| Anthurium andraeanum, a kind of perennial herb which belongs to Anthurium genus of Araceae family, is generally used as cutting flower maretials because of its peculiar patterns and the long flowering period. Potted plants are also widely used as indoor decoration flowers due to their beautiful plant types. However, anthurium is highly vulnerable to bacterial blight in production process, even a devastating loss. That is a serious obstacle on domestic and foreign production of anthurium. Therefore, research of genetic engineering and variety breeding for disease resistance are quite important in theory and practice.Based on previous researchers, this study was mainly arm to the regeneration system for genetic engineering in anthurium, genetic transformation system for agrobacterium mediated method, synthetise and modification of antibacterial peptide gene, vector construction of plant expression for target gene and its transformation into Agrobacterium tumefacien. The main research results were as follows:1. Optimized a regeneration system in Anthurium andraeanum. The effects of different hormones on adventitious bud differentiation of anthurium callus were investigated through 1/2MS medium containing different auxins(2, 4-D, NAA, IBA) and cytokinins(6-BA, TDZ) and their concentration by using the pooted cultivar named SYN-A in Anthurium andraeanum. The results showed that adventitious bud differentiation of callus rate had reached above 76.67% in all treatments. The differentiation rate of adventitious buds of callus could reach 100% and coefficient of redifferentiation was 31.06 under the optimum culture medium with 1/2MS+ 6-BA 0.5 mg/L +2,4-D 0.1 mg/L.2. Proved the sensitivity difference to three kinds of antibiotics in anthurium. The results showed that the explants of anthurium callus was more sensitive to hygromycin than kanamycin. The former was more suitable for screening transgenic tissue of anthurium, and the suitable concentration was 20 mg/L. Secondly, callus was not sensitive to cefalexin. In the experiment, 500 mg/L cefalexin could achieve good antibacterial effect, and explants grew well.3. Established the genetic transformation system for agrobacterium mediated method in anthurium. With callus as receptor material and Agrobacterium tumefacien EHA105 as tested strain, the influences of pre-culture time, infection time, ultrasonic time and co-culture time on genetic transformation were discussed by orthogonal tests. The results showed the influence extent is co-culture time > pre-culture time > infection time > ultrasonic time. The genetic transformation system applicable for Anthurium andraeanum was established a callus of 1 cm × 1 cm × 0.5 cm was used as the acceptor material, pre-cultured for 3 days, then infected for 20 minutes in bacterial suspension, during which time used the ultrasonic treatment for 20 seconds when Agrobacterium tumefacien EHA105 solution was OD600 0.6, cleaned the callus and set it in the antibacterial medium for few days, then transfered into screening culture until the formation of new callus.4. Constructed the plant expression vector carrying the antimicrobial peptide fusion gene and obtained an engineering agrobacterium. Signal peptide of pathogenesis related protein PR1 a were attached with antibacterial peptide Shiva-1, forming the fusion gene, which named Aa AMP were modified and optimized by Anthurium andraeanum preference codon and synthesized with specific sites of restriction endonuclease. The fusion gene was inserted into the intermediate vector p CAMBIA1301 and obtained the plant expression vector p CAMBIA1301-Aa AMP, which was used to be transformed into Agrobacterium tumefacien EHA105. |
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