Impact of Plant Growth Regulators on Callus Induction in Cannabis sativa L.

Callogenesis is a fundamental step in plant biotechnology and tissue culture, providing the basis for multiple scientific and practical applications. In this study, the impact on callogenesis of different plant growth regulators was studied on <i>Cannabis sativa</i> L. (a non-commercial genotype of hemp), with the objective of identifying the most suitable combination for the establishment of vigorously growing, friable calli. Forty-nine media combinations were evaluated using four PGRs: two auxins (2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid) and two cytokinins (6-benzylaminopurine, kinetin). Parameters such as percentage of callus induction, proliferation, colour, texture, and growth area were assessed. Three media were identified for further spectrophotometric assays and targeted gene expression analysis: the first containing 2,4-dichlorophenoxyacetic acid 1.5 µM and benzylaminopurine 1.5 µM, the second with 2,4-dichlorophenoxyacetic acid 1.5 µM and kinetin 1.5 µM and the third supplemented with 2,4-dichlorophenoxyacetic acid 4.5 µM and kinetin 1.5 µM. The last medium proved to be superior in terms of vigour, friability and phenolic content and showed increased expression of genes involved in the early steps of the phenylpropanoid pathway. These findings highlight the central role of auxin-cytokinin interactions in regulating both callus formation and secondary metabolism. The optimised medium opens the way to subsequent biotechnological applications relying on the cultivation of plant cell suspension cultures.