油菜下胚轴农杆菌介导法转化影响因素探讨

Study on the Major Factors Affecting Transformation Efficiency by Agrobacterium-mediated in Brassica napus

利用甘蓝型油菜下胚轴为外植体,对油菜农杆菌介导转化中的主要因素进行了研究,并由此建立了油菜下胚轴农杆菌介导的遗传转化体系。研究发现,在农杆菌介导的油菜下胚轴转化中,较好的转化体系是:外植体在农杆菌侵染前进行3d的预培养。农杆菌的浸染浓度为OD600=0.2～0.4。共培养时的pH值为5.2。在转化后的分化培养基中附加30μmol/LAgNO3。

The hypocotyls segments were transformated by Agrobacterium mediated transformation in Brassica napus. Some factors affect shoot regeneration of transformated cells were studied. Our study revealed that, for Agrobacterium -mediated transformation, the hypocotyls segments were preconditioned on preconditioning medium for 3 days prior to infect with A.tumefaciens and the addition of 30 μmol/L AgNO_3 in the regeneration medium were prerequiste for obtained high transformation efficiency of explants. Explants were submerged in the A.tumefaciens suspension at the concentration of OD_(600)=0.2-0.4 for 5 minutes and placed on the co-cultivation medium pH 5.2 could result in high transformation efficiency of explants.