Effect of Cytokinins on Morphogenesis and Accumulation of Phenolic Compounds in Hyssopus officinalis: A Comparative Study in vitro

Hyssopus officinalis L. is a valuable essential oil plant of the Lamiaceae family, which owes its pharmacological properties to phenolic compounds, flavonoids, and hydroxycinnamic acids. Cultivation in vitro makes it possible to control morphogenesis and the accumulation of secondary metabolites by adding growth regulators to the nutrient medium. Since this requires knowledge of optimal concentrations, the aim of this in vitro study was to investigate the effect of cytokinin-type growth regulators on the micropropagation and content of bioactive substances in H. officinalis.The research featured in vitro microplants of H. officinalis (cv. Lekar). The seeds were sterilized using H₂O₂, AgNO₃, NaClO, and H₂SO₄ as disinfectants. They were then sown on a Murashige and Skoog medium without growth regulators. The resulting seedlings were cultured on an medium supplemented with different concentrations of cytokinins (6-benzylaminopurine, kinetin, thidiazuron, and 6-(γ,γ-dimethylallylamino)purine). The experiment lasted for 30 days under a 16/8-h light/dark regimen at 25 ± 1°C. The obtained microplant extracts underwent spectrophotometry to determine the total contents of phenolic compounds, flavonoids, hydroxycinnamic acids, and antioxidant activity. Statistical data processing was performed using a one-way analysis of variance and hierarchical cluster analysis.
The treatment of H. officinalis seeds with concentrated sulfuric acid resulted in 98% sterility and 99% germination, exceeding previously published data. Among the growth regulators, 6-benzylaminopurine at 0.1 mg/L was found to be the most effective for morphogenesis, resulting in the highest shoot length (4.6 cm), shoot number (3.2), and node number (9.6). Cluster analysis confirmed that concentrations of 0.1 mg/L 6-benzylaminopurine, 1.0 mg/L kinetin, and 0.1 mg/L thidiazuron were optimal for microplant development. In terms of bioactivity, 6-benzylaminopurine (0.1 mg/L) maximally increased the content of phenolic compounds (5-fold), hydroxycinnamic acids (5-fold), and antioxidant activity, while thidiazuron (0.1 mg/L) selectively stimulated flavonoid accumulation.
The novel methods of seed sterilization combined with specific growth regulators for H. officinalis ensured the production of plant material with a targeted biochemical profile.

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