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Micropropagation allows large-scale plant multiplication and germplasm preservation, representing an added value in forest breeding strategies to combat desertification and/or protect endangered species. We developed a large-scale micropropagation protocol of Olea maderensis (a native endangered wild olive of Madeira Archipelago) using OMG medium (rich in Fe, Mg and Mn) supplemented with zeatin for elongation and with NAA for rooting. We now describe the performance of micropropagated plants during five-period field acclimatization: (1) in vitro, (2) growth-cabinet, (3) greenhouse, (4) open-greenhouse, and (5) field mountain in Porto Santo Island. One hundred OG4 plants were acclimatized, showing >95% surviving rates. During acclimatization, several physiological parameters were evaluated; water content remained higher in in vitro/greenhouse conditions, decreased in field leaves. Soluble protein contents decreased during the first acclimatization periods increasing thereafter. Membrane permeability slightly increased during the field acclimatization. Chlorophylls content increased in in vitro leaves, while during acclimatization, mostly chl b decreased, increasing chl a/chl b ratio. F-0 decreased in first acclimatization periods, increasing thereafter, while the other parameters (F-v; F-m; F-v/F-m) decreased. Nutrient contents decreased in plants transferred to poor field soil conditions, reaching values similar to mother plant leaves. Overall, with the exception of PSII fluorescence, field acclimatized plants had similar values to mother plants, showing a good adjustment to stressful field conditions. This protocol is being used in large-scale micropropagation within a reforestation program, and is an example of R&D technologies with immediate application on protection of endangered ecosystems.