Droplet-vitrification cryotherapy for eradication of apple stem grooving virus and apple stem pitting virus from "Marubakaido" apple rootstock

The apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) are among the major viral species that infect apple trees and are responsible for substantial damage and loss. The aim of this study was to assess the effectiveness of droplet-vitrification cryotherapy technique in eradication of ASGV and ASPV in Malus prunifolia "Marubakaido" apple rootstock plants. The axillary shoot tips were excised from ASGV- and ASPV-infected plants and incubated for one day on preculture medium. Subsequently, they were exposed to plant vitrification solution 2 (PVS2) for 0, 20, 40, 50, 60 or 80 min at 22 degrees C and immersed in liquid nitrogen for 1 h. Cryopreserved shoot tips had the highest survival (75 and 65%) and regrowth (53 and 58%) percentages with PVS2 exposure duration of 20 and 40 min, respectively. After six months of growth in a greenhouse, RT-PCR analyses revealed that all the tested plants were free of ASPV and 70% were free of ASGV. The high eradication percentage of ASGV and ASPV indicated that the droplet-vitrification cryotherapy technique is an effective tool to produce ASGV- and ASPV-free propagating material of "Marubakaido" apple rootstock.