SYNERGISM IN REPLICATION OF CYMBIDIUM MOSAIC POTEXVIRUS (CYMMV) AND ODONTOGLOSSUM RINGSPOT TOBAMOVIRUS (ORSV) RNA IN ORCHID PROTOPLASTS

An in vitro orchid protoplast isolation method to study replication ki netics of CymMV and ORSV was developed. This method allows the isolati on of viable and raphid-free petal protoplasts from an orchid hybrid, Dendrobium Sonia (Dendrobium Caesar x Dendrobium Tomie Drake). The opt imum field strength for both viral RNA to achieve good efficiency of e lectroporation was 750 V/cm and the optimum viral RNA concentration re quired was 1 mu g and 4 mu g per 2 x 10(6) protoplasts for CymMV and O RSV, respectively. Autoradiographs of Northern blots depicting the vir al genomic and subgenomic RNA in the extracts, referred to as the ''Re plication Footprint Profiles'' (RFP) of specific CymMV/ORSV virus were prepared at different time intervals. Viral RNA synthesis reached a m axi mum at 18 h for CymMV and 24 h for ORSV. When CymMV and ORSV viral RNA were electroporated into the protoplasts simultaneously, detectio n signals of both the positive and negative strand viral RNA increased as compared to the singly infected protoplasts. Thus, synergism in re plication of CymMV and ORSV was observed in orchid protoplasts.