| Stilbene synthase gene (STS) and its specific promoter were previously isolated from Chinese wild Vitis pseudoreticulata accession'Baihe-35-1',which were related to disease resistance. The promoter was Added different enhancement components for construction of three enhanced expression vectors. In order to provide theoretical and technical basis for obtaining disease resistance transgenic grapevine, in this study, these four vectors were tried to transform into Vitis vinifera by genetic engineering technology.Regeneration of"Carignane","Flame Seedless","Thompson Seedless","Long-bunch Thompson Seedless"were established by somatic embryogenesis, respectively, based upon the regeneration systems via floral organ culture. The vectors harboring stilbene synthase gene were introduced into grape and tomato by Agrobacterium-mediated method, respectively. The main results are as follow:1.anthers,ovary and small buds collected from five grape varieties were inoculated in four different medium as explants to induce callus. In which, medium NN69 + 2.0 mg·L-1 6-BA + 1.0 mg·L-1 2,4-D + 60 g·L-1 Sucrose + 3 g·L-1 phatagel and medium MS + 0.55 mg·L-1 2,4-D + 0.5 mg·L-1 NOA+1.24 mg·L-1 4-CPPU + 30 g·L-1 Sucrose + 3 g·L-1 phatage are effective for callus induction,up to 6.2%.2.somatic embryogenesis were induced from callus of five grape varieties except from'Cabernet Sauvignon'on medium X6 + 60 g·L-1 Sucrose + 7 g·L-1TC-Agar + 0.5 g·L-1AC. Moreover, the somatic embryos before heart-shaped period were induced secondary somatic embryos on the same medium.3.Somatic embryos could regenerate plantlets using about one week as the following steps: dark cultured in liquid MS with shaking at 110rpm 25℃for 3 days, then transferred to solid medium WPM+ 0.15 mg·L-1IBA + 30 g·L-1 Sucrose +7 g·L-1 agar + 0.5 g·L-1AC under low light treatment for 3 days, change to strong light.4.The vector harboring STS and its specific promoter was successfully transformed to tomato'Micro-Tom'by Agrobacterium-mediated method. Four lines including 54 transgenic plants were obtained by PCR and PCR-Southern detection.5.Resveratrol contents were determined by HPLC from transgenic and wild type tomato plants mock and 12 h post inoculation of Botrytis cinerea. Results showed that resveratrol content of transgenic plants that transformed with vector harboring Enhancer were highest in mock and Botrytis cinerea inoculated plants, which is 4.4 times more than tomato transformed with vector harboring no enhanced element .while it was undetectable in wild type.6.The disease resistance index of transgenic tomato plants was measured after inoculation of Botrytis cinerea. Results showed that resistance of all transgenic plants was increased to some degree, in which resistance of the plants transformed with vector harboring Enhancer is strongest. |
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