In Europe, Douglas-fir is a major species for reforestation with increasing demand for its wood. Therefore, adaptation of new varieties to climate change is one challenging question for ongoing breeding programs. Efficient selection and vegetative propagation of improved varieties appeared to be key issues to maintain productivity in plantation forestry (Lelu-Walter et al. 2013). Somatic embryogenesis, coupled with cryopreservation, is a promising clonal propagation system of selected trees that has been successfully improved in Douglas-fir (Reeves et al. 2018; Lelu-Walter et al. 2018). Repetitive somatic embryogenesis from cotyledonary somatic embryos (SEs) has been obtained for two unrelated Douglas-fir genotypes, producing secondary and tertiary embryogenic lines. Each one exhibited significantly higher embryogenic potential for both genotypes compared to primary or secondary lines, respectively (increase of 63 to 727%). The origin of such differences in embryogenic potential is unknown. Our objective was to study changes induced in embryonal masses (EMs) after repetitive somatic embryogenesis at the histo-cytological and molecular levels (LC-MS-based proteomics). Repetitive somatic embryogenesis improved the EM structure by increasing frequency of small, singulated immature SEs together with reducing the size of polyembryogenic centers. Each cycle of embryogenesis induced a modification of the expression of proteins related with biological processes and already known to be involvedin somatic embryogenesis but quite new for EMs i.e.plant development, defense response, metabolism, proteolysis, and stress. The innovative implementation of protein networks in our proteomic analyses had been very conclusive. The networks revealed a global down or up pattern of protein expression after the first or second cycle of somatic embryogenesis, respectively. For both patterns, interactions with different plant growth signaling molecules (flavonoids and associated compounds, jasmonic acid, ABA, auxin, salicylic acid, lignin) were predominant. It is shown that cells have the ability to use different protein regulatory pathways to result in increased embryogenic potential (cotyledonary SE production). In Douglas-fir and in conifers, this is the first report describing cellular and molecular changes in EMs obtained after two successive cycles of repetitive somatic embryogenesis (Gautier et al. 2018). The results contribute to a better understanding of the cytological and proteomic changes associated with to enhanced embryogenic potential of secondary and tertiary embryogenic lines.< Réduire