Eradication of latent viruses from apple cultivar ‘Monalisa’ shoot tips using droplet-vitrification cryotherapy

Authors: BETTONI, JEAN CARLOS - University Of Santa Catarina; SOUZA, JULIANA - University Of Santa Catarina; Volk, Gayle; COSTA, MURILO DALLA - Epagri; NASCIMENTO DA SILVA, FABIO - University Of Santa Catarina; KRETZSCHMAR, AIKE ANNELIESE - University Of Santa Catarina

Submitted to: Scientia Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/11/2019
Publication Date: 2/28/2019
Citation: Bettoni, J., Souza, J.A., Volk, G.M., Costa, M., Nascimento Da Silva, F., Kretzschmar, A. 2019. Eradication of latent viruses from apple cultivar ‘Monalisa’ shoot tips using droplet-vitrification cryotherapy. Scientia Horticulturae. 250:12-18. https://doi.org/10.1016/j.scienta.2019.02.033.
DOI: https://doi.org/10.1016/j.scienta.2019.02.033

Interpretive Summary: Plant viruses significantly impact crop production and limit the movement of plant materials across international borders. Cryotherapy is an application whereby plant viruses are eradicated from 1 millimeter plant shoot tips after liquid nitrogen exposure. This study determined whether apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) could be eliminated from the apple cultivar 'Monalisa'. Shoot tips excised from infected in vitro plants were treated with cryoprotectants, exposed to liquid nitrogen, and rewarmed. They were then grown into plantlets in tissue culture and transferred to the greenhouse. After 6 months of growth in a greenhouse, all of the plants regenerated from shoot tips that were exposed to liquid nitrogen were free of ASPV, 95% were free of ACLSV, and 35% were free of ASGV. This promising cryotherapy procedure may facilitate the production of virus-free plants.

Technical Abstract: In apple, virally transmitted diseases cause economic losses as a result of their direct effect on production and yield and by causing an increased susceptibility to other phytopathogenic agents. This study evaluated the effectiveness of a droplet-vitrification cryopreservation technique in eradicating latent viruses: Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) from in vitro axillary shoot tips excised from ‘SC417 Monalisa’ apple cultivar shoot tips. In vitro stock cultures, four weeks after subculture, infected with ASGV, ASPV and ACLSV were used as source material. Axillary shoot tips were excised from stock cultures, precultured in MS+2M glycerol + 0.8M sucrose, and then exposed to plant vitrification solution 2 (PVS2) for between 0 and 80 min (six treatments) at 0 °C or room temperature prior to liquid nitrogen (LN) exposure. Shoot tips were then warmed and recovered on growth medium. The survival and regrowth of cryopreserved (+LN) and non-cryopreserved (-LN) 'Monalisa' shoot tips were evaluated and the efficiency of virus eradication was determined using RT-PCR after recovered plants were grown in a greenhouse for 6 months. With this protocol, the highest degree of regrowth (45%) were obtained after 20 to 40 min PVS2 exposure at room temperature. After 6 months of growth in a greenhouse, all of the plants regenerated after cryopreservation were free of ASPV, 95% were free of ACLSV, and 35% were free of ASGV. This promising cryotherapy procedure may facilitate the production of virus-free plants.