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A great emphasis of plant research is being placed on developing crops with increased nutritional value. Generating plant materials suitable for controlled studies as potential tools for pre-breeding is still the main hurdle. Improvements are needed in generating different allele combinations to stack various nutrients into a single genotype, without losses in fruit yield or quality, and in testing the specific effects of nutrients in their original matrix, avoiding the noise caused by the characteristic mix of compounds. An elegant approach in both pre-breeding and diet supplementation tests is the use of near-isogenic lines (NILs). Here, we tap on the large pool of monogenic mutants and natural genetic variation available in tomato to create a series of NILs in the genetic background of the cultivar Micro-Tom (MT). We describe the introgression of the mutations Anthocyanin fruit (Aft), atroviolacium (atv), Aubergine (Abg), Beta-carotene (B), old-gold crimson (og) and high pigment 1 and 2 (hp1, hp2) and characterize their fruit metabolic profiles in single, double and triple mutant combinations. We show that Brix can be raised without yield penalty, along with increases in lycopene, β-carotene and ascorbic acid, and a concomitant enhancement of anti-oxidant capacity. As proof-of-concept of the suitability of stacking alleles for breeding nutrient-rich tomatoes, we introduce three mutations leading to uniformly purple fruits and enhanced nutrient contents from MT into a commercial cherry tomato cultivar. [ABSTRACT FROM AUTHOR]