甘薯SAAT法遗传转化条件的优化  

Optimization of Genetic Transformation Using SAAT in Sweetpotato

作　　者：王欣[1] 周忠[2] 李强[1] 顾向华[1] 马代夫[1] 

机构地区：[1]江苏徐州甘薯研究中心,江苏徐州221121 [2]徐州师范大学生命科学学院,江苏徐州221116

出　　处：《江苏农业学报》2006年第1期14-18,共5页Jiangsu Journal of Agricultural Sciences

基　　金：国家"863"计划(2004AA241180)

摘　　要：SAAT即声波辅助的农杆菌遗传转化方法。在遗传转化中,超声波处理可以在外植体上产生许多易于农杆菌进入的微小伤口和通道,从而提高转化率。该研究首次将SAAT法应用于甘薯遗传转化并对遗传转化体系进行优化,根癌农杆菌菌株为EHA101/pROA93(nptⅡ和gus),受体材料为甘薯品种栗子香的胚性悬浮细胞。结果表明,悬浮培养105 d,继代培养3 d,将经超声波处理30 s的胚性悬浮细胞侵染到OD600值稀释至0.5的对数生长期的EHA101菌液中10 m in,经4 d共培养后,GUS瞬时表达率最高,达到86.7%,稳定表达率为37.9%;转化时加入乙酰丁香酮亦能提高转化率;转基因植株筛选的适宜选择压为Kan 10 mg/L;适宜的抑菌剂为Carb 100 mg/L。During the sonication-assisted Agrobacterium-mediated transformation （SAAT） ,supersonication produces many small fissures and channels throughout the tissue allowing the Agrobacterium tumefaciens to get easily access to internal plant tissues, and increases transformation efficiency, In the study SAAT was first used in genetic transformation of sweetpotato and the system of transformation was optimized, The Agrobacterium tumefaciens strain EHA101 harbors the binary vector pROA93 containing gus（ β-glucuronidase gcne） as a report gene and npt Ⅱ （ chimerical neomycin phosphotransferase gene） as a selectable marker. The receptor was embryogenic suspension cells of sweetpotato cv Lizixiang, The results demonstrated that embryogenic suspension cells,which were suspension-cultured for 105 days, precuhured for 3 days and ultrasound-treated for 30 seconds,were suitable for transformation, The results also indicated that the embryogenic suspension cells infected by Agrobacterium tumefaciens strain EHA101 with OD600 = 0. 5 for 10 minutes and then co-cultured for 4 days achieved the highest transient and stable GUS expression rates of 86, 7% and 37.9% respectively. The optimal concentration of kanamycin was 10 mg/L for the selection of transgenic explants and the suitable concentration of carbencillin was 100 mg/L for the control of Agrobacterium tumefaciens growth.

关 键 词：甘薯 遗传转化 声波辅助农杆菌遗传转化 

分 类 号：S531.035.3[农业科学—作物学]