| In Xinjiang Oil sunflower is suffered from many kinds of Fungi such as Sclerotinia sclerotiorum (Lib) de Bary,which can affect its yield and deteriorate its quality. Traditional method of breeding has such limitations such as long period, low efficiency, lack of resistant genes, so the resistance of breed is easily to be degraded. Moreover, the hinder of hybridization make the method hardly to be employed to obtain fine varieties with good resistance. With the developing genetic engineering technology, Agrobacterium-mediated transformation is emerged as the main breeding means to improve varieties Gastrodia Antifungal Protein (abbreviated as GAFP) is a protein with broad anti-fungal activity, it can strongly inhibit many specific pathogens responsible for plants fungal diseases, therefore, this protein is of great application value in the field of resisting fungal diseases in the plant genetic engineering.Our dissertation established a highly efficient regeneration system of Oil sunflower by the means of genes engineering, and transfered the GAFP genes into Oil sunflower, which mediated by Agrobacterium tumefaciens. The main factors influencing regeneration and transformation rate were discussed and the best conduction of regeneration and transformation were determined. The positive regeneration was obtained and confirmed from polymerase chain reaction. The results were summarized as follows:1 Development of the regeneration system of organ cell:(1) Optimization of the disinfect substances and the disinfecting time. The better disinfecting method is finally chosen as: After remove the shell, the breed is rinsed by 70% C2H5OH for 30s, then is immerged into 0.1% HgCl2 for 5 minutes.(2) Selection of explant kind. The kind of cotyledon and the cultured time were investigated. The better explants were cotyledons that were cultured 6 hours.(3) Demonstration of the best shoot-inducing medium.The optimum inducing medium for shoot is the improved MS (B5 organic compounds) + 4 mg/L 6-BAP + 6-9 mg/L AgNO3+ 3% glucose + 2.6 g/L Phytagel . The addition of AgNO3 solution can improve the rates of inducing shoot, 6-9 mg/L AgNO3 was found to be the best condition. Meanwhile, the test strains only exhibit insignificant influence on the rates of regeneration. The high frequency of regeneration is 7B7 (84.4%) and the second is 6B6 (83.3%), respectively.2 Establishment of the Agrobacterium-mediated transformation:(1) Optimization of transformation condition of oil sunflower. The cotyledons of oil sunflower were immersed in Agrobacterium tumefaciens for 10-15 minutes, co-cultivated in the medium including 100μmol/L AS at pH 5.4 for 2 days in the dark, the temperature was maintained at 22±1oC, and thus the higher performance of regeneration was obtained.(2) Determination of best antibiotics concentration and antibacterial type. The optimum concentration of kanamycin used in resistant shoots selection was 40 mg/L and cefotaxime used in resistant germ culture was 300mg/L, respectively.3 Detection of transforming regeneration oil sunflower of Gastrodia Antifungal Protein (GAFP):Two pairs of primer were used to perform polymerase chain reaction amplificaition, respectively. 4 of 87 regenerated plants were positive and the positive rate calculated from the number of infected explants was 4.6%. The results of PCR demonstrated the integration of target gene into oil sunflower chromosome primarily. Our dissertation provided a new approach to the anti-fungal disease of oil sunflower method by use of plant genetic engineering technology.
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