Study On Transformation System Of Rabbit Defensin NP-1 Gene To Rapeseed Mediated By Agrobacterium Tumefaciens

Rapeseed is an important oilseed plant in China. However, this crophas been suffering from the disease of Sclerotinia sclerotiorum.Introducing resistant genes into rapeseed varieties by transformationtechnology is one of the most important way to resolve rapeseed disease.Rabbit defensin NP-1, a broad-spectrum antibacterial peptide, have beensuccessfully introduced into tobacco, tomato such as lily plants, whichdisplayed strong resistance to different kinds of diseases. Transferringrabbit defensin NP-1gene into rapeseed has not yet been reported. Thisstudy begins with establishing highly effective regeneration systems, thenintroducing rabbit defensin NP-1 gene into five rapeseed strains viaAgrobacterium. The results are as following:1. Optimization of regeneration systems of cotyledon petiolesCotyledon petioles from 5 day-old and 7 day-old seedings of fivestrains screened repeatedly on MS containing different concentration6-BA and NAA. The optimum medium was MS supplying with 4.0mg/L6-BA and 0.02mg/L NAA. Regeneration shoot frequency amounting to87.7-98.3%.2. Optimization of A.tumefaciens-mediated transformation systemsof cotyledon petioles.Cotyledon petioles were pre-cultured on MS containing 4.0mg/L6-BA for 2d before infection via Agrobacterium. After infected into Agrobacterium suspension of OD₆₀₀ 0.4 for 5min, the cotyledon petioleswere co-cultured on MS containing 4.0mg/L 6-BA and 200umol/L AS for48h in darkness. When the co-culture was over, the cotyledon petioleswere transferred to the delay selection media for 5d and cultured ongradation Kan(5,10,15mg/L) selection media, the obtained shoots weretooted and transplanted. The Kan-resistant plantlets were obtainedultimately.