<p>Increasing global demand for energy along with climate change claims for sustainable and renewable energy sources. In this study, we investigated transcriptomic changes in two cardoon (<i>Cynara cardunculus</i> L. var. <i>altilis</i>) calli cell culture lines engineered for production of valuable fatty acids, therefore suitable for large-scale production of biofuel precursors, as alternative to triacylglycerols (TAGs) engineered plants. Namely, to gain insight in the molecular effects of genetic transformation, we analyzed the transcriptome of <i>SAD</i>-OE line (overexpressing stearic acid desaturase), and <i>FAD</i>-KD line (a fatty acid desaturase RNA interference line, FAD2.2-RNAi) accumulating oleic and linoleic acids, respectively. Differentially expressed genes (DEGs) involved in fatty acid metabolism and streamed pathways such as glycolysis, pyruvate, glycerolipid, and glycerophospholipid metabolism, were enriched in the KEGG analysis in both lines. In addition to analyzing DEGs, we used qRT-PCR to monitor key regulatory and biosynthetic genes involved in TAGs formation and assembly, aiming to understand their transcriptional dynamics over ten days of cell growth in liquid culture. In transgenic scaled up lines, upon observing increased lipid accumulation, we investigated how genetic transformation influenced sugar metabolism. Fructan depletion analysis further supported their role as carbon sources for TAG biosynthesis. Confocal microscopy confirmed a significantly higher accumulation of oil bodies in transgenic lines compared to wild-type (WT) calli. Overall, this study presents a comprehensive characterization of a cardoon cell-based platform for large-scale vegetable oil production. Unlike whole plants, these engineered cell cultures do not suffer from severe oxidative stress or growth and metabolic impairments, making them a promising alternative for sustainable fatty acid biosynthesis.</p>