Integrated analysis of antioxidant responses, phenolic profiles and Phytohormonal changes in Thymus daenensis treated with Paclobutrazol under in vitro conditions

Background Paclobutrazol (PBZ), a triazole-type plant growth regulator, is widely used to modulate plant growth and stress tolerance by influencing phytohormonal signaling and oxidative metabolism. However, its effects on medicinal plants, particularly Thymus daenensis , under in vitro conditions remain largely unexplored. This study aimed to investigate the impact of different concentrations of PBZ (0, 2.5, 5, and 10 mg/L) on growth traits, antioxidant defense, phenolic metabolism, oxidative stress markers, and endogenous phytohormones in T. daenensis cultured in vitro . Results PBZ treatments induced pronounced changes in growth performance. The fresh weight of shoots progressively decreased with increasing PBZ concentrations, reaching 46% below the control at 10 mg/L, whereas root fresh weight markedly increased by 66% and 330% at 2.5 and 10 mg L⁻¹, respectively. Shoot length remained unaffected at 2.5 mg/L but declined significantly at 5 and 10 mg/L, while root length increased at 2.5 mg L⁻¹ but decreased at higher doses, with the strongest reduction at 10 mg/L. Consequently, the shoot-to-root fresh weight ratio rose at 2.5 and peaked at 5 mg/L, but declined at 10 mg/L, indicating a biomass allocation shift under elevated PBZ levels. In parallel, PBZ significantly modulated enzymatic antioxidant activities, with the greatest enhancement at 5 mg/L, and stimulated phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) at moderate concentrations. Hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents declined at 2.5 and 5 mg/L, suggesting reduced oxidative damage, while DPPH radical scavenging capacity and total flavonoid content peaked at 5 mg/L but decreased at 10 mg/L. Hormonal analysis revealed a concentration-dependent reduction in gibberellic acid (GA), indole-3-acetic acid (IAA), and brassinosteroids (BR), with the lowest values at 10 mg/L, whereas abscisic acid (ABA) progressively increased, reaching its maximum at the highest PBZ concentration. Conclusion These findings demonstrate that PBZ exerts a dose-dependent modulation of growth, antioxidant metabolism, and hormonal balance in T. daenensis under in vitro conditions. Moderate concentrations (particularly 5 mg/L) enhance antioxidant capacity, phenolic metabolism, and biomass allocation toward roots, while higher doses suppress shoot growth and accentuate hormonal shifts (reduced GA, IAA, BR; elevated ABA). Overall, PBZ shows promise as an elicitor to improve physiological resilience and potentially the medicinal quality of T. daenensis during micropropagation.