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The production and commercialization of natural antioxidants is gaining increasing importance due to their wide range of biological effects and applications. In vitro cell culture is a valuable source of plant bioactive compounds, especially those highly dependent on environmental factors. Nonetheless, research on the accumulation in plant cultured cells of water-soluble antioxidant vitamins, such as the ascorbic acid (AsA), is very limited. Tomato fruits are a main dietary source of vitamin C and in this work, we explored the potential of in vitro cultured cells for AsA accumulation. Specifically, using a full factorial design, we examined the effect of the source explant, the time in tissue culture and the genetic difference present in two Introgression Line (IL7-3 and IL12-4) that harbor Quantitative Trait Loci (QTLs) for ascorbic acid in fruits. Moreover, we performed an expression analysis of genes involved in AsA metabolism to highlight the molecular mechanisms that can account for the difference between fruit explants and calli. Our work indicated that cultured tomato cells accumulate AsA well beyond the amount present in fruits and that the three factors under investigation and their interaction significantly influence AsA accumulation. The time in tissue culture is the main single factor and, different from the expectations for secondary metabolites, explants from unripe, mature green fruits provided the highest increase in AsA. Moreover, in controlled conditions the genetic differences between the ILs and the control genotype are less relevant for calli cultivated for longer time. Our work showed the potential of tomato cell culture to produce AsA and prompt further refinements towards its possible large-scale exploitation.