阔叶猕猴桃遗传转化技术参数的优化

Study on the optimum technological parameters for genetic transformation of Actinidia latifolia

为了提高阔叶猕猴桃的遗传转化效率,以阔叶猕猴桃叶柄为试材,通过根癌农杆菌介导法进行了遗传转化技术参数的研究。结果表明,叶柄预培养3～4d、用农杆菌悬浮液(D600nm值0.5)感染10min、共培养48h、共培养时在培养基中加入200μmol/L乙酰丁香酮处理可以获得较高的gus基因表达率。在供试的1200个叶柄中获得了49个抗性芽,转化频率为4.1%。对转基因抗性材料进行PCR检测和gus基因组织化学染色,证实了外源基因已整合到阔叶猕猴桃的基因组中,并得到稳定表达。

In order to improve the genetic transformation efficiency of Actinidia latifolia Merr., its petiole explants were transformed by Agrobacterium tumefaciens for studying the optimum technological parameters. Results showed that the petiole explants pre-cultured for 3-4 days, infected for 10 min with Agrobacterium tumefaciens （concentration： 600nm 0.5 ）, and co-cultured for 48 h in the medium with 200μmol/L acetosyfingone could get the highest level of gus gene expression. 49 kanamycin-resistant shoots were obtained from 1 200 petiole explants and transgenic frequency was 4.1%. Successful transformation was confirmed by PCR and histochemical analysis of gus activity in leaves of kanamycin-resistant shoots. The resuits showed that gus gene was integrated into A. latifolia genomes and had stable expression.