Studies On Agrobacterium-Mediated Transformation Of Apple And Muskmelon With DHAR Gene

Under the adverse circumstance forces, plants can produce massive ROS which has very strongly poisonous function to the plant.The ROS scavenging system maintains its normal physiological function of plants. Melon and apples are our favorite fruits because they have a nice taste with high contents of nutritive components. Reseachers hope to enhance the ROS elimination enzyme activity through the genetic engineering method. Thus sharpens the ability of the anti- oxidation forces and breeds the variety which has the high resistance. It would be also helpful to get more information about the role of DHAR playing in AsA-GSH cycle and its metabolism pathway.The main results were as follows:1 A efficient regeneration system of plantlets was established with cotyledon and leaves in melon (Cucumis melo L. cv. Xitianyihao). These factors that include concentration of plant growth regulators, age, ways of inoculation and period of dark were studied. With the cotyledons growing for 5-7 d as accepter, the best medium of adventitious bud regeneration is MS+0.8 mg/L6-BA.With the leaves growing for two weeks as accepter, the best medium of adventitious bud regeneration is MS+1.0 mg/L6-BA. Culturing in normal photoperiod, abaxial side touching media, the differentiation rate of two explants was maximum reaching 100%.The optical concentrations of plant growth regulators were 0.05 mg/L6-BA during the stage of bud stretching. In the m edium 1/2MS+ IAA 0.2 mg/L, the elongated shoots rooted at the rate of 100%.2 Plant expression vector carrying dehydroascorbat reductase gene (DHAR, from Malus domestica Boekh. cv. Gala, Genbank accession number: DQ322706) was constructed, identificated and then introduced into Agrobacterium tumefaciens.3 With the cotyledon explants, we studied the factors that affected transformation ratio of Agrobacterium-mediated method. The optimal conditions which based on the results of PCR and the number of hygromycin B -resistant plants were investigated. The result showed that the suiltable conditions of transformation contained 4 mg/L Hyg, 500 mg/L Cef, 2 d preculture, 3 d coculture, 8 min infection, 0.6 OD600. Thus a simple and efficient genetic transformation system was developed, and the resistent melon plants.were abtained.4 With six lines of those positive melon plants, we assayed the AsA and DHA and found that the amounts of AsA and DHA improved strongly. Most of the lines, the ASA/DHA ratio did not change obviously. The results showed that the DHAR gene had integrated into melon and the overexpressing of DHAR gene could increase the amounts of AsA in melon.5 On the basis of the system of efficient regeneration and transformation with NagafuNo.2 leaves, we transformed DHAR gene into the NagafuNo.2 and obtained 1 positive plant by PCR preliminarily.