苹果属植物再生体系优化及长富6号遗传转化体系的建立

Optimization of Malus plant regeneration system and establishment of genetic transformation system for apple cultivar Nagafu No.6

以苹果栽培品种长富6号和砧木湖北海棠、八棱海棠3种苹果属植物的无菌生根苗和继代苗叶片为材料,研究不同苹果属植物生根苗与继代苗叶片间的再生差异和暗培养时间对苹果属植物生根苗叶片再生的影响;以长富6号生根苗叶片为受体材料,对影响长富6号遗传转化的因素进行优化,对获得的抗性芽进行PCR和RT-PCR检测。结果表明:3种苹果属植物生根苗叶片的再生能力优于继代苗,暗培养21 d时,3种苹果属植物生根苗叶片的再生状况最佳;在菌液OD600为0.5、浸染时间9 min、共培养时间3 d和潮霉素选择压1.0 mg/L体系中抗性芽诱导率最高。用PCR和RT-PCR对所获得的抗性芽检测,结果初步证实外源基因已成功导入长富6号并在转录水平上表达。

Three Malus plants, Nagafu No. 6, M. hupehensis Rehd. , and M. robusta Rehd. were selected as mate- rials to study their regeneration using rooted seedlings and secondary seedling leaves as explant, and the effect of different dark cuhure time on regeneration was also investigated. The results showed that rooted seedlings of three Malus plant were better for regeneration than secondary seedings when cultured in dark for 21 d. The factors effecting genetic transformation for Nagafu No. 6 were discussed using rooted seedlings as explant, and hygromycin-resistant shoots were confirmed by PCR and RT-PCR. The transformation efficiency was the highest when the explant was dipped in Agrobacterium tumefaciens solu- tion with OD600 0.5 for 9 min, and was co-cultured for 3 d in 1 mg/L hyg syste PCR and RT-PCR analysis confirmed that the exogenous gene was transferred into Nagafu No. 6 and expressed in transcription level.