农杆菌介导将Bt杀虫蛋白基因导入优良玉米自交系的研究

Transfer of Insecticidal Protein Gene from Bacillus thuringiensis into Conventional Maize Inbred-line Mediated by Agrobacterium tumefaciens

以杂交育种中广泛使用的优良玉米自交系 34 0、4112为材料 ,用带有质粒pGBIL0 4(Pactin -Bt-Tnos)的根癌农杆菌LBA44 0 4转化其幼胚及其初始愈伤组织 ,共培养 3天后 ,在含PPT的培养基上连续筛选培养 3代 ,然后分化获得再生植株。PCR检测证明目的基因已整合到再生植株的基因组中。实验结果表明幼胚预培养后形成的新鲜的初始愈伤组织是比较适宜的转化受体 ,结果还发现将共培养温度降到 2 2℃可以提高农杆菌介导的玉米遗传转化的筛选频率。

Excellent inbred-lines of maize,340 and 4112,which were used largely in hybridized combination were transformed with Agrobacterium tumefaciens.The immature embryos and their original calli were infected by A.tumefaciens LBA4404 containing plasmid pGBIL04.After 3 days of co-cultivation,the immature embryos and calli were continuously selected on the medium containing phosphinothricin (PPT) for 3 generations,then plants were regenerated.It was proved by PCR analysis that the target Bt gene had been integrated into the genome of regenerated plants.The results showed that fresh original calli from the immature embryos after pre-culture were suitable acceptors.The results also showed that it could increase the frequency of selection by properly lowering the co-culture temperature to 22℃.