System Optimization Of Agrobacterium-Mediated Transfer Δ~6-fatty Acid Desaturase Gene Into Cotton

The Δ6-fatty acid desaturase gene cloned from Nonea capsica (Willd.) G.Don(GenBank，DQ367892) was cloned into the plant expression binary vectorpCAMBIA2301.1（pCAMBIA2301.1-D6D）and transformed into Agrobacteriumtumefaciens EHA105by electro-transformation.The hypocotyls of Xinluzao19and Xinluzao23were used for the experiments.The effects of various concentrations of kanamycin were evaluated on hypocotyls todetermine the appropriate screen concentration. Our analysis revealed that less than5%hypocotyls can induce callus in the presence of25mg/L kanamycin and that nocallus in the presence of30mg/L kanamycin. Therefore,30mg/L kanamycin wasused in subsequent transformation experiments.GUS gene as the report gene,both the transient expression of GUS gene in thetransformed hypocotyls of cotton and the callus ratio from hypocotyls were examinedto analyze the influences of Agrobacterium density, infection time, acetosyringone(AS) concentration,sonication treatment,co-cultivation temperature and co-cultivationtime on the Agrobacterium-mediated transformation efficiency. As results, the optimalinfection combination for Xinluzao19and Xinluzao23were Agrobacterium densityat OD600=0.5with15min infection,the perfect acetosyringone concentration was50~100μmol/L, the appropriate co-cultivation temperature and time were incubated at24°C for72h.By GUS dyeing the Kanamycin-resistant calli and most of them were blue. D6Dgene specific primers XAM/APAL PCR amplification analysis showed that the D6D gene wastransferred into the kanamycin-resistant calli, the result shown the D6D gene wasintegrated into the genome of kanamycin-resistant calli. Agrobacterium-mediatedtransformation of D6D gene into shoot-tip meristem got kanamycin–resistant shoots,which showed GUS expression in leaves,successful transformation wasascertained by PCR amplification of D6D gene in the genome of transgenic plants.We have researched the main factors influencing the transformation efficiencyand established a highly efficient Agrobacterium-mediated genetic transformationsystem for Xinjiang Cotton.This study would provide technique base for XinjiangCotton resistant breeding and improve germplasm resources using Geneticengineering technology.