Influence of nanoparticles and growth regulators on in vitro propagation of Kaempferia galanga L.: assessment of molecular clonal fidelity, phytochemical evaluation, and micro-anatomical characterization

Abstract

The present study explores the role of zinc oxide nanoparticles (ZnONPs) and silver nanoparticles (AgNPs) in enhancing the micropropagation efficiency of Kaempferia galanga L., a medicinal plant known for its rich phytochemical profile. ZnONPs and AgNPs were synthesized and characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and ultraviolet-visible (UV-vis) spectroscopy to confirm their purity and crystalline structure. Their effects were evaluated by supplementing Murashige and Skoog (MS) medium with various concentrations of nanoparticles and conventional plant growth regulators, including 6-benzylaminopurine (BAP), kinetin (KN), α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA). AgNPs significantly accelerated shoot induction, with primordia appearing within 10 days, compared to 2–3 weeks in other treatments. The best shoot multiplication occurred in the medium appended with 2.5 mg L⁻¹ BAP, 2.5 mg L⁻¹ ZnONPs, and 0.5 mg L⁻¹ NAA. Rooting was most effective with 2.0 mg L⁻¹ IBA, yielding an average of 11.7 roots per plantlet. The genetic fidelity of regenerated plantlets was confirmed using inter-simple sequence repeat (ISSR), start codon targeted (SCoT), and inter-primer binding site (iPBS) markers, showing 93–96% monomorphism, indicating high clonal uniformity. Comparative foliar micromorphology between the mother plant and the regenerants further confirmed phenotypic stability. Biochemical assays revealed high antioxidant activity and elevated phenolic and flavonoid contents in in vitro plantlets. Liquid chromatography-mass spectrometry (LC-MS) and total chemical constituent (TCC) analyses confirmed key bioactives such as kaempferol, ethyl cinnamate, methyl cinnamate, p-cymene, and germacrene. Overall, nanoparticle supplementation improved regeneration and preserved phytochemical integrity in K. galanga.