Transformation Of Tunisia Soft-seed Pomegranate With Cold-resistance Gene Mediated By Agrobacterium Tumefaciens

At present, Tunisia soft-seed pomegranate has become the fine varieties ofpomegranate cultivation in the south of the Yellow River, because its early fruit andhigh yield, beautiful appearance of fruit shape, fruit cracking of light, thin, sweet andjuicy, especially the very soft nucleoli and mature period coincides with theMid-Autumn. But due to bad cold-resistance, frost damage phenomena in theproduction and cultivation has already become a problem that can not be ignored. Thetraditional breeding program has been hampered by the long juvenility,self-incompatibility, linkage between genes etc. The genetic engineering provided afast and effective way to improve the traits of plants. In this study, cold-resistant genewas introduced into the Tunisia soft-seed pomegranate by theAgrobacterium-mediated transformation. The main aim of the study is to creat coldresistance, soft-seed and succulent new germplasm. The main results are as follows:1. In term of the ability of genetic transformation, the LBA4404AND EHA105agrobacterium strains has been studied in this paper. Experimental results show thatthe contamination rate and browning rate with LBA4404strains infection was higherthan those in EHA105, but in term of the contamination rates and browning rates, theresistant shoots with lba4404strains infection has been got more than eha105strain.For this reason, for the optimization of agrobacterium strains still need further study.2. Through investigating the co-culture temperature in Tunisian soft-seedspomegranate genetic transformation experiments, Experimental results show that thecontamination rates and browning rates of callus have improved significantly underthe optimum co-culture at19°C. Then the callus finally got resistant acllus and theresistant shoots after co-culture and selective culture. Consequently, the optimizationof co-culture temperature of Tunisian soft-seeds pomegranate genetic transformationexperiments is19°C.3. In the Tunisian soft-seeds pomegranate genetic transformation experiments, theleaves and callus were used to be explant for genetic transformation of cold-resistantgene. The research result shows, Tunisian soft-seeds pomegranate callus were used tobe explant for agrobacterium infection, and finally got resistant acllus and differentiate into resistant shoots. Consequently, callus were used to be good explantfor agrobacterium infection.4. Optimization of regeneration of resistant shoots in Tunisian soft-seeds pomegranatetissue culture. The results showed that the most appropriate media for the first time ofsubculture were MS medium supplemented with0.5mg/L NAA,1.5mg/L6-BA,0.3mg/L GA,1.0mg/L KT,1.0g/L PVP,50mg/L Kan,500mg/L Cef. The mostappropriate media for the resistant shoots of callus were MS medium supplementedwith0.5mg/L NAA,1.0mg/L6-BA,0.3mg/L GA,1.0mg/L KT,1.0g/L PVP,50mg/L Kan,500mg/L Cef.5. Molecular analysis of plasmid DNA of LBA4404bacteria strain. The stripe wereamplification by agarose gel electrophoresis. The plasmid DNA could be used todetect the resistant acllus and the resistant shoots.