Key message <p><b>Manipulating nitrogen and micronutrient concentrations in MS medium significantly enhances Ilex paraguariensis shoot regeneration and reduces oxidative stress, offering a more efficient approach for large-scale plant production</b></p> Abstract <p><i>Ilex paraguariensis</i>, commonly known as yerba mate, is an economically important plant cultivated in South America. Its propagation is hindered by low rates of rooting by stem cuttings in traditional vegetative methods and high contamination levels in tissue culture. This study aimed to optimize in vitro shoot production from nodal explants. of yerba mate by adjusting the concentrations of NH₄NO₃, KNO₃, and micronutrients in the Murashige and Skoog (MS) culture medium using a Box-Behnken experimental design. The results identified the best-performing combination for shoot regeneration: NH₄NO₃ at 0.5× MS or 0.2× MS, KNO₃ at 0× MS or 0.10× MS, and micronutrients at 1.5× MS, achieving shoot regeneration rates of up to 30%. Lower KNO₃ concentrations favored regeneration, while interactions between NH₄NO₃, KNO_3, and micronutrients significantly influenced shoot formation. However, oxidation remained high (90%) and was not directly affected by nutrient composition, and 26.93% of explants were lost due to contamination. Among the surviving explants, a higher NH₄NO₃ / KNO_3, ratio was correlated with a lower accumulation of phenolic compounds, but no direct impact on oxidation was observed. These findings reinforce the importance of precise nutrient optimization in improving shoot formation from axillary buds efficiency, bringing the possibility to future large-scale production of genetically uniform plants for food and medicinal applications.</p>