农杆菌介导小麦遗传转化的影响因素

Influencing Factors of Agrobacterium tumefaciens Transforming into Wheat Callus

[目的]对农杆菌菌株、小麦品种、培养基状态及报告基因选择等进行优化筛选,为进一步完善农杆菌介导小麦遗传转化方法提供参考。[方法]用携带质粒pCAMBIA1304的农杆菌菌株LBA4404、EHA105,对温麦19、郑离03和郑离06小麦的幼胚愈伤组织进行遗传转化,从农杆菌菌株、小麦品种、共培养基状态及报告基因选择等方面进行探讨。[结果]农杆菌菌株EHA105转化效率较高,最高达到22.58%;姊妹系郑离03与郑离06的转化率差别极大,郑离06转化率最高而郑离03最低;农杆菌与愈伤组织共培养时,使用MS半固体培养基的转化率高于MS固体培养基;GFP和GUS都可以用作判断转化率的报告基因,GFP优势更明显。[结论]高毒性农杆菌菌株EHA105转化效率明显高于菌株LBA4404;小麦基因型是影响转化效率的重要因素之一;农杆菌与愈伤组织共培养时使用半固体培养基可提高转化率;通过GFP瞬时表达,可有效进行小麦转化效率的研究。

[ Objective] The types of Agrobacterium turnefaciens, the varieties of wheat, the state of co-cuhivating media and reporting gene were optimized in order to supplement the way of genetic transformation through Agrobacterium tumefaciens transforming into wheat. [ Method] The callus of three wheat varieties Wenmail9, Zhengli03 and Zhengli06 was transformed using the Agrobacterium tumefaciens LBA4404 and EHA105 with the plasmid pCAMBIA1304. The types of Agrobacterium tumefaciens, the varieties of wheat, the state of eo-euhivating media and the effect of reporting gene were studied. [ Result] The highest transformation ratio of EHA105 was 22.58%. The sister varieties of Zhengli03 and Zhengli06 had great differences in the genetic transform efficiency. The efficiency of Zhengli06 was the highest while that of Zhengli03 was the lowest. The transformation efficiency of MS half solid-state co-cultivating media was higher than MS solid-state co-cultivating media. GFP and GUS had the same result as the reporting gene and GFP had more superiority. [ Conclusion] The transformation efficiency of EHA105 with powerful virulence was higher than LBA4404. The gene types of wheat had great influence on the transformation ratio. The half solid-state co -cultivating media could improve the efficiency. The genetic transformation efficiency could be deduced effectively by GFP reporter.