The Establishment Of Regeneration System Of Two Large-flowered Clematis

Clematis is a kind of valuable ornamental plant distributing all over the world with graceful posture and colorful blossoms.Particularly,the application value of large-flowered group is prominent.Thus,establishing an efficient propagation and regeneration method is necessary.In this study,excised stems with buds of Clematis‘Ville de Lyon'and C.‘Rhapsody'through direct organogenesis pathways were used to establish regeneration system.In addition,internodes,leaf blades,petioles of C.‘Ville de Lyon'were used as materials to study the effects of hormone types and concentrations on callus induction,proliferation and adventitious bud differentiation.The results are as follows:?1?Explants disinfection:To obtain bacteria-free explants,a comparative study of one-step and two-step disinfection methods on stems of C.‘Ville de Lyon'was conducted.Results showed stems with buds using two-step sterilization disinfected 6 min and 5 min by 0.1%HgCl₂was better than one-step.By using two-step disinfection method,the pollution rate was 5.56%and the germination rate was 78.89%.Besides,the suitable disinfection time for leaf blades was 6 min and 5 min;the internodes was 8 min and 5 min;4min and 5min was appropriate for petioles.In C.‘Rhapsody',the suitable disinfection time for stems was 6min and 5 min with two-step sterilization,the contamination rate reduced to 6.67%and germination rate was 77.78%.?2?Callus induction,proliferation and differentiation of C.‘Ville de Lyon':MS+0.10 mg/L NAA+1.00 mg/L 6-BA+1.00 mg/L 2,4-D was suitable for callus induction of leaf blades,petioles and internodes,and the induction rate was 80.00%,73.33%and 66.67%,respectively.MS+0.10mg/L NAA+2.00 mg/L 6-BA+2.00 mg/L 2,4-D was suitable for callus induction of petioles,and the induction rate was 86.67%.For callus proliferation,MS+0.10 mg/L NAA+2.00 mg/L 6-BA+2.00 mg/L 2,4-D was the proper culture medium.The growth-curve of callus of C.‘Ville de Lyon'was similar to the sigmoid curve,5²0 d was the peak growth of callus and 20³0d was slow and stable.Therefore,3⁴ weeks was the appropriate subculture cycle of callus.The suitable medium for callus differentiation was MS+0.1 mg/L NAA+1.0 mg/L 6-BA+0.5 mg/L KT,the differentiation rate was 63.33%,and the light condition was favorable for the growth of differentiated buds.?3?Anti-browning of callus:1.00 g/L Ac and 0.10 g/L Vc were the suitable anti-browning agents in C.‘Ville de Lyon',the browning rate was13.33%and 16.67%respectively,much less than 55.33%in control.Under the illumination,the callus presented dense,green;while loose,white callus was observed in dark condition.?4?Induction and proliferation of adventitious buds:MS+0.1 mg/L NAA+1.0 mg/L 6-BA was the optimal medium for germination of C.‘Ville de Lyon',and the germination rate was 93.33%with the shoot forming index of 1.70;while MS+0.1 mg/L NAA+3.0 mg/L 6-BA was the suitable medium for germination of C.‘Rhapsody',and the germination rate was96.67%with the shoot forming index of 1.82.MS+0.10 mg/L NAA+1.00mg/L 6-BA+1.00 mg/L KT was the suitable medium for C.‘Ville de Lyon',proliferation coefficient was 4.07;while MS+0.15 mg/L NAA+2.00 mg/L6-BA+0.05 mg/L KT was the suitable medium for C.‘Rhapsody',and the proliferation coefficient was 3.93.?5?Rooting culture:The suitable basic medium for rooting was 1/2 MS medium,and 1/2 MS+0.3 mg/L NAA+3.0 mg/L IBA was the optimal rooting medium for C.‘Ville de Lyon',the rooting rate was 93.33%.While1/2 MS+0.2 mg/L NAA+1.0 mg/L IBA was the optimal rooting medium for C.‘Rhapsody',and the rooting ratio was 96.67%.?6?Acclimatization and transplantation:73.0%of C.‘Ville de Lyon'plantlets survived following acclimatization and transfer to a potting mixture of perlite and peat?1:1,v/v?.The plantlets survival rate of C.‘Rhapsody'in the same substrate was 71.0%.