Incorporating Chitinase Gene And β-1,3-Glucanase Gene Into Rapeseed (Brassica Napus L.)by Agrobacterium Tumefaciens

Rapeseed (Brassica napus L.) is one of the most important industrial crops in the world. However, this crop has been suffering from the disease of Sclerotinia sclerotiorum for a long time. After long time practice, it seemed that it is diffcult for breeders to find a new anti-fungal materials resisting to S. sclerotiorum by traditional breeding methods. 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. In this study, a regeneration system and a transformation via A. iumefaciences system were investigated. Based on the former, Maize Chitinase gene , Wheat β-1 ,3-Glucanase gene and their bivalent expression vector were introduced into rapeseed respectively. Transgenic plants were confirmed in a series of examinations. The results are as following: 1. Regeneration systems of hypocotyls Hypocotyls from 6 days old seedings were cut into O.5.8cm in length and pre-cultured on MS containing 1 .OmgIL 2, 4-D and O.2mgIL BA for 3 d. Subsequently, hypocotyls were transferred on MS supplied with 3.Omg/L BA 0.1 mgIL NAA and 6mgIL AgNO3 and Shoots formed after 50 days culture. Four varieties of B. napus cultivars were employed in the experiment. Regenerationfrequency ranged from 64% to 84%. 2. A.tumefaciences mediated transformation system of hypocotyls Hypocotyls segments from D2 were pre-cultured on MS containing I .Omg/L 2, 4-D and 0.2mgIL BA for 3d. After quickly dipped into the Agrobacterium suspension of OD600 0.3 0.4 for 30sec , the hpocotyls were co-cultured on the same medium covered with a sterile filter paper disc for 2d in darkness. When the co-culture was over, the explants were washed in liquid MS containing SOOmg/L Cef for 2 3 times to kill the overgrown Agrobacterium. Subsequently, the explants were transferred to the delay selection media for 7d and cultured on gradation Kan (5.. 10.. lSmgtL) selection media. Transformed frequency amounted to 1.6? 8.4%. 3. Detection of transgenic plants The PCR assay of three kinds of Kan-resistant shoots showed that the target gene had been integrated into rapeseed accompanying with NPT II gene. Crude enzyme liquid was extracted from Chitinase gene modified plants .The result that Chitinase activity in transformants is higher than that in control was detected in Fe2(KCN)3 color reagent solution examination and glycol Chitin plate examination. It indicated that exogenous DNA can be expressed in transgenic plants. Resistance was observed in transgenic plants of Chitinase gene when they were challenged with S. sclerotiorum...