Abstract:Calli derived from leaf explant of sugarcane F134 pollen plant were used as the material for isolating protoplasts. Protoplasts were cultured in modified MS medium by means of liquid-thin layer culture. The first cell division of protoplasts was observed within a week in culture. Calli were formed about 5–6 weeks after culture. Selected calli consisting of embryonic cell masses were transferred onto differentiation medium without 2,4-D or with lower concentration of 2,4-D, but supplemented with BA. About 2–3 weeks, coleoptiles have been developed in some calli, radicls or roots were differentiated in some others. The process of somatic embryogenesis from protoplasts traced systematically from the first division to the formation of calli.