农杆菌介导GFP基因转化苹果茎尖碎片获得转基因植株

Transgenic plants from fragmented shoot tips of apple via Agrobacterium-mediated transformation with GFP gene

【目的】为解决基因型依赖性比较强的苹果品种遗传转化困难等问题,研究不同伤害处理方式下苹果茎尖碎片的再生能力及其对苹果转化率的影响。【方法】以苹果‘姬神’试管苗为试材,运用农杆菌介导法对苹果茎尖和离体叶片进行遗传转化。【结果】将茎尖纵切为4部分时,可以高比率的形成愈伤组织(88.4%)和再生不定芽(59.8%)。在20 mg·L-1的卡那霉素筛选压下,300个茎尖外植体中有50.4%的茎尖碎片形成愈伤组织并获得了19株表达GFP的转基因植株。而在同样的筛选压下有22.6%农杆菌介导的离体叶片形成愈伤组织,但不能获得转基因植株。降低筛选压可以提高植株再生率,但其中包含大量嵌合体,转化效率并没有明显提高。【结论】苹果茎尖做为转化受体基因型依赖性较低,要优于苹果离体叶片。

[Objective] It is difficult to introduce a new gene into some apple varieties which are strongly dependent on genotype. In order to resolve this problem , the effects of different damage treatment on regeneration ability of apple shoot tips and transgenie conversion rate were studied. [Method]The GFP gene was introduced into the shoot tips and the leaves of Jishen apple cuhivar by Agrobacterium-mediated ge netic transformation. [Result]The results showed that, when shoot tips were cut vertically into 4 parts, a high percentage of callus formation（88.4%） and adventitious shoot regeneration （59.8%） was observed. Un- der 20 mg. L-1 Kanamycin selection pressure, over 50.4% fragmented shoot tips developed callus and 19 transgenic plants with GFP（green fluorescent protein） expression were obtained from about 300 explants. No transgenic plant was obtained fromAgrobacterium-mediated transformed leaves, even though 22.6% of which formed callus after co-cultivation and selection. Our experiments also showed that with lower selec- tion pressure, the transformation efficiency could be improved generally accompanied by a high numbers of chimeric shoots. [Conclusion]As transformation receptor, apple stem tips is better than the apple in vitro leaves because the apple stem tips are weakly dependent on genotvpe.