Establishment Of Regeneration System Of Tomato And Melon In Vitro And The Study On Transformation Of RCK Gene Into Tomato

Tomato and melon both are dicotyledonous plants with sarcocarp wich have the development of flesh fruit. The genome of both had been sequenced, and would complement Arabdopsis in the study of genome and gene fuction. The RCK gene in Arabidopsis is necessary for normal crossovers in meiosis. Studies showed that RCK mutants were defective in crossover formation, synapsis, and bivalent formation during meiosis, and it led to the reduction of bivalents and the formation of univalents, and it reduces the fertility of the plants.This thesis aims to study regeneration system in vitro of tomato and melon, and study transformation of RCK gene into tomato, so as to establish high effective transformation system. They would provide basis for functional identification of RCK gene in tamato and melon.1Study on the regeneration system of tomatoThe sterilizing conditions of Zhongshu NO.4, Zhongza105and Yellow Cherry tomato are all as follows:the seed was sterilized with75%alcohol for30s, and then sterilized with0.1%HgCl2for5min. In this way the germination rate was higher and sterile. The influence of various plant hormones on tomato regeneration is analyzed, and we can draw the following conclusions:cotyledon explants grow well when added2.0mg/L6-BA and0.2mg/L IAA in regeneration medium for Zhongshu NO.4,1.0mg/L6-BA and0.2mg/L IAA in regeneration medium for Zhongza105,1.0mg/L ZT and0.1mg/L IAA in regeneration medium for Yellow Cherry. Explants grew well using6or7d sterile plantlet in seedling age test. The optimum rooting media was1/2MS added0.1mg/L IAA or0.5mg/L NAA for Zhongshu NO.4,0.5mg/L NAA for Zhongza105,0.1mg/L IAA or0.5mg/L-1.0mg/L NAA for Yellow Cherry. Explants of Zhongshu NO.4tomato cannot produce any callus and adventitious buds when Hyg is higher than5mg/L.2The anatomical study on adventitious bud regeneration of Zhongshu NO.4tomatoThe cotyledon explants of Zhongshu NO.4tomato were used, and growth process of explants callus and adventitious bud was studied by the method of paraffin section. Callus grows constantly along with culture, and then adventitious buds occured in explants. There are many callus and adventitious buds in both ends of explants nearby incision, and there were hardly any callus and adventitious buds in middle of explants. Malformation in growing point is the main cause of the deformed buds, deformed buds just including single protuberance when first born, without completed buds structure. 3Study on the regeneration system of melonThe sterilized conditions of Baibao208melon are as follows:sterilized with75%alcohol for30s, and then sterilized with0.1%HgCl2; for6min. And Lanhanguawang melon is sterilized with75%alcohol for30s and0.1%HgCl2:for8or10min. The optimum differentiation medium was MS added2mg/L6-BA and0.2mg/L IAA for Baibao208,1.5mg/L6-BA and0.1mg/L IAA for Lanhanguawang to grow adventitious buds. The elongation culture media of Baibao208, Lanhanguawang need0.3-0.5mg/L6-BA in medium. Seedling age test get that explants grow well using6d sterile plantlet for Baibao208, and it is7d for Lanhanguawang. Several plant hormones on rooting of melon were analyzed, when added0.3mg/L IAA to rooting medium of Baibao208, the root grow well, and Lanhanguawang need0.1or0.3mg/L IAA.4Study on RCK gene transforming Zhongshu NO.4tomato.Transforming Zhongshu NO.4tomato with Agrobacterium containing RCK gene, this way we can not only get resistant plants but also find several factors in transformation test. The infection conditions of Zhongshu NO.4tomato to grow well are as follows:pre-cultured8d, diluted20or30-fold of Agrobacterium re-suspension solution, infected2-10min. The optimal antibiotic concentration is400mg/L Cef to control Agrobacterium. Hyg has a great influence to adventitious buds generation, and play a role in the selection of resistant seedlings.In summary, the vitro regeneration system of Zhongshu NO.4, Zhongza105, Yellow Cherry tomato and Baibao208, Lanhanguawang melon had established. The study on RCK gene transforming Zhongshu NO.4tomato was successfully. We get regenerated plants of Zhongshu NO.4, Zhongza105, Yellow Cherry tomato and Baibao208, Lanhanguawang melon. Resistance plants of Zhongshu NO.4tomato had been obtained. Finally, this study optimizes many kinds of conditions in regeneration and transformation process.