Abstract

Cannabis is well known for its pharmacological and therapeutic properties. As an outcrossing species, seed propagation induces high genetic variability; micropropagation offers a promising solution to ensure stability and uniformity. This study aimed to optimize an in vitro micropropagation protocol for axillary buds of two Moroccan C. sativa cultivars, Beldia and Khardala. The effects of basal medium composition, sucrose concentration and plant growth regulators (PGRs) on growth and development were evaluated. Effect of various strength of Murashige and Skoog (MS) medium (1/2 - 1/1) and four sucrose levels (0%, 1.5%, 3% and 4.5%) were tested. Cytokinins, including benzyladenine (BA), 6-benzylaminopurine (BAP) and n⁶-(Δ²-isopentenyl) adenine (2IP) at 0, 0.25, 0.5 and 1 mg/L, as well as other plant growth regulators, including gibberellic acid (GA3), thidiazuron (TDZ) and zeatin (ZEN) at 0.5 mg/L, were applied individually. For rooting, microshoots were transferred to MS medium containing 0.5 mg/L indole-3-acetic acid (IAA) or 1.5 and 2.5 mg/L indole-3-butyric acid (IBA). MS medium Murashige and Skoog promoted both shoot development and callus formation. Supplementation with 1.5% or 3.0% sucrose resulted in high regeneration rates (67.71–77.43%), whereas 4.5% sucrose induced osmotic stress, reducing growth and regeneration. The influence of different PGRs was examined as well. ZEN (0.5 mg/L) and 2IP were the most effective treatments, inducing high regeneration in both C. sativa varieties over one month. ZEN promoted strong organogenesis, with producing 8 nodes and developing up to 19 leaves, 3 shoots and shoot lengths of 7.22 cm. In contrast, TDZ (0.5 mg/L) resulted in lower regeneration (~75% in Khardala), reduced shoot formation (1.5 shoots) and shorter shoots (3.81 cm), while callus formation remained relatively high. For rooting, AIB at 1.5 mg/L was the most effective, achieving acclimatization rates above 90%. Correlation analysis highlighted cultivar-specific responses: Beldia was more sensitive to low cytokinin levels, while Khardala showed a more stable response across treatments. These results underline the importance of genotype-specific strategies and the appropriate selection of PGRs to improve C. sativa micropropagation protocols.