Optimization of In Vitro Propagation of Artemisia pontica Through Integrated Morphophysiological, Biochemical, and SEM Analysis Under GA3 and MeJA

Abstract

Efficient micropropagation systems are essential for the large-scale production of uniform and high-quality plant material in aromatic species. In this study, the effects of gibberellic acid (GA3; 1.4, 2.8, and 5.6 µM) and methyl jasmonate (MeJA; 2.2, 4.4, and 8.8 µM) were evaluated on the in vitro performance of Artemisia pontica across two successive subcultures. Morphological, physiological, and biochemical parameters were assessed, and the most effective treatment was further investigated through scanning electron microscopy (SEM) to evaluate leaf trichome characteristics. GA3 treatments significantly enhanced shoot growth, shoot number, and relative growth rate, with the strongest response observed at 2.8 µM. This concentration also promoted higher chlorophyll content and antioxidant activity, indicating improved physiological status and metabolic performance of plantlets. In contrast, MeJA treatments, particularly at 8.8 µM, reduced growth performance and pigment accumulation, suggesting a less favorable physiological status for micropropagation. Multivariate analysis (PCA and hierarchical clustering) revealed a clear separation among treatments, with GA3 at 2.8 µM associated with a coordinated increase in growth-related and antioxidant traits. SEM analysis showed that GA3 influenced leaf epidermal structure, increasing the density of T-shaped, non-glandular trichomes and the diameter of glandular secreting trichomes, suggesting structural adjustments linked to metabolic activity. Overall, the results indicate that GA3 at 2.8 µM represents the most effective supplementation under the tested conditions, promoting a balanced improvement in shoot proliferation, physiological performance, antioxidant activity, and selected structural traits.