Synergism in replication of cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) RNA in orchid protoplasts

Abstract

An in vitro orchid protoplast isolation method to study replication kinetics of CymMV and ORSV was developed. This method allows the isolation of viable and raphid-free petal protoplasts from an orchid hybrid, Dendrobium Sonia (Dendrobium Caesar x Dendrobium Tomie Drake). The optimum field strength for both viral RNA to achieve good efficiency of electroporation was 750 V/cm and the optimum viral RNA concentration required was 1 μg and 4 μg per 2 x 106 protoplasts for CymMV and ORSV, respectively. Autoradiographs of Northern blots depicting the viral genomic and subgenomic RNA in the extracts, referred to as the 'Replication Footprint Profiles' (RFP) of specific CymMV/ORSV virus were prepared at different time intervals. Viral RNA synthesis reached a maximum at 18 h for CymMV and 24 h for ORSV. When CymMV and ORSV viral RNA were electroporated into the protoplasts simultaneously, detection signals of both the positive and negative strand viral RNA increased as compared to the singly infected protoplasts. Thus, synergism in replication of CymMV and ORSV was observed in orchid protoplasts.