Regeneration and Transformation of Oilseed (Brassica napus) Using CaMV ۳۵S Promoter- β-glucuronidase Gene

CaMV ۳۵S promoter activity has not been analysed in different stages of growth in oil-seed (B. napus). Higher plants lack intrinsic β-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. The binary transformation vector pVW۴۳۲ (Buchanan-Wollaston, unpublished), carrying the GUS gene fused to the CaMV ۳۵S promoter, was used to test transformation efficiencies and promoter activity in B. napus. The effect of different components of culture media was tested. Having studied different antibiotics, two selectable markers, and Agrobacterium strains, the combination of ۵۰۰ mg/l carbenicillin with ۲۵ mg/l kanamycin was the optimum level for inhibition of Agrobacterium strain C۵۸pGV۳۱۰۱ and the selection of transformants, respectively. Dif-ferent developmental stages of plants were analysed by protein assay. DNA extraction and PCR amplification confirmed the presence of the gene. High level of β-glucuronidase activity, hence CaMV ۳۵S promoter activity, was observed for one of the transgenic plants. The β-glucuronidase activity altered from maturity to senescence indicating that the CaMV ۳۵S promoter has different activity at different developmental stages in B. napus. Therefore, the growth stage of B. napus may influence the CaMV ۳۵S promoter activity.

کلیدواژه ها:

CaMV ۳۵S Promoter ، β-glucuronidase (GUS) activity ، B. napus ، Growth stages ، Transformation