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作物学报 ›› 2010, Vol. 36 ›› Issue (10): 1691-1697.doi: 10.3724/SP.J.1006.2010.01691

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

根癌农杆菌介导的橡胶树花药愈伤组织遗传转化体系的建立

黄天带,李哲,孙爱花,周权男,华玉伟,黄华孙*   

  1. 中国热带农业科学院橡胶研究所/国家橡胶树育种中心/农业部橡胶树生物学重点开放实验室/省部共建国家重点实验室培育基地/海南省热带作物栽培生理学重点实验室，海南儋州571737
  • 收稿日期:2010-02-05 修回日期:2010-04-20 出版日期:2010-10-12 网络出版日期:2010-07-05
  • 通讯作者: 黄华孙, E-mail: xjshhs@163.com
  • 基金资助:

    本研究由国家自然科学基金项目（30960322），中国热带农业科学院橡胶研究所基本科研业务费专项资金项目（XJSYWFZX2008-01）和国际科技合作目（2008DFA32020）资助。

Establishment of Agrobacterium tumefaciens -mediated Anther Calli Transformation System in Hevea brasiliensis

HUANG Tian-Dai,LI Zhe,SUN Ai-Hua,ZHOU Quan-Nan,HUA Yu-Wei,HUANG Hua-Sun*   

  1. Rubber Research Institute of Chinese Academy of Tropical Agricultural Sciences / National Rubber Tree Breeding Centre / Key Laboratory of Rubber Biology / Ministry of Agriculture / State Key Laboratory Breeding Base of Cultivation ＆ Physiology of Tropical Crops, Danzhou 571737, China
  • Received:2010-02-05 Revised:2010-04-20 Published:2010-10-12 Published online:2010-07-05
  • Contact: HUANG Hua-Sun,E-mail:xjshhs@163.com

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1655

被引次数

50

摘要： 为建立根癌农杆菌介导的橡胶树遗传转化体系，以花药愈伤组织为受体，研究了农杆菌菌株、共培养温度、共培养时间、乙酰丁香酮(AS)等因素对遗传转化效率的影响。结果表明，农杆菌菌株、共培养温度和共培养时间对转化效率具有显著影响，AS不影响转化效率。2.2万个花药愈伤组织经EHA105菌株侵染后，转入未添加AS的培养基，22℃共培养6 d，通过50 mg L–1卡那霉素抗性筛选、叶片GUS染色、uidA和Npt II基因PCR特异扩增、PCR产物测序及Npt II基因Southern检测，鉴定出11株转基因植株，并通过嫁接和次生体胚发生，获得来自8个转基因株系的681株转基因植株，移栽成活253株。

关键词: 橡胶树, 根癌农杆菌, 花药愈伤组织, 遗传转化, 次生体胚发生

Abstract: Developping a new Hevea variety by crossing breeding will take 20-30 years, but genetic transformation provides an effective alternative for shortening breeding cycle. Until now, two kinds of recipient materials, anther compact embryogenic calli (ACEC) and maintained friable embryogenic calli (MFEC), have been used successfully for Hevea genetic transformation. However, few transformed plants are produced from ACEC-derived embyos, and plants regenerated from MFEC are abnormal, showing that they have poorer performance in growth, yield and disease resistance than the budded control. In order to establish Agrobacterium tumefaciens-mediated Hevea factors influencing transformation frequency, including strains of Agrobacteriumembryos regeneration. GUS transient expression frequency was significantly affected by Agrobacterium strains and co-culture temperature/time, which was 5.07 blue spots/callus for EHA105, higher than that for GV3101 and LBA4404. The highest GUS transient expression frequency was 8.43 and 19.10 blue spots/callus under 22℃ co-cultured temperature and 6 days co-cultured time respectively. Twenty-two thousand ACEC were transformed by the strain EHA105 with pCAMBIA2301, and then transferred to the medium without AS and incubated at 22℃ for 6 days in the dark. Subsequently, 11 transformants were identified by GUS staining, PCR amplification of target genes, sequencing of PCR products and Southern hybridization. Among them, three planted directly in the field died; three as scions were budded onto seedlings, producing five budded transgenic plants; five were multiplicated at embryo phase by secondary embryogenesis, producing 676 transgenic plantlets, among which 248 survived in the field. The results showed that the present transformation system and the plant propagation by transgenic embryos secondary embryogenesis are efficient for the production of transgenic rubber plants., co-culture temperature/time and acetosyringone (AS), and the methods for anther calli transformation system, we surveyed the

Key words: Hevea brasiliensis, Agrobacterium tumefaciens, Anther calli, Genetic transformation, Secondary embryogenesis

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