Establishment of a TRV-based virus-induced gene silencing system using the vacuum infiltration in Tartary buckwheat

A TRV-based and bud-vacuum-infiltration-mediated VIGS system achieves an effective gene silencing across multiple varieties under defined optimal conditions in Tartary buckwheat (TB). TB is a valuable grain rich in bioactive flavonoids, but the absence of a genetic transformation system has hindered in vivo functional verification of its genes. Virus-Induced Gene Silencing (VIGS) offers a rapid and efficient alternative for gene studies. This study established and optimized a VIGS system for TB using the phytoene desaturase gene (FtPDS) as a reporter, whose silencing causes photobleaching (albinism). Bioinformatic analysis revealed that FtPDS has a 6,127 bp genomic sequence and a 1,713 bp cDNA, having high similarity to rice phytoene desaturase (OsPDS). We compared two infiltration methods for VIGS delivery: leaf injection into TB cotyledons versus bud vacuum infiltration of germinated seeds, and found the bud vacuum method was more effective, yielding a higher albinism rate (28.9% vs. 12.3%). Using response surface methodology, we determined optimal conditions including germination length at 1.5 cm, the Agrobacterium OD₆₀₀ at around 1.0, the infiltration duration for 5 min and post-infiltration cultivation temperature at 20 °C, achieving a silencing efficiency ranging from 27% to 62.05%. The applicability of this system was validated by silencing FtUFGT163 and FtMYB5, key genes in rutin biosynthesis. Silencing FtUFGT163 reduced its expression by 81.7%-89.74% and decreased rutin content by 22.22%-36.18%, while FtMYB5 silencing lowered its expression by 76.77%-85.64% and reduced rutin content by 22.19%-33.89%. Additionally, FtMYB5 silencing downregulated several genes in the flavonol synthesis pathway, including FtC4H, FtCHS, FtCHI, FtF3H, FtPAL, Ft4CL, FtF3'H, and FtFLS, confirming its regulatory role. Overall, this efficient and stable VIGS system provides a powerful platform for functional genomic studies in TB.