Establishment and optimization of the virus-induced gene silencing (VIGS) system in Angelica sinensis (Oliv.) diels

As a renowned traditional medicinal plant in China, Angelica sinensis requires in-depth functional genomic studies to decipher the molecular mechanisms governing its growth and development. Virus-induced gene silencing (VIGS) represents a powerful tool for such investigations owing to its straightforward methodology, short experimental cycle, and capacity for rapid transient gene functional validation. However, the current absence of a robust VIGS system in A. sinensis significantly impedes progress in gene function research. In this study, based on the whole-genome data from A. sinensis, three candidate reporter genes, AsPDS, AsCLA1–1 and AsCLA1–2, were cloned for VIGS vector construction and inserted into a tobacco rattle virus (TRV) silencing vector. Four Agrobacterium-mediated transformation methods, namely, root incision soaking, ultrasound assisted infiltration, vacuum infiltration, and quartz sand abrasion, were evaluated for the silencing of three reporter genes in A. sinensis. The root incision soaking method induced the earliest appearance of the albino phenotype while maintaining a relatively high survival rate. Among the tested genes, AsCLA1–1 demonstrated consistently high silencing efficiency (>50 %) across all methods and was consequently established as the optimal reporter gene. Two-way analysis of variance (ANOVA) revealed a highly significant interaction (P < 0.01) between Agrobacterium concentration (OD₆₀₀) and cocultivation time. Through multiple statistical comparison methods, the optimal parameters were determined to be an OD₆₀₀ = 1.60 and 14 h cocultivation. This optimized regimen achieved silencing efficiencies of 62 % at 14 d and 70 % at 28 d, while maintaining excellent plant viability. In summary, this study successfully established and optimized a VIGS system for A. sinensis, providing an effective methodology for investigating the functions of key genes involved in its medicinal compound biosynthesis and growth regulation while also offering a valuable technical framework for establishing VIGS systems in other root and rhizome medicinal plants.