Genetic Transformation Of Sad To Rape (Brassica Napus L.) Mediated By Agrobacterium Tumefaciens

Rape (Brassica napus L.) is a kind of very important economic crop and oil plant at home and abroad. But everywhere the rape were threatened by the illness caused by bacteria and fungi.Plant defensins are small, basic peptides that exhibit antimicrobial activity against a broad range of bacteria and fungi. Because of their three-dimensional folding pattern, they can inhibit the growth of many fugi without damaging plant. Plant defensins can protect plant seeding and fight against pathogens which attack plant. Luo et al. (2005) had cloned the Sad gene successfully. By prokaryotic expression, the cDNA of Sad gene was inserted into the protein expression vector, GTK to induce Sad gene expression. And they had transferred to Arabidopsis by plant method to study Sad function.In the thesis, restorer line of "SHUZA No.9" which is a new sort of hybrid rape (brassica napus) is used as my experimental material, the hypocotyls were used as explants and were transformed by Sad gene via Agrobacterium tumefaciens-mediation method. Transgenic plants were obtained after co-culture, antibiotic screen and root regeneration. The transgenic plants were detected with PCR. The results suggest that foreign Sad gene has already been combined into the rape genome and were expressed. The factors that influence the frequency of agrobacterium mediated transformation were also discussed.The details are shown as followings:1. An optimized system of re-generation is formed, through comparing and observing the different situations of the induction and differentiation of the explant. Experiment shows the following results: The differentiation frequency of hypocotyl is higher than that of cotyledon and the 0.5-1cm hypocotyl which doses to the growth ponit is the best; hypocotyls were infecting for 1 min as OD₆₀₀ of Agrobacterium suspending liquid is 0.5; hypocotyls were co-cultered with Agrobacterium for 2 days. The transformation frequency of explants was promoted when 20 mg/L acetosyringone was added in Agrobacterium suspension liquid and co-cultured medium. AgNO₃ can increase the differentiation frequency of hypocotyl. the differntiation frequency of bud is the highest when MS＋3mg/L6－BA＋1mg/L 2, 4-D: it was useful for anti-Kan positive shoot to produce when initial concentration of kanmaycin was 10mg/L.2. Agrobacterium tumefaciens which contains the vector pBI121-Sad is used to transform the plants of rape (Brassica napus). The condition of transforming is also optimized, and the best conditions of culture media are conformed as followings:â… medium of shooting: MS;â…¡medium of pre-culture& co-culture :MS+2mg/L6-BA+1mg/L2,4-D+2.5mg/LAgNO₃+19.62mg/LAs;â…¢medium of inducing callus& differentiation:MS+2mg/L6-BA+2.5mg/LAgNO₃+500mg/L Carb+10mg/L Kanâ…£medium of rooting:1/2MS+0.15mg/LNAA+250mg/Lcefâ…¤medium of Agrobacterium activation: LB+40mg/LRif+20mg/LStr+50mg/Lkanâ…¥medium of Agrobacterium suspendingMS+19.62mg/LAs3. Resistant seedlings are obtained after being selected by kan, and elementarily proved to be Sad gene via PCR test.