A High-Efficiency Transient Expression System Reveals That CjMYB5 Positively Regulates Anthocyanin Biosynthesis in Camellia japonica

The establishment of a transient expression system in petals is significant for elucidating gene functions in flowering trees characterized by a prolonged juvenile phase. Genetic improvements in Camellia japonica have been hindered due to the absence of a functional validation platform. In this study, we explored an Agrobacterium-mediated and readily observable transient expression system in camellia petals to systematically optimize four critical factors affecting transformation efficiency. As a result, the bud stage, ‘Banliuxiang’ genotype, OD600 of 1.0, and 1-day co-cultivation achieved the highest intensity of transient expression, and overexpression of the Ruby1 reporter gene induced substantial anthocyanin synthesis, manifested as distinct red pigmentation. Furthermore, the optimized transient expression system revealed that the R2R3-MYB transcription factor CjMYB5, which interacted with CjGL3, promoted anthocyanin biosynthesis in camellia petals by transactivating key DFR structural genes. This transient expression platform not only advances functional genomics studies in ornamental woody species but also lays a foundation for molecular breeding programs in C. japonica.