Establishment Of In Vitro Regeneration System And Genetic Transformation Of Disease-resistance Transcription Factor Pti4 Gene In Pepper

Pepper (Capsicum annuum L.) is one of the most important vegetables, and accepts favour broadly because of its special flavour. It is cultivated broadly in many countries and regions in the world at present. But its output and quality have been severely fluenced due to plant diseases. So improvement of its disease-resistance has always been an important research field. Conventional breeding methods are often limited by germplasm resources scarcity and long breeding cycle, but plant gene engineering can overcome these problems to some extent. In systemic acquired resistance, synergistic expression of many PR (pathogenesis-related) gene can improve plant broad-spectrum disease-resistance. So introduction of transcription factor gene which controls disease-resistance gene expression make synergistic expression of PR gene become one kind of more effective ways to improve plant disease-resistance by gene engineering. When protein product of disease-resistance transcription factor Pti4 gene in tomato over-express, PR gene which has GCC-box or similar GCC-box sequence in the promoter region will obviously express. Namely introduction of exogenous Pti4 gene activate expression of a lot of PR gene and improve comprehensive disease-resistance ability of transgenic plant.The target of this study is to establish in vitro regeneration system in pepper, and intrduct disease-resistance transcription factor Pti4 gene in tomato into pepper by agrobacterium-mediated transformation, and carry out PCR detection in transgenic plant. The main results are as follows:1. Establishment of in vitro regeneration system in pepper1) By compring ability of adventitious bud guidance between'Erjintiao'and 'Chuannongpaojiao No.1', it finds the genetype has bigger effect;2) With seedling age' increasing, differentiation rate of adventitious bud and the average number of adventitious bud per explant both increase at first and then decrease, 12～16 day is the best seedling age of explant in pepper;3) Flamingo-bill explant is better than cotyledon with petiole, and cotyledon with petiole is better than cotyledon;4) The best differentitation medium of Erjintiao'is MS+0.2mg/LIAA+5.0mg/L6-BA +10.0mg/LAgNO3+6.5g/LAgar+30.0g/LSugar; The best differentitation medium of 'Chuannongpaojiao No.1'is MS+0.5mg/LIAA+5.0mg/L6-BA+6.0mg/LAgNO3+6.5g/L Agar+30.0g/LSugar;5) 2.0mg/L GA3 is the best concentration of adventitious bud elongation;6) The best rooting medium is MS+0.2mg/LIAA+0.1mg/LNAA+6.5g/LAgar+ 30.0g/LSugar.2. Genetic transformation in pepperIntrduct exogenous target gene Pti4 into pepper by agrobacterium-mediated transformation, and get 52 Km-resistance plants in pepper. Use NPTⅡprimer to carry out PCR detection in these plants (blank as negative control, agrobacterium bacterial liquid with trager gene Pti4 as positive control),45 Km-resistance plants amplified the expected gene fragment, conversion rate is about 86.5%.