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The present investigation is an attempt towards finding an effective sterilization protocol for two, field grown sugarcane varieties, CoS 88230 and CoS 767 from Meerut region and to establish the callus culture protocol. Four sterilizing agents EtOH, NaOCI, HgCI... and H... O... were used alone and in combination. The finding suggests that the use of only one sterilizing agent is not successful for the sterilization of leaf sheath explant of sugarcane. HgCI...(0.1%) for 5 min and EtOH (90%) for 10 min was found best when used along with prior washing and surface sterilization with 20 and bavistin. An increased response of callus initiation was achieved on MS medium supplemented with 2, 4-D (1 mg L...) and antibiotic amoxicillin (2 mL L...). present research attempt will allow mass multiplication of economically important sugarcane varieties through tissue culture technology can be used further to complete the demand of …