Abstract: Protocol for induction of callus and regeneration response of two sugarcane varieties (Isd-16, Isd-28) was established through callus culture using leaf sheath. Multiple shoot regeneration at various frequencies was observed using different concentrations and combinations of growth regulators. The highest percentage of callus induction was observed in the medium containing 3.0 mg l -1 2,4-D with 10% coconut water (CM). The best response in terms of multiple shoot formation was observed that on MS medium supplemented with BAP 1.0 mg l -1 +IBA 0.5 mg l -1. NAA (3.0 mg l -1) was found effective in the production of roots. The variety Isd-16 showed better response than the variety Isd-28 towards shoot multiplication. Seventy percent of the plantlets produced from in vitro culture method survived in the ex vitro condition.