农杆菌介导红三叶草遗传转化体系的建立

Establishment of Agrobacterium-mediated Genetic Transformation System of Trifolium pratense L.

以红三叶草品种‘Altaswede’作为受体材料,以GUS基因为报告基因,研究了影响农杆菌介导红三叶草遗传转化的几种因素以及几种侵染方法对提高转化效率的影响。建立了农杆菌介导的红三叶草快速高效遗传转化体系,并获得了转基因抗性苗。研究结果表明,以上胚轴为外植体,在OD600值为0.6的菌液中侵染20 min,真空度6×10-2Pa处理9 min,共培养5 d后转入含卡那霉素50 mg/L的分化培养基中,4周后60%的外植体分化出不定芽并生根成苗。PCR分子检测表明有80%的植株为GUS阳性,转化率约为50%。

Altaswede was used as transformation material and the GUS gene was taken as report gene. Some factors and infection methods, which may affect the transformation frequency, were researched. An efficient agrobacterium-mediated genetic transformation system of Trifolium pratense was established and some transgenic plants were obtained. The best result was obtained when epicotyls were as explants. The epicotyls of red clover were firstly infected with agrobacterium suspension （OD600,0. 6） for 20 minutes and under 6 × 10^-2 Pa for 10 minutes, and then co-cultured for 5 days without light. The explants were further cultured on the differentiation medium containing Kanamycin （50 mg/L）. Shoots could be obtained from 60% of the explants and the seedlings with roots occurred after 4 weeks. GUS gene had been detected in 80% of the transgenic plants by PCR. The transformation frequency was up to 50%.