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

Tobacco leaf explants were transformed by bacteria <em>Agrobacterium tumefaciens</em> (<em>A. t</em>.) and plasmid pBIN mgfp5-ER, which has a single copy of the green fluorescent <em>gfp</em> gene and <em>A. t</em>.-pART27 2mgfp5-ER, which has two copies of the <em>gfp</em> gene. Both plasmids have a built-in selection <em>nptII</em> 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 <em>A. t.</em>-pART27 2mgfp5-ER and after 6 days in cells transformed with <em>A.t.-</em>pBIN mgfp5-ER. After infection by <em>A. t.</em>-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 <em>A. t.-</em>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 <em>nptII</em> gene and length of 422 bp specific for <em>gfp</em> marker gene