农杆菌介导GUS基因对多年生黑麦草转化的研究

通过检测愈伤组织中GUS基因的瞬间表达,研究农杆菌LBA4404/pCAMBIA1301介导多年生黑麦草的转化体系。通过对多年生黑麦草瞬间表达率的比较,确立了其遗传转化的最佳优化条件。研究发现,多年生黑麦草不同品种的转化率在25%~45%之间变化。多年生黑麦草遗传转化最佳优化条件是预培养10d的胚性愈伤组织、浓度为0.5~0.8OD的农杆菌菌液以及2d共培养时间。在共培养基中添加100μmol/L乙酰丁香酮能有效地提高植物瞬间表达率。两种侵染处理方法比较结果为滤纸滴加法比浸泡法更优。转化后对愈伤组织的干燥处理能抑制农杆菌过度繁殖,能改善愈伤状态,有利于提高转化率。

Study on Agrobacterium-mediated GUS transformation for Lolium perenne L.

Agrobacterium tumefaciens mediated transformation system of Lolium perenne was studied by using GUS gene transient expression method.Mature seed derived callus from L.perenne was transformed by GUS gene containing A.tumefaciens LBA4404/pCAMBIA1301.Frequency of the transformation was detected,and the optimal conditions for L.perenne genetic transformation were determined.It was found that the transformation frequency varied from 25%～45% according to different genotypes(variety).The optimal conditions to get higher transformation efficiency were 2 days co-cultivation with 0.5～0.8 OD A.tumefaciens solution in dark after 10 days pre-culturing.The transformation frequency was significantly improved by adding 100 μmol/L acetosyingone into co-cultivation medium.Adding A.tumefaciens solution on surface of filter paper was found to be a better infecting method of A.tumefaciens rather than suspending in A.tumefaciens solution.The partial desiccation treatment after transformation was beneficial to prevent over-growth of A.tumefaciens and increase the transformation efficiency.