Researchers Develop One of the Most Advanced Cannabis Protoplast Isolation and Transfection Systems to Date

The work comes from researchers at the University of Guelph and the University of Agriculture in Krakow. The team reports that while cannabis has long been considered difficult to regenerate from protoplasts, they were able to establish a reliable, step-by-step system that produced abundant, viable cells and successfully guided them through cell wall re-synthesis, division, and microcallus formation. Protoplast yield reached 2.2 × 10⁶ per gram of fresh leaf material, with nearly 79% remaining viable. More than half re-formed their cell walls, and close to 16% proceeded to divide.

The study also found that several factors are essential for success, including the age of the donor tissue, the composition of the enzyme blend used during isolation, and the length of the enzymolysis phase. Embedding the protoplasts and providing a nutrient-rich culture medium with the right combination of plant growth regulators proved critical for early development.

The researchers also demonstrated the system’s usefulness for genetic work. Polyethylene glycol-mediated transfection achieved a 28% transfection rate, and the resulting 10-day cultures maintained a 17% plating efficiency. Microcalli generated through this process were able to proliferate across six regeneration media containing different concentrations of 6-benzylaminopurine and thidiazuron, continuing to grow and green over a two-month period. The study additionally reported the formation of somatic embryo-like structures from protoplast-derived callus.

According to the authors, the protocol provides a strong foundation for functional gene studies and could support future advances in protoplast fusion, genome editing, and regeneration research in cannabis.