Optimization of Genetic Transformation System of Peppers by Green Fluorescent Protein

Abstract

【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.

Keywords

• pepper
• flamingo-bill explant
• green fluorescent protein
• genetic transformation
• agrobacterium tumefaciens
• tissue culture