Application of thermotherapy combined with shoot tip culture and in vitro micrografting for virus elimination in olive tree (<i>Olea europaea</i> L.) cultivars

The objective of this work was to evaluate the effectiveness of thermotherapy alone or combined with either shoot tip culture or in vitro micrografting treatments and shoot tip culture alone in eliminating olive leaf yellowing-associated virus (OLYaV) and strawberry latent ringspot virus (SLRSV) from naturally infected olive (Olea europaea L.) cultivars to obtain virus-free stock plants. Reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the efficiency of virus elimination in the in vitro regenerated plants at six months following the acclimation to ex vitro conditions. All treatments proved effective in obtaining virus-free plants for all selected cultivars except for the heat therapy used alone which did not completely remove the virus from infected propagative material. The establishment of in vitro cultures of shoot tips from plants exposed to an alternating day/night temperature of 38/30°C or from vitroplants maintained at a constant temperature of 38°C resulted in OLYaV elimination rates of 67-100% and 60-100%, respectively. The combination of in vitro thermotherapy with shoot-tip grafting yielded up to 100% of OLYaV-negative plants, although grafting success rates were relatively low (29-33%). Instead, in vitro culture of shoot-tip after eight consecutive cycles of sub-culturing without heat therapy, led to the elimination of OLYaV/SLRSV from 17% of co-infected plants and OLYaV from 20% of single-infected plants. These results indicate that the combined application of treatments are effective methods for virus elimination in olive trees and could be used to ensure the production of healthy nursery stock plants.