为建立一套高效的野葛(Pueraria lobata)毛状根遗传转化体系, 本研究以野葛组培苗为外植体, 探讨不同因素对野葛毛状根遗传转化效率的影响。结果表明, 基因型是野葛毛状根遗传转化体系高效建立的主要限制因子; 发根农杆菌菌株K599为最适宜的菌株; 以培养5−13代组培苗继代培养8天的第1−2节位刚展开的幼嫩叶片为最佳外植体材料, 预培养3天, 菌液侵染15分钟, 毛状根诱导率最高, 可达22.4%。野葛毛状根继代增殖的最佳培养方式为固体培养基培养, 其生长的毛状根鲜重是液体培养基生长的75倍; PCR检测和荧光显微观察结果显示, GFP及rolB基因在野葛毛状根基因组中稳定表达, 共转化频率为80%。本研究初步建立了发根农杆菌介导的野葛毛状根遗传转化体系, 为野葛基因功能鉴定奠定基础。

To establish an efficient Agrobacterium rhizogenes-mediated transformation system of Pueraria lobata, in this study, tissue cultured seedlings were used as explants to explore the effects of different factors on efficiency of Agrobacterium rhizogenes-mediated transformation system of P. lobata. The results indicated that genotype was the most important limiting factor of all. A. rhizogenes K599 was identified to be the most suitable strain. The optimal explant material was immature leaves that had just unfolded from the first to second nodes of the 5th to 13th generation tissue culture seedlings subcultured for 8 days. After 3 days of pre-culture and 15 minutes of bacterial infection, the highest induction rate of hairy roots could up to be 22.4%. The optimal culture method for proliferation of hairy roots in P. lobata was solid medium culture, and the fresh weight of hairy roots grown on solid medium was 75 times that of hairy roots grown on liquid medium. The PCR analysis and fluorescence microscopy assays showed the expression of GFP and rolB gene in the hairy roots of P. lobata was stable, and the rate of co-transformation was 80%. In our study, an A. rhizogenes-mediated genetic transformation system of P. lobata was preliminarily established, which laid a foundation for gene function identification in P. lobata.