Synergistic effects of plant growth regulators, riboflavin, and iron nanoparticles on secondary metabolites in micropropagated Ceratonia siliqua L.

Abstract

Abstract Plant growth regulators (PGRs) play a crucial role in regulating the biochemical and physiological processes. Recently, nanoparticle-based approaches have attracted increasing attention for improving in vitro rooting efficiency. Ceratonia siliqua L. is a significant tree species utilized for landscaping and medicinal purposes; however, its conventional propagation is limited, making micropropagation a viable alternative. This study aimed to optimize in vitro multiplication, rooting, and secondary metabolite accumulation of C. siliqua through the use of different PGR combinations, Murashige and Skoog (MS) medium strength, and riboflavin during the multiplication stage, while evaluating the effect of iron oxide nanoparticles (Fe₂O₃ NPs) exclusively during the rooting stage. During establishment and multiplication, explants were cultured on full- or half-strength MS media supplemented with riboflavin in combination with benzyladenine (BA), naphthaleneacetic acid (NAA), and gibberellic acid (GA₃). Rooting experiments were conducted using indole-3-butyric acid (IBA; 2, 3, and 4 mg L − 1 ) applied alone or in combination with Fe₂O₃ NPs (5 and 10 mg L − 1 ). Chemical analyses were performed only on responsive multiplication treatments and compared with the mother plant. The highest shoot number (3.33 shoots per explant), shoot length (46.33 mm), and leaf number (42.67 leaves per explant) were recorded on full-strength MS medium supplemented with 1 mg L − 1 riboflavin, 1 mg L − 1 BA, 0.1 mg L − 1 NAA, and 0.1 mg L − 1 GA₃, which also resulted in the highest flavonoid content in the multiplied shootlets. For rooting, the highest rooting percentage and root number were obtained with 3 mg L − 1 IBA alone or in combination with 5 mg L − 1 Fe₂O₃ NPs. Regarding secondary metabolites, shoots on ½ MS with 1 mg L⁻¹ riboflavin and 0.5 mg L⁻¹ BA yielded high phenolic content, nearly matching the mother plant, while significantly surpassing it in DPPH antioxidant activity. HPLC confirmed this enhanced bioactivity via the unique accumulation of gallic acid, rutin, and vanillin.