| Citrus is one of the most important economic fruits in the world and the south China and also is a perennial and woody fruit tree characterized by long juvenile period, genetic heterozygosity and polyembryony, which made the breeding of this specie become difficult. In recent years, transgene technology had brought a novel way for the improvement of cultivars.At the present time, more and more attention were paid on the issue of the biosafety of transgenic crops. The application of selective marker gene with biology safety in the genetic transformation was an effective solution.The sweet orange cultivar of"Xuegan"(C. sinensis L.Osb.) was used as the material to optimize the genetic transformation system, based on the PMI (Phospho-mannose-isomerase) selection system established earlier. LEAFY gene of Arabidopsis and its ortholog LLFY in longan were both transformed into"Xuegan"genome for getting short juvenile varieties. Additionally, the exploring of the transformation using method of prickling mediated by Agrobacterium tumefaciens was also carried out, providing the new mehtod for the transgene of Citrus and woody trees. The main results were as follows:1. Optimization of genetic transformation system of"Xuegan"using PMI selection marker. At the condition of 0.6 at OD600 of Agrobacterium mixture, explants pretreatment with 1mg·L-1 2,4-D for 3 h, infection for 30 min, co-cultivation for 4d, 20g·L-1 mannose for selection, the regeneration rate was markedly increased after transformation. Supplement with 2mg·L-1 BA and 0.5mg·L-1 NAA on the media were benefit for the selection of resistant big plantlets.2. Transformation of LFY and LLFY into"Xuegan". Using the genetic transformation system optimized, LFY and LLFY were transformed into"Xuegan". Transgentic plantlets were obtained. Medium for seedling strengthening were added with 20g·L-1 mannose and 10g·L-1 sucrose to screen resistant transgenic plantlets frequently and effectively. CPR and PCR positivity rates of resistant plantlets transformed with LFY and LLFY were 3.7% and 7.4% for the big ones, respectively, suggesting the target genes were successfully integrated into"Xuegan"genome. The transgenic plantlets were induced to root using method of filter paper bridge and transplanted.3. Genetic transformation of"Xuegan"using method of prickling mediated by Agrobacterium tumefaciens was carried out. To inoculate A.tumefaciens into the embryonic apical meristerm of the soaked seeds, a region on the seed suiface where a shoot would later emerge was pierced twice up to a depth of about 1mm with a needle dipped in the A. tumefaciens inoculum.Germinated seedings were transplanted.One transgenic plantlet was obtained, which was confirmed by PCR assay and PCR-Southern blot, with the transformation rate of 4.35%, suggesting the feasibility of this method on the genetic transformation of"Xuegan"and providing the new method for the transgene of woody trees.4. Construction of plant expression vector of homologous genes of LLFY and SLLFY in longan. LLFY gene of"Honghezi"and SLLFY gene of"Sijimi"were inserted into the pC2300-35S-OCS vector between 35S promoter and OCS terminator to produce the plant expression vectors of pC2300-35S-LLFY-OCS and pC2300-35S-SLLFY-OCS respectively for the research of the function of the two genes.
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