苹果叶盘法基因转化中抗生素种类和浓度的筛选

The Selection of Antibiotics and Their Concentration during Gene Transformation via Leaf Disc Regeneration System in Apple

以苹果品种嘠拉试管苗为试材,研究了卡那霉素(Km)、潮霉素(Hm)对继代苗生长和离体叶片再生的影响,以确定叶盘法基因转化的选择压和转化体的筛选浓度;同时研究了不同浓度的头孢霉素(Cef)和羧苄青霉素(Carb)对苹果离体叶片再生的影响及对农杆菌EHA105和LBA4404的抑菌效果;以确定侵菌共培养后合适的抑菌抗生素种类和浓度。结果表明:Km10mg/L、Hm4.0mg/L完全抑制了叶片不定芽的分化,可作为苹果叶盘法转化时抗性芽的选择压;Km50mg/L、Hm5.0mg/L达到继代苗的半致死浓度,可作为转化后抗性苗的筛选浓度;由于抗性芽再生的选择压与抗性苗筛选浓度相差较小,Hm相对于Km更适合作为苹果基因转化的选择标记;培养基中附加Cef300mg/L能完全抑制农杆菌生长,对叶片不定芽的再生影响不大;而附加Carb则需400mg/L以上才能抑制农杆菌生长,同时严重抑制了叶片再生。因此,宜选用Cef作为苹果基因转化的抑菌抗生素。

It was studied that the effect of Kanamycin and Hygromycin on the growth of subcultured explants and the efficiency of adventitious bud regeneration from leaf disc, and the effect of the concentration of Cefotaxime and Carbenicillin on the efficiency of regeneration and the preventing growth of Agrobacteriua strains EHA 105 and LBA4404. The results showed that the supplying of Kanamycin 10 mg/L or Hygromycin 4.0 mg/L in the media could restrict the adventitious bud regeneration in a greatest extent, and it could be used as a selecting pressure for the regeneration of adventitious buds with antibiotic-resistant. The supplying of Kanamycin 50 mg/L or Hygromycin 5.0 mg/L in the media could led half of the subculture plantlets to death, and it could be used for the selection of transgenic plantlets with antibiotic-resistant. The Hygromycin is considered to be more useful than Kanamycin as a selecting marker during gene transformation in apple, since the difference of the concentrations of Hygromycin used for the adventitious bud regeneration and the plantlets selection is less than that of Kanamycin. The supplying of Cefotaxime 300 mg/L could eliminated the growth of Agrobacteria eventually without affecting the adventitious bud regeneration from leave. But the supplying of Carbenicillin should be more than 400 mg/L to obtain the well controlling, and that seriously limited the adventitious bud regeneration. Then, it suggests that Cefotaxime should be used for the eliminating of Agrobacteria strains EHA105 and LBA4404.