| Begonia wallichiana L.is a kind of perennial herb belonging to the Begonia genus of Begonia family.It has large plant size,strong stems and white flowers,some of which appear to be similar with that of woody plants.What’s more,it also has large amounts of flowers and fruits,great vitality and short regeneration cycle,which make the plant an excellent ornamental material for genetic transformation.Many of the famous cultivated types also belong to Begonia genus.This study was carried out for a better genetic transformation research on this species,providing new references for the improvement of this techniques in Begonia and other ornamental plants at the same time.B.wallichiana was used as the main material in this study to figure out a suitable system for efficient regeneration and genetic transformation.Molecular technology was used in the identification of Hyg-resistant plant materials.The results were as follows.1.Study on the aseptic germination technology of B.wallichiana seeds.The seeds of B.wallichiana were used as test material,the effects of sterilization methods,vernalization time,and basic medium on germination were discussed.The results showed that after disinfected with 0.1%potassium permanganate for 1 min and 10%sodium hypochlorite solution for 10 mins,clean materials and high germination rate can be obtained.Seeds without vernalization were difficult to germinate and grow.10 d was a suitable vernalization time for seeds to germinate and grow healthily.MS was a suitable medium for germination.2.Study on the highly efficient regeneration system of B.wallichiana.The sterile seedling leaves of B.wallichiana were used as explants to carry out the study,while MS and 1/2MS were used as the basic medium and different 6-BA concentrations of 0.5,1.0,1.5,2.0 mg/L and NAA concentrations of 0.1,0.2,0.3,0.4,0.5 mg/L.The results showed that a suitable combination was MS with 1.5 mg/L 6-BA+0.2 mg/L NAA.A large amount of adventitious buds can be formed within one month with a 100%rooting rate.3.Study on the genetic transformation system of B.wallichiana with leaf-disk method.The sterile seedling leaves of B.wallichiana were used as explants,the concentration of bacteriostatic antibiotics(Cef),concentration of screening antibiotics(Hyg),infection time and method,concentration of bacteria solution,co-cultivation time,the effect of recovery culture and screening culture and the effect of gradient screening after recovery were explored.The results showed that the suitable concentration of Cef varied from 100 to 300 mg/L according to the conditions of the explants,while the minimum Hyg concentration for screening was 5 mg/L.Infected for 1-2 h while shaking the bacterial solution and co-cultivated for 2 d,gradient screening after recovery is a suitable system for genetic transformation of B.wallichiana.4.Identification of transgenic resistant plants of B.wallichiana.The above genetic transformation system was used to do the GFP gene test and the regenerated plants obtained before and after recovery were used as materials.Duplex PCR method was used to test the GFP gene and agrobacterium plasmid.The result showed that the attachment rate of agrobacterium before recovery was 100%.87.5%of the regenerated plantlets was infected successfully by agrobacterium while 12.5%were confirmed with GFP gene in them.The rate of Hyg-resistant explants after first screening was 40-60%with 10%of which sure with GFP gene.The final Hyg-resistant rate was 50%after screening for the third time,and 16.7%of the explants were determined with GFP.Results of fluorescence microscopy showed that the GFP gene was expressed dispersedly in the plant like little spots near the main leaf veins. |
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