Developing a homology-driven gene silencing system in <i>Iris japonica</i>: a versatile tool for comparative functional studies

<italic>Iris japonica</italic>, an evergreen ornamental species known for its beautiful flowers and year-round evergreen foliage, is difficult to genetically manipulate due to the lack of an efficient genetic transformation system. This limitation hinders the functional verification of key genes in <italic>I. japonica</italic>. To address this, a Virus-Induced Gene Silencing (VIGS) system based on Tobacco rattle virus (TRV) was developed, enabling efficient, transient gene silencing in <italic>I. japonica.</italic> In this system, the <italic>phytoene desaturase</italic> (<italic>PDS</italic>) gene, a common reporter gene used for VIGS, was selected to assess the silencing efficiency. The recombinant vector pTRV2-<italic>IjPDS</italic> was used to infect <italic>I. japonica</italic> plants. The <italic>PDS</italic> gene silencing resulted in distinct photobleaching symptoms, demonstrating the successful silencing of the target gene. The presence of the TRV vector was verified by using a GFP-tagged pTRV2-GFP vector, and GFP expression was detected using fluorescence visualization under ultraviolet (UV) light and confirmed by laser confocal microscopy. Real-time PCR was used to quantify the reduction in <italic>IjPDS</italic> gene expression, confirming the successful silencing. The study further optimized the VIGS system by evaluating the impact of seedling ages on silencing efficiency, identifying one-year-old seedlings as the most effective for gene silencing (36.67%). This TRV-based VIGS system provides a robust tool for functional gene analysis in <italic>I. japonica</italic> and offers a new approach to studying the rules of key genes in its biological processes, with potential applications in ornamental plant breeding and genomics research.