根癌农杆菌介导转化籼稻影响因素的研究

选用我国籼型杂交稻中的几个重要亲本 ,以携带有双元载体 p CAMBIA1 3 0 1的根癌农杆菌 EHA1 0 5为载体 ,以GUS基因的瞬间表达为依据 ,研究根癌农杆菌介导转化籼稻的影响因素 ,确立对转化频率有重要影响的几个条件。结果表明 :经短期预培养 (4～ 5d)的幼胚是较为理想的转化受体材料 ;在共培养基中添加较高浓度的乙酰丁香酮 (3 0 0～ 4 0 0μmol/ L)可提高 GUS基因的瞬间表达频率 ;以 CC培养基作为共培养后抗性愈伤筛选的基本培养基有利于抗性愈伤组织的筛选培养。按此确立的条件 ,在特青、龙特甫 (B)、珍汕 97(B)、盐恢 559等籼稻品种或亲本中的 GUS瞬间表达率高达 80 % ,抗性愈伤组织转化率高达 70 %

STUDY ON SOME FACTORS INFLUENCING AGROBACTERIUM- MEDIATED TRANSFORMATION OF INDICA RICE

Several important parents of hybrid rice were used in this study and transferred with the grobacterium tume faciens strain EHA105 harboring Ti plasmid pCAMBIA1301, which contains a hygromycin resistant gene as the selectable marker and a β-glucuronidase gene (GUS) as the reporter gene. Based on the transient expression of GUS reporter gene,the factors influencing transformation of Indica rice mediated by Agrobacterium were carefully investigated and several crucial parameters of efficient transformation were established. The results showed that the immature embryos precultured for 4～ 5 days were ideal as the start explants for transformation. And the efficiency of transient expression of GUS gene was increased in the presence of 300～ 400 μmol/L acetosyringone in the co-cultivation medium. The CC medium was an efficient selectable medium for screening hygromycin-resistant calIi after co-cultivation. Under these conditions, the rate of transient expression of GUS gene could reach more than 80% and that of stable transformation measured as hygromycin resistant calli 70％.