农杆菌介导石斛兰遗传转化的研究

 利用根癌农杆菌介导转化石斛兰类原球茎（PLBs），研究了石斛兰对潮霉素的敏感性，并通过gus瞬时表达率比较不同类型农杆菌、侵染液浓度及乙酰丁香酮（AS） 等对石斛兰转化的影响。结果表明：带有pCAMBIA1301的根癌农杆菌（Agrobacterium tumefaciens）EHA105（简称为E1301）比带有相同表达载体的LBA4404（简称L1301）对石斛兰的转化效率更高，前者约为后者的6倍；较高浓度的侵染液转化效率更高（OD₆₀₀为1.0或1.2时的转化效率约为OD₆₀₀为0.6或0.8时的2倍）；在农杆菌生长至OD₆₀₀值为0.8时，添加AS可提高转化效率；而当OD₆₀₀为0.6、1.0和1.2时，添加AS反而不同程度地降低转化效率；在各种处理中，OD₆₀₀为1.0时不添加AS的转化效率最高，达44.4％。经过5~6个月选择培养，抗性小苗经GUS染色、PCR 和 Southern杂交检测证明为转基因植株。

Study on Agrobacterium-mediated Transformation of Dendrobium

Dendrobium ekapol 'Panda 1' was transformed by Agrobacterium tumefaciens. The sensitivity of Dendrobium to hygromycin and transformation effective factors such as different strains of Agrobacterium、bacterium concentration and acetosyringone (AS) were studied by transient gus expression. The results showed that the transformation efficiency of Dendrobium transformed by E1301 (EHA105 equipped with pCAMBIA1301) was 6 times higher than L1301 (LBA4404 equipped with the same vector). Higher concentration of bacterium was more effective to transform Dendrobium (the transformation efficiency at OD₆₀₀ 1.0 or 1.2 was twice as OD₆₀₀ 0.6 or 0.8). When the concentration of bacterium was 0.8 (OD₆₀₀), the transformation efficiency was increased with the addition of AS. At other concentrations (OD₆₀₀ 0.6, 1.0 or 1.2), the transformation efficiencies were reduced with AS. Among all the treatments, the concentration of bacterium of OD₆₀₀ 1.0 without AS gave the highest transformation efficiency of 44.4%. Subjected to GUS histochemical staining, PCR and Southern blot analysis, the hygromycin-resistant shoots were proved to be transformed shoots.