根癌农杆菌介导小麦幼胚遗传转化的影响因素

Factors Influencing Transformation of Wheat (Triticum aestivum L.) Immature Embryos Mediated by Agrobacterium tumefaciens

利用携带pC3 3 0 1质粒 (含bar和gus基因 )的超毒根癌农杆菌菌株EHA1 0 5对普通小麦 (TriticumaestivumL .)扬麦 1 5 8进行了遗传转化 ,对筛选中的抗生素浓度、菌液浓度、共培养温度和时间、幼胚预培养时间、乙酰丁香酮 (AS)浓度、洗涤用液及筛选方式等影响转化的几个重要因素进行了讨论。对从 2 94个小麦幼胚外植体中转化得到的 5株成活植株进行了PCR和Southernblot分析 ,结果表明其中 2株小麦基因组中整合了外源DNA ,转化频率为 0 .68%。

Immature embryos of wheat (Triticum aestivum L.) cv. Yangmai158 were transformed using Agrobacterium tumefaciens strain EHA105 carrying binary vector pC3301 which carrying bar gene and gus gene. Both bar gene and gus gene were promoted by CaMV35S promoter. Factors that influence Agrobacterium- mediated transformation of wheat, including concentration of antibiotics during selection (Table 2), concentration of bacterium suspension, temperature and time of co-culture (Table 3), pre-culture time of immature embryos (Table 4), concentration of AS (Table 5), washing methods (Fig.2) and selection methods (Table 6, Fig.3), were investigated. Two transgenic wheat plants from 294 explants were obtained in this study (Fig.1, Fig.4) and the integration of transgene was confirmed by PCR and Southern blot analysis (Fig.5). The transformation frequency was 0.68%.