Integrative metabolomic and genetic analysis of in vitro regenerated Populus alba in response to nano hydroxyapatite

Nano-elicitation has gained increasing attention as a strategy to enhance plant growth and metabolic performance in vitro. However, the genetic and metabolomic consequences of nano-hydroxyapatite (nHAp) application in woody plant species remain poorly understood. This study examined the influence of biosynthesized nHAp on growth performance, genetic variation, metabolomic remodelling, and metabolic pathway regulation in in vitro regenerated Populus alba . The biosynthesized nHAp exhibited a rod-like morphology with average dimensions of 60 nm × 213 nm and a negative surface charge (zeta potential − 22.1 mV). The effects of nHAp were examined at three concentration levels: 20, 40, and 60 mg/L. Supplementation with nHAp significantly improved vegetative growth parameters, including weight, shoot and root length, leaf number, and adventitious root formation, compared to the control. Random Amplification Polymorphic DNA (RAPD) and Start Codon Targeted (SCoT) analysis revealed genotypic variation, with polymorphism percentages of 39.16% and 34.4%, respectively. Based on 1 H and 1 H– 13 C Heteronuclear Single Quantum Coherence (HSQC) Nuclear Magnetic Resonance (NMR) data, thirty-five metabolites were identified. Treatment with nHAp resulted in significant metabolomic changes, characterized by marked alterations in the abundance of primary and secondary metabolites and the enrichment of their biosynthetic pathways. Sucrose served as a key metabolic biomarker, detected exclusively in control samples but depleted in nHAp-treated samples. Conversely, nHAp-treated samples were characterized by the accumulation of phenylalanine, quinic acid, and other cell wall precursors. Trigonelline and catechol concentrations were significantly upregulated with the addition of 40 mg/L nHAp. The highest nHAp concentration (60 mg/L) significantly increased the levels of γ-aminobutyric acid, asparagine, catechol, and cellobiose. Pathway analysis identified significant enrichment, with the aminoacyl-tRNA biosynthesis and ABC transporter pathways being the most significantly enriched. These findings confirm that nHAp treatment enhances vegetative growth parameters and induces both genetic and metabolomic variation in nodal cuttings of Populus alba . The metabolic reprogramming, specifically the shift from sucrose storage to structural carbohydrate and amino acid synthesis, underscores the potential of nHAp as effective nano-elicitors in plant biotechnology.