disruption in Arabidopsis. Nature, 1997, 389(6653): 802-803.

[64] Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S. Efficient gene targeting by homologous recombination in rice. Nature Biotechnology, 2002, 20(10): 1030-1034.

[65] Zhu T, Peterson D J, Tagliani L, Clair G S, Baszczynski C L, Bowen B. Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proceedings of the National Academy of Sciences of the USA, 1998, 96(15): 8768-8773.

[66] Boynton J E, Gillham N W, Harris E H, Hosler J P, Johnson A M, Jones A R, Randolph-Anderson B L, Robertson D, Klein T M, Shark K B. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science, 1988, 240(4858): 1534-1538.

[67] Maliga P. Progress towards commercialization of plastid transformation technology. Trends in Biotechnology, 2003, 21(1): 20-28.

[68] Kumar S, Dhingra A, Daniell H. Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant Molecular Biology, 2004, 56: 203-216.

[69] Darbani B, Eimanifar A, Stewart C N, Camargo W N. Methods to produce marker-free transgenic plants. Biotechnology Journal, 2007, 2(1): 83-90.

[70] Ding Z S, Zhao M, Jing Y X, Li L B, Kuang T Y. Efficient Agrobacterium-mediated transformation of rice by phosphomannose isomerase/mannose selection. Plant Molecular Biology Reporter, 2006, 24: 295-303.

[71] Endo S, Kasahara T, Sugita K, Matsunaga E, Ebinuma H. The isopentenyl transferase gene is effective as a selectable marker gene for plant transformation in tobacco (Nicotiana tabacum cv. Petite Havana SRI). Plant Cell Reports, 2001, 20: 60-66.

[72] Zhu Z G, Wu R. Regeneration of transgenic rice plants using high salt for selection without the need for antibiotics or herbicides. Plant Science, 2008, 174(5): 519-523.

[73] Daniell H, Muthukumar B, Lee S B. Marker free transgenic plants: Engineering the chloroplast genome without the use of antibiotic selection. Current Genetics, 2001, 39(2): 109-116.

[74] 杨晓杰, 刘传亮, 张雪妍, 李付广. 转基因植物中T-DNA整合的分子特征及表达. 基因组学与应用生物学, 2010, 29(1): 125-130.

Yang X J, Liu C L, Zhang X Y, Li F G. Molecule characterization and expression of T-DNA integration in transformed plants. Genomics and Applied Biology, 2010, 29(1): 125-130. (in Chinese)