Micropropagation of the Endemic Stachys pinardii Boiss. and Comparative Investigation of the Phytochemical Composition of Cultured and Wild-Grown Plants

This study aimed to establish an efficient micropropagation protocol for the endemic plant Stachys pinardii Boiss. and to characterize differences in specialized metabolite profiles between in vitro-derived and wild plants under the tested conditions. Nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different cytokinin (BAP, KIN) and auxin (IAA, NAA) combinations, with morphogenetic data analyzed by one-way ANOVA followed by Duncan’s Multiple Range Test and Tukey’s HSD test (n = 30 per treatment); air-dried aerial parts (upper shoots) of in vitro-derived and wild-grown plants were compared phytochemically using GC–MS for compound identification and GC–FID for relative quantification based on peak-area normalization (n = 3 repeated analyses of each extract). The BAP (1.0 mg/L) + IAA (2.0 mg/L) combination produced both the highest shoot number (11.43 ± 0.20 shoots per explant) and the greatest shoot length (3.36 ± 0.05 cm) among the tested treatments. GC–MS identification and GC–FID relative quantification revealed a marked difference in phytochemical composition: the relative percentage of nerolidol was 45.4% in the optimized in vitro treatment, compared with 10.3% in wild-grown plants, while, in the hexane extracts, carvacrol showed its highest relative percentage in wild-grown plants (12.0%) and was not detected in the optimized in vitro treatment. These findings indicate that in vitro culture systems can serve not only as a tool for ex situ conservation but also as a controlled platform for characterizing differences in specialized metabolite profiles under defined culture conditions, providing a basis for further investigation into the biotechnological potential of S. pinardii.