Study On Transformation Of Brassica Napus L. With Oxalate Oxidase Gene

However, this crop has been suffering from the destructive disease of Sclerotinia sclerotiorum for a long time. In China, Sclerotinia sclerotiorum is the first one of the three most serious diseases of rape and affects the rape production greatly. After long time practice,no completely resistant cultivars have been identified in rapeseed, as well as in the closely related species. Resistance to Sclerotinia sclerotiorum in rapeseed is quantitative trait and easily affected by environment condition. There is no accepted widely identification resistance method. These questions make breeders difficult to select really resistant plants Fortunately the successe on plant genetic engineering has brought light to the difficulty. People began to resort to it and several successful results have been reported recently. It is a fundamental way to seek control method based on the determinant factor of pathogenesis. Aiming at oxalic acid, the major pathogenicity factor of S. sclerotiorum, the introduction and expression of genes encoding enzymes that degrade oxalic acid is a potential powerful strategy to enhance resistance to the fungi that secrete oxalic acid. In order to study the possibility of utilizing oxalate oxidase gene (OxO) to increase plant's resistance and obtain the resistant rape, Brassica napus were transformed by using Agrobacterium tumefaciens harbouring the plasmid containing OxO from wheat. The main work and results were as follows: 1. Establishment of Agrobacterium tumefaciens-mediated transformation systems Brassica napus 03603,03709,P137,04Y335 were used for transformation experiment. The cotyledon petiole and hypocotyls from Brassica napus were transformed as acceptor. Optimum Agrobacterium tumefaciens-mediated transformation systems were established. Optimum medium for Brassica napus transformation: I.pre-culture medium: MS+1 mg/L 2.4-D+0.2 mg/L 6-BA II.differentiation medium F₁: MS+2.5 mg/L 6-BA+0.2 mg/L NAA+400 mg/L Cb+6 mg/L AgNO₃, pH 5.8 III.differentiation medium F2: MS+2³ mg/L 6-BA+0.01 mg/L NAA+400 mg/L Cb+6 mg/L AgNO3, pH 5.8 IV.selective medium S1,S2,S3: F1+20 mg/L Km,F1+25 mg/L Km,F1+30 mg/L Km â…´.selective medium S4,S5,S6: F2+20 mg/L Km,F2+25 mg/L Km,F2+30 mg/L Km â…µ.root-inducing medium: 1/2 MS+0.1 mg/L IBA+300 mg/L Cef,pH 5.8 or 1/2 MS+0.1 mg/L NAA+300 mg/L Cef,pH 5.8 2. Obtaining of kanamycin-resistant regenerated plants Bacterial culture (optimum density: OD600 0.3～0.4) of Agrobacterium strain was diluted to 3～5 times, explants immerged in it for 3～5 min. After cocultured with Agrobacterium, selected by kanamycin, 203 kanamycin-resistant regenerated plants of Brassica napus were obtained. 3. Molecular biology characteristic of kanamycin-resistant regenerated plants PCR identification of the kanamycin resistant regenerated plants with gene-specific primers showed 104 of 203 kanamycin-resistant regenerated plants of Brassica napus gave the expected fragment of 470 bp as the positive control. OxO had been integrated into Brassica napus genomes. 4. Identification of resistance against oxalic acid of positive plants Treated with oxalic acid, transgenic plants showed a stronger tolerance against oxalic acid than wild type, so the introduction and expression of OxO could enhance the resistance to fungi that secrete oxalic acid. It has been demonstrated that representation of Brassica napus treated with oxalic acid is highly identical to that of infected by Sclerotinia sclerotiorum, so the oxalate oxidase can surely improve the resistance of rape plant to Sclerotinia sclerotiorum. 5. Artificial vernalization of four cultivars of transgenic rapeseeds Artificial vernalization technique of four oriental cultivars of transgenic rapeseeds (Brassica napus L.) in seedling stage was studied.The result are four oriental cultivars can flower by proper low temperature treatment days, as well as aseptic seedlings. 6. Identification of resistance in the field of T1 of transgenic rapeseeds The resistance of rapeseed maturing stage was evaluated with mycelial toothpicks to induce stems disease.The lesion length was significantly different from 6d to35d, lesion length of susceptible plants increased linearly however,that of resistant plants increased steadily.so, resistant plants about 150 from 1000 materials are obtain.