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Peer-reviewedCC BYBiotechnology journal

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

  • Sarah Pullano1
  • Aida Shahabi1
  • Parmis Shariat Torbaghani1
  • Ivan Monsalvo1
  • Madison Sandler1
  • Adam Sumner2
  • Alex Harris2
  • David Haak2
  • Bastiaan Bargmann2
  • Nik Kovinich1
  1. 1Department of Biology, York University, Toronto, Ontario, Canada.
  2. 2College of Agricultural and Life Sciences, Virginia Tech, Blacksburg, Virginia, USA.
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Abstract

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.

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Cite

BibTeXRIS
BibTeX
@article{pullano2026integrated,
  title = {{An Integrated Transient Expression and UPLC-PDA Approach for Functional Genomics and Metabolic Analysis of Cannabis sativa.}},
  author = {Sarah Pullano and Aida Shahabi and Parmis Shariat Torbaghani and Ivan Monsalvo and Madison Sandler and Adam Sumner and Alex Harris and David Haak and Bastiaan Bargmann and Nik Kovinich},
  journal = {Biotechnology journal},
  year = {2026},
  month = oct,
  doi = {10.1002/biot.70316},
  url = {https://europepmc.org/article/MED/42875966},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - An Integrated Transient Expression and UPLC-PDA Approach for Functional Genomics and Metabolic Analysis of Cannabis sativa.
AU  - Sarah Pullano
AU  - Aida Shahabi
AU  - Parmis Shariat Torbaghani
AU  - Ivan Monsalvo
AU  - Madison Sandler
AU  - Adam Sumner
AU  - Alex Harris
AU  - David Haak
AU  - Bastiaan Bargmann
AU  - Nik Kovinich
PY  - 2026
DA  - 2026/10/01
JO  - Biotechnology journal
DO  - 10.1002/biot.70316
UR  - https://europepmc.org/article/MED/42875966
AB  - 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.
SN  - 1860-6768
SN  - 1860-7314
ER  -