Development of callus culture protocol for secondary metabolite production from hemp (Cannabis sativa L.)

The hemp contains cannabinoids and other important secondary metabolites of commercial value [ 1 ]. Plant tissue culture, which is an alternative soilless, aseptic method for the production of bioactive compounds from plants, is remarkable in terms of providing continuous and more standardized production [ 2 ]. In this study, it was aimed to determine the callus induction ability of hemp genotype in media containing different growth regulators (PGRs), to determine the most effective cytokinin-auxin PGR combination on callus biomass production and to determine the subculturing capacity of calli. Three different explant sources; hypocotyl, cotyledon and root were taken and cytokinin [Kinetin, Zeatin, Tidiazuron (TDZ) and 6-Benzylaminopurine (BAP)] and auxin [naphthalene acetic acid (NAA)] PGRs were combined at different concentrations and cultured in Murashige and Skoog (MS) nutrient medium. 100% callus formation was observed in all applications and applications were subcultured every 4 weeks. Callus biomasses were recorded between 0.15-12.63 g at 8, 12, 16, 20 and 24 weeks. For each application, the growth indexes of calli at different biomasses (0.5, 1.0, 2.0 g) were calculated in subculturing, and the highest growth index was recorded as 8.58. In TDZ and NAA applications, callus biomass progressed slower compared to other applications, and in the 12th week, approximately 65% of the applications had callus growth index below 1 and were excluded from the experiment. In the 24 th week, callus growth indexes were determined between 0.05-8.44. As a result, the highest yield was determined in cotyledon explants and in the application of Zeatin and NAA PGR ([ Fig. 1 ]). Fig. 1 Callus regeneration from cotyledon explants on MS medium supplemented with Zeatin and NAA (Initial fresh weight: a. 1.0 g, b. 0.5 g, c. 2.0 g). Acknowledgement : This study was produced from the doctoral thesis supported by Scientific Research Projects Organization of Gazi University (TDK-2024-9682). Publication History Article published online: 05 March 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany