Meristem Tip Culture for In Vitro Eradication of Grapevine Leaf Roll-associated Virus-1 (GLRaV-1) and Grapevine Fan Leaf Virus (GFLV) from Infected Flame Seedless Grapevine Plantlets

Grapevine (Vitis vinifera cv. Flame Seedless) was found infected with viral diseases; showing thicker leaves than normal, brittle, with margins rolled downwards and yellowish, which were identified as Grapevine Leaf roll-associated Virus-1 (GLRaV-1). Other symptoms were observed on leaves. The infected leaves showed malformation with abnormal gather primary veins, giving the leaf the appearance of an open fan, including yellowing and mosaic pattern on leaves, or bright yellow bands along major veins. Fan-shaped leaves were associated with mosaic or vein banding symptoms, these observations were identified as Grapevine Fan leaf Virus (GFLV). Both viruses were detected using Double Antibody Sandwich – Enzyme Linked Immuno Sorbent Assay (DAS-ELISA) and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The use of tissue culture was investigated as a mean to eliminate the two viruses. Virus-free plants were produced within six months using meristem tip culture. Woody plant (WP) medium supplied with benzyl amino purine BAP (4.44 µM) was used for shoot proliferation, and indole butyric acid IBA (0.2 µM) for plantlets rooting. Before acclimatization, the plantlets were submitted to DAS-ELISA and RT-PCR in order to evaluate virus eradication. GLRaV-1 and GFLV free plants (95 and 90 %, respectively) were obtained from the optimum size (0.5 mm) of meristem tips (as indexed by DAS-ELISA). Of these, 82.5 and 75 % plants were found negative for GLRaV-1 and GFLV, respectively, as diagnosed by RT-PCR. RT-PCR and meristem tip culture were found to be reliable methods for virus indexing and elimination of GLRaV-1 and GFLV