根癌农杆菌介导苹果愈伤组织遗传转化体系的优化

Optimization of Agrobacterium tumefaciens mediated genetic transformation system of apple callus

【目的】以‘王林’苹果愈伤组织为转化研究的材料,在已经建立的遗传转化基础上,优化根癌农杆菌介导的‘王林’愈伤组织的遗传转化系统。【方法】在遗传转化的过程中,通过对根癌农杆菌的侵染时间、共培养以及抗性的选择,优化遗传转化体系并且增加转化效率。【结果】选择生长20 d的愈伤组织,在根癌农杆菌OD 600=0.8时侵染20 min,共培养3 d、转入卡那霉素30 mg/L和头孢噻肟250 mg/L的培养基上进行筛选,可显著提高遗传转化效率。【结论】将McmiR399d作为标记基因进行转化,优化苹果‘王林’愈伤组织的遗传转化系统,从而得到过表达和沉默的McmiR399d‘王林’愈伤组织。

【Objective】In this experiment,the method of genetic transformation mediated by Agrobacterium tumefaciens was optimized in the callus of Malus domestica cv.‘Orin’.【Methods】The genetic transformation system was optimized and the transformation efficiency was increased by the selection of the bacterial solution concentration,soaking time,co-culture and screening resistance.【Results】The callus growing for 20 days was selected,infected for 20 minutes at the concentration of Agrobacterium tumefaciens solution OD 600=0.8,co-cultured for 3 days,and screened on medium with kanamycin concentration of 30 mg/L and cephalosporin concentration of 250 mg/L,which could significantly improve the efficiency of genetic transformation.【Conclusion】McmiR399d was transformed as a marker gene,the genetic transformation system of the callus of Malus domestica cv.‘Orin’was optimized to obtain the over-expressed and silenced McmiR399d the callus of Malus domestica cv.‘Orin’.