Regeneration Of Cabbage(Brassica Oleracea L.Var. Capitata L.) In Vitro And It’s Rapd Analysis Of Somaclonal Variation

The cabbage (Brasssica oleracea L.Var. capitata L.) is one of the most important vegetables, in recent years, with the rapid development of molecular biotechnology, plant genetic engineering technology provides a new way for improving cabbage traits effectivelly. The establishment of a high-frequency plant regeneration system is very necessary for genetic transformation and the rapid asexual multiplication.In this study, cabbage hypocotyl protoplasts and no vaccine hypocotyls were used as materals to establish efficient plant regeneration system, and obtain the largest regeneration coefficients, which will provide the foundation of genetic transformation and the improvement and innovation of plant breeding. Moreover, RAPD molecular markers were used to analyze the somaclonal variation among the cabbage regenerated plants. The results are as follows:1. The main factors effected on cabbage(Brasssica oleracea L.Var. capitata L.) hypocotyl protoplast isolation, purification and cultivation were studied, in order to establish a practical technology system of isolation, purification, collection, culture and eventually a complete plant regeneration for cabbage protoplast of two early maturing varieties. The results are as follows:The frequency of cell division and the plating rate of ’QL50’were higher than’XX50’, that more suitable for asymmetric cell fusion and genetic transformation research; Protoplasts of ’XX50’were isolated by enzyme digestion from hypocotyl of4day-old seedling. The optimum enzyme combination was attained with2.5%Cellulase R-10+0.05%Pectolase Y-23+9CPW+5mmol·L-1MES, the plating rate after40days is0.43%; Protoplasts of’QL50’were isolated by enzyme digestion from hypocotyl of3day-old seedling. The optimum enzyme combination was attained with2%Cellulase R-10+0.05%Pectolase Y-23+9CPW+5mmol·L-1MES, the plating rate after40days is0.57%. In the basic medium of improved B5supplemented with2,4-D0.5mg-L-1,6-BA 0.2mg·L-1, NAA0.2mg·L-1, the cells divided luxuriantly. Regenerated plants were formed from callus after bud induction and root initiation.2. hypocotyl of cabbage(Brasssica oleracea L.Var. capitata L.) cultivars were used as explants to study in vitro regeneration of adventitious buds so as to choose optimal hormone combination and seedling age and concentration of AgNO3.. The results were as follows:The adventitious shoot could not be induced from the hypocotyl in MS medium added with hormone (6-BA or NAA) only and not added hormones.’QL50’has a highest regerenation rate in the medium MS+6-BA2mg-L-1+NAA0.02mg·L-1+AgNO36mg·L-1, is93%, the optimal seedling age was9days old, the average regeneration coefficients were9.17.’Gan19cms’ has a highest regerenation rate in the medium MS+6-BA2mg-L"1+NAA0.2mg·L-1+AgNO36mg·L-1, is73.8%, the optimal seedling age was6days old, the average regeneration coefficients were4.27. The effect of adding0.5mg·L-1NAA in1/2MS medium on rooting culture was better, rooting rate of adventitious bud reached100%, and the root can grow strongly.3. Used protoplast regenerated plants and hypocotyl regenerated plants of cabbage as materials. Using RAPD molecular markers to analyze the somaclonal variation in cabbage regeneration process. The results were as follows:14Primers which were screened out of50primers, this primers amplified253repeatable loci which including73polymorphic loci. This polymorphic loci showed absence of DNA fragments and newly amplified bands. The percentage of polymorphic bands was28.85%, Each primer produced5.21polymorphic bands on average. According to RAPD molecular markers. Genetic similarity of17varieties of cabbage were computed and cluster analysis diagram is drawn using UPGMA. The results show that:Somaclonal variation between the range of0.8340～0.9842. the average genetic similarity was0.9304, genetic variation exists regenerated plants. RAPD molecular markers suitable for screening somaclonal variation initial.