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 microgram and 4 micrograms per 2 x 10(6) 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.

Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids

“…Nevertheless, callus induction is time consuming and impractical for protoplast isolation. Hence, flower petals have been selected as an alternative for protoplast isolation for ornamental plants such as Rosa rugose [32], Dendrobium [33] and Phalaenopsis [34]. In addition to species types and source materials, enzyme combination and the concentration of osmotic pressure regulating substance D-mannitol also affect the effective release of protoplasts [22,35].…”

Differential Roles of AC2 and AC4 of Cassava Geminiviruses in Mediating Synergism and Suppression of Posttranscriptional Gene Silencing

“…In PVY and PVX synergism, HC-Pro of PVY, the determinant of the synergistic effect, was found to be a suppressor of gene silencing, permitting viruses to accumulate beyond the normal host-mediated limits (1,35,49). Synergism at the level of replication between cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus has been studied using protoplasts as tools (20). In this paper, we report that the severe cassava mosaic disease associated with synergism between the Cameroon strain of ACMV (ACMV- [CM]) and East African cassava mosaic Cameroon virus (EACMCV) is a two-way process involving the DNA-A components of both viruses in a double infection.…”

An efficient protocol for plant regeneration from protoplasts of the moss Atrichum undulatum P. Beauv in vitro

“…It entails brief, high intensity pulse to create transient pores in the cell membrane to facilitate the entry of exogenous molecules like DNA (Kisaka et al, 1998;Chadha et al, 2000;John, 2003), RNA (Hu et al, 1998) etc. To our knowledge, there were no reports on the transformation of protoplasts via electroporation in mosses.…”