This study was aimed to optimize the organogenesis in rough lemon (Citrus jambhiri Lush), an important citrus rootstock of India. Organogenesis was induced in epicotyl segments of rough lemon seedlings. Three important factors influencing organogenesis in vitro viz. hormone combination, cut modes and photoperiod were evaluated. Etiolation of seedlings had a positive effect on bud formation and higher number of buds per explant was obtained from etiolated seedlings. Among the cut modes, transverse cut performed better for its effect on the number of regenerated buds and shoots per explant. The best response regarding the number of adventitious buds formed per explant was observed with BAP at the concentration of 2.0 mg/l along with malt extract (500 ppm) and additional dose of 25 g/l sucrose.Key words: Citrus, epicotyl, hypocotyl, etiolation, transverse cut, longitudinal cut, photoperiod.