Stable Integration of Transgenes in Tobacco

Abstract

Genetic transformation has been used to introduce various genes, coding for accumulation of osmoprotectants -proline or fructan in commercially grown tobacco cultivars in order to improve the abiotic stress tolerance. Various approaches, including screening for resistance to antibiotics, PCR and freezing stress were used to select valuable transgenic lines. Southern blot analysis showed that they contained one to three copies of the integrated genes. The number of genetic loci was estimated by segregation in T1 progeny and stable transgenic lines were selected in T2 progeny.

Transgenic tobacco plants accumulating osmolytes show reduced oxidative damage under freezing stress

“…Synthesis of fructan was achieved by transfer of SacB gene, isolated from Bacillus subtilis [36] and synthesis of glycine betaine by transfer of codA gene isolated from Arthrobacter globiformis [11]. The genes were introduced into plant genome and effectively expressed [23,24]. Among the several homozygous lines [24], we used: AtPro26 (with P5CS gene form A. thaliana for proline accumulation), VacPro29 (with P5CS gene form V. aconitifolia for proline accumulation), Fru52 (with SacB gene for fructan accumulation) and GB9 (with codA gene for glycine betaine accumulation).…”

“…The genes were introduced into plant genome and effectively expressed [23,24]. Among the several homozygous lines [24], we used: AtPro26 (with P5CS gene form A. thaliana for proline accumulation), VacPro29 (with P5CS gene form V. aconitifolia for proline accumulation), Fru52 (with SacB gene for fructan accumulation) and GB9 (with codA gene for glycine betaine accumulation). Untransformed plants were designated as WT.…”

Improved Abiotic Stress Tolerance in Plants by Accumulation of Osmoprotectants—Gene Transfer Approach

“…They are known to be of high quality and wide-spread use in Bulgaria (78). The plants were transformed to accumulate proline, fructan or glycine betaine (78,80). We used constructs harboring Arabidopsis-or Vigna-derived genes (AtP5Cs, VacP5Cs) for D 1 -pyroline-5-carboxylate synthetase production, SacB gene coding for levansucrase from Bacillus subtilis or the codA gene coding for choline oxidase from Arthrobacter globiformis.…”

“…The genes were isolated earlier (26, 40; 66, 81) and kindly provided to us. We developed a specific procedure with several steps of selection, including osmotic stress in vitro and freezing of potted seedlings at transplanting stage, complemented with various physiological studies (24,78,79,80) This procedure allowed us to select stable transgenic lines able to survive freezing stress at controlled and field conditions in several subsequent progenies. The transgenic seeds were able to germinate after long exposure to chilling in vitro better than the wild-type seeds.…”