A versatile protoplast system and its application in Cannabis sativa L.

Cannabis sativa L. (cannabis) is one of the earliest domesticated crops cultivated for medicinal, food, industrial, and recreational use throughout the world. A growing number of jurisdictions have lifted the prohibition on medical and recreational cannabis with legislation that allows researchers to conduct important and urgently needed research, such as understanding the molecular basis of agronomically important traits. However, a dearth of suitable molecular genetic techniques for the analysis of cannabis species has hampered the elucidation of gene function. Here, we developed a robust protoplast transient transformation system by optimizing conditions of protoplast isolation and polyethylene glycol–mediated transformation using cannabis cotyledons. A protoplast isolation yield of up to 1.15 × 10 7 cells per gram of fresh material and 98.5% viability and a transformation efficiency of 75.4% were achieved. This optimized protocol for protoplast isolation and transformation was successfully applied to eight representative cannabis varieties, which highlight the versatility and robustness of this technique. Using this protocol, the nuclear localization of a basic helix–loop–helix transcription factor (CsMYC2) was visualized, and the transcriptional activity of CsMYC2 was assessed. These results demonstrate the effectiveness of our cannabis protoplast isolation and transient transformation method that should serve as a powerful molecular research tool in a wide range of molecular studies.