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Title: Cryotherapy by encapsulation-dehydration is effective for in vitro eradication of latent viruses from ‘Marubakaido’ apple rootstock

Author
item BETTONI, JEAN - University Of Santa Catarina
item COSTA, MURILO - Epagri
item SOUZA, JULIANA - University Of Santa Catarina
item NICKEL, OSMAR - Embrapa
item Volk, Gayle
item DA SILVA, FABIO - University Of Santa Catarina
item KRETZSCHMAR, AIKE - University Of Santa Catarina

Submitted to: Journal of Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/21/2018
Publication Date: 3/10/2018
Citation: Bettoni, J.C., Costa, M.D., Souza, J.A., Nickel, O., Volk, G.M., Da Silva, F.N., Kretzschmar, A.A. 2018. Cryotherapy by encapsulation-dehydration is effective for in vitro eradication of latent viruses from ‘Marubakaido’ apple rootstock. Journal of Biotechnology. 269:1-7.

Interpretive Summary: Viral pathogens decrease apple orchard productivity and must be removed from imported apple tree cultivars before they can be released from quarantine. Traditionally, viruses are eradicated from trees using meristem excision and thermotherapy procedures that are very difficult and time-consuming. This research tested the possibility of using cryotherapy, whereby plant pathogens are eliminated from plant tissues after liquid nitrogen exposure, to eradicate three viruses (Apple stem pitting virus, ASPV; Apple chlorotic leaf spot virus, ACLSV; Apple stem grooving virus, ASGV) from apple rootstock cultivar 'Marubakaido'. No ASPV and ACLSV were detected in plants grown for 8 months from shoot tips that were excised from infected plants, treated with cryoprotectants, and exposed to liquid nitrogen. Ten percent of the recovered plants were still infected with ASGV after infected shoot tips were excised, treated with cryoprotectants, and exposed to liquid nitrogen. Plants recovered from shoot tips infected with ACLSV, ASPV, and AGSV that were excised, treated with cryoprotectants, and regrown without liquid nitrogen exposure remained infected. This research suggests that cryotherapy methods may be effective for apple cultivar virus eradication.

Technical Abstract: Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) are several major viral pathogens of apple trees, responsible for substantial damage to the world's apple industry. This study aimed to evaluate the effectiveness of encapsulation-dehydration cryopreservation technique to eradicate these viral pathogens from in vitro shoot tips excised from ‘Marubakaido’ apple rootstock cultures. Axillary shoot tips were excised from in vitro cultures, encapsulated in alginate beads, precultured in MS salts, dehydrated in a laminar flow hood, immersed in liquid nitrogen, then warmed and recovered on medium. After LN exposure, in vitro rooting and acclimatization, recovered ‘Marubakaido’ plants exhibited 52% survival and 35% regrowth without callus formation. After 8 months of regrowth, PCR analyses revealed that all the plants were free of ACLSV and ASPV, but 2 out of 20 recovered plants were still infected with ASGV. This is the first report in Brazil of the application of cryotherapy to eradicate viral complexes in Malus. Cryotherapy can facilitate the production of virus-free plants by producing high quality plant material.