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In vitro organogenesis of Lisianthus [Eustoma grandiflorum (Raf.) Shinn] derived from leaf explant as explant source was successfully studied. High callus formation up to 50% in leaf explants was established on Gamborg B5 medium supplemented with 0.1 mg L-1 α-naphatelene acetic acid (NAA) and 0.5 mg L-1 N6-benzylamino purine (BAP). The callus was initiated on the Gamborg B5 medium fortified by 0.1 mg L-1 2,4-dichloropenoxy acetic acid (2,4-D) and 0.5 mg L-1 BAP and successfully produced 18.7 shoots per explant in 28 days under light incubation. The best growth performance of plantlet without rosette problem was recorded on Murashige and Skoog (MS) medium augmented with 10 mg L-1 giberellic acid (GA3) with 3 shoots per planlet and 9.3 cm shoot height after 4 week in culture. The shoots were easily rooted on MS medium containing 2.0 mg L-1 indole-3-acetic acid (IAA) and 1 mg L-1activated charcoal (AC) with 1.9 number of roots per shoot and 1.8 cm root length after 4 weeks in culture. Plantlets derived from the study were successfully acclimatized during 6 weeks on a mixture of burned rice husk and cocopeat (1:1, v/v) with 80% survivability. Results of the study give high chance in producing well planting materials for growing E. grandiflorum cultivation in Indonesia.