Expression and molecular analysis of DsRed and fluorescent genes in tobacco (Nicotiana tabacum L.) / Izražanje in molekulska analiza DsRed in gfp fluorescentnih genov pri tobaku (nicotiana tabacum L.)

Abstract

Agrobacterium-mediated transformation of tobacco leaf disks with Agrobacterium tumefaciens (A. t.) strain LBA4404 and two plasmids (pCAMBIA1390-DsRed and pART27 2mgfp5-ER) was used for introducing red fluorescent gene (DsRed), green fluorescent gene (gfp) and corresponding selection genes (hptII for resistance to antibiotic hygromycin and nptII for resistance to kanamycin) into leaf discs of tobacco (Nicotiana tabacum L.). Epifluorescent microscopy with the appropriate set of filters did not reveal phenotypic expression of the DsRed gene in 6.9 % of regenerants and the gfp gene in 1.3 % of regenerants that were successfully grown on selective medium. The duplex PCR method also did not confirm the presence of fragments specific to DsRed or gfp genes in these regenerants, while the presence of fragments characteristic of selection genes hptII and nptII was confirmed. A built-in nptII gene mutation, a deletion, was detected in one regenerant. Out of the 139 regenerants generated after the transformation of A. t.-pCAMBIA1390-DsRed, 38 or 25.5 % successfully grew only on non-selective medium; after transformation with A. t.-pART27 2mgfp5-ER 9 or 5.4 % of the 161 generants grew successfully. PCR analysis confirmed in all regenerants the presence of fragments characteristic of both transgenes, which were not expressed or were silenced. The effectiveness of transformation after infection with A. t.-pCAMBIA1390-DsRed was 93.1 %, and 98.7 % after infection with A. t.-pART27 2mgfp5-ER. We established that both fluorescent genes are suitable for setting up a transformation system. The antibiotics hygromycin and kanamycin successfully prevented the growth of untransformed tissues, but the antibiotic timentin successfully prevented the growth of bacteria A. t. after the transformation.

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

“…For the separation of DNA fragments, horizontal electrophoresis was used on a 1.4 % gel according Oven and Luthar (2013).…”

“…Bacterial suspensions of A. t., with the appropriate plasmid included, were incubated and prepared for transformation and co-cultivated according to Oven and Luthar (2013). Then, the leaf explants were transferred onto selective MSr medium with the addition of [Fe-Na 2 -EDTA 0.1 mg/l, thiamine 0.1 mg/l, 6-benzylaminopurine (BAP) 1.0 mg/l, 1-naphthaleneacetic acid (NAA) 0.1 mg/l, agar 8 g/l; pH 5.8] (Stolarz et al, 1991) without acetosyringone and with the addition of timentin 150 mg/l to prevent the growth of A t. bacteria and an appropriate selection antibiotic 300 mg/l of kanamycin for the selection of tobacco transformants after infection both with A. t.-pBIN mgfp5-ER or A. t.-pART27 2mgfp5-ER.…”