Study On The Transformation Of Apple And Tomato With Apple Genes DHAR And MDHAR Mediated By Agrobacterium Tumefaciens

L-Ascorbic acid (AsA), also known as vitamin C or ascorbate, is one of the most abundant antioxidants and cofactor for several enzymes. It has been reported that AsA is necessary for plant growth and development, and is also well known that it have an important role in resistance to oxidative stress. Moreover, it is the main source of natural AsA to human because humans are incapable of synthesizing AsA and must secure it by means of dietary uptake to be healthy.The most important pathway for AsA recycling is the ascorbate-glutathione system (AsA-GSH). In this cycle, AsA is oxidized to monodehydroascorbate (MDHA) radical while ascorbate peroxidase (APX, EC 1.11.1.11) is using AsA as electron donor to scavenge hydrogen peroxide (H2O2). The MDHA can spontaneously be degraded to AsA and dehydroascorbate (DHA) or be enzymatically reduced to AsA by NADPH or NADH-dependent-monodehydroascorbate reductase (MDHAR, EC 1.6.5.4). The DHA can be also reduced to AsA in a reaction mediated by dehydroascorbate reductase (DHAR, EC 1.8.5.1), using GSH as the reducing substrate. The resulting oxidized glutathione (GSSG) is then reduced back to GSH by a NADPH-dependent glutathione reductase (GR, EC 1.6.4.2). While DHA could not be completely reduced back to AsA in time, it would be further oxidized to 2, 3-diketogulonic acid or non-renewable oxalic acid and/or tartaric acid and lost. Thus, DHAR and MDHAR are important for maintaining AsA level and redox status because of recapturing it before lost. Overexpression of DHAR resulted in increase in the endogenous AsA pool size and the AsA redox state in leaves of tobacco and improved their tolerance to stress. In this study, the leaves of apple"Greensleeves"and tomato"S yi 12 fen"were infected by Agrobacterium tumefaciens carrying apple DHAR and MDHAR gene .The transformed tomato lines had higher enzymatic activity than the wild lines. The main results were as follows:1 The leaf of apple cultivar."Greensleeves"was used for transformation.Different combintions of plant growth regulators and leaves at different age were studied to induce the adventitious buds. The results showed that the optunium medium was: MS+5.0 mg/L 6-BA+1.0 mg/L NAA +30 g/L sucrose +7.0 g/L agar (pH5.8), and the youngest unfolded leaf has higher regeneration rate than old leaf. The'right-side-up'leaves have higher regeneration rate than the ones'right-side-down'.2 Agrobacterium-mediated transformation of leaves from apple cultivar'Greensleeves'was conducted and we obtained hygromycin-resistant plantlets.3 Cotyledon and hypocotyl of tomato'S yi 12 fen'was used for transformation. Different combinations of plant growth regulators were studied to induce the adventitious buds. The best medium for Cotyledon and hypocotyl to induce adventitious buds was: MS +2.0 mg/L ZT + 0.2 mg/L IAA + 30 g/L sucrose + 7.0 g/L agar (pH 5.8), and the regeneration efficiency were 99% and 75% respectively.4. Agrobacterium-mediated transformation of cotyledon from tomato cultivar's yi 12 fen'was conducted by apple DHAR and MDHAR gene and 12 and 2 homomycin-resistant lines were obtained respectively. The PCR results showed that exogenous gene was successfully integrated into the genomic DNA of tomato cultivar's yi 12 fen'.5 The activity of transgenic lines of tomato was higher than the control plants to some extent but the results were not all the same consistently.