Studies On Establishment Of Tomato High-Frequency Regeneration System And Transformation Of Etr1 And IPT Genes

This essay aims at establishing the high-frequency regeneration system of Cherry tomato (Lycopercicon esculentum Mill) cultivars comparing kinds, concentrations,different combinations of hormones and other additives (glutamine, trptophane,proline,yeast extract and silver nitrate )effects on the regeneration of tomato cotyledons and hypocotyls.We set up a good kind genetic transformation system with cotyledon as explants which is suit for the hereditary and translation of the tomato and optimized the transformation system.We constructed a binary vector carrying etr1 and IPT genes under the drive of double CaMV 35S promoters and GH promoter respectively. IPT and etrl were tranferred into tomatoes with agrobacterium-mediated transformation. Transgenic plants grew well in the medium with PPT 3mg/L.Transgenic tomato plants were confirmed by PCR analysis and RT-PCR analysis.By PCR analysis,the results proved that the foreign genes had been integrated into the genome of cherry tomato.By RT-PCR analysis, we knew that the etr1 gene had been transcripted. The activity of phosphinothricin acetyitransferase of transgenic plants are strengthened in comparison with controls.The main results of this study were as fallowings:1 Treating the tomato seeds in the. 40 C water bath before sterilization helped the seeds germination.The treated seeds had a high rate of germination and almost germinated together.2 Taking orthogonal experiment, we got the optimal combination and concentration was BA1.0mg/L and IAA0.2mg/L.2,4D kept down the regeneration of cotyledons distinctly.3 The concentration of sugar effects calli formation and adventitious buds differation. 3% sugar is the best.4 Adding different additives(glutamme, trptophane, proline, yeast extract and silver nitrate) into the regeneration medium infected the ability of calli formtion and differentiation.Different additives had different effects:glutamine 50mg/L promoted the differentiation of explants and the formation of clustered budsjyeast extract 500mg/L or trptophane 20mg/L restrained the differentation of calli not the formation;proline 200mg/L restrained the formation markably and got small browning. calliand no aadventitious buds; silver nitrate 5mg/L browned the cut of cotyledons and formed fewer and smaller calli. From these,the effects of additives had concentration particularity and species particularity.5 Optimizing the agrobacterium-mediated transformation system, we got some results.-OD6000. 1-0.2 was the fitful bacteria concentration for invading; the explants and the bacteria had been best co-cultured for 48 hours; put the cotyledons upside down when culturing and co-culturing but inverted them when cultured inmedium with antibiotics; choosing after being cultured in mediun without PPT for 7 days were better;the optimal choice press is PPT3mg/L.6 By PCR analysis, the foreign genes had been integrated into the genome of cherry tomato.7 By RT-PCR analysis, the etrl had been transcripted.8 The activity of phosphinothricin acetyltransferase of transgenic plants are strengthed in comparison with controls.