Ziziphora clinopodioides Lam., a valuable medicinal plant, exhibits significant potential for flavonoid production, which signif- icantly contributes to its therapeutic properties. In the initial phase of this ongoing study, phenolic derivatives were analyzed in 13 populations during two vegetative growth stages under greenhouse conditions. In the next phase, the effects of precursors [phenylalanine (Phe) and naringenin (Nar)], biotic elicitor [yeast extract (YE)], and abiotic elicitors [salicylic acid (SA) and me- thyl jasmonate (MeJA)] were evaluated on the accumulation of specific flavonoids and phenolic acids in a selected population. Younger plants (2-MO growth stage) exhibited higher phenolic content than older ones (5-MO growth stage). Selected popula- tions (P1, P4, P5, and P10 for shoots; P4, P8, P11, and P13 for roots) were subjected to further LC–MS/MS analysis, revealing that P5 shoots had the highest potential for producing quercetin (QUE), rutin (RUT), and apigenin (API). Treatment with 5 mM Phe resulted in its highest level in 96 h post-treatment, reaching 3.49- and 6.66-fold higher levels than the control, respectively. Nar at concentrations of 0.4- and 0.8-mM enhanced API and QUE production by 1.91- and 3.28-fold, respectively. YE notably increased API and ACA (acacetin) levels, while SA resulted in the highest accumulation of QUE and RUT, highlighting its crucial role in flavonol biosynthesis. YE and Nar significantly enhanced FNSII gene expression, with Nar being the most effective. This makes Nar an effective candidate for increasing flavonoid production in Z. clinopodioides. This finding may have important implica- tions for industries seeking to elevate flavonoid content, particularly the medicinal compound ACA, in plant-based products.

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