Optimization of Genetic Transformation System of Peppers by Green Fluorescent Protein
[Objective]The genetic transformation of pepper mediated by Agrobacterium was a very difficult process,and no efficient transformation system had been established so far.Green fluorescent protein(GFP)gene is a common reporter gene in plant genetic transformation.In the study,GFP was used as the reporter gene to optimize the Agrobacterium-mediated genetic transformation system of pepper.[Method]By using GFP expression system,three types of explants(cotyledons,hypocotyls and Flamingo-bill explants)from 3 pepper varieties('HP''8214'and'L55')were counted according to the adventitious bud differentiation rate,adventitious root differentiation rate and fluorescence positive rate.The effects of factors including infection concentration and infection time,pre-culture time and co-culture time on adventitious bud differentiation rate,adventitious root differentiation rate and fluorescence positive rate were explored.[Result]The adventitious bud differentiation rate of Flamingo-bill explants was significantly higher than that of the hypocotyl and cotyledon explants,among which the'L55'Flamingo-bill explant had the highest adventitious bud differentiation rate of 77.59%.Therefore,the'L55'Flamingo-bill explant was selected for subsequent research.Under 4 different combinations of Agrobacterium infection concentration and infection time,all of them could produce adventitious buds,adventitious roots and GFP-expressing calluses.When the infection concentration of Agrobacterium was OD600=0.05 and the infection time was 30 min,the adventitious bud differentiation rate and fluorescence positive rate of Flamingo-bill explants were the highest,reaching 48.39%and 4.84%,respectively.Under 6 different pre-culture and co-culture time combinations,the Flamingo-bill explants also produced adventitious buds and adventitious roots,and the highest adventitious bud differentiation rate and fluorescence positive rate were 48.44%and 12.50%,respectively,under the treatment of pre-culture for 1 d and co-culture for 1-2 d.Finally,PCR detection of adventitious roots and calluses with GFP fluorescence expression showed that the 3 target genes GFP,Kan and Cas9 all presented signal in the fluorescence positive tissues,indicating that the T-DNA insertion mediated by Agrobacterium was successful and the transformation was stable.[Conclusion]The explant type of pepper,Agrobacterium infection concentration,infection time,pre-culture time and co-culture time all have effects on the genetic transformation efficiency of pepper.Screening appropriate transformation conditions with GFP as a reporter gene could improve the efficiency of Agrobacterium-mediated genetic transformation of pepper.
万方数据
维普
