A rapid and highly efficient tobacco ringspot virus (TRSV)-induced gene silencing system based on vacuum infiltration and tenoxicam co-cultivation in melon

1. A rapid and high-efficiency TRSV-mediated VIGS method for melon was developed using vacuum infiltration of germinated seeds. 2. This method achieved 95.2% silencing efficiency with 80% transformation frequency, completing the entire process from seed treatment to observable phenotype within just 11 days. 3. Tenoxicam (TNX) supplementation during co-culture significantly improved transformation efficiency by suppressing immune gene expression. Melon is a globally important cucurbit crop, but its functional genomics are hindered by inefficient genetic transformation. Virus-induced gene silencing (VIGS) enables rapid gene analysis and high-throughput screening. In this study, we evaluated the silencing efficiency of three viral vectors delivered via vacuum infiltration and cotyledon injection. We developed an optimized tobacco ringspot virus (TRSV)-mediated VIGS system using vacuum infiltration, which exhibited remarkable silencing efficiency and accelerated phenotypic manifestation in melon. The reporter gene CmPDS (phytoene desaturase) was effectively silenced, resulting in complete photobleaching across the entire leaf surface. This method achieved 95.2% silencing efficiency with 80% transformation frequency, completing the entire process from seed treatment to observable phenotype within just 11 days. Supplementing with tenoxicam (TNX, oxicam-type nonsteroidal anti-inflammatory drugs NSAIDs) during co-culture significantly enhanced transformation frequency to 93.3% across diverse genotypes. qRT-PCR showed TNX may boost transformation by attenuating plant immunity. To validate the system's broad applicability, we silenced the Mg-chelatase H subunit ( CmChlH ) gene, resulting in the expected yellow-leaf phenotype. The VIGS system developed herein provides a powerful tool for investigating gene function during early melon development. Also, this work establishes a foundational framework for VIGS system construction and accelerates genetic research in other cucurbit species.