Eradication of viruses in microplants of three cultivated potato species (Solanum tuberosum L., S. phureja Juz. & Buk., S. stenotomum Juz. & Buk.) using combined thermo-chemotherapy method

A modified method of combined chemo-thermotherapy for eradication the most damaging viruses in potato plants is proposed. The materials for study included 91 clones of 85 accessions of three South-American cultivated species Solanum phureja (18 accessions), S. stenotomum (26 accessions), S. tuberosum ( S. tuberosum ssp. andigenum and S. tuberosum ssp. tuberosum ) (41 accessions) from the VIR field collection (N.I. Vavilov All-Russian Institute of Plant Genetic Resources). The results of ELISA for 91 clones from the field collection and RT-PCR analysis of the corresponding microplants, when introducing clones into in vitro culture, have shown that all material was infected by viruses (potato viruses Y, X, S, M and potato leafroll virus - PVY, PVX, PVS, PVM and PLRV) to varying degrees. Single infections were detected in 25.3 % of the 91 clones, and the remaining clones were multi-infected and contained several viruses in various combinations. Based on the termo- and chemotherapy protocols as well as complex therapy used in leading potato genebanks (Leibniz Institute of Plant Genetics and Crop Plant Research - IPK, Germany; International Potato Centre - CIP, Peru), we have developed a modified method of combined therapy comprising three successive cycles of in vitro plant cultivation on Murashige and Skoog basal medium (MS) supplemented with ribavirin (30 mg/l) at 37 °С during 4 weeks. Between the cycles, the microplants were incubated for 2 days at 26 °С on the same medium with ribavirin. Each clone was tested for the presence of viruses by RT (revers transcription) PCR before and after the combined thermo-chemotherapy. In RT-PCR, 42 clones (46 %) were completely free from tested viruses, 44 % were free from some viruses, and 10 % of the clones were unable to improve. On the whole of 91 clones, PLRV was eliminated from 72.7 %, PVY from 71.4 %, PVM from 63.9 % and PVS from 57.4 % of microplants. Differences in elimination frequency between the viruses were not significant (р > 0.05). Also, there were no significant differences in elimination of different viruses between the accessions of different cultivated species. The difference of this modification from IPK protocols (Germany) lays in simultaneous effect of high temperature (37 °C) and ribavirin (instead of sequential application), and, unlike the CIP (Peru) scheme, it comprises less steps.