Production of Tobacco Plant Resistant to PVX by RNA Silencing Mechanism

One of the most pathogenic viruses in Solanaceous plants is Potato X virus (PVX) which causes systemic mosaic spots and economic loss in Nicotiana tabacum. Use of the tobacco resistance varieties is the best method in management of this disease. In this study, to establish the tobacco plants resistant to PVX, an RNAi construct was designed for a 400 bp fragment of PVX’s P25 gene which is a suppressor of RNA Silencing in plant. The P25 fragment was amplified with specific primers by PCR and was cloned in two steps (in sense and antisense directions) into the pFGC5941 plasmid. Then, the RNAi construct was transformed from recombinant plasmid into the tobacco explants (Samsun) by means of Agrobacterium cells. The transgenic plants were identified by selection on Basta containing media and propagated, followed by molecular and phenotypic analyses. PCR test using plant’s DNA confirmed the presence of the transgene in transgenic plants. The next generation of the transgenic plants was inoculated with PVX in greenhouse. ELISA using anti-PVX-CP antibody showed that three transgenic lines are resistant to PVX. Molecular confirmation of three tobacco resistant lines was performed by RT-PCR using plant RNA and CP-specific primers and PVX did not detected in transgenic plants. This study resulted in production of three tobacco lines resistant to PVX. Study of their resistance in the field condition is in progress.