Combined ribavirin treatment and cryotherapy for efficient Potato virus M and Potato virus S eradication in potato (Solanum tuberosum L.) in vitro shoots

Laboratory of Germplasm Cryopreservation group at the Institute of Plant Biology and Biotechnology. From left to right: Saule Zholamanova (Master’s student), Dr. Svetlana Kushnarenko (Associated Professor, Head of the Laboratory) and Dr. Natalya Romadanova.

Alena Alexandrova (PhD-student), M. Aitkhozhin Institute of Molecular Biology and Biochemistry

Dr. Oksana Karpova, M. Aitkhozhin Institute of Molecular Biology and Biochemistry

Moldir Aralbayeva (B. Sc.), is now on leave to care for a child

Plant germplasm held in field collections accumulate many insect-borne viruses, making them unsuitable for distribution or for planting stock. Elimination of these viruses from in vitro cultures provides healthy materials for germplasm distribution. Standard virus elimination techniques include meristem culture, thermotherapy, and chemotherapy. Cryotherapy of in vitro-grown shoot tips is a promising new technique for plant virus eradication (Wang and Valkonen 2008). In this paper cryotherapy, chemotherapy and a combination of both methods were tested on several potato cultivars. An in vitro collection of 33 potato cultivars and hybrids were 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. 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 investigated 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 cryotherapy resulted in 100% virus-free potato shoots. This study of potato accessions demonstrates that cryotherapy in combination with chemotherapy is a promising approach for plant virus eradication.

Svetlana Kushnarenko, Natalya Romadanova, Moldir Aralbayeva, Saule Zholamanova, Alena Alexandrova, Oksana Karpova. Combined ribavirin treatment and cryotherapy for efficient Potato virus M and Potato virus S eradication in potato (Solanum tuberosum L.) in vitro shoots. In Vitro Cellular & Developmental Biology-Plant, 53:435-432, 2017.

Advances in Understanding the Fundamental Aspects Required for Successful Cryopreservation of Australian Flora

(left to right) Dr Bryn Funnekotter and Dr Eric Bunn (Kings Park and Botanic Garden)

Prof. Ricardo Mancera (Biomedical Science, Curtin University)

Australia is host to an amazing diversity of plant species, many of which require conservation efforts. Cryopreservation provides the greatest long-term storage option as a conservation tool for storing a range of plant germplasm; such as seeds, seed axes, protocorms, shoot tips and callus from tissue culture as well as somatic embryos. However, while cryopreservation has proven capable of delivering viable long-term storage with some plant taxa, the process of deriving protocols is still largely an incremental process. Only a better understanding of the fundamental aspects relating to cryopreservation will lead to improved overall success. This review focuses on some of the recent work investigating these fundamental aspects required for successful cryo-storage, including issues with plant physiology (such as genetic stability, the composition of the proteome and metabolome, cell membrane characteristics and antioxidant defences) and how the stresses imposed by cryopreservation (such as the excision damage, desiccation, CPA toxicity, ice crystal damage and cooling to cryogenic temperatures) interact, all of which may contribute to the cryo-capability of a species. This information can then be used for a more targeted approach, reducing time and resources currently spent on developing successful cryopreservation protocols.

Bryn Funnekotter, Ricardo L. Mancera & Eric Bunn. Advances in understanding the fundamental aspects required for successful cryopreservation of Australian flora. In Vitro Cellular & Developmental Biology-Plant. 53: 289-298, 2017.