A newly developed two-step ex vitro transformation method has significantly improved the efficiency and speed of generating transgenic hairy roots in cannabis, according to researchers from Université Laval.
Hairy root induction and RUBY gene expression using the two-step ex vitro method in cannabis. (a) The emergence of red hairy roots expressing RUBY gene in cannabis mother plant. (b) A fully developed composite cannabis plant (bar = 1 cm)
The method, detailed in the journal BMC Biotechnology, achieved a 90% transformation efficiency—surpassing both traditional in vitro and one-step ex vitro techniques—and shortened the time required for producing genetically modified hairy roots by up to 29 days.
Cannabis sativa has long been prized for its diverse applications, from fiber and food to medicinal and psychoactive uses. However, due to decades of prohibition and social stigma, modern genetic and biotechnological research into cannabis has lagged behind that of other crops. With recent waves of legalization and a projected $74.29 billion global market by 2029, cannabis research is entering a new era of scientific rigor and industrial application.
Central to this advancement is the production of secondary metabolites—compounds like cannabinoids, terpenes, and flavonoids that give cannabis its therapeutic and psychoactive properties. Traditionally harvested from female flowers, these compounds can now also be synthesized through more controlled and efficient methods such as hairy root (HR) cultures. These cultures, induced by Agrobacterium rhizogenes, are prized for their rapid biomass growth and ability to stably produce target metabolites without requiring full plant cultivation.
The researchers at Université Laval sought to optimize HR transformation techniques by comparing three approaches: traditional in vitro, one-step ex vitro using plant cuttings, and a novel two-step ex vitro method using the living mother plant. They employed the RUBY reporter gene system to visually identify successful transformations through the expression of a vivid red pigment, allowing easy detection without chemical testing.
Their findings showed that the in vitro method had the lowest efficiency, with only 33% HR induction and extended timelines of up to 35 days for full development. While the one-step ex vitro method fared better—with 100% root induction using certain bacterial strains—it still fell short in transformation efficiency (max 56.25%) and took about 30 days for full development.
In contrast, the two-step ex vitro technique not only matched the 100% root induction rate but also achieved a 90% transformation efficiency. Roots expressing the RUBY gene began appearing as early as 14 days post-inoculation, and robust transgenic growth was seen by day 22. This approach, using mature cannabis mother plants and direct application of A. rhizogenes to the plant’s stem nodes, allowed for faster development, lower contamination risk, and minimal technical handling—key advantages for commercial scalability and high-throughput studies.
“This method stands out as a significant improvement over previous transformation systems in cannabis,” the researchers wrote, noting that its simplicity and reproducibility make it an attractive option for producing high volumes of transgenic material. Such a system is particularly valuable for applying CRISPR/Cas-based gene editing to enhance cannabinoid synthesis or study gene functions.
While the study used the AJ-441 cultivar—containing 7% THC and 0.1% CBD—the authors suggest that further trials are needed across a wider range of cannabis genotypes to confirm consistency. They also noted that future efforts to scale the protocol for industrial or research use may benefit from automated systems to manage inoculation, environmental conditions, and data collection.
In conclusion, this two-step ex vitro transformation method presents a new benchmark for cannabis biotechnology. It offers a streamlined, effective, and contamination-resistant approach for genetic transformation, supporting both functional genomics research and commercial applications like metabolite production and gene-editing trials.
