Removal of leafroll viruses from infected grapevine plants by droplet vitrification

A robust method for removing all microorganisms from infected tissue is important for cultivar imports, germplasm maintenance and to produce healthy grafted material for the grapevine industry. In the droplet vitrification method of cryopreservation, plant tissue pre-treated with a vitrification solution is placed on aluminium foil in a droplet of vitrification solution and directly immersed in liquid nitrogen. Only highly cytoplasmic, non-vacuolar meristematic cells survive freezing. Therefore, cryopreservation can be considered a precise method of meristem culture and is developing into a new method for virus eradication in horticultural species called cryotherapy. To test the suitability of cryotherapy for virus eradication, we used 'Chardonnay' and 'Lakemont Seedless' infected with Grapevine leafroll associated virus-3 (GLRaV-3, an Ampelovirus), 'Pinot gris' and 'Sauvignon blanc 316' infected with Grapevine leafroll associated virus-2 (a Closterovirus), and another clone of 'Sauvignon blanc' infected with both Grapevine leafroll associated virus-1 (an Ampelovirus) and GLRaV-3. Plants regenerated after cryo-treatment tested negative (DAS-ELISA) for all three viruses, whereas untreated control plants tested positive. Droplet vitrification has the potential to be a novel and precise tool for virus eradication and establishment of high-health grapevine germplasm collections. In addition to removing virus infections, the method provides a cost-effective way of maintaining clonal plant material.