Establishment Of In Vitro Regeneration System And Protoplast Isolation Of Orange-Heading Chinese Cabbage

The effects of hormone, AgNO₃, ABA and genotypes, seedling age on adventitious bud regeneration and different auxin on adventitious root regeneration of orange-heading Chinese cabbage 01S303-1 were studied. As the result, the regeneration system from cotyledon were established. In addition, the effects of seedling age, enzyme combination, enzymatic time and mannitol concentration on protoplast yield of orange-heading Chinese cabbage 01S303-1 were studied. The results were as follows:1. There were only a few adventitious buds from cotyledon and hypocotyl when thry were cultrued in MS medium added cytokinin(6-BA or TDZ) and auxin(NAA) together.2. AgNO₃ could raise the frequency of adventitious bud from cotyledon and hypocotyl of orange-heading Chinese cabbage by a large amount. The regeneration frequency of bud from cotyledon was only 2.5% in MS+7 mg/L 6-BA+2 mg/L NAA, while the frequency increased to 57.5% when the medium was added 2 mg/L AgNO₃; As for the hypocotyl, the regeneration frequency of bud increased to 27.5% when 6 mg/L AgNO₃ was added. The regeneration frequency of bud from cotyledon was only 0% in MS+0.1 mg/L TDZ+1.5 mg/L NAA, while the frequency increased to 82.5% when 10 mg/L AgNO₃ was added; As for the hypocotyl, The regeneration frequency could increased to 25% when the MS+0.05 mg/L TDZ+4 mg/L NAA was added 10 mg/L AgNO₃.3. The effect of ABA on adventitious bud regeneration from cotyledon varied from medium to medium. The frequency of bud regeneration from cotyledon was promoted by ABA in MS+7 mg/L 6-BA+2 mg/L NAA+2 mg/L AgNO₃. The frequency could increased to 85% when 0.3 mg/L ABA was added. Whlie the bud regeneration from cotyledon was restrained by ABA in MS+0.1 mg/L TDZ+1.5 mg/L NAA+10 mg/L AgNO₃.4. The regeneration frequency of adventitious bud also was affected by explant types,seedling age and genotypes. The frequency of adventitious bud from cotyledon was higher than from hypocotyl; there was the higher frequency of adventitious bud from hypocotyl in the 6-day-old seedlings than in the older or junior seedling. The bud regeneration ability varied from strain to strain. The results showed that the frequency of adventitious bud of 01S303-1 is the highest, 04S1361 and 04S1363 were in the medium and the 04S26 is the lowest.5. Both IAA and NAA could promote the adventitious root regeneration of orange-heading Chinese cabbage. The regeneration frequency of it reached to 93.9% in MS+ 0.3 mg/L NAA.6. The protoplast isolation of orange-heading Chinese cabbage was studied on seedling age, enzyme combination, enzymatic time and mannitol concentration. The result showed that the best protoplast yield obtained when the 8-day-old cotyledon was dissociated in enzyme solution composed of 0.6%(w/v) Cellulase Onozuka R-10, 0.1%(w/v) Pectinase Y-23 and 10% mannitol for an hour.