An Integrated Transient Expression and UPLC-PDA Approach for Functional Genomics and Metabolic Analysis of Cannabis sativa.

Cannabis sativa (Cannabis) is a high-value medicinal crop whose functional genomics and metabolic engineering remain constrained by limited genetic tools. The species is regarded as recalcitrant to stable transformation, and existing transient expression systems exhibit low efficiency or are restricted to hemp cultivars. Here, we report two Agrobacterium-mediated agroinfiltration protocols enabling rapid, robust, and reproducible transient gene expression in drug-type, high-THC Cannabis tissues. Using the RUBY betalain reporter system, we demonstrate transformation efficiencies of up to 100% in mature leaf disks and 81%-100% in whole seedlings. By independently comparing Agrobacterium strains and vector configurations, we demonstrate that transformation efficiency and transgene expression are influenced by distinct experimental variables, with EHA105 providing the highest transformation efficiency and p35S-based vectors producing the strongest reporter expression. Complementing these genetic tools, we developed a UPLC-PDA method enabling simultaneous detection and quantification of cannabinoids and cannflavins. While not intended to replace mass spectrometry-based analyses, the method provides an accessible and cost-effective platform for comparative metabolite profiling during functional genomics and metabolic engineering studies. Together, these advances establish an integrated workflow linking high-efficiency transient gene expression with streamlined biochemical analysis, providing a practical foundation for gene function studies, pathway interrogation, and biotechnology-driven trait engineering in Cannabis.