Abstract The present study reports that a revised nutrient concentration in the basal medium improved shoot bud induction and subsequent plant regeneration in barley (Hordeum vulgare L. var. BL-2). Cultures were raised from immature embryos on MSB5 medium supplemented with picloram. Concentrations of five nutrients were varied. The effect of these nutrients was investigated on (1) induction, (2) induction and subculture, and (3) induction, subculture and regeneration stages. The basal MSB5 medium was not optimal for each phase of barley culture. Decreased ammonium nitrate, increased potassium dihydrogen phosphate, sodium molybdate, cobalt chloride, and addition of glycine enhanced shoot bud induction and plant regeneration. The different media that were optimal for immature embryo culture were: MSB5 medium supplemented with 20.70 μM picloram, 10.30 mM NH4NO3, 6.25 mM KH2PO4, 2.06 μM Na2MoO4, 0.55 μM CoCl2, and 26.64 μM glycine (for induction); MSB5 medium supplemented with 12.47 μM picloram, 10.30 mM NH4NO3, and 0.55 μM CoCl2 (for subculture); and MSB5 medium supplemented with 0.2 μM picloram and 10.3 mM NH4NO3 (for regeneration). Primary cultures required 6 wk (without transfer) for morphogenic callus formation. Callus required 4 wk of subculture and another 4 wk on regeneration medium for optimal plant regeneration. The revised medium could also promote regeneration of the recalcitrant barley genotype RD-2552. Histological analysis showed that the major pathway of differentiation was through shoot bud formation.