Elimination of Grapevine fanleaf virus (GFLV) and Grapevine leaf roll-associated virus-1 (GLRaV-1) from Infected Grapevine Plants Using Meristem Tip Culture

摘要

In this study, we reported the detection of GLRaV-1 and GFLV in grapevine (Vitis vinifera L. cv. Thompson seedless) by DAS-ELISA and RT-PCR and in vitro production of virus-tested grapevine by meristem tip culture. Grapevine (Vitis vinifera L. cv. Thompson seedless) was found to be infected with viral diseases showing thicker leaves than normal, brittle, with margins rolled downwards and yellowish, which identified as Grapevine leaf roll-associated virus-1 GLRaV-1). Other symptoms observed on infected leaves were, malformation with abnormal gathered primary veins, giving the leaf the appearance of an open fan, including yellowing and mosaic pattern or bright yellow bands along major veins. Fan-shaped leaves may or may not be present with mosaic or vein banding symptoms which identified as Grapevine fanleaf virus (GFLV). Detection of the viruses was carried out by Double Antibody Sandwich-Enzyme-Linked Immunosorbent Assay (DAS-ELISA) and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Theuse 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 medium supplied with Benzylamino Purine (BAP) (1.5 mg L~(-1)) for shoot proliferationand Indol Butyric Acid (IBA) (0.05 mg L~(-1)) for plants rooting. Before acclimatization, the plantlets were submitted to DAS-ELISA and RT-PCR in order to evaluate virus eradication. GFLV-and GLRaV-1-free plants (92.5 and 95%, respectively) were obtained from the optimum size (1 mm) of meristem tips (as indexed by DAS-ELISA). Of these, 85 and 87.5% plants were found negative from GFLV and GLRaV-1, respectively, as indexed by RT-PCR. Virus indexing by RT-PCR was found to be a reliable method, thus proving the efficiency of this method (meristem tip culture) for GLRaV-1 and GFLV elimination.