Transformation Of A Trivalent Antifungal Recombinant Into Pepper (Capsicum Annuum L.)

Pepper (Capsicum annuum L.) is one of the most important and popularvegetable crops around the world for its rich nutrient and economic values. However,the productivity and quality are greatly affected by pathogen attack. As it is difficultyto breed high quality variety by using the conventional methods, people swerve togenetic engineering technology to solve the problem. Pepper genetic transformationhas been explored many researchers in recent years, but it developed slowly due to thedifficulty on the regeneration of the transgenic plants. Cotyledons form Bulgariapointed pepper and Chiyan No. 6 were used in the experiment, factors influencingtissue culture of pepper were investigated and the efficient plant regeneration systemswere established respectively. On the base of this work, the trivalent expressionvectors containing Chitinase(Chi)/β-1,3-Glucanase(Glu.)/rahpanussativus-antifungalprotein(Rs-AFP2)genes were transferred into peppers. It was confirmed that thesegenes were integrated into the peppers genome by the method of Kan, PCR, dotblotting and PCR-Southern blotting. Fungal growth of Phytophthora capsici Leonianwas strongly inhibited on the leaf of transgenic pepper, while no resistance with thecontrols. The main results were following:1. High efficient regeneration system of peppers were establisheda. Bulgaria pointed pepperThe differentiation medium of adventitious buds: MB+sugar 30 g/L+agar 8g/L+LC 1.0 mg/L+IAA 0.1 mg/L+ABA 0.3 mg/L+Spm 100μmol/L+AgNO₃ 5mg/L+PD 6 ml/L (pH 5.8～6.0), the differentiation rate of adventitious buds was100%, the rate of superior buds was 83.3%.The elongation medium of adventitious buds: MB+sugar 30 g/L+agar 8 g/L+LC 0.1 mg/L+IAA 0.2 mg/L+GA₃ 1.0 mg/L+Spm 100μmol/L+AgNO₃ 5 mg/L+PD 6 ml/L (pH 5.8～6.0), the elongation rate of adventitious buds was 78.5%.Rooting medium of regenerated plant: 1/2 MS+sugar 30 g/L+agar 8 g/L+IAA1.0 mg/L+NAA 0.5 mg/L (pH 5.8～6.0), the rooting rate was 100%.b. Chiyan 6The differentiation medium of adventitious buds: MB+sugar 30 g/L+agar 8g/L+LC 1.0 mg/L+IAA 1.0 mg/L+ABA 0.3 mg/L+Spm 100μmol/L+AgNO₃ 5mg/L+PD 6 ml/L (pH 5.8～6.0), the differentiation rate of adventitious buds was100%, the rate of superior buds was 83.3%.The elongation medium of adventitious buds: MB+sugar 30 g/L+agar 8 g/L+ LC 0.2 mg/L+IAA 0.6 mg/L+GA₃ 2.0 mg/L+Spm 100μmol/L+AgNO₃ 5 mg/L+PD 6 ml/L (pH 5.8～6.0), the elongation rate of adventitious buds was 78.5%.Rooting medium of regenerated plant: 1/2 MS+sugar 30 g/L+agar 8 g/L+IAA0.2 mg/L+NAA 0.1 mg/L (pH 5.8～6.0), the rooting rate was 100%.2. Construction of pepper transformation systemFactors influencing Agrobacterium-mediated pepper transformation wereinvestigated, the optimal conditions for transformation were that pH value ofAgrobacterium solution was 5.2, cotyledons were pre-cultured for 2 days, mixed withcell suspension of A. tumefacient for inoculation in 7～8 min, co-cultivated with A.tumefacient on the differentiation medium for 2 days.3. Obtained transgenic pepperThe trivalent expression vectors were introduced into peppers. It was confirmedthat the three genes were integrated into the pepper genome by PCR analysis, dotblotting and PCR-Southern blotting.Fungal growth of Phytophthora capsici Leonian was strongly inhibited on theleaf of transgenic pepper, but no resistance with the controls.