根癌农杆菌介导蓝猪耳转化的影响因素研究

Optimization of Agrobacterium-mediated Transformation System for Torenia fournieri

研究了根癌农杆菌介导蓝猪耳转化的影响因子。结果表明,以蓝色花类型蓝猪耳5～6周的叶片为外植体,在OD600为0.5～0.6的活化菌液中浸染5～10min,暗培养4d后,在愈伤组织诱导培养基(MS+BA1.0mg/L+2,4-D0.1mg/L)上生长14d,获得抗性愈伤组织;经芽诱导培养基(1/2MS+BA1.0mg/L+NAA0.1mg/L)培养28d得到抗性芽;生根培养基(1/2MS)上培养14d得到抗性植株。经PCR检测证实外源基因已整合到蓝猪耳基因组中,转化率达13%～14%。Cef和Hyg浓度对转化影响较大,转化的不同阶段其适宜浓度不同。

An efficient and stable Agrobacterium-mediated transformation system for Torenia was established through investigating the factors influencing the transformation efficiency. Genetic transformation of Torenia was performed by using 5-45 week leaflet as explants. After incubation in Agrobacterium suspension of OD600 at 0.5-0.6 for 5-10min, the explants were co-cultivated on co-culture medium containing 200μmol/L acetosyringone for 4d under dark. The explants were then transferred to the calla induction agar medium MS ＋ BA 1.0mg/L ＋ 2,4-D 0.1g/L, and cultivated for 14d to obtain the resistant calla. The resistant calla were transferred to the shoot induction agar medium 1/2MS ＋ BA 1.0mg/L ＋ NAA 0.1mg/L, and cultured for another 28d to obtain resistant shoots. The 0.5cm or taller resistant shoots were finally transferred to rooting agar medium, and the resistant plantlets were achieved after 14d cultivation. Hygromycin- resistant plants were confirmed to be positive transformants by PCR. The transformation frequency was up to 13% - 14% overall.