农杆菌介导的优良玉米自交系遗传转化体系的建立

Establishment of the System of Conventional Maize Inbred-lines Transformation Mediated by Agrobacterium tumefaciens

以杂交育种中广泛使用的优良玉米自交系340、4112为材料 ,用带有质粒pGBIL04(actin.Bt.35S .bar)的根癌农杆菌LBA4404转化其幼胚及其初始愈伤组织 ,经PPT抗性筛选后分化再生植株。PCR分子检测初步证明Bt基因已整合到再生植株基因组中 ,转化率为1.68 %～4.44%。本实验利用转化受体的抗性筛选频率对转化体系进行了优化 ,结果表明 :比较适宜的感染液浓度为OD600=0.5;预培养有利于幼胚转化 ,预培养时间以刚刚诱导出新鲜稳定的初始愈伤为宜 ;

Elite inbred-lines of maize, 340 and 4112, which were widely used in hybridization were transformed with Agrobacterium tumefaciens. The immature embryos and their initial callus were infected by A. tumefaciens LBA4404 (pGBIL04).Selected on the medium containing phosphinothricin (PPT), the resistant calli were differentiated to plants. It was proved that the target Bt gene had been integrated into the genome of regeneration plants after PCR analysis and transformation frequency was 1.68%～4.44%. The factors influencing maize transformation by A. tumefaciens were studied. The results showed that the frequency of selection was highest when OD600 of engineering bacterial suspension was 0.5. Pre-culture was favorable to immature embryos' transformation.It was feasible that the immature embryos had been induced to form initial callus. The frequency of selection and PCR+via A. tumefaciens increased along with lowering the co-culture temperature to 22℃.