Optimization of in vitro micropropagation, assessment of genetic fidelity, and phytochemical evaluation of Arnica montana L. under pH and osmotic stress

Arnica montana L., a perennial rhizomatous medicinal plant from the Asteraceae family, has a long history of use in traditional medicine across Europe and North America. Its various parts contain valuable metabolites, especially phenolic compounds, which are reported to possess anti-inflammatory, anticancer, and antioxidant properties. To support sustainable cultivation and conservation, optimizing propagation methods is essential. This study aimed to optimize direct in vitro micropropagation and assess the effects of acidity (pH) and osmotic stress on the proliferation of A. montana plantlets and their phenolic content. The experiment was conducted in a completely randomized design. Shoot tips were cultured on Murashige and Skoog (MS) medium with different concentrations of benzylaminopurine (BAP) and kinetin, combined with indole-3-butyric acid (IBA) and naphthaleneacetic acid (NAA). The highest shoot-forming capacity (6.6) and shoot proliferation percentage (83.3%) were obtained in medium containing 4.4 μM BA and 2.7 μM NAA. Rooting was performed on half-strength MS with different concentrations of indole-3-acetic acid (IAA), IBA, NAA, and 2,4-dichlorophenoxyacetic acid (2,4-D), which resulted in the highest root formation (11.4) in 2.7 μM NAA. Reduced pH improved shoot/root induction and dry weight, while osmotic stress from mannitol and sorbitol negatively affected proliferation. RAPD-PCR analysis using Operon primers (OPB-03, OPR-06, OPI-12, OPA-13, OPI-18, OPG-19) and ISSR markers (IS-05, IS-07, IS-23, UBC-810, UBC-866, UBC-880) confirmed the genetic uniformity of regenerated plantlets. Acclimatization success in greenhouse conditions was 70%. Notably, stressed plantlets showed significantly higher phenolic content than control and in vivo plants. These findings demonstrate the potential of optimized in vitro culture as an effective strategy for large-scale propagation and enhanced production of bioactive phenolic compounds in A. montana .