Production of virus-free plants is necessary to control viral diseases, import novel cultivars from other countries, exchange breeding materials between countries or regions and preserve plant germplasm. In vitro techniques represent the most successful approaches for virus eradication. In vitro thermotherapy-based methods, including combining thermotherapy with shoot tip culture, chemotherapy, micrografting or shoot tip cryotherapy, have been successfully established for efficient eradication of various viruses from almost all of the most economically important crops. The present study reviewed recent advances in in vitro thermotherapy-based methods for virus eradication since the twenty-first century. Mechanisms as to why thermotherapy-based methods could efficiently eradicate viruses were discussed. Finally, future prospects were proposed to direct further studies.

Reddish-purple coloration on the leaf blades and downward rolling of leaf margins are typical symptoms of grapevine leafroll disease (GLD) in red-fruited grapevine cultivars. These typical symptoms are attributed to the expression of genes encoding enzymes for anthocyanins synthesis, and the accumulation of flavonoids in diseased leaves. Drought has been proven to accelerate development of GLD symptoms in virus-infected leaves of grapevine. However, it is not known how drought affects GLD expression nor how anthocyanin biosynthesis in virus-infected leaves is altered. The present study used HPLC to determine the types and levels of anthocyanins, and applied reverse transcription quantitative polymerase chain reaction (RT-qPCR) to analyze the expression of genes encoding enzymes for anthocyanin synthesis. Plantlets of Grapevine leafroll-associated virus 3 (GLRaV-3)-infected Vitis vinifera ‘Cabernet Sauvignon’ were grown in vitro under PEG-induced drought stress. HPLC found no anthocyanin-related peaks in the healthy plantlets with or without PEG-induced stress, while 11 peaks were detected in the infected plantlets with or without PEG-induced drought stress, but the peaks were significantly higher in infected drought-stressed plantlets. Increased accumulation of total anthocyanin compounds was related to the development of GLD symptoms in the infected plantlets under PEG stress. The highest level of up-regulated gene expression was found in GLRaV-3-infected leaves with PEG-induced drought stress. Analyses of variance and correlation of anthocyanin accumulation with related gene expression levels found that GLRaV-3-infection was the key factor in increased anthocyanin accumulation. This accumulation involved the up-regulation of two key genes, MYBA1 and UFGT, and their expression levels were further enhanced by drought stress.

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 ascorbic 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 solution 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 identify that only freezing in liquid nitrogen results in the death of all cells in areas of shoot tips harbouring 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.

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