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 °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.

中文翻译：

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液滴玻璃化冷冻疗法根除“Marubakaido”苹果砧木中的苹果茎沟病毒和苹果茎坑病毒

苹果茎沟病毒 (ASGV) 和苹果茎凹坑病毒 (ASPV) 是感染苹果树的主要病毒种类之一，是造成大量损害和损失的原因。本研究的目的是评估液滴玻璃化冷冻疗法技术在根除 Malus prunifolia “Marubakaido”苹果砧木植物中的 ASGV 和 ASPV 中的有效性。从 ASGV 和 ASPV 感染的植物中切下腋芽尖端，并在预培养基上培养一天。随后，将它们在 22°C 下暴露于植物玻璃化溶液 2 (PVS2) 0、20、40、50、60 或 80 分钟，然后浸入液氮中 1 小时。PVS2 暴露时间分别为 20 和 40 分钟时，冷冻保存的芽尖具有最高的存活率（75% 和 65%）和再生率（53% 和 58%）。在温室中生长六个月后，RT-PCR 分析表明，所有测试的植物都不含 ASPV，70% 的植物不含 ASGV。ASGV和ASPV的高根除率表明，液滴玻璃化冷冻治疗技术是生产“Marubakaido”苹果砧木无ASGV和ASPV繁殖材料的有效工具。