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根癌农杆菌介导转化诸葛菜获得转基因植株

周冀明, 卫志明, 许智宏, 刘世贵, 罗鹏

中科院上海植物生理研究所植物分子遗传国家重点实验室. 上海 200032；四川大学生物工程系. 四川, 成都610064；四川大学生物系.四川, 成都610064

摘要

以诸葛菜下胚轴和子叶为材料, 在附加BA和NAA的MS培养基上诱导芽再生,在1/2　MS培养基上诱导生根,获得完整再生植株,建立了诸葛菜组织培养高频再生体系,再用根癌农杆菌介导转化诸葛菜下胚轴和子叶,在附加一定量的氨苄青霉素、头孢霉素和卡那霉素的相应培养基上进行筛选, 并培养再生成苗,获得完整抗性再生植株,移植到盛有土壤的花盆中均可存活,生长正常。将再生植株叶片,进行GUS、NPTII酶活性测定和Southern blot 分子杂交,证实外源基因已稳定整合到植物基因组中,并高效表达。

关键词： 根癌农杆菌; 诸葛菜; 下胚轴; 子叶; 转基因植株

Agrobacterium-mediated transformation of Orychopheagmus violaceus and regeneration of transgenic plants

Zhou Jiming, Wei Zhiming, Xu Zhihong, Liu Shigui, Luo Peng

National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology, The Chinese Academy of Sciences. Shanghai 200032； China；Department of Biology, Sichuan University.　Chengdu 610064, Sichuan

Abstract

Excised hypocotyls and cotyledons of Orychophragmus violaceus were used as explants for tissue culture. They were cultured on MS medium supplemented with BA 2 mg/L or with BA 3 mg/L and NAA 0.2 mg/L.Regenerated buds were excised when they were 2cm long, and transferred onto 1/2MS medium with IBA 0. 03 mg/L, then whole　plantlets were regenerated at a frequency of 100%. Genetic transformation of O. violaceus was studied by coculture with Agrobacterium tumefaciens strain A208se(pTiT37,pROA93),After 2-3d of coculture the hypocotyls and cotyledens were transferred onto selection medium containing kanamycin(Km) 25　mg/L and 6-aminopenicillanic acia(Ap) 250　mg/L.8 weeks later, shoots emerged, and were excised and transferred onto the rooting medium containing AP 25　mg/L and Cephazoline 100 mg/L,and roots were formed within 4-5 weeks.When the whole plants were transplanted into pots they grew well. The frequency of plant regeneration of hypocotyls was about 30%, and that of cotyledens was about 51%. The regenerated plants showed high enzymatic activities of #beta# glucuronidase and neomycine phosphotransferase II. Southern blot analysis confirmed that NPTII gene had been stably integrated into the chomosomal genome of O.violaceus.The transformation frequency of hypocotyls was 10%, and that of cotyledens was 5.5%.This is the first report about the regeneration of transgenic plants of O.violaceus.

Key words: Agrobacterium tumefaciens;Orychopheagmus violaceus;Hypocotyl;Cotyleden;Transgenic plants

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引用本文：

周冀明, 卫志明, 许智宏, 刘世贵, 罗鹏. 根癌农杆菌介导转化诸葛菜获得转基因植株[J]. 生理学报 1997; 49 (1): .

Zhou Jiming, Wei Zhiming, Xu Zhihong, Liu Shigui, Luo Peng. Agrobacterium-mediated transformation of Orychopheagmus violaceus and regeneration of transgenic plants. Acta Physiol Sin 1997; 49 (1): (in Chinese with English abstract).