Monthony, A.S.; Jones, A.M.P. Enhancing Protoplast Isolation and Early Cell Division from Cannabis sativa Callus Cultures via Phenylpropanoid Inhibition. Plants2024, 13, 130.
Monthony, A.S.; Jones, A.M.P. Enhancing Protoplast Isolation and Early Cell Division from Cannabis sativa Callus Cultures via Phenylpropanoid Inhibition. Plants 2024, 13, 130.
Monthony, A.S.; Jones, A.M.P. Enhancing Protoplast Isolation and Early Cell Division from Cannabis sativa Callus Cultures via Phenylpropanoid Inhibition. Plants2024, 13, 130.
Monthony, A.S.; Jones, A.M.P. Enhancing Protoplast Isolation and Early Cell Division from Cannabis sativa Callus Cultures via Phenylpropanoid Inhibition. Plants 2024, 13, 130.
Abstract
De novo regeneration of Cannabis sativa L. (cannabis) using tissue culture techniques remains unreliable and infrequent. Conventional methods for the regeneration and transformation of cannabis have not achieved the reliability and replicability needed to be integrated into research and breeding programs. Protoplast systems are effective for gene expression studies, transformation and genome editing technologies, and opens the possibility of somatic hybridization to create interspecific hybrids. To date, leaf-derived protoplast have been isolated for transient gene expression studies, but attempts at protoplast-to-plant regeneration have not been reported. The present study aims to test the efficacy of using a callus culture system, as an abundant source for protoplast isolation and lays the groundwork for a protoplast-to-plant regeneration system. Using hypocotyl-derived callus cultures, which are known to have relatively greater regenerative potential, the efficacy of protoplast isolation and initial cell division were assessed. In this study, the effect of 2-aminoindane-2-phosphonic acid (AIP) in callus culture media and the effect of subculture frequency on protoplast yield were assessed. This study found that inclusion of AIP at 1 mM resulted in a 334% increase in protoplast yield compared with AIP-free medium, representing the first known use of AIP in cannabis tissue culture. Inclusion of AIP led to a 28% decrease in total soluble phenolics and 52% decrease in tissue browning compared to the control medium. Lastly, a two-phase culture system for protoplast regeneration was tested. At a concentration of 2.0×10^5 protoplasts per mL, division was observed, providing the first know report of cell division from cannabis protoplasts and setting the stage for future development of a protoplast-to-plant regeneration system.
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