Agrobacterium-mediated transient expression in Torreya grandis cones: A simple and rapid tool for gene expression and functional gene assay

Agrobacterium-mediated transient gene expression is a powerful tool for characterizing gene function in plants, particularly in fruit trees with long juvenile periods. However, few studies have systematically explored transient expression in nut trees, such as T. grandis. In this study, we developed a rapid and easily manipulated transient transformation system using Agrobacterium-mediated injection infiltration in T. grandis cones. Cones at the enlargement stage (30 days after aril protrusion) were used, and the bacterial solution was gently injected into the kernel at a depth of 1 cm, filling nearly the entire cone, as indicated by a red dye. Using the green fluorescent protein (GFP) gene as a reporter for Agrobacterium-mediated transformation, we confirmed GFP expression in T. grandis cone successfully through confocal microscopy, western blot, and PCR. Transformation conditions, including bacterial density and days after agroinfiltration, were optimized for higher transformation efficiency, with the optimal conditions being an OD₆₀₀ of 0.6 and an agroinfiltration period of 6 - 9 days. Transient overexpression of TgWRKY47, a cell size regulator, altered cell size in T. grandis cones, indicating that this Agrobacterium infiltration method can be used for gene functional analysis. Furthermore, confocal microscopy revealed that TgWRKY47-GFP was localized in the nucleus along with a nuclear marker, demonstrating its applicability for protein subcellular localization. In summary, this method presents a versatile tool for efficient and targeted transient gene functional analysis and subcellular localization in T. grandis cones, offering new insights into nut tree biology research.