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Background: The Pehuén or Monkey puzzle tree (Araucaria araucana) is an evergreen coniferous tree, which has been historically used for social, medicinal, and nutritional purposes. We have recently showed the value of A. araucana seeds as a rich source of micronutrients and antioxidants. This endemic species present in a reduced area in Argentina and Chile is endangered because of the low germination rate and the overexploitation of its edible seeds. Thus, the massive extraction of its seeds is ecologically non-viable resulting in limited availability of its active metabolites. However, biotechnological approaches are attractive strategies of production of valuable metabolites and healthy specimens of endangered plants. The aim of this work was to develop a protocol for in vitro production of antioxidants derived from A. araucana seeds and to obtain healthy plants by optimized seed germination. Results: Calli of Pehuén seeds were induced in Murashige and Skoog medium with different combinations of auxins and cytokinins, in light and dark conditions. Callus from embryonic axes developed in medium with 1 mg/l α-naphthaleneacetic acid and 1.5 mg/l 6-benzylaminopurine in light conditions had efficient biomass production, antioxidant activity, high phenolic, and flavonoid content and no cytotoxicity on mammalian cells. Additionally, 100 % germination was obtained in vitro and healthy plants were acclimatized to non-sterile conditions. Conclusion: In conclusion, in vitro culture of A. araucana could provide new and sustainable options for production of its valuable metabolites with possible therapeutic and nutritional uses. Also, optimized plant germination and acclimatization of endangered species can contribute to the preservation of pristine environments. [ABSTRACT FROM AUTHOR]