CRYOPRESERVATION Combined ribavirin treatment and cryotherapy for efficient Potato virus M and Potato virus S eradication in potato (Solanum tuberosum L.) in vitro shoots Svetlana Kushnarenko 1 & Natalya Romadanova 1 & Moldir Aralbayeva 1 & Saule Zholamanova 1 & Alena Alexandrova 2 & Oksana Karpova 2 Received: 14 February 2017 /Accepted: 25 June 2017 / Editor: Barbara Reed # The Society for In Vitro Biology 2017 Abstract Potato germplasm held as field collections accumu- late virus infections, making them unsuitable for distribution as planting stock. Elimination of these viruses from in vitro cultures provides healthy materials for germplasm distribu- tion. An in vitro collection of 33 potato cultivars and hybrids was evaluated for five viruses: Potato leafroll virus (PLRV), Potato virus M (PVM), Potato virus S (PVS), Potato virus X (PVX), and Potato virus Y (PVY) by ELISA and RT-PCR. PLRV was not detected in any accessions. Seven accessions were singly infected by PVM, 15 were mix-infected by PVM and PVS, and four by PVM and PVY. One accession had both PVS and PVX, and one was mix-infected by PVM, PVS, and PVY. Two accessions were singly infected by PVY, and three were virus-free. Cryotherapy, chemotherapy, and a combina- tion of both methods were tested on several potato accessions. For cryotherapy of shoot tips, the PVS2-vitrification protocol was used. Chemotherapy using prolonged culture with 100 mg L -1 ribavirin on PVM and PVS eradication was in- vestigated both alone and combined with cryotherapy. Cryotherapy alone eliminated single PVM infection in 38.6% of shoot cultures, but totally virus-free shoots were not found in mix-infected accessions. Treatment with ribavirin alone was only effective for eliminating both PVM and PVS after three subcultures on ribavirin, or ribavirin followed by cryotherapy. Three subcultures on ribavirin followed by cryo- therapy resulted in 100% virus-free potato shoots. Future studies will include cultivars infected with PVX and PVY. This is the first report of combined chemo- and cryotherapy for virus elimination in potato. Keywords Chemotherapy . Shoot tips . Cryopreservation . Virus eradication Introduction Plant germplasm held in field collections accumulate many insect-borne viruses that are detrimental to plant growth. These infections make the collections unsuitable for distribution or for planting stock (Waterworth and Hadidi 1998). Standard virus elimination techniques in- clude meristem culture, thermotherapy, and chemothera- py. Cryotherapy of in vitro-grown shoot tips is a promis- ing new technique for plant virus eradication. Cryotherapy employs cryopreservation of shoot tips in which small meristematic cells are successfully vitrified and continue to grow after rewarming, while large virus laden cells die (Wang and Valkonen 2008). Uneven dis- tribution of Raspberry bushy dwarf virus (RBDV) in rasp- berry (Rubus idaeus) shoot tips was clearly demonstrated by Wang et al. (2008) with immunohistochemistry meth- odology. Immunostaining shoot tip sections with antibod- ies to RBDV coat protein revealed the viruses in the basal part of meristem and all leaf primordia cells, and only a few cell layers in the apical dome remained unstained, which indicated that these meristematic cells were RBDV-free (Wang et al. 2008). These meristematic cells were usually not damaged by the cryopreservation procedure and had normal cytoplasm without plasmolysis signs, as shown by Kushnarenko et al. ( 2010 ) on apple ( Malus spp. ) shoot tips. The cells * Svetlana Kushnarenko svetlana_bio@mail.ru 1 Institute of Plant Biology and Biotechnology, Almaty, Republic of Kazakhstan 2 M. Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty, Republic of Kazakhstan In Vitro Cell.Dev.Biol.—Plant DOI 10.1007/s11627-017-9839-0