RESEARCH ARTICLE Shoot tip cryotherapy for efficient eradication of grapevine leafroll-associated virus-3 from diseased grapevine in vitro plants Wen-Lu Bi 1† | Xin-Yi Hao 1† | Zhen-Hua Cui 2 | Ranjith Pathirana 3 | Gayle M. Volk 4 | Qiao-Chun Wang 1 1 State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, China 2 College of Horticulture, Qingdao Agriculture University, Qingdao, China 3 New Zealand Institute for Plant & Food Research Ltd., Palmerston North, New Zealand 4 USDA-ARS National Laboratory for Genetic Resources Preservation, Fort Collins, Colorado Correspondence Qiao-Chun Wang, State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China. Email: qiaochunwang@nwsuaf.edu.cn 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 pre-culture of 1.0-mm shoot tips containing five to six leaf primordia (LPs) for 3 days with a pre-culture medium containing 0.3 M sucrose, 0.16 mM glutathione and 0.14 mM ascor- bic acid, treatment of the pre-cultured 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 plant vitrification solu- tion 2 (PVS2), prior to freezing in liquid nitrogen of dehydrated shoot tips contained in 2.5-μL PVS2 droplets on aluminium foil strips. Virus localisation 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 iden- tify that only freezing in liquid nitrogen results in the death of all cells in areas of shoot tips har- bouring 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. KEYWORDS cryotherapy, grapevine leafroll disease, histological study, shoot tip, virus localisation, Vitis 1 | INTRODUCTION Grapevine (Vitis) is a highly valuable fruit crop that is grown through- out the world. Viral diseases have long been recognised as a major constraint for sustainability of grapevine production (Alabi et al., 2016; Maree et al., 2013; Naidu, Maree, & Burger, 2015; Naidu, Rowhani, Fuchs, Golino, & Martelli, 2014). Grapevine leafroll disease (GLD) is among the most serious viral diseases (Alabi et al., 2016; Atallah, Gomez, Fuchs, & Martinson, 2012; Maree et al., 2013; Naidu et al., 2014, 2015). Grapevine leafroll-associated virus-3 (GLRaV-3), a type member of the genus Ampelovirus, has been identified as the main aetiological agent inducing GLD (Maree et al., 2013; Naidu et al., 2014, 2015). GLD has been shown to decrease photosynthesis and carbohydrate metabo- lism (Atallah et al., 2012; Bertamini, Muthuchelian, & Nedunchezhian, 2004), delay fruit ripening (Atallah et al., 2012; Naidu et al., 2014, 2015), reduce berry weight and quality and affect wine quality (Akbas, Kunter, & Ilhan, 2009; Alabi et al., 2016; Atallah et al., 2012; Kovacs, Hanami, Fortenberry, & Kaps, 2001; Martínez et al., 2016; Naidu et al., 2014, 2015). GLRaV-3 can be transmitted by several species of mealybugs and scale insects in a semi-persistent manner, and by vegetative propaga- tion (Naidu et al., 2015). These transmission pathways allow GLRaV-3 to be easily transmitted from plant to plant, making Vitis species vul- nerable to the virus infection. A survey of 19 major grapevine-growing provinces in China showed that all samples tested were infected with † These two authors contributed equally to the present study. Received: 11 June 2018 Accepted: 27 August 2018 DOI: 10.1111/aab.12459 Ann Appl Biol. 2018;1–10. wileyonlinelibrary.com/journal/aab © 2018 Association of Applied Biologists 1