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<p><i>Euphorbia milii</i> is a prominent medicinal species with established therapeutic potential; however, the systematic chemical transition occurring during its in vitro morphogenesis remains largely unexplored. While previous studies have addressed isolated aspects of micropropagation or basic phytochemical screening, this research presents the first integrated study combining optimized in vitro regeneration with advanced LC-ESI-MS/MS metabolomics and GNPS molecular networking. We utilized this multi-platform approach to perform a comparative evaluation of the metabolic profiles across the mother plant (MP), regenerated leaves (RL), callus leaf (CL) and callus stem (CS). Optimal callus induction from leaf explants required MS media supplemented with 1&#xa0;mg/L BA (6-benzyladenine) + 2&#xa0;mg/L NAA (naphthalene acetic acid). Single node callus induction was successful with 1&#xa0;mg/L or 0.5&#xa0;mg/ L BA + 2&#xa0;mg/L or 0.5&#xa0;mg/L NAA (respectively) and 0.5&#xa0;mg/L 2,4-D with 1&#xa0;mg/L Kin (<i>N</i>6-furfurylaminopurine). Indirect regeneration was established on MS medium supplemented with 0.5&#xa0;mg/L BA + 0.5&#xa0;mg/L NAA. Quantitative analysis revealed that the MP extract possessed the highest total phenolic content, which was further validated by targeted MRM-LC-ESI-MS/MS quantification of key phenolics. Untargeted profiling led to the characterization of 52 constituents for the first time in the <i>Euphorbia</i> genus. Hierarchical clustering analysis (HCA) demonstrated that while the RL extract maintained the highest compositional similarity to the MP, the dedifferentiation process significantly altered the constituents’ profile in CL and CS. This study underscores the utility of molecular networking in mapping the metabolic shifts of in vitro systems, establishing them as effective tools for specialized metabolite discovery and plant conservation.</p>

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Integrated micropropagation and metabolomic mapping of Euphorbia milii tissues and callus cultures

  • 期刊论文

  • 发表日期：

    2026年6月30日

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  • Mona A. Raslan,
  • Heba D. Khlifa,
  • Heba-tollah M. Sweelam

摘要

Euphorbia milii is a prominent medicinal species with established therapeutic potential; however, the systematic chemical transition occurring during its in vitro morphogenesis remains largely unexplored. While previous studies have addressed isolated aspects of micropropagation or basic phytochemical screening, this research presents the first integrated study combining optimized in vitro regeneration with advanced LC-ESI-MS/MS metabolomics and GNPS molecular networking. We utilized this multi-platform approach to perform a comparative evaluation of the metabolic profiles across the mother plant (MP), regenerated leaves (RL), callus leaf (CL) and callus stem (CS). Optimal callus induction from leaf explants required MS media supplemented with 1 mg/L BA (6-benzyladenine) + 2 mg/L NAA (naphthalene acetic acid). Single node callus induction was successful with 1 mg/L or 0.5 mg/ L BA + 2 mg/L or 0.5 mg/L NAA (respectively) and 0.5 mg/L 2,4-D with 1 mg/L Kin (N6-furfurylaminopurine). Indirect regeneration was established on MS medium supplemented with 0.5 mg/L BA + 0.5 mg/L NAA. Quantitative analysis revealed that the MP extract possessed the highest total phenolic content, which was further validated by targeted MRM-LC-ESI-MS/MS quantification of key phenolics. Untargeted profiling led to the characterization of 52 constituents for the first time in the Euphorbia genus. Hierarchical clustering analysis (HCA) demonstrated that while the RL extract maintained the highest compositional similarity to the MP, the dedifferentiation process significantly altered the constituents’ profile in CL and CS. This study underscores the utility of molecular networking in mapping the metabolic shifts of in vitro systems, establishing them as effective tools for specialized metabolite discovery and plant conservation.