Studies On High Efficient In Vitro Shoot Regeneration From Hypocotyl Explants Of Chinese Cabbage

The hypocotyls of Chinese cabbage [ Brassica campestris ssp. pekinensis (L.) Olsson ] were used as explants in this experiment. The effects of different hormone combinations, seedling age, hypocotyl sources, inoculation methods and different cultivars on shoot regeneration from hypocotyls were studied to obtain a high-frequency regeneration system from hypocotyls of Chinese cabbage. Differences of changes of soluble protein content, activities of peroxidase (POD), superoxidase (SOD) and catalase (CAT) in hypocotyl explants of Chinese cabbage ' cv. Guilong No. 5 ' inoculated with three different methods on the same medium were compared. At the same time, changes of contents and balance of endogenous hormones and polyamine were investigated in hypocotyl explants inoculated with their abaxial ends up. The results were as follows.Compared to 6-BA, TDZ was more effective in adventitious shoot regeneration from hypocotyls. The shoot regeneration percentage from hypocotyls reached 87.8% and there were 15.1 shoots regenerated per explant in the MS medium containing 0.5 mg·L^-1 NAA, 0.3 mg·L^-1 TDZ and AgNO₃ 5 mg·L^-1. There was no significant difference among hypocotyls of 3 to 5 days of seedling age in their regeneration frequencies which were above 80% respectively, increasing of seedling age resulted in a reduction of shoot differentiation frequency, which was only 51.1% at the seedling age of 7 days. The order of shoot regeneration ability of hypocotyls from different position was: the upper > the middle > the lower. Adventitious shoots appeared from hypocotyls inoculated with their abaxial ends up after 8-10 days of culture, shoot regeneration percentage were higher and there were more shoots regenerated per explant, which showed the higher regeneration ability than those were either inoculated with their abaxial ends down or placed horizontally. Different cultivars showed difference in shoot regeneration ability.Physiological indexes of soluble protein content, activities of POD, SOD and CAT in hypocotyl explants changed rhythmically as adventitious shoots were differentiated. Soluble protein content, activities of POD and SOD were in the trend of increase but activity of CAT decreased before shoot primordia occurred. However, these indexes showed contrary changing trends after adventitious shoots were differentiated. Hypocotylsinoculated with different methods showed differences in changes of physiological indexes. The above results indicated that morphological changes were strongly associated with physiological changes during the process of adventitious shoot regeneration.Endogenous hormone and polyamine contents and balance in hypocotyl explants also changed rhythmically as adventitious shoots were differentiated. Before shoot primordia occurred ( 0 to 8 days after inoculation), endogenous zeatin ribosides ( ZRs ) and abscisic acid ( ABA ) contents were increased, while indole-3-acetic acid ( IAA ) content was decreased. After shoot promordia occurred ( on the 8th day of inoculation ), the contrary changing patterns of ZRs, ABA and IAA contents were observed. During the early stage of culture (0 to 8 days after inoculation), ABA/IAA and ZRs/IAA ratios were increased, then gradually decreased after adventitious shoots occurred. Endogenous putrescine ( Put ), spermine (Spm) and spermidine ( Spd ) contents were increased at the early stage and then gradually declined during the culture course, these indexes reached their peaks on the 8th day of culture. Similar changing patterns were also observed in the ratios of Spm/Put and Spd/Put. The above results indicated that endogenous hormone and polyamine contents and their balance were closely associated with the in vitro adventitious shoots regeneration from hypocotyl explants of Chinese cabbage.