Optimization Of Regeneration In Vitro Culture In Rapeseed And The Genetic Transformation Of Sclerotinia Resistant Gene-BnPGIP

Rapeseed is the second largest oil crops in the world, and is also one of the important oil crops in China. Sclerotinia stem rot(Sclerotinia sclerotiorum) is the major disease of oilseed rape, up to now no immune variety is found in rapeseed and its related crops. Now gene engineering was used to transform resistant gene into rapeseed in order to enhance the sclerotinia resistance of rapeseed. It was reported that PGIP gene of plants had an important effect on the process of plants and fungus interaction. Our research also found that the expression of PGIP in sclerotinia resistant rapeseed Ning RS-1 was increased significantly when it was inoculated with sclerotinia sclerotium. In order to further study the defensive effect of PGIP gene on sclerotinia stem rot in rapeseed, the transgenic of over-expression of BnPGIP was done to verify the function of this gene. For this purpose, this study conduct the optimization of vitro culture regeneration system and agrobacterium-mediated transformation system in rape, and the transformation of PGIP over-expression was carried out in rapeseed. The results were as follows.1. In order to optimize the vitro culture regeneration system in rapeseed (Brassica napus L), the key factors affecting the regeneration frequency, which include varieties, seedling age, pre-culture time and hormone concentration and proportion, are analyzed in this paper. Results show that the regeneration frequency of different varieties have significant difference, the explant regeneration frequency of Westar is higher than that of Ningyou 18 and Zhongshuang 11. There are differences between different explants, the regeneration frequency of cotyledon petiole is significantly higher than that of hypocotyls. The seedling age of 4d and pre-culture time in 2d is suitable for vitro culture. The optimal concentration of hormone 2,4-D is 1.0mg/L in pre-culture medium. For cotyledon petiole, the optimal ratio of 6-BA/NAA in differentiation medium is 10:1, and the regeneration frequency reach to 70.79%; for the hypocotyls, the optimal ratio of 6-BA/NAA ratio is 9:1, and the regeneration frequency reach to 96.73%.2. The research for vitrificated regeneration seedling of rapeseed get out of vitrification.During the process of the regeneration seedling cultivation, the problem of vitrificated plants is very serious, especially in the phase of the bud induction. By experimental study that pick out vitrification seedlings from the culture medium, put in the culture bottles which are germ free and don’t have medium, deal with bright light-"hunger" at 25℃ about 2-3d, and then transfer to the normal cultivation, found that vitrificated regeneration can vitrificate and transfer to the normal plant. The probability vitrification seedling which dealt with hungry and bright light turn into normal seedlings as high as 90%.3. The difference research of agrobacterium infection of different rapeseed varieties. Taking the petioles and hypocotyls of Ningyou 18, Zhongshuang Hand Westar as the testing materials, then do the translating experiment with agrobacterium which contain GUS gene infection, and set up the plants which have not been infected by agrobacterium as comparison. The expression of GUS gene transient detection shows that the GUS positive rate as high as 77.78% for Westar’s hypocotyls and 6.25% for petioles. No blue reaction is found for both the petioles Ningyou 18 and of Zhongshuang 11; the GUS positive rate for hypocotyls of Zhongshuang 11 is about 4.16%. The research shows that among this three varieties, Westar is relatively easy to be infected and its seed germination rate is higher, so Westar is more suitable for agrobacterium mediated genetic transformation experiment; the conversion efficiency of the petioles is higher compared with its hypocotyls of the Westar.4. The research of the effect of the BnPGIP gene on rapeseed conversion. Using agrobacterium EHA105 containing over- expression vector to infect hypocotyls and petioles of Westar. We finally received 30 regeneration plants that resistant to Hyg after screening under resistance cultivation using the optimized regeneration system of rapeseed. In order to further determine whether the purpose gene is switched to the regeneration seedlings or not, we collected the DNA from 30 received seedlings for the PCR detection. The PCR detection results show that 17 are PCR positive.