结球甘蓝下胚轴原生质体培养再生植株体系的优化研究

Plant Regeneration from Hypocotyl Protoplast Culture of Cabbage(Brassica oleracea L.var capitata)

以结球甘蓝‘新夏50’的无菌苗下胚轴为材料,对影响原生质体分离、纯化与培养的主要因素进行研究,建立适合结球甘蓝原生质体游离、纯化、收集、培养以至再生出完整植株的实用技术体系,为其非对称细胞融合及品种改良与创新等研究奠定基础。结果表明:2.5%纤维素酶R-10+0.05%果胶酶Y-23+9CPW+5mmol/L MES的混合酶液,从4d苗龄的下胚轴上分离出高产率的原生质体。在改良B5+0.5mg/L 2,4-D+0.2mg/L 6-BA+0.2mg/L NAA的液体培养基上,原生质体分裂旺盛。形成愈伤组织后经芽诱导和生根培养,获得了再生植株。倍性检测结果表明,不同原生质体所获得的24株再生植株中,19株为正常二倍体,4株为嵌合体,1株为四倍体。

The main factors affect cabbage hypocotyl protoplast isolation,purficatlon and cultlvation were studied,in order to establish a practical technology system of isolation, purification, collection, culture and eventually a complete plant regeneration for cabbage protopiast. Then establish a basis for latter research such as asymmetric cell fusion and finally provide the conditions for cabbage variety improvement and inno vation. The results are as follows：Protoplasts were isolated by enzyme digestion from hypocotyl of 4 day old seedling of head cabbage. The optimum enzyme combination was attained with 2.5% cellulase R-10＋0. 05% pectolase Y-23＋9CPW＋5 mmol/L MES. In the basic medium of improved B： supplemented with 0.5 mg/L 2,4-D）, 0. 2 mg/L 6-BA, 0. 2 mg/L NAA, the cells divided luxuriantly. Regenerated plants were formed from callus after bud induction and root initiation. In 24 regenerated plants, 19 were normal diploid, 4 were diploid/telraploid chimera and 1 was tetraploid.