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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。
The optimum concentration of antibiotics (Km) for callus proliferation, differentiation and seedling rooting were screened from the embryogenic calli of sugarcane, and the factors influencing Agrobacterium-mediated genetic transformation of sugarcane were: pre-culture time, infection Time and co-culture time were studied. Finally, the optimum antibiotic (Km) concentration of the three stages of proliferation, differentiation and seedling rooting of sugarcane were 300 mg / L, 40 mg / L and 20 mg / L, respectively. Agrobacterium tumefaciens pretreatment time, infection time and co-culture time were determined 4d, 30min, 4d. PCR analysis showed that 61.6% of the resistant plants amplified the expected fragment, indicating that the foreign gene has been integrated into the sugarcane genome. It provided a good technical support for further functional verification of sugarcane gene and transgenic research.