During different phases of in vitro culture, plant tissues may be exposed to some stresses that never encounter in their natural habitats. The most significant stresses which interfere with in vitro culture are pathogenic contamination and browning disorder. Since browning sign is occurred during all phases of in vitro culture of Spartium junceum L., the present study was done preventing explants from browning during disinfection and callogenesis phases using exposure time of sterilants (ethanol 0, 30, 60 s and home bleach 0, 10, 15 min), antioxidant compounds (PVP 0.5%, Activated charcoal 0.1%, Curcumin 0.1%), Running water (30 and 60 min) plant growth regulators (2,4-D 0, 0.5, 1 and 2 mg L-1 and BA 0, 0.1 and 0.2 mg L-1), and by changing light/dark conditions was designed. The results showed that ethanol 70% (30 s) in combination with home bleach 20% (10 min) had the best effect in control contaminations and browning sign in nodal explants of S. junceum. The application of PVP 0.5% in medium was the best treatment to control of browning nodal explants in callus induction phase. The highest callus formation and the lowest explant browning were obtained on the medium supplemented with 0.5 mg L-1 2,4-D under the darkness condition. According to the results of this study, how disinfection methods, culture medium compositions and light conditions were effective on the browning and callogenesis of Spartium junceum L.