The impact of plasmid on regeneration and expression efficiencies of gfp gene in tobacco (Nicotiana tabacum L.)

Abstract

Tobacco leaf explants were transformed by bacteria Agrobacterium tumefaciens (A. t.) and plasmid pBIN mgfp5-ER, which has a single copy of the green fluorescent gfp gene and A. t.-pART27 2mgfp5-ER, which has two copies of the gfp gene. Both plasmids have a built-in selection nptII gene for resistance to the antibiotic kanamycin. The presence of the green fluorescent mGFP-ER protein was detected with the epifluorescent microscope in the individual cells 3 days after transformation with A. t.-pART27 2mgfp5-ER and after 6 days in cells transformed with A.t.-pBIN mgfp5-ER. After infection by A. t.-pART27 2mgfp5-ER, in most cases the regeneration was direct, without intermediate stages of callus and faster, as the first globular structures were formed 10–12 days after transformation and a 204 % regeneration was achieved, while the first globular structure, after infection with A. t.-pBIN mgfp5-ER, occurred after 18 days and formed more callus and the regeneration was only 78.4 %. The duplex PCR analysis, performed on all 149 resulting regenerants, confirmed the presence of fragments of length 650 bp specific to the selection nptII gene and length of 422 bp specific for gfp marker gene.