Development of virus-induced gene silencing and Agrobacterium-mediated transient expression systems for gene function analysis in Chinese Cymbidium

Chinese orchids (Cymbidium spp.) are high-value ornamental plants, renowned for their elegant flowers, leaf variegation, and distinctive fragrance. However, their low transformation efficiency and prolonged regeneration periods have hindered the functional characterization of key genes in Cymbidium. To address these challenges, we developed a Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) system and a highly efficient Agrobacterium-mediated transient expression system using the Ruby reporter. These systems facilitate efficient transient gene silencing and overexpression in Cymbidium, enabling functional characterization of target genes. Phytoene desaturase (PDS), a visual marker commonly used for VIGS, and the Ruby reporter were selected to validate the system's efficiency. Infiltration with pTRV2-GFP confirmed that TRV can establish systemic infection and movement within Cymbidium plants. Furthermore, inoculation with pTRV2-CsPDS induced distinct photobleaching in leaves, and quantitative real-time polymerase chain reaction analysis confirmed the successful silencing of the endogenous PDS gene. We further optimized the VIGS conditions and identified the optimal parameters as an optical density at 600 nm (OD600) of 1.0 under a negative pressure of 0.06 MPa, followed by dark incubation at 25 °C for 24 h, which yielded the highest infiltration efficiency and plant survival rate. Using the Ruby reporter system, we also demonstrated efficient transient overexpression in Cymbidium petals and fleshy rhizomes. Additionally, we established that the optimal infiltration conditions for rhizomes were an Agrobacterium density of OD600 = 0.6 and a 10-min infiltration period. Collectively, the TRV-based VIGS system and the Ruby-based transient expression system offer reliable tools for comparative functional studies, enabling the dissection of molecular mechanisms that underlie key biological processes in Cymbidium.