农杆菌介导霞多丽葡萄胚性细胞系遗传转化条件的优化

为建立葡萄(Vitis vinifera)遗传转化技术体系,以霞多丽葡萄(Chardonnay)胚性细胞系为靶组织,采用GUS检测法,对影响农杆菌介导葡萄遗传转化效率的主要因素进行了研究。结果表明,超声波处理时间长短对转化效率有较大影响,在所试的0.5、1、5、10 min 4种不同时间的超声波处理中,以5 min时转化效率较好,平均达到9.11个蓝色斑点;AS浓度对转化效率有明显差异,当浓度为50μmol/L时,平均蓝色斑点数为8.89个,100μmol/L时,达到12.44个;DTT质量浓度对转化效率也有较大影响,在所试的3种质量浓度(1,2,3 mg/L)中,以3 mg/L为最佳,有16.67个蓝色斑点。通过GUS瞬时表达检测,确立了农杆菌介导葡萄胚性细胞系遗传转化的几个最适影响因素,从而为葡萄遗传转化技术体系的建立奠定了基础。

Optimizing conditions for Agrobacterium-mediated transformation of Chardonnay grape cultivar's embryogenic cells

To establish an efficient transformation of grape(Vitis vinifera),the factors affecting the Agrobacterium-mediated transformation efficiency were studied by GUS assay using the embryogenic cells as the target tissue.The embryogenic cells were sonicated and inoculated with Agrobacterium EHA105/pCAMBIA1301,and placed on co-culture medium containing different concentrations of acetosyringone and dithiothreitol.The transient expression of the gene gus was assayed at 3 or 4 d after co-cultivation for transformation.The highest transformation efficiency was obtained when the embryogenic cells were sonicated for 5 min.The concentration of acetosyringone also affected the transformation efficiency.The average blue spots were determined to be 8.89 at 50 μmol/L AS,and 12.44 at 100 μmol/L AS.The addition of DTT to the co-culture medium also enhanced transient GUS expression.The blue spots were reached up to 16.67 at 3 mg/L of DTT.In this study,the optimum factors affecting the Agrobacterium-mediated transformation of grape embryogenic cells were established by GUS transient expression assay.These results will provide useful information for establishment of an efficient transformation method of grape.