Toward scalable micropropagation of Cannabis sativa (chemotype III): protocol optimization and preliminary evaluation of cannabinoid stability

Abstract

A micropropagation protocol was optimized for Cannabis sativa L. cv. Charlie’s Dream (chemotype III; Δ9-THC ≪CBD) to generate clonal plants for cannabinoid stability assessment. Axillary buds were cultured on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP) or thidiazuron (TDZ) (0.5–2.5 µM), with or without vitamin supplementation. Shoot number, shoot length, and foliar area were evaluated. Rooting responses were tested using 1-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) under both in vitro and ex vitro conditions. Resins extracted from dried flowers of donor and micropropagated plants were analyzed for CBD and Δ9 -THC contents by gas chromatography coupled to mass spectrometry (GC/MS). Vitamin supplementation did not improve growth, whereas TDZ and BAP showed differential effects over time. At 15 days postinitiation of micropropagation (dpim), explants treated with 0.5 µM TDZ produced more shoots and greater foliar area than those treated with BAP. By 30 dpim, explants treated with BAP achieved values similar to those with TDZ, whereas 2.5 µM TDZ promoted callus formation and vitrification. Shoot elongation was significantly enhanced when gibberellin (7 µM) or red light was combined with 0.5–1 µM BAP, but not with TDZ. In vitro rooting was unsuccessful; however, ex vitro rooting improved after pretreatment with 2.5 µM IBA. Based on these findings, the optimized micropropagation workflow consisted of: (i) shoot multiplication on MS + BAP (0.5–1 µM) for 30 days; (ii) shoot elongation under MS + BAP (0.5–1 µM) supplemented with gibberellin (7 µM) or red light; and (iii) ex vitro rooting after IBA (2.5 µM) pretreatment. GC-MS analysis demonstrated that the CBD/Δ9 -THC ratio remained stable between donor, first-generation (G1), and second-generation (G2) plants, supporting chemotype stability. This approach reduces medium complexity, improves scalability, and provides a reproducible framework suitable for industrial applications in high-value crops.