水稻悬浮细胞再生及根癌农杆菌介导转化的影响因素(英文)  

Factors Influencing Agrobacterium-mediated Transformation and Regeneration of Rice Suspension Cells

作　　者：PROMMEE Wittaya 王梅珍[1] 朱登云[1] 赵倩[1] 敖光明[1] 于静娟[1] 

机构地区：[1]中国农业大学生物学院农业生物技术国家重点实验室,北京100193 [2]泰国塔切纳苏拉塔尼橡胶研究中心,塔切纳84170

出　　处：《中国生物化学与分子生物学报》2009年第11期1010-1016,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：Supported by Important National Sciences&Technology Specific Projects for Transgenic Research(No.2008ZX003-002)~~

摘　　要：已经成功报道的农杆菌介导的水稻遗传转化多以活力较高的胚性愈伤为材料,很少以水稻悬浮细胞作为受体.另外,利用农杆菌转化多数都是通过浸泡的方式进行侵染.本实验利用滴加浸染法进行农杆菌介导转化水稻悬浮细胞,探讨影响DNA转化效率的因素.研究显示,在转化前,将水稻悬浮细胞在愈伤诱导培养基上培养1～2周,诱导产生直径为2～3mm的微小愈伤组织对转化非常重要.微小愈伤组织大小不应小于2mm;对悬浮细胞短时间培养不但会缩短植株再生时间,而且会提高转化效率.此外,侵染农杆菌的浓度、侵染时间和不同侵染方法也影响T-DNA插入基因组的效率.用1mlA600值为0.5浓度的农杆菌悬液滴加在水稻悬浮细胞诱导的愈伤,培养3d或直到可见农杆菌菌落,此方法可以得到较高转化效率.将再生的潮霉素抗性的转化植株在含有50mg/L潮霉素的分化和生根培养基中筛选得到,并对转化植株gus基因的表达进行PCR检测.结果显示,用A600值为0.5浓度的农杆菌浸泡侵染20min和滴加浸染法,分别得到PCR阳性植株率为70%和92%.Callus immerse inoculation was used in Agrobacterium-mediated DNA transformation. Unlike the, callus derived suspension cells, actively growing embryogenic callus was successfully used for rice Agrobacterium-mediated transformation in many reports. In our experiments, callus derived from suspension cells and drop-wise co-cultivation inoculation were used. We analyzed several influencing parameters for the effects of transformation. The data showed that pre-cuhure of the suspension cells in callus induction medium for 1 to 2 weeks until the microcallus derived from suspension reached 2 - 3 mm （no smaller than 2 mm） in diameters was critical for transformation. The shorter duration in suspension cells culture favored more rapid plant regeneration and higher frequencies of transformation. In addition, the concentration of Agrobacterium suspension, inoculation time and different inoculation method influenced the T-DNA delivery to the genome. Inoculating plate drop-wise with 1 ml of Agrobacterium suspension cells and co-cultivation for 3 days or until visible Agrobacterium colonies showed up resulted in a high transformation potential for the rice suspension cells. The regenerated transformed plants with the hpt gene were selected on regeneration medium and rooting medium containing 50 mg/L hygromycin and, further confirmed for the gus gene by PCR. The ratios of obtained gus positive transgenic plants were 70% and 92 % in appropriate Agrobacterium suspension （A600 = 0.5） for 20 min or drop-wise inoculation with Agrobacterium suspension, respectively.

关 键 词：根癌农杆菌 水稻悬浮细胞 微小愈伤组织 转化条件 滴加浸染共培养 

分 类 号：Q785[生物学—分子生物学] Q813