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The aim of this study was to evaluate the growth characteristics and pigment content of tomato plantlets grown under various ratios of red (R) (661 nm) and blue (B) (449 nm) LED light. In this study, three different ratios of R and B (RB) light such as 5:01, 10:01, and 19:01 along with R (100%) were used. The photosynthetic photon flux density (PPFD), and photoperiod of the growth chamber was 120 ± 5 μmol m^−2s^−1 and 16/8 h (day/night), respectively. Tomato plantlets were cultured for six weeks in the growth chamber. It was shown that tomato plantlets had higher photosynthesis rate, higher pigments content, higher growth characteristics (e.g., number of leaves, leaf area, shoot number, root number, root length, dry, and fresh mass), and greater surviving rate under the R:B = 10:01 ratio among the treatments. The plantlets showed at least a threefold decrease in photosynthesis rate, as well as a significant abnormal stem elongation when grown under 100% R light. It is concluded that the RB ratio of 10:01 showed excellent performance in all growth parameters. This result has shown that the optimum lighting environment improves tomato plantlet cultures in vitro.