Previous studies have reported pigments in plant tissue cultures (e.g., betalains in Beta vulgaris) and the effects of blue light, sucrose concentration, and temperature on pigment biosynthesis like anthocyanin. Preliminary studies on Gomphena globosa L. (Amaranthaceae) calli cultures demonstrated pigments (a mixture of at least seven betacyanins) in morphologically undifferentiated calli, indicating this system is suitable for studying cell differentiation control. The aim of this work was to determine the optimum conditions for G. globosa callus growth and characterize factors involved in pigment formation. We investigated the effects of hormone concentration, light wavelength, and carbon source (sucrose) concentration on callus growth and pigment production. Results showed the best callus growth (friable, no differentiation) was achieved with Murashige and Skoog medium containing 10⁻⁶M 2,4-dichlorophenyoxyacetic acid (2,4-D) + 10⁻⁸M benzyladenine (BA), 3% sucrose, and incubation under blue light. Higher sucrose concentrations (5% or 7%) increased dry weight, likely due to starch accumulation rather than cell number. Blue light induced greater pigment production compared to white or red light. Callus incubated at 24℃ exhibited more pigmentation than at 28℃, though growth decreased at the lower temperature. Maximum pigment content was observed around the 24th day of incubation. Media with 10⁻⁵M naphthalene acetic acid (NAA) + 10⁻⁹M BA (3% sucrose) led to poor propagation but differentiation into roots (more abundant in 5% or 7% sucrose), while 10⁻⁵M NAA + 10⁻⁸M BA resulted in shoot predominance with little root differentiation.