| Micro-Tom is a miniature dwarf tomato. Compared with common tomato, its small size enables it to grow at a high density, and it has a short life cycle and can be transformed efficiently. With such advantageous features, Micro-Tom has been used extensively in plant functional genomics research. In this study, we established an efficient regeneration system, a genetic transformation system for Micro-Tom. Some transgenic plants were obtained and had been confirmed by Real-time PCR assay. The results were summarized below:(1) Establishment of a high frequency plant regeneration system for Micro-Tom.Several factors influencing the efficiency of plant regeneration, including explant types and orientation of cutting, concentrations and of plant growth regulators and concentrations of sucrose, were studied. It was found that the buds frequency of cotyledon explant, especially transversely cutted, is much higher than hypocotyls; more shoots were regenerated from MS supplemented with 2.0 mg.L-1 ZR and 0.5 mg.L-1 IAA with 20 g.L-1 sucrose. The medium containing 0.2 mg.L-1 IBA was the optimum rooting medium.(2) Establishment of an efficient genetic transformation system for Micro-TomWe establish a genetic transformation system based on kanamycin selection. Cotyledon segments of Micro-Tom were firstly co-cultured with Agrobacterium tumefaciens GV3101 harboring neomycin phosphotransferase gene (nptII)-containing plant expression vectors in darkness at 28℃on kanamycin free MS for 10 min, then after cultured on kanamycin free MS for 2 day the explants were transferred to subculture on MS supplemented with 100 mg.dm-3 kanamycin and 500 mg.dm-3 Cefotaxime and cultured under 16-h photoperiod. As a result, a transformation efficiency of 11.25% was achieved using the optimized transformation procedure. Meantime, the transformation system with hygromycin selection was studied and the optimal concentration of hygromycin was set at 10 mg.L-1.(3) Development of detection assay for transgenic plant materials.In this study, a reliable real-time PCR analysis method was established for transformation analysis in plant materials. Two pairs of primers, corresponding to CRTISO gene (internal gene of plant) and NPTII gene were selected as for target and internal gene respectively. According to△Ct assay, the copy number of inserted gene in transgenic plant can be calculated approximately. The DNA template, from a boiling protocol, has to be diluted in 100-1000 times, while for DNA template obtained by routine DNA extraction, the optimum concentration is 0.25-25 ng.μL-1.
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