根癌农杆菌介导的籼稻蜀恢527遗传转化方法的探究

Studies on Methods of Agrobacterium-mediated Transformation of Indica Rice(Shuhui 527)

籼稻(Indica rice)组织培养过程中愈伤组织转化率较低,尤其是对优良品种成熟胚愈伤高频再生体系的建立和影响转化的主要因素还缺乏研究。在一定程度上制约着籼稻基因工程的进展。以籼稻品种重穗型恢复系蜀恢527成熟种子为材料,研究了不同温度和光照条件对其成熟胚愈伤诱导及生理状态的影响;并在分化培养基中添加不同的激素和调整激素间浓度配比从而筛选出蜀恢527最佳分化培养基。结果表明:在32℃光照条件下,蜀恢527成熟种子在N6D诱导培养基上诱导愈伤组织效果最佳,继代2次后胚性愈伤质量状态最好。以基本培养基NBS中添加0.02 mg/L NAA+2 mg/L KT+3 mg/L 6-BA+50 mg/L VC+0.5 mg/LIAA为蜀恢527成熟胚遗传转化的最佳分化培养基,分化频率较高,分化率达到36.42%。利用优化的籼稻遗传转化体系,通过根癌农杆菌介导法将水稻转录因子OsbZIP39转入籼稻基因组中,经PCR分子初步检测得到了抗性植株。

The callus transformation rate of Indica rice in tissue culture is relatively poor. Especially, the establishment of highly efficient regeneration system of superior varieties and the factors which affect its transformation remain unknown. This situation restricts at certain extent the progress of Indica rice genetic engineering. The seeds of restorer line Shuhui527 were used in this study to investigate the effects of quality and physiological state of theirs mature embryo callus under different temperatures and light conditions. Meanwhile, transformation efficiency influenced by differentiation medium supplemented with different hormones and the ratio between hormones were further studied. The results showed that under light condition at 32℃, the mature embryo callus growth is the best, under N6 D medium callus quality is the best after twice subcultures. The NBS medium adding 2.0 mg/L KT ＋ 0. 02 mg/L NAA ＋ 3 mg/L 6-BA ＋ 50 mg/L VC ＋ 0. 5 mg/L IAA was found most suitable for Shuhui 527 genetic transformation, and the differentiation rate reached 36. 42%. Using our new system, rice transcription factor OsbZIP39 was introduced into Indica rice genome by Agrobacterium-mediated transformation. Resistant plants were obtained preliminarily by PCR analysis.