农杆菌介导玉米遗传转化体系的优化

Optimization of Agrobacterium Tumefaciens Mediated Maize Transformation System

以自交系18H、Mo17、9046和178的幼胚为材料,进行愈伤组织诱导,用根癌农杆菌EHA105和LBA4404对获得的II型胚性愈伤组织进行转化,导入TERF1-LeERF2基因,并对农杆菌介导玉米遗传转化体系的条件进行了优化。结果表明:使用毒性较强的根癌农杆菌EHA105侵染愈伤组织;农杆菌浓度在OD600=0.6,侵染时间20min;侵染后的愈伤组织经7d的恢复培养后,先进行低浓度选择压的筛选培养,再进行高浓度选择压的筛选培养,以及在筛选培养基中添加10mg/LAgNO3提高了抗性愈伤组织的获得率。获得植株经PCR鉴定表明,外源基因已经整合到玉米自交系18H的基因组中。

Immature embryos of maize inbred lines 18H, Mo17,9046 and 178 were transformed by Agrobacterium tumefaciens LBA4404 and EHA105. Factors influencing maize transformation mediated by Agrobacterium tumefaciens were studied. The results showed that the rate of resisitance callus acquirement could be significantly enhanced by the following method：（ 1 ） callus was infected by highly toxic Agrobacterium EHA105; （2） Agrobacterium concen- tration at OD600 = 0. 6 and 20 minutes infection time were used ; （3） after 7 days on the delay medium culture, in- creasing concentration was used in selection; （4） 10 mg/L AgNO3 in the selective medium was added. Through optimizing the Agrobacterium mediated transformation system, the transformation frequency was distinctly improved. The result of PCR amplification indicated that the target gene was introduced into the maize inbred line 18H.