Establishment of a protoplast isolation and transient transformation system for tung tree (Vernicia fordii)

Tung tree (Vernicia fordii) serves as an economically important woody oilseed plant. High-quality protoplasts are essential for applications such as somatic hybridization, studying cell signal transduction and rapid gene function analysis. A reliable protocol for protoplast isolation and transient gene expression was developed using petals, anther-derived callus, and root tips as source materials. Through systematic optimization of key parameters such as enzyme combinations, osmotic pressure, digestion duration, and centrifugation speed, we successfully established tissue-specific isolation protocols. To ensure the availability of widely applicable experimental materials, protoplasts isolated from anther-derived callus were used to optimize transient transformation conditions. Under the optimized parameters (20 µg plasmid, 40% PEG4000, and 30-min incubation), the transformation efficiency reached 44.91%. Using this system, we conducted multiple experimental validations, including subcellular localization of VfWOX1.2 protein, detection of its protein expression, analysis of its transcriptional regulation of phytohormone biosynthetic genes, and assessment of stress-responsive gene expression under various treatment conditions. Collectively, these results confirm the utility of this platform for functional gene studies in tung tree. This study establishes a robust technical foundation, which is helpful for overcoming the limitation posed by the absence of a stable genetic transformation system and will facilitate future research on gene function in woody species.