农杆菌介导甘蔗愈伤组织遗传转化体系的优化

Optimization of Genetic Transformation System of Sugarcane Callus Mediated by Agrobacterium

以甘蔗胚性愈伤组织为材料,对愈伤组织增殖、分化、幼苗生根的最适抗生素(Km)浓度进行筛选,并对影响农杆菌介导的甘蔗遗传转化因素:预培养时间、侵染时间和共培养时间等进行了研究。最后确立了甘蔗愈伤组织增殖、分化及幼苗生根三个阶段的最适抗生素(Km)浓度分别为300 mg/L,40 mg/L,20 mg/L。确定了农杆菌浸染的预培养时间、侵染时间和共培养时间分别为4d,30min,4d。PCR检测表明,61.6%的抗性植株可扩增出预期的片段,表明外源基因已经整合到甘蔗基因组中。为进一步的甘蔗基因功能验证及转基因研究提供良好的技术支撑。

To establish high-efficiency sugarcane transformation system, the optimal concentration of antibiotic （Kin） was screened for subculture and differentiation of sugarcane calli, as well as seedling rooting, respectively. The factors that influenced sugarcane transformation, such as pre-cuhure time, infection time and co-culture time were also studied. The results showed that the optimal concentrations of antibiotic （ Km ） for subculture, differentiation and seedling rooting were 300 rag/L, 40 mg/L and 20 rag/L, respectively. The optimal pre-culture time, infection time and co-culture time were 4d, 30min and 4d, respectively. PCR detection showed that 61.6% of resistance plants were transformed plants. This work lays a solid foundation for further study on gene function and transgenic research of sugarcane.