Shoot tip cryotherapy for efficient eradication of grapevine leafroll-associated virus-3 from diseased grapevine in vitro plants

Authors: BI, WEN-LU - Northwest Agricultural & Forestry University; HAO, XIN-YI - Northwest Agricultural & Forestry University; CUI, ZHEN-HUA - Agricultural University Of China; PATHIRANA, RANJITH - Plant And Food Research; Volk, Gayle; WANG, QIAO-CHUN - Northwest Agricultural & Forestry University

Submitted to: Annals of Applied Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/27/2018
Publication Date: 10/10/2018
Citation: Bi, W., Hao, X., Cui, Z., Pathirana, R., Volk, G.M., Wang, Q. 2018. Shoot tip cryotherapy for efficient eradication of grapevine leafroll-associated virus-3 from diseased grapevine in vitro plants. Annals of Applied Biology. 173(3):261-270. https://doi.org/10.1111/aab.12459.
DOI: https://doi.org/10.1111/aab.12459

Interpretive Summary: Grapevine leafroll virus is a serious grapevine disease both in the U.S and internationally. It is transmitted vegetatively during the propagation process. This research uses grape (Vitis) cryopreservation technologies to eradicate the disease from shoot tips as a result of cryoexposure. Shoot tips from infected plants were precultured with sucrose and antioxidants, treated with the cryoprotectant PVS2, and then exposed to liquid nitrogen. They were then recovered and assessed for the presence of the virus. The virus was not present in the apical dome or in young leaf primordia of shoot tips. The virus was present in more mature tissues that did not survive the liquid nitrogen exposure. Regrowth levels were 43 to 59% and all plants recovered after cryotherapy were free of grapevine leafroll virus. Thus, cryopreservation methods could be used to clean-up Vitis plants that are infected with grapevine leafroll virus.

Technical Abstract: We describe a droplet-vitrification cryotherapy method for the eradication of Grapevine leafroll-associated virus-3 (GLRaV-3) from diseased in vitro shoots of Vitis plants. The procedure involved preculture of 1.0 mm-shoot tips containing 5-6 leaf primordia (LPs) for 3 days with a preculture medium containing 0.3 M sucrose, 0.16 mM glutathione and 0.14 mM ascorbic acid, treatment of the precultured shoot tips for 20 min at room temperature with a loading solution composed of 2 M glycerol and 0.4 M sucrose, and exposure to PVS2, prior to freezing in liquid nitrogen of dehydrated shoot tips contained in 2.5-µL PVS2 droplets on aluminum foil strips. Virus localization showed GLRaV-3 was not present in apical dome (AD) and LPs 1-4, but it was detected in the basal shoot tip region, approximately 0.5 mm from the AD, as well as in LP 5 and more mature tissues. Histological observations identify that only freezing in liquid nitrogen results in the death of all cells in areas of shoot tips harboring virus, whereas PVS2 treatment does not. Thus, freezing in liquid nitrogen is a necessary step that eradicates GLRaV-3. This cryotherapy procedure produced shoot regrowth levels that ranged from 43% to 59%, and all plants recovered after cryotherapy were free of GLRaV-3 in two wine, one table, and one rootstock cultivars. Thus, this procedure can be considered to be efficient and wildly applicable for eradication of GLRaV-3 from Vitis spp.