CsCBDAS2-Driven Enhancement of Cannabinoid Biosynthetic Genes Using a High-Efficiency Transient Transformation System in Cannabis sativa ‘Cheungsam’

<i>Cannabis sativa</i> produces pharmacologically valuable cannabinoids. In this study, we developed and optimized a transient transformation system using <i>Cannabis sativa</i> 'Cheungsam' to facilitate gene functional analysis. Various experimental conditions, including plant developmental stages, light conditions, <i>Agrobacterium</i> strains, tissue types, and physical treatments such as sonication and vacuum infiltration, were systematically evaluated using GUS histochemical staining and qPCR analysis. Among these, 7-day-old seedlings cultured under dark conditions and transformed with the GV3101 strain exhibited high transformation efficiency. Leaf tissue showed a higher GUS staining proportion and GUS staining area compared to hypocotyl and cotyledon tissues. The application of a combination of sonication and vacuum infiltration techniques resulted in the most intense GUS expression. Using the optimized protocol, we introduced a recombinant vector carrying <i>CsCBDAS2</i>, a key gene in cannabidiol (CBD) biosynthesis. qPCR analysis revealed that <i>CsCBDAS2</i> overexpression led to significant upregulation of multiple upstream CBD biosynthetic genes (<i>CsOAC</i>, <i>CsGOT</i>, <i>CsPT1</i>, <i>CsPT4</i>, <i>CsCBDAS1</i>, and <i>CsCBDAS2</i>) and the transcription factor (TF) <i>CsWRKY20</i>, suggesting coordinated co-expression and potential involvement of a transcriptional feedback loop. These results demonstrate the effectiveness of our transient transformation system and provide insights into the regulatory mechanisms of cannabinoid biosynthesis in <i>cannabis</i>.