农杆菌介导紫花苜蓿子叶遗传转化的影响因素研究

以5-7d的紫花苜蓿无菌苗为材料进行卡那霉素抗性筛选，用RG-2质粒（合VP7基因和卡那霉素抗性基因）和农杆菌（EHA101、EHA105）构建重组质粒，对无菌苗和重组质粒进行共培养，研究菌株类型、菌液浓度对转化效率的影响。结果表明，菌株EHA105对紫花苜蓿的转化率达4％，重悬菌液的0D60。以0．3～0．5为宜，农杆菌侵染时间以10～12min较好；PCR检测结果表明，37株卡那霉素抗性植株中30株为阳性，说明VP7外源基因已整合到受体植物基因组中。

Study of Influencing Factors on Genetic Transformation Efficiency of Medicago sativa L. Cotyledon Mediated by Agrobacterium

With 5-7 days old aseptic seedling of Medicago sativa L. as the material, their kanamycin resistance was selected. The recombinant plasmid was constructed with RG-2 plasmid (containing VP7 gene and kanamycin resistance gene) and agrobacterium (EHA101、EHA105), the recombinant plasmid and the aseptic seedling was co-cultured, and the effects of bacteria strain type and bacterial concn. on transformation efficiency were studied. The results showed that the transformation efficiency of EHA105 on M. sativa reached 4%. The optimum cocn. of resuspended bacterial liquid was OD600 of 0.3-0.5 and the optimum infection time was 10-12 min. PCR detection results showed that 30 plants showed positive in 37 kanamycin resistance plants. Its indicated that the foreign gene VP7 had already integrated into genome of receptor plants.