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Creeping ground-cover chrysanthemum, as one of new and attractive ground-cover type plants with all-around of virescence, beautification, color and fragrance, was widely cultivated in gardens and greenbelts. In this experiment, using creeping ground-cover chrysanthemum ’yuhuaxunzhang’ and Agrobacterium tumefaciens strain LB4404 as materials, the transformation of DmDREBa gene to ’yuhuaxunzhang’ mediated by Agrobacterium tumefaciens was studied, which included the establishment of efficient and reliable invitro regeneration system, the optimization of transformation system, the hpt gene PCR assay and southern blot analysis of leaves from transformants. In addition, studies on the tolerance of transgenic plants under cold, drought and salt conditions and the mechanisms about stress in creeping ground-cover chrysanthemum was also carried out, the detailed results were listed as follows:1. The establishment of regeneration system of creeping ground-cover chrysanthemum.Using leaf disks of three varieties of creeping ground-cover chrysanthemum ’yuhuaxunzhang’,’yuhuajingui’ and ’yuhuayingui’ as explants, factors that effect rate of regeneration were studied, including cultivars, plant growth regulators, growth degree of leaf, darkness culture. The results showed that the regeneration ability of cultivar ’yuhuaxuanzhang’ was better than the other two cultivars; the optimum medium was MS medium with supplement of 1.0mg·l-1 6-BA and 1.0mg·l-1 NAA, shoot regeneration rate was 91.1%, No.of buds per explant7.27, roots were obtained when shoots were transferred onto 1/2MS medium with supplement of 0.1mg·l-1 NAA, the rooting rate was 100%, which was enough for genetic transformation; The two to forth leaves from top aged 25days was the best explants for regeneration; a period of darkness culture could effectively prevent callus browning and promote regeneration rate.2. The optimization of transformation system mediated by Agrobacterium tumefaciens.Factors that influence percentage of transformation including pre-culture time, agro infection time, co-culture time, postponed antibiotics selecting time, concentration of AS in the medium, transformants filtration method and bacterium control method were studied. Te effective genetic transformation was performed, with the optimized pre-culture time 3d, the suitable agroinfection time 10min(OD600=0.5), the time for appropriate co-culture and postponed antibiotics selecting both 3d. Browning of callus was accelerated when Acetosytingone(AS) was used in the transformation experiments; Both leaf and root were very sensitive to Hygromycin (Hyg), leaf could be killed with Hyg concentration 10mg·l-1 and rooting could be completely restricted with 8mg·l-1.In practice, concentration of Hyg for selecting was a little lower than critical concentration, so we chose 6～8mg·l-1 as the optimal concentration of Hyg for callus and shoot selecting and 7mg·l-1 for root selecting. During the screening course, intergrating gradual-reducing method with long-playing selecting for times, more resistant shoots were obtained. Leaf was also sensitive to Carbenicillin (Carb), Carb concentration 350mg·l-1 could completely control the growth of bacterium whereas inhibit the shoot regeneration. While lower concentration with 250 mg·l-1 couldn’t kill the bacterium completely but higher regeneration rate.With gradual-reducing bacterium controlling method, transformants regeneration rate was increased.3. The selcetingof resistance plants, the regeneration and identification of transgenic plantsThe indirect method of selecting resistance plants was that after co-culture, explants were cultured on the MS medium supplemented with 1.0mg·l-1 6-BA,1.0mg·l-1 NAA and 350mg·l-1 Carb for 3 days, then cultured on the MS medium supplemented with 1.0mg·l-1 6-BA,1.0mg·l-1 NAA,8mg·l-1 Hyg and 250mg·l-1 Carb in darkness until resistance callus was obtained, then transferred the whole callus on the MS medium supplemented with 1.0 mg·l-1 6-BA,1.0mg·l-1 NAA,6mg·l-1 Hyg and 150mg·l-1 Carb until resistance shoots were obtained, then cut the resistance shoots cultured on the MS medium supplemented with 1.0 mg·l-1 6-BA and 1.0mg·l-1 NAA until resistance shoots grown up to 2-3cm, rooted on the 1/2 MS medium supplemented with 0.1mg·l-1 NAA and 7mg·l-1 Hyg. Twenty-eight transgenic plants were obtained by HPT gene PCR method, eight of them was analyzed by southern blot, it indicated that 2～4copies of DmDREBa were randomly inserted into six transgenic plants. The identified transgenic plants survived after seedling training and transplant.4. The analysis of tolerance under cold, drought and salt stress in overexpression of DmDREBa plants.Six DmDREBa plants were analyzed under cold in the To generation, the LT50 tempera ture of six transgenic emergent rhizomes were -18.92℃,-19.49℃,-20.22,-20.36℃,-20.36℃,-20.57℃respectively, higher than that of untransformed control plant (-17.43℃). Two of the six transgenic plants whose LT50 temperature was significantly lower than untransformed control plant were analyzed for stress tolerance.Under low temperature, activity of POD and content of proline were all remarkable increased in overexpression of DmDREBa plants, while malondialdehyde remarkable lower than untransformed control plant. For the drought stress treatment, the survival rates of transgenic plant 35S:TDa4 and 35S:TDa5 were 43.8% and 31.3%, clearly higher than that of untransformed control plant (6.3%), and the DmDREBa plants had higher fresh weight retentionrate, activity of SOD and content of proline and lower content of malondialdehyde. For the salt stress treatment, the survival rates of transgenic plant 35S:TDa4 and 35S:TDa5 (50.0% and 33.3%) were significantly higher than that of untransformed control plant (11.1%), and the activity of POD and content of proline were all increased in DmDREBa plants, while malondialehyde lower than untransformed control plant.Our data suggest that the multiple stress tolerance of DmDREBa plants was all increased comparing to the untransformed control plants under cold, drought and salt conditions, and the tolerance under drought stress was higher enhanced than that of salt stress in overexpression of DmDREBa plants in the same growth state.
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