The coat protein (CP) of certain plant viruses may reassemble into empty virus-like particles (VLPs) and these protein cages may serve as potential drug delivery platforms. In this paper, the production of novel VLPs from the Hibiscus chlorotic ringspot virus (HCRSV) is reported and the capacity to load foreign materials was characterized. VLPs were readily produced by destabilizing the HCRSV in 8 M urea or Tris buffer pH 8, in the absence of calcium ions, followed by removal of viral RNA by ultrahigh-speed centrifugation and the reassembly of the CP in sodium acetate buffer pH 5. The loading of foreign materials into the VLPs was dependent on electrostatic interactions. Anionic polyacids, such as polystyrenesulfonic acid and polyacrylic acid, were successfully loaded but neutrally charged dextran molecules were not. The molecular-mass threshold for the polyacid cargo was about 13 kDa, due to the poor retention of smaller molecules, which readily diffused through the holes between the S domains present on the surface of the VLPs. These holes precluded the entry of large molecules, but allowed smaller molecules to enter or exit. The polyacid-loaded VLPs had comparable size, morphology and surface-charge density to the native HCRSV, and the amount of polyacids loaded was comparable to the weight of the native genomic materials. The conditions applied to disassembly–reassembly of the virions did not change the structural conformation of the CP. HCRSV-derived VLPs may provide a promising nano-sized protein cage for delivery of anionic drug molecules.

Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for Cymbidium orchids. Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. It requires optimal D-mannitol concentration (0.5 M), enzyme concentration (1.2 % (w/v) cellulose and 0.6 % (w/v) macerozyme) and digestion time (6 h). With this protocol, the highest yield (3.50 × 10⁷/g fresh weight of orchid tissue) and viability (94.21%) of protoplasts were obtained from flower petals of Cymbidium. In addition, we achieved high transfection efficiency (80%) through the optimization of factors affecting polyethylene glycol (PEG)-mediated protoplast transfection including incubation time, final PEG4000 concentration and plasmid DNA amount. This highly efficient protoplast-based transient expression system (PTES) was further used for protein subcellular localization, bimolecular fluorescence complementation (BiFC) assay and gene regulation studies of flowering related genes in Cymbidium orchids. Taken together, our protoplast isolation and transfection protocol is highly efficient, stable and time-saving. It can be used for gene function and molecular analyses in orchids and other economically important monocot crops.

Transgenic plants expressing artificial microRNAs (amiRNAs) have been shown to confer specific resistance to corresponding viruses. Here, we generated Nicotiana benthamiana transgenic lines containing Oryza sativa miR528 as backbone, expressing amiRNAs targeting RNA-dependent RNA polymerase (RdRp) gene of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). The amiRNA transgenic lines could express amiR-CymMV and confer high percentage resistance to CymMV, while lack of detectable level of amiR-ORSV expression in amiR-ORSV transgenic N. benthamiana plants led to weak resistance to ORSV infection. In this project, we provide the first report of CymMV-resistant transgenic N. benthamiana plants based on amiRNA strategy. We believe that this amiRNA approach can be extended to generate CymMV-resistant transgenic orchids.