农杆菌侵染小麦的优化方案  

Optimization of Agrobacterium Tumefaciens Infection Process for Wheat (Triticum aestivum)

作　　者：陈明利[1] 刘香利[1] 唐广立[1] 徐波[1] 李彦杰[1] 郭蔼光[1] 

机构地区：[1]西北农林科技大学生命科学学院,杨陵712100

出　　处：《分子植物育种》2007年第4期577-582,共6页Molecular Plant Breeding

基　　金：科技部转基因专项基金(JY-03-13-01)资助。

摘　　要：本实验选用优质高产小麦品种洛阳8716和绵阳19幼胚愈伤作为受体材料,以pCAMBIA3301为载体,GV3101,EHA105,LBA4404农杆菌为供体材料进行侵染过程的优化研究。采用3种菌株,4个浓度梯度:OD600=0.2,0.5,0.7,1.0;3个时间参数:10min,30min,60min;一共36个处理,每个处理4个重复。结果显示,农杆菌GV3101和EHA105,OD6001.0×10min和OD6000.5×30min两个侵染处理结果比较好,瞬时表达率达到50%～70%。以小麦幼胚愈伤组织为受体,预培养3～5d,在24±1℃下,共培养3￣4d和350mg/L羧苄青霉素除菌及5mg/LPPT筛选处理后,转化效果较好,平均转化率为0.45%。In this experiment immature embryo calli ofWheat ‘Luoyang8716＇ and‘ Mianyang 19＇ wasusedto be infected byA. tumefaciens strains （GV3101, EHA105 and LBA4404） with the pCAMBIA3301 vector. Three strains, four concentration gradient ofA.tumefaciens （OD600： 0.2, 0.5, 0.7, 1.0）. and three time parameters （10, 30, 60 rain） were employed robe optimized in this study. Totally 36 treatments with four repeats were carded out. The results showed that the combinations of infection ’ OD600 1.0 × 10 min＇ and ‘ OD600 0.5 × 30 min＇ were demonstrated to be optimal for A. tumefaciens infection. The efficiency of instantaneous expression was even up to 50%-70%. Under the condition that precultured for 3-5 d in the dark, co-cultivated embryo calli with A. tumefaciens at 24± 1 ℃ for 3-4 d, after debacterium at 350 mg/L carbenicillin and selection at 5 mg/L PPT, transgenic plant was determined by PCR amplification of transgene fragments and indicated an transformation average efficiency of 0.45%.

关 键 词：小麦 幼胚 农杆菌 侵染 

分 类 号：S512.1[农业科学—作物学]