摘要
采用携带gus和 (或 )bar基因双元表达载体 (p330 1,pBTAaB)的 3个根癌农杆菌 (Agrobacteriumtumefaciens)菌株(AGL 1,EHA10 5和LBA4 4 0 4 )对普通小麦 (TriticumaestivumL .)冬性栽培品种农大 170和农大 14 6的幼胚及幼胚愈伤组织进行了遗传转化。结果表明 ,菌液浓度OD60 0 1.0和侵染时间 1h对外植体的生存和转化最为有利 ;侵染前对外植体进行高渗处理较明显地提高了抗性愈伤获得率 ;乙酰丁香酮 (AB)对小麦转化的作用随菌株和外植体的不同而异 ;菌株 /质粒组合、受体基因型及外植体的类型、年龄和生理状态对转化效率有很大的影响。条件优化后 ,得到大量具有PPT抗性的愈伤和一些抗性植株。抗性愈伤的GUS染色阳性率在 5 0 %～ 6 0 %之间 ,所检测的抗性苗呈GUS阳性。对 6株抗性苗的PCR和Southern检测初步证明 ,外源基因已经整合到其中 3株的基因组中。
Immature embryos and embryo derived calli from two cultivars of winter wheat ( Triticum aestivum L.), BAU146 and BAU170, were transformed with three strains of Agrobacterium tumefaciens, AGL 1, EHA105 and LBA4404 harboring expression vector p3301 or pBTAaB. Both vectors contained bar gene and p3301 contained also gus gene with an intron. The highest explant survival rate and transformation efficiency was obtained when the bacterial cell density was OD 600 1.0 with 1 h of infection incubation. Higher osmotic treatment of the explants before inoculation had a positive effect on transformation, while addition of acetosyringone showed ambiguous one, depending on the explant types and bacterium strains. The efficiencies of transformation and transgenic plant regeneration were varied greatly with the bacterium strain, receptor genotype, explant type and its age and physiological state. After optimizing these factors, a large number of PPT resistant calli and some of PPT resistant plants were obtained. The resistant plantlet tested and 50% to 60% of the resistant calli were GUS positive. The integration of foreign DNA into the genome of transgenic plants (3 out 6) was further confirmed by PCR and Southern Blot analysis.
基金
国家转基因植物研究和产业化专项 (J99 B 0 15 )
"863"计划 (2 0 0 1AA2 12 12 1)资助项目~~
