农杆菌介导大豆子叶节遗传转化的研究

Study of the Agrobacterium-mediated Transformation Systems of Soybean Cotyledonary Node

直接以农杆菌转化系统为平台 ,以栽培大豆 (GlycinemaxL )吉林 35、中黄 2 8、南农、铁丰 2 9、铁丰 30和开育 12的子叶节为外植体 ,用LBA4 4 0 4农杆菌 (含pPC KSA质粒 )研究了影响大豆子叶节转化的因素。结果表明 ,大豆品种之间转化存在着差异 ,吉林 35转化率最高 ,平均转化率为 2 16 % ,单次最高转化率达 6 12 % ;中黄 2 8次之 ,平均转化率为1 9% ,单次最高转化率达 3 33% ;南农转化率为 1 0 5 % ;铁丰 2 9居第 4位 ,转化率为 0 95 % ;铁丰 30和开育 12无转化植株。用 4d苗龄的子叶节 ,农杆菌浸染 30min ,共培养 3d比较合适。萌发培养基和不定芽诱导培养基对转化率的影响存在交互作用 ,对于吉林 35来说 ,大豆萌发培养基 (G)和不定芽诱导培养基 (Y)的最佳组配方式为G2 +Y1(转化率 6 12 % )和G1+Y2组合 (转化率 5 2 6 % )。农杆菌介导的转化系统中 ,转化植株的筛选方式对转化率影响很大 ,筛选培养初期 ,采用比较低的筛选浓度 ,在继代培养过程中逐次提高筛选压力 ,可以获得较好的转化效果。

Suitable soybean genetic transformation system has not been established by now. The factors influencing Agrobacterium-mediated soybean cotyledonary node transformation were studied in this research so as to improve the transformation frequency of soybean. The results showed that the transformation frequency was different among cultivars of soybean. Jilin 35 was the highest in transformation rate, Zhonghuang 28 was the second and followed by Nannong and Tiefeng 29, and there was no transformed plant in Tiefeng 30 and Kaiyu 12. The average and the highest transformation rate were 2.16% and 6.12% for Jilin 35, and 1.9% and 3.33% for Zhonghuang 28, respectively (Table 1). The suitable seedling age was about 4 days-old (Fig.5) and the appropriate duration of infection and co-culture with Agrobacterium were about 30 minutes and 3 days respectively (Fig. 4). There was an interaction between the germination medium and shoots regeneration medium. for transformation frequency. The better combination pattern of germination medium and shoots regeneration medium. were G2+Y1 (transformation rate was 6.12%) and G1+Y2 (transformation rate was 5.26%) in this research (Fig.6). The screening method had a significant effect on increasing the transformation frequency of the Agrobacterium-mediated transformation system (Table 2). Kanamycin was used as a screening agent in this study. Higher transformation rate was obtained by using the screening model S1 which the kanamycin concentration increased gradually from 60 mg/L to 100 mg/L when subculture in the shoots regeneration stage. It was suggested that the lower concentration of screening agent should be used in the earlier screening stage, then the higher concentrations increased gradually in the later stages.