Tobacco (Nicotiana benthamiana Domin) Plant Transformation With and Without Selectable Marker Gene for Multiple Virus Resistance

Abstract

Transformation of tobacco plants (Nicotiana benthamiana Domin), containing non-translatable coat protein (cp) gene fragments of Potato virus y (pvy), Potato leafroll virus (plrv) and Potato virus X (pvx) with and without selectable marker gene was conducted using Agrobacterium-mediated transformation. Preliminary identifications of the transformants were done based on the expression of the reporter gene, Beta-Glucuronidase (gus) gene. gus activity test was conducted in the first (T0) generation while the plants were in test tubes and after transferring to a greenhouse for marker-free and marker-aided transformations, respectively. In the transformation with marker gene, three lines, which later on (in T1 generation) became virus resistant and pcr-positive for the insert, were generated from gus-negative mother plants. Although regeneration of plants from marker-free transformation was relatively easy, it was not possible to get transgenic plants using histochemical gus staining selection system.