The Protoplast Culture And Plant Regeneration Of Head Cabbage

In this experiment,6 varieties with different maturity and genotypes were tested for the exploration of protoplast culture. The main factors effected on cabbage 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. Then establish a basis for latter research such as asymmetric cell fusion, and finally provide the conditions for cabbage variety improvement and innovation. The results are as follows:1. In the optimization of protoplast isolation condition, the optimum enzyme combination was attained with 2% Cellulose R-10+0.5% Pectolase Y-23+9CPW+ 5mmol/L MES. Using it to purifying hypocotyls'protoplasts from cabbage with 4day's seedling age, better results could be get after 16 hours. The protoplast production was 16.85 X 105 per gram, and the protoplast viability was 86.3%. The optimum centrifugation condition was 1000rpm,4min.2. At the same time, a series of optimizing conditions for protoplast culture were researched. YP+ NAA 0.2mg/L+2,4-D 0.5mg/L+6-BA 0.2mg/L could get the optimum protoplast culture effect with the frequency of cell division after 10 days and the plating rate after 40 days respectively were 20.8% and 0.53%. By double culture method, the cell division frequency was similar to that by liquid superficial culture method, while plating efficiency was significantly greater than it. In the MS medium containing NAA 0.025mg/L+2,4-D 0.025mg/L+6-BA O.lmg/L, micro callis from hypocotyls'protoplasts could get better callus proliferation. In the MS medium supplemented with 6-BA 2mg/L, ZT 0.5mg/L, regenerated callus from cabbage hypocotyls'protoplast were better differentiated. In the process of cabbage protoplast culture and regeneration, AgNO3 7.5mg/L added in to the shoot differentiation medium can increase the differentiation of regenerated green shoots, the sprouting rate increased from 51.3% to 65.2% and the browning rate decreased by about 5 percentage. Regenerated green shoots in good growth state were chose for rooting in the 1/2MS rooting medium with IB A 0.2mg/L, all of them could be rooting for complete regeneration, plant regeneration rate was 100%.3. The differences of regenerated plants were tested. All of the QL regenerated plants were basically the same to the stock plants in morphology, only a few strains of the phenotype is slightly different. Ploidy results from flow cytometry cell test showed that in all protoplast regenerated plants,79.6% were normal diploid,9.1% were haploid plants,6.8% were single/diploid chimera plants and 4.5% were tetraploid plants. DNA mutation of obtained regenerated plants was studied by RAPD molecular markers, the results showed that, the average similarity between cabbage protoplast regenerated plants and their stock plant was 0.8709.4. By the comparison of effects of protoplast culture of these varieties, a result was preliminary get that early maturity varieties may be more suitable for the protoplast culture.