Optimization Research On Rapid Propagation And Transformation System Of Tomato

Using tomato variety "Zhongshu No.4" as the materials, the paper optimized the regeneration system of adventitious bud by tissue culture and established a rapid propagation system. The transformation plant were obtained by infecting tomato cotyledons with the Agrobacterium strain EHA105 carrying the binary plasmid pCAMBIA3301 (containing the GUS gene and the Bar gene). It laid the foundation for improving tomato traits through genetic engineering technology in the future. The detail results are presented as follows:1. Using tomato variety "Zhongshu No.4" as the materials, the regeneration system of adventitious bud by tissue culture was established by researching the factors related to regeneration from the cotyledon in tomato. Firstly, before sowing, the seeds were soaked in cold water for 10 h. After sowing, the seeds were placed under dark for 3 d till germination, then were transfered to light for 8-9 d and the sterile seedlings were obtained. The sterile seedlings also could been gotten by the embryo, which would take 5 d and shorter than the former and the plantlet were similar to the former. The effect of ZT and IAA by filtration sterilization was better than high pressure sterilization; The experiment studied the effects of different organic additives, The results showed that the best medium for the regeneration of tomato cotyledons was N&N, followed by DeLong and MES, MS was the lowest. The middle section of the cotyledon was the best explants; Placing the cotyledon upside down is more benefit to induce the adventitious buds than upside; The best medium for the regeneration from tomato cotyledons is MS (Phyto Technology Laboratories-M519)+ZR.; The best enrichment medium is MS+NAA 0.01 mg/L+6-BA 2.0 mg/L.2. In order to establish a fast propagation system, the paper studied the components:seedling time, multiplication capacity and the cost of every plant. Statistical analysis showed that developing from a seed into a aseptic seedling needs 5 d and inducing callus and adventitions buds need 25 days, adventitions buds elongating need 10 d, taking another 7 d the roots can be seen. The plants could been transplanted to the soil in the greenhouse after hardening for 3 d. It taked stotally 56 d from the seed to the plantlets. One seed can rapid propagate 7 plants, it cost approximately RMB 0.35-0.4.3. The paper researched the best optimum concentration of PPT, the best concentration for callus induction was 0.8 mg/L and for roorting was 0.5 mg/L; The Agrobacterium growth curve showed that the bacteria shaking for 8 h enter into logarithmic growth phase, and the stable growth period after 10 h. The Agrobacterium was of high activity when cultured between 8 h to 10 h and suitable to infect the explants. The transgenic tomato were obtained in the experiment, PCR test results to resistant plants have indicated that the target gene have been integrated into the tomato. This research provides some references for the transformation of tomato in the future.