Abstract:

The germinating seeds of white clover were used as transformation receptor, and npt-Ⅱ

gene was chosen as a selection gene. External factors that affect the efficiency of

genetic transformation were studied and optimized. A high-frequency transformation system

of white clover had been established by adopting Agrobacterium tumefaciens soaked seeds

transformation technology. The seeds of white clover germinated for 1 hour, then adding

Agrobacterium with concentration of 0.9(OD600) under 5 min+30 s vacuum treatment. After

24 hours infection in oscillating culture and 1 day co-cultivation, the transferred

plants were screened by 50 mg/L kanamycin. This transformation system was so effective,

that positive transformation rate reached 45.5%. The transgenic plants were also examined

by PCR analysis and GUS staining. It was proved that npt-Ⅱ and GUS gene had been

integrated into the genome.

Key words: white clover, soaking seeds with Agrobacterium tumefaciens, genetic transformation

摘要：

以白三叶萌动种子作为转化受体,利用npt-Ⅱ筛选基因对影响农杆菌介导的遗传转化外部因素进行深入

探索,通过优化转化条件,建立了农杆菌浸种途径介导的白三叶遗传转化体系。白三叶种子催芽1 h后,

加入OD600值为0.9的农杆菌菌液,附加5 min+30 s真空处理方式,振荡侵染24 h,共培养1 d后,用50

mg/L卡那霉素筛选抗性植株,该体系能够获得较好的转化效果,阳性转化率为45.5%。通过PCR检测和GUS

组织化学染色法,初步验证npt-Ⅱ和GUS基因已经整合到植物基因组中。

关键词: 白三叶；农杆菌浸种法；遗传转化