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

Pullano S.; Shahabi A.; Torbaghani P.; Monsalvo I.; Sandler M.; Sumner A.; Harris A.; Haak D.; Bargmann B.; Kovinich N. · Biotechnology journal · 2026

**Background & Methods** This review presents two Agrobacterium-mediated agroinfiltration protocols for transient gene expression in cannabis tissues, combined with a UPLC-PDA analytical method for metabolite detection. The study independently evaluated multiple Agrobacterium strains and vector configurations to optimize transformation efficiency in drug-type, high-THC Cannabis cultivars, moving beyond previous limitations restricting work to hemp varieties. **Key Findings** • Transient transformation efficiencies reached 100% in mature leaf disks and 81-100% in whole seedlings using optimized agroinfiltration protocols, with EHA105 strain and p35S-based vectors providing superior performance compared to alternative configurations. • The RUBY betalain reporter system successfully demonstrated robust and reproducible transgene expression, establishing a practical genetic tool for functional genomics studies in cannabis tissues previously considered recalcitrant to transformation. • A novel UPLC-PDA method enables simultaneous detection and quantification of cannabinoids and cannflavins, providing a cost-effective, accessible platform for comparative metabolite profiling without requiring mass spectrometry equipment. **Dosage & Administration** Not reported. **Safety & Adverse Effects** Not reported. **Evidence Quality** This is a methodological review synthesizing technical advances rather than a clinical trial. The study represents a significant improvement in cannabis genetic tools but carries several limitations: (1) protocols were optimized for specific cultivars and may require adaptation for broader cannabis varieties; (2) the UPLC-PDA method is explicitly positioned as complementary to rather than replacive of mass spectrometry-based analyses, potentially limiting detection sensitivity for trace metabolites; (3) no in vivo efficacy data or long-term phenotypic outcomes are presented; (4) the work focuses on technical feasibility rather than functional characterization of specific genes or pathways. Evidence quality is moderate, suitable for establishing research foundations but requiring subsequent functional validation studies. The integration of transient expression systems with streamlined metabolic analysis provides valuable practical frameworks for cannabis research but does not directly inform clinical applications or therapeutic efficacy.

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