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Coffee Coffea arabica L. depends on abundantly distributed rainfall, and drought negatively impacts plant development, fruit production, bean quality, and, ultimately, beverage quality. Plant biotechnology by means of genetic manipulation and plant regeneration by the somatic embryogenic process is an alternative technology to overcome these problems. In the present work, we used the molecular approach of the Trehalase gene silencing to allow trehalose accumulation favoring plants surviving in extreme drought/salt environments. We used a cassette containing the antisense C. arabica L. Trehalase gene under the RD29 promoter from A. thaliana and the NOS terminator to genetically modify an embryogenic coffee C. arabica L. cv Typica line under osmotic stress supplemented with mannitol (0.3 M) and sorbitol (0.3 M). Osmotic stress-tolerant somatic embryos lines were recovered and regenerated into plants. Tolerant somatic embryo lines showed a higher rate of competence to induce secondary SE capacity and plants robustness. These lines showed a down-regulation of the Trehalase; accumulation of trehalose, sucrose, starch, and proline; higher photosynthetic rate; improved water-use efficiency; and appropriated vapor deficit pressure under soil conditions. A transcriptome analysis was performed from highly competent somatic embryogenic lines to understand the molecular mechanisms underlying osmotic-stress tolerance. From the up-regulated genes, a PPI network made by STRING v12.0 with high confidence (0.700) revealed the presence of the 10 modules: the cell cycle, chromatin remodeling, somatic embryogenesis, oxidative stress, generic transcription pathway, carbon metabolism, phenylpropanoid biosynthesis, trehalose biosynthesis, proline biosynthesis, and glycerolipid metabolism. Full article