Biotechnological Approaches on Two High CBD and CBG Cannabis sativa L. (Cannabaceae) Varieties: In Vitro Regeneration and Phytochemical Consistency Evaluation of Micropropagated Plants Using Quantitative 1H-NMR
2020
Kostas Ioannidis | Evangelos Dadiotis | Vangelis Mitsis | Eleni Melliou | Prokopios Magiatis
High cannabidiol (CBD) and cannabigerol (CBG) varieties of Cannabis sativa L., a species with medicinal properties, were regenerated in vitro. Explants of nodal segments including healthy axillary bud, after sterilization, were placed in Murashige-Skoog (MS) culture medium. The shoots formed after 30 days were subcultured in full- or half-strength MS medium supplemented with several concentrations of 6-benzyl-amino-purine (BA) or thidiazuron (TDZ). The highest average number and length of shoots was achieved when both full and half-strength MS media were supplemented with 4.0 &mu:M BA. The presence of 4.0 &mu:M TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 &mu:M and 2.0, 4.0 &mu:M respectively, displayed the maximum shooting frequency. The new shoots were transferred on the same media and were either self-rooted or after being enhanced with different concentrations of indole-3-butyric acid (IBA) or &alpha:-naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 &mu:M IBA or 4.0 &mu:M NAA resulted to the optimum rooting rates. The maximum average number and length of roots per shoot was observed when the culture media was supplemented with 4.0 &mu:M IBA or NAA. Approximately 92% of the plantlets were successfully established and acclimatized in field. The consistency of the chemical profile of the acclimatized in vitro propagated clones was assessed using quantitative 1H-NMR high throughput screening. In each variety, analysis of the micropropagated plant in comparison with the mother plant showed no statistically significant differences (p &le: 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.
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