The Genetic Transformation Of Capsicum Annuum L. With The Nicotiana Protein Kinase Gene(NPK1)

Pepper (Capsicum annuum L.) is one of the most important vegetables for its rich nutrient with fruit and economic value. Stresses of freezing, heat, salinity, dry and many diseases seriously affect the productivity of pepper. It has being studied to improve the stress tolerance and diseases resistance of pepper.The NPK1 gene from tobacco, which encodes a protein kinase related to MAPKKK plays important roles in embryogenesis and the oxidative stress signalling pathway. In this research, we cloning of the tobacco NPK1 gene, study the effect of establishment of high efficiency regeneration system and determination of parameters involved in Agrobacterium mediated transformation. Screening and characterization of transforming were systematically studied. Cotyledons of pepper were successfully transformed with the tobacco NPK1 gene by Agrobacterium mediated transformation. The results were summarized as follow:1. In vitro plant regeneration was achieved from eight chilli pepper materials (Capsicum annuum L.). The effects of genotype, various explant types (cotyledons, hypocotyls) and hormone was evaluated. It was shown that the high 6-BA/ IAA ratio was conducive to the differentiation and the low 6-BA/ IAA ratio was suitable for the elongation of the regenerative buds; different pepper materials differed greatly in their regeneration capacities. The cotyledons had stronger regenerative capacity than the hypocotyls, and the explants from 12-16 day old seedlings had a high differentiation rate. Higher generation frequency was obtained from materials of B17, P41 and P54. Comparatively speaking , the optimal medium for the differentiation of pepper buds was Murashige and Skoog (MS)medium supplemented with B5 organic nutrient, 0.5mg/L IAA and 5.0mg/L 6-BA. The best media for shoots elongation was Murashige and Skoog (MS)medium supplemented with B5 organic nutrient medium with 0.5mg/L IAA , 1.5mg/L 6-BA and 2.0mg/L GA3 and the rooting medium was Murashige and Skoog (MS)medium supplemented with B5 organic nutrient medium with 0.2 mg/L IAA, 0.1 mg/L NAA. 2. The efficient genetic transformation system for pepper is that 14d cotyledon explants were preconditioned for 2 days on Murashige and Skoog (MS)medium supplemented with B5 organic nutrient, 0.5mg/L IAA and 5.0mg/L 6-BA and inoculated with the cultures of Agrobacterium strain LBA4404 for 8-10 min, followed by co-cultivation with Agrobacterium tumefaciens on Murashige and Skoog (MS)medium supplemented with B5 organic nutrient, 0.5mg/L IAA and 5.0mg/L 6-BA for 2 days and delay selection on Murashige and Skoog (MS)medium supplemented with B5 organic nutrient +0.5mg/L IAA +5.0mg/L 6-BA +250mg/L Cef for 2 days. The explants were then placed on Murashige and Skoog (MS)medium supplemented with B5 organic nutrient +0.5mg/L IAA +5.0mg/L 6-BA +250mg/L Cef +50mg/L Km for selection.3. We obtained 48 Km-resistant regenerated plants and DNA from the plants was subjected to PCR analysis 17 showed. Then RT-PCR analysis 14 showed.