Evaluation of Tobacco Rattle Virus-based Vectors for Gene Silencing and Editing in Tomato

This study established an optimized protocol for tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) and virus-induced gene editing (VIGE) in tomato using phytoene desaturase (PDS) as a visual marker. Five tomato genotypes and three concentrations of Agrobacterium were evaluated for their VIGS efficiency. Line M82 exhibited the strongest silencing response, while the Agrobacterium concentration showed no significant difference, indicating a genotype-dependent effect. For VIGE, a TRV vector carrying a PDS -targeting sgRNA was agroinfiltrated into a transgenic MicroTom line overexpressing Cas9 . Photobleaching and a sequence analysis confirmed somatic editing near the PAM site with a frequency of up to 92%. However, no heritable mutations were recovered in the progeny. Tissue culture regeneration was tested, and CLN1558A, MicroTom, and M82 showed efficient shoot and root formation, with complete regeneration achieved within 4 months. These findings provide a practical framework for TRV-mediated gene silencing and editing in tomato, thus highlighting the importance of genotype selection and regeneration strategies for further breeding applications.