Optimization of protoplast isolation from Cannabis sativa L. ‘Hang Kra Rog’
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Abstract
This study investigated the effects of enzyme type and concentration, osmotic environment, and digestion time on protoplast yield and viability. The results indicated that both the type and concentration of cellulase significantly influenced protoplast yield and viability. Specifically, Cellulase R-10 outperformed Cellulase RS, with a 2.0 % Cellulase R-10 yielding the highest protoplast density (4.51 ×106 cells/g fresh weight) and a viability of 95.80%. For pectinolytic enzyme, macerozyme at a concentration of 0.3% was optimal, yielding a higher protoplast yield (4.38 ×106 cells/g fresh weight) compared to higher concentrations. The optimal osmotic environment was achieved with 0.4 M mannitol, which maintained a viability rate above 94% and produced a maximum yield of 4.38 × 106 cells/g fresh weight and a viability of 95.93%. Higher mannitol concentrations led to a continuous decline in both yield and viability. Furthermore, an enzymatic digestion time of 16 hours was found to be the optimal, resulting in a maximum yield of 4.42 ×106 cells/g fresh weight without reducing cell viability. In conclusion, the combination of 2.0% Cellulase R-10, 0.3% macerozyme, and 0.4 M mannitol, and 16 hours provides a robust and reproducible protocol for cannabis protoplast isolation. This is providing a foundation for advanced cellular and molecular studies in cannabis biotechnology.
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