Micropropagation, callus induction and cell culture establishment of Zataria multiflora (Lamiaceae): An efficient biotechnological platform for the production of rosmarinic acid

A promising and efficient in vitro protocol with a conservation perspective for the production of rosmarinic acid (RA), a well-known natural antioxidant, from Zataria multiflora (Lamiaceae) was presented. Tha plant shoo tips were regenerated on Murashige and Skoog (MS) medium fortified with various plant growth regulators. The highest shoot-forming capacity (15.93) was occurred on the MS medium with 1.5 mg L¹ 6-benzylaminopurine (BAP) plus 0.5 mg L¹ indole-3-butyric acid (IBA), while the best root-forming capacity (10.10) was found on the half-strength MS medium with1.5 mg L¹ IBA. Callus was induced from in vitro leaves on B5 and MS media with varying concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), either alone or in combination with BAP and kinetin (KIN). The highest callus induction (96.66 ± 0.39 %) was found on B5 medium with 1.0 mg L¹ 2,4-D plus 0.5 mg L¹ BAP. The greatest fresh weight of callus (2750 ± 6.94 mg) was recorded on B5 medium with 0.75 mg L¹ BAP and 200 mg L¹ casein hydrolysate. Cell culture was performed on B5 medium containing 0.75 mg L¹ BAP. The established cell culture followed a general sigmoid growth pattern. Maximum specific cell growth rate and doubling time were 0.29 g day¹ and 8 day, respectively. The highest RA production was observed at the third week culture, reaching 73.75 ± 1.20 mg g⁻¹ DW which was 9.16-fold higher than the mother plant (8.06 ± 0.19 mg g¹ DW). The findings underscore the potential of in vitro culture techniques for enhancing the production of rosmarinic acid in Z. multiflora.