Developing and applying a virus-induced gene silencing system for functional genomics in walnut (Juglans regia L.) mediated by tobacco rattle virus

Walnut (Juglans regia L.) is a high-value tree species planted worldwide, but the incomplete less developed genetic transformation system limits its gene function analysis. In this study, virus-induced gene silencing (VIGS) mediated by tobacco rattle virus (TRV) technology was applied to walnut seedlings to degrade the transcript of target gene. Different infiltration methods were used to explore the effects of infection mode, Agrobacterium cell density, silencing fragment length, and walnut cultivars. The results showed that spray infiltration of seedlings resulted in a photobleaching phenotype of the whole plant. Leaf injection was a more effective way of infiltration. The optimal combination was the Agrobacterium cell density at OD₆₀₀ = 1.1 with target fragment = 255 bp for the treatment of walnut early-fruiting cultivar 'Xiangling.' This combination can reach up to 48 % of gene silencing efficiency. Based on this optimized VIGS system, silencing a walnut chlorophyll synthesis-related gene, JrPOR (Protochlorophyllide reductase), to further validate the system's effect. The results showed that the expression of JrPOR was significantly repressed, and the chlorophyll level of the silenced plants was significantly decreased compared with the control. The above results indicate that the walnut TRV-VIGS system has been successfully established and can be used for reverse genetic studies, providing an option for verifying gene function in walnut.