| Soybean is one of the important sources of supply for edible oil and protein extracted from plant But it was easily suffered from many kinds of fungi diseases, which were much devastating to decrease the yield and quality of soybean. During the past years. control of the disease has been mainly depended on the traditional breeding of crops and extensive utilization of chemical pesticides, a practice that lead to tedious breeding cycles environmental pollution and insecticide resistance. Sustainable growth of soybean will require new disease control strategies to respond to increased disease pressure and reduced availability of pesticides. The research aimed at obtaining the transgenic soybean with broad fungi-resistant traits by gene engineering techniques. Whereas, some obstacles still exists in the genetic transformation of fungi-resistant gene into soybean, such as narrow antifungal pedigree for single resistant genex few regeneration systems suitable for transformation and low frequency of genetic transformation.Concerns over the above problems, with four main soybean cultivars in Dongbei district in China, this study was made by taking the gene engineering approach with integrated strategies to construct plant expression vector containing bivalent fungi-resistant genes of chitinase gene (chi) and ribosome inactivating protein gene (rip), transfer the two target genes into soybeans for the first time with Agrobacteriian-mediated transformation and particle bombardment, also, optimize the conditions of regeneration and transformation of soybeans, establish the effective systems of plant regeneration and genetic transformation, and obtain transgenic soybean integrated with the two target genes identified by molecular blotting ways. This theme selected has a high academic value and practical significance, which will set a base for molecular breeding in disease controlling for soybeans and other pulses, and also will supply gene resources for sexual breeding approach . The main results were summarized as follows:1. This study constructed plant expression vector containing fungi-resistant genes of chi gene and rip genea. This study constructed 5 plant expression vectors, including one mediate clone vector pUR01 one mediated expression vector pARIP two plant expression vectors pBIRTP and pBchE one bivalent plant expression vector pBRC.b. Plasmid pBRC carried rip gene and chi gene, which will greatly broaden antifungalpedigree with their expression at the same time.c. pARJOP carrying rip gene and pBch carrying chi gene were used for particle bombardment.d. pBIRIP and pBchE contained their own fungi-resistant gene respectively, and pBIRIP also carried Gits gene. They all could be used for the study of genetic transformation of fungi-resistant genes into plants.2. This study reconstructed promoter for target genesThe target genes in vectors pARU\ pBIRIP > pBchE and pBRC were all designed to be driven by the directed duplicate of CaMV35S promoter, which would greatly increase the expression efficiency of target genes in transgenic plants.3. This study established organogenesis regeneration system from cotyledonary nodes of soybeana. The differentiation medium of multiple shoots was MSB+1 .5mg/L BA+0.2mg/L IBA, 0.8%agar, pH5.8.b. The percentage of shoots differentiation and No. of shoots on each explant varied from different genotypes. The percentage of differentiation of Heinong 3 5 > Hefeng 35 ^ Dongnong42andJilin30was98.5%> 993%, 73.4%> 40.6%, andtheNo.of shoot on each explant was 4.9, 5.Q, 3.0 and 3.0 respectively. The highly efficient genotypes of regeneration were Heinong 35 and Hefeng 35.c. The optima] medium of root regeneration from shoots was l/2MSB+1.5mg/L IBA+2% sucrose., 0.8%agar, pH5.8.d. The rate and duration of root regeneration varied from different genotypes. The rate of root regeneration of Hefeng 35 Heinong 35 Dongnong 42 and Jilin 30 was 95.1% 62.2%, 60.8% 79.8%, and the duration of root regener...
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