GENETIC ENGINEERING OF POTATO FOR BROAD SPECTRUM RESISTANCE AGAINST RNA VIRUSES

Abstract

Pathogens and pests always have been and still is a potential threat to agricultural production worldwide. Potato (Solanum tuberosum L.) is nutritionally a balanced staple food; therefore, it has potential to minimize the pressure on cereal crops in Pakistan. Viral diseases are major problem in stable crop production, especially in vegetative propagated plants such as potato where diseases are easily transmitted from one clonal generation to the next. Conventional methods of virus control are limited to use of virus-free seed tubers and chemical control of insect vectors. However, development of resistant cultivar is the only effective, economical and environmentally safe method of disease control. The use of RNA silencing has become the tool of choice for gene silencing in many crop species. The key element of this technology is the discovery of double-stranded RNA (dsRNA), diced into small interfering RNAs (siRNA), which is a potent trigger for RNA silencing. By arranging transgenes as inverted repeats encoding self-complementary hairpin RNA (hpRNA), which is diced into siRNA after transcription, it is possible to obtain strong silencing of expression of homologous RNA. Using this technology efficient simultaneous knock-down of multiple genes of three different viruses have been achieved by using a single RNAi construct in potato. In this study, the transgenic resistance in potato was obtained based on the construction of hairpin RNA plant expression cassettes I and II containing the sequences of different genes of three important potato viruses. The cassette I containing the short sequences of Nucleocide Triphosphate binding helicase (NTP) gene of Potato Virus X (PVX), Helper Component Protease (HC-Pro) gene of Potato Virus Y (PVY) and Coat Protein (CP) gene of Potato Leaf Roll Virus (PLRV), while the expression cassette II contains the sequences of CP genes of PVX, PVY and PLRV. The sense and anti-sense fragments of these genes were fused separately to form a chimeric N gene and arranged in an RNAi vector as inverted repeats, under the CaMV 35S promoter, separated by intron. These expression cassettes were transformed separately in potato cv Kuroda and Desiree through Agrobacterium mediated transformation by using Agrobacterium tumefacience strain LBA 4404. Fourteen independent transgenic lines of each cassette were developed and transferred to containment after analysis of T0 transgenic plants by PCR and Southern hybridization for the presence of transgenes. The transgenic expression of these cassettes showed that up to 20 % of the transformed plant lines were resistant and 46 % were tolerant to all three viruses. The analysis of the resistant plants showed accumulation of siRNA as compared to susceptible transgenic and non-transformed control plants. This indicates that the resistance is due to simultaneous RNA silencing of the three target genes in each construct. Overall, the work presented here demonstrates a simple procedure to obtain broad spectrum virus resistance in two commercial potato cultivars Kuroda and Desiree by RNA silencing technology. At present, another independent study is being conducted to multiply and evaluate the field performance of putative transgenic potato lines after obtaining approval of National Biosafety Committee (NBC) of Environment Protection Agency (EPA), Government of Pakistan. In future, studies to improve frequency of developing multiple virus resistant plants could be attempted by extending the transgenes construct with a large number of smaller fragments of target genes. Moreover, it is possible that present strategy can be extended to other plant species to obtain broad spectrum resistance against many other devastating