| Summary: | <i>Eryngium foetidum</i> L., a biennial herb with diverse applications in food and traditional medicine, holds economic and pharmacological significance. Given its growing commercial interest, implementing biotechnological approaches like plant tissue culture is vital for sustainable propagation and metabolite production. In this study, we investigated the <i>in vitro</i> photoautotrophic potential of <i>Eryngium foetidum</i>, examining growth, chlorophyll <i>a</i> fluorescence, photosynthetic pigments, and anatomical features under sucrose concentrations (0 and 30 g L<sup>−1</sup>) and gas exchange rate (14 and 25 μL L<sup>−1</sup> s<sup>−1</sup> CO<sub>2</sub>). Acclimatization and survival rates of plants after <i>ex vitro</i> transfer were also assessed. <i>Eryngium foetidum</i> exhibited robust growth in both photoautotrophic and photomixotrophic conditions, with natural ventilation significantly enhancing plant development. Chlorophyll <i>a</i> fluorescence and photosynthetic performance were influenced by sucrose and gas exchange, highlighting the importance of these factors in plant micropropagation. Moreover, the species demonstrated remarkable plasticity during acclimatization, with high survival rates and rapid inflorescence development. The research provides valuable insights into optimizing <i>in vitro</i> cultivation conditions for <i>Eryngium foetidum</i>, emphasizing the potential for large-scale clonal propagation and exploring secondary metabolites. The observed phenotypic plasticity underscores the adaptability of the species to diverse environments. These biotechnological strategies open avenues for future studies, including the application of elicitors for enhanced secondary metabolite production.
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