The Use Of The PMI/Mannose Selection System To Recover Transgenic Chrysanthemum Morifolium With PtDHAR Gene

Chrysanthemum morifolium is commonly used in ornamental gardens and flower. The significant features are low power plant, coverage, easy to manage the extensive breeding, wide, and resistant to strong, a great variety, spend more dense, long flowering period, ornamental value. In saline and Northeast China, as well as the city of saline, most intense cultivation of Chrysanthemum morifolium affected by salt of the exhibit leaf chlorosis, bad growth, bud is blocked or even death. This study aims to take advantage of the security token of the transformation method to obtain a conversion plant has produced salt more lines, rich in plants species, improve income in flower growers.This study draws Gateway technology to phosphomannose isomerase gene PMI is safe for planting in the cloned tenuiflora salt tolerance gene PtDHAR, built for the purpose of gene pCAMBIA1301-PMI-PtDHAR dual-element expression vector, and the carrier to Agrobacterium strain EHA105 by Agrobacterium-mediated method, the salt tolerance gene PtDHAR turn into Chrysanthemum morifolium'Huoyan'notation genome, alkali more lines, on large daisy in the country, the better for landscaping services.The major findings are as follows:(1) Building genetic transformation vector. From the Escheriachia coli PMI by PCR cloning, gene-digestion method to PMI-T carrier and carrier-based, pCAMBIA1301 into pCAMBIA1301-PMI carrier; use Gateway technology to build pCAMBIA1301-PMI-PtDHAR dual-element expression vector, and the carrier to Agrobacterium strain EHA105 by PCR validation results showed that pCAMBIA1301-PMI-PtDHAR carrier has successfully transferred to Agrobacterium strain EHA105.(2) Chrysanthemum morifolium'Huoyan'on Mannose and cephalosporin sensitivity. Study results indicate that leaves explants on Mannose is very sensitive, mannose concentration to 10g/L at apparent suppression leaves explants of differentiation and adventitious buds of produce that can be used as genetic transformation when leaves explant culture of critical tolerance filter concentration; differentiation and proliferation factor as adventitious buds cephalosporin Cef concentration but steady decline,300mg/L Cef concentration can be used as leaf explant cultured in total suppression of Agrobacterium tumefaciens inhibitory concentration.(3) Established in Chrysanthemum morifolium'Huoyan'genetic transformation system. To pre-training 1d of the leaf explants were transformed material, with a value of 0.4 OD600 Agrobacterium infection 10 min,23-25℃in the dark were cultured under the conditions of 2d, for 2d delayed train to 300mg/L of Cef concentration inhibitory concentration, using selective pressure increments were screened, initial screening medium mannose concentration 8g/L, post-screening medium concentration of mannose increased to lOg/L, continuous screening to produce adventitious buds. To shoot into the mannose concentration of 10g/L, Cef concentration of 100mg/L of rooting medium to filter selection, until the growing robust and resistant plants produce root.(4) Transformed plants testing. To 9 resistant adult plant which obtains carries on the PCR examination, finally indicated that obtained 2 to transfer the PtDHAR gene adult plant.