【作者】 张丽丽；

【导师】 师校欣； 杜国强；

【作者基本信息】 河北农业大学 ， 果树生物技术， 2007， 硕士

【摘要】 本研究以苹果（Malus domestica Borkh）主栽品种嘎拉（Gala）组培苗为试材，系统研究了影响叶片离体再生及叶盘法遗传转化效率的若干因素，优化了苹果离体叶片再生不定芽系统和遗传转化系统；以农杆菌EHA105为介导将目的基因——β-1，3-葡聚糖酶抗病基因导入苹果品种，成功获得了转化植株，为今后苹果遗传转化及抗病性研究提供了依据。试验结果表明：1．苹果不同外植体再生不定芽能力不同，以叶片的再生效果优于白化茎段。2．继代苗苗龄对嘎拉叶片不定芽再生效率无明显影响，但考虑到取材的难易，以继代培养20～30d为宜。3．苹果离体叶片再生过程中，前期暗培养可以显著提高叶片再生频率。暗培养时间以14～21d为宣，随暗培养时间延长再生频率虽没有降低趋势，但是再生芽黄化现象严重，不利生长。4．接种方式对嘎拉苹果的叶片再生效率影响不显著，但对再生芽数的影响显著，以近轴面朝上接种再生芽数多。5．在根癌农杆菌EHA105介导的遗传转化体系中，以叶片为转化受体，适宜的卡那霉素（Kan）选择压为10mg／L，抑菌抗生素为头孢霉素（Cef）300mg／L。6．研究了影响叶盘法遗传转化的因素，优化了遗传转化途径：外植体在分化培养基上预培养1～2d；农杆菌悬浮至对数生长期，菌液浓度为OD₆₀₀=0.6～0.8时侵染外植体10min；共培养1～2d；然后脱菌、选择同时进行，每隔20d左右更换一次选择培养基。7．共有19个转化株系通过了Kan抗性检测，具体包括三个方面：①转化株在附加Kan50mg／L的继代培养基上能够连续增殖继代，生长正常。②转化株叶片在附加或未加Kan50mg／L的再生培养基上的再生能力无明显差异。③转化株在附加Kan50mg／L的生根培养基中能正常生根。8．通过Kan抗性筛选的转化株系经GUS检测，均出现蓝色斑点，进一步经PCR扩增检测，转化株系扩增出的特异条带和阳性对照一致，表明Glu基因已整合至嘎拉苹果基因组DNA中。更多还原

【Abstract】 In this study, the disease-resistant geneβ-1, 3-glucanase was transferred into apple cultivarGala via Agrobacterium tumefaciens strain EHA 105. The adventitious bud regenerationsystem and genetic transformation system have been modified by studying the effect ofexplant types and status on the efficiency of regeneration and transformation. Transgenicplants were obtained and examined successfully. The main results were as followed:1. The efficiency of adventitious bud regeneration was varied from the explants, with thatof the leaf disc was higher than that of the albino stem segments.2. The efficiency of adventitious bud regeneration was not remarkable when the leaveswere sampled from the plantlets subcultured for different days. The highest efficiency wasobtained when the leaves were sampled from the plantlets that were subcultured for 20～30days.3. During the course of adventitious bud regeneration, the dark incubation can improve theregeneration rate significantly. The best dark incubating time was 14～21d days for theregeneration rate.4. The inoculation method effect on regeneration rate is not significantly. But the effect onnumber of regeneration buds is significantly. The number of bud regenerated were morewhen adaxial side upward in medium.5. The optimal concentration of Kanamycin was 10 mg/L when then leaves were used asthe explants for the-transformation mediated by Agrobacteria of strain EHA 105,at thesame time, the optimal concentration of Cefotaxime was 300mg/L.6. By comparing the factors on agrobacterium-mediated gene transformation, it suggeststhe optimal protocol for gene transformation in apple would be: at first, the explants shouldbe pre cultured for one day or two on the inducing medium; the bacterium concentrationfor inoculation should be around OD₆₀₀=0.6～0.8 around, the co-cultivation time is about 10minutes, co-cultured less for two days, and then, the explants were transferred onto theinducing medium. Changing the new medium about every 20 days. 7. There were nineteen transgenic plantlets passed the kanamycin-resistant test. Thekanamycin-resistant test of transgenic plantlets was based on the three main aspects:①Thetransgenic plantlets were able to be subcultured on media supplemented with 50mg/Lkanamycin, continuously.②The leaf pieces of the transgenic plantlets showed as sameregeneration ability on media containing 50mg/L kanamycin as on media withoutkanamycin.③The transgenic plantles were able to root normally on media supplementedwith 50mg/L kanamycin.8. Choose the plantlets which pass the kanamycin-resistant test for further tested by GUSanalysis. The result showed that all those transgenic plantlets were showed blue spots Then,the transgenic plantlets were further tested by PCR analysis. The plantlets showed specificpositive band as same as the plasmid control. The result indicated that the Glu gene hasbeen integrated into the gala apple genome.更多还原