根癌农杆菌介导寒富苹果转化体系的建立

Establishment of Agrobacterium tumefaciens-mediated transformation system for Hanfu apple

以寒富苹果组培苗叶片为外植体,利用植物表达载体上含潮霉素抗性基因的根癌农杆菌EHA105(pCAMBI-A1301)和含卡那霉素抗性基因的根癌农杆菌EHA105(pCAMBIA2301)对影响寒富遗传转化效率的因素进行系统研究。结果表明,寒富叶片对潮霉素反应敏感,在附加潮霉素的培养基上寒富叶片褐化较为严重。潮霉素和卡那霉素适宜的筛选质量浓度分别为4 mg.L-1和25 mg.L-1。农杆菌介导寒富苹果转化体系的建立以MS+TDZ 2.0 mg.L-1+NAA 0.5 mg.L-1培养基为叶片离体再生芽培养基。适宜的转化条件为:菌液浓度D 600nm=0.5、叶片外植体在菌液浸泡8 min、共培养时间为3～4 d、推迟4 d进行筛选培养。抗性基因对转化效率具有明显的影响,EHA105(pCAMBIA2301)的平均抗性芽再生率(0.96%)比EHA105(pCAMBIA1301)的平均抗性芽再生率(0.58%)高出几乎50%,EHA105(pCAMBIA2301)的抗性芽再生率最高达1.87%。GUS组织化学染色和PCR鉴定结果表明本研究获得了寒富苹果转基因植株。

Several factors that affect the efficiency of the Agrobacterium tumefaciens-mediated transformation of Malus do-mestica cv.Hanfu were studied using leaves of in vitro plants and Agrobacterium strain EHA105 containing a binary vector pCAMBIA1301 inserted the hygromycin（Hyg） resistance gene or a binary vector pCAMBIA2301 inserted kanamycin（Kan） resistance gene.The result showed that the leaves of Hanfu apple cultivar browned seriously in medium with Hyg which indi-cated that they are sensitive to Hyg.The suitable concentration of antibiotics was 4 mg.L-1 Hyg or 25 mg.L-1 Kan.The medi-um of MS supplemented with 2.0 mg.L-1 TDZ and 0.5 mg.L-1 NAA was used as the regeneration medium in the transformation of Hanfu apple.The highest transformation efficiency was obtained when the D600nm of Agrobacterium tumefaciens solution was 0.5,the explants were infected in the bacterium solution for 8 min,the co-cultivated period was 3 to 4 days,and the time of delayed selection was 4 days.It was found that the resistance gene inserted in the plasmid strongly affected the transformation efficiency.The average rate of resistant bud regeneration was 0.96% for the treatment with EHA105（pCAMBIA2301）,near 50% higher than that（0.58%） with EHA105（pCAMBIA1301）.The highest rate of resistant bud regeneration reached 1.87% for the treatment with EHA105（pCAMBIA2301） in this study.Transgenic plants were identified and confirmed by GUS histochemical assay and PCR analysis.