A novel protocol for protoplast isolation, transfection, and culture in Cannabis sativa L

Protoplasts are a valuable tool for studying gene expression and applying genome editing techniques. Given the high medicinal and industrial potential of Cannabis sativa L., developing an efficient protoplast-to-plant regeneration protocol is highly desirable. Due to its recalcitrant nature, a complete plant regeneration from cannabis protoplasts has not yet been achieved.

This study details a robust protocol for cannabis protoplast isolation, purification, transient transfection, and culture, additionally reporting somatic embryo-like structures derived from protoplast-derived callus. We demonstrated that the age of donor material, the composition of the enzyme solution, and the duration of enzymolysis are crucial for efficient protoplast isolation. Protoplast embedding, coupled with a rich culture medium and plant growth regulators, proved critical for initiating cell wall re-synthesis, cell division, and microcallus formation. Protoplasts isolated using the reported protocol were abundant (2.2 × 10⁶ protoplasts/1 g of fresh weight), viable (78.8% viability) and able to undergo cell wall re-synthesis (56.1% of viable cells), followed by cell divisions (15.8% plating efficiency). Polyethylene glycol-mediated transfection yielded a 28% transfection efficiency and 17% plating efficiency in 10-day cultures. Protoplast-derived microcalli successfully proliferated on six regeneration media containing various concentrations of 6-benzylaminopurine and thidiazuron, exhibiting further proliferation and greening within two months.

This system provides a reliable protocol for isolation, transfection and culture of cannabis protoplasts. It also offers a framework for investigating gene function, as well as advancing protoplast fusion and genome editing technologies for this species.