A growth regulators-free anthocyanin-producing habituated callus line of Tarenaya rosea was establishment through the selective subculture of cell aggregates from a 2,4-D-dependent anthocyanin callus line to a growth regulators-free MS medium (MS0). After the establishment of the habituated line, new culture conditions were evaluated in order to increase de pigment production. Calluses were transferred to MS0 containing different sucrose concentration (30; 50; 70; 90 g.L-1), total nitrogen concentration (50; 60; 70 mM), NH4+ to NO3 - ratio (1:1; 1:2; 1:4; 1:6), and total mineral salt concentration (MS; MS1/2; MS1/4). The most suitable culture conditions to pigment induction have combined to create two new culture formulations, named M1 (70 mM total nitrogen + 70 g.L-1 sucrose) and M2 (1:4 NH4 +:NO3 - ratio + 70 g.L-1 sucrose). Calluses cultivated on M1 and M2 reached an increase on anthocyanin productivity of 3- and 4-fold, respectively, when compared to cultures maintained in standard MS0 medium. The present work demonstrated the feasibility of eliminating the supplementation with 2,4-D in callus cultures of T. rosea without the loss of anthocyanin production. Moreover, the manipulation of basal medium and sucrose concentration contributed with the increment in anthocyanin content.