Generation of double-virus-resistant marker-free transgenic potato plants

Viruses are very widespread in nature, and they cause severe diseases and yield losses in potato production. The transfer of the dsRNA-producing gene could confer a high level virus resistance by specific targeting of cognate viral RNA. In this study, we constructed a marker-free expression vector of a chimeric gene derived from the coat protein sequence of Potato virus X (PVX) and the nuclear inclusion protein sequence of Potato virus Y (PVY) in the form of an intramolecular dsRNA. Then this chimeric gene was introduced into potato cv. Zihuabai, a popular variety in China, via Agrobacterium tumefaciens-mediated transformation. Marker gene-free transgenic plants resistant to both PVX and PVY were obtained and confirmed by RT-PCR and DAS-ELISA detection. Northern blot analysis showed that transgene-derived mRNA was cleaved into short interfering RNAs (siRNAs), and that the virus resistance was mediated by RNA silencing. One important aspect of the study is that the transgenic viral sequence is not translated and the actual RNA transcript is cleaved, which possibly limit the environmental risks, such as transcapsidation and recombination of the transgene with an incoming virus. In addition, the biosafety risk resulting from marker genes can be avoided because of the absence of marker genes in transgenic plants.