以近轴端子叶为外植体,研究了菌液浓度、菌液浸泡时间、共培养时间及推迟筛选对根癌农杆菌介导的苹果属植物平邑甜茶转化效率的影响,建立了平邑甜茶高效的转化体系.结果表明:较优转化体系是将成熟胚接种在胚萌发培养基上培养,7～10d后剪取近轴端子叶,子叶外植体在OD600=0.5的EHA105菌液中浸泡10min后在再生培养基上共培养5d,然后在附加20mg· L-1Kan和400mg· L-1 Cef的再生培养基上选择培养,在此转化体系上平邑甜茶抗性芽再生率达9.0％.对27个Kan抗性株系的叶片进行GUS组织化学染色分析,23个株系呈现出GUS染色阳性反应.本研究为平邑甜茶的基因工程育种奠定了重要基础.%The factors that affect transformation efficiency, including the concentration of Agrobacterium, inoculation time, co-culture time and delayed selection were studied in Agrobacterium-mediated transformation of Malus hupehensis var. Pingyiensis using cotyledon explants. A high-efficiency transformation system for M. Hupehensis var. Pingyiensis was developed. In this protocol, the mature embryos were cultured on embryo germination medium for 7-10 days and then the paraxial cotyledons were cut from the germinated embryos, the cotyledon explants were dipped in the solution (Odgjo=0.5) of Agrobacterium tumefaciens strain EHA10S for 10 min and then were co-cultured on regeneration medium for 5 days, and the resistant buds were regenerated from explants cultured on regeneration medium supplemented with 20 mg-L"1 Kan and 400 mg-L"1 Cef. The rate of Kan resistant buds reached 9.0%. Among 27 Kan resistant lines, 23 lines were GUS-positive in the GUS histochemical test. The establishment of high-efficiency transformation system laid a sound foundation for improving M. Hupehensis var. Pingyiensis by gene engineering.
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