Gladiolus is a non-native, costly but an important flowering plant in South Asia. Considering its lower production rate and economic importance, micro-propagation technology was optimized in this study to establish a protocol for
pathogen free clonal genotypes. Taken the results together, the best response was observed at 27°C as growing temperature, table sugar (sucrose) as a carbon source, apical meristem of 3mm for shoot formation as an explant. The best plant regeneration was observed for shoot apical meristem (within 7.8 days after inoculation) by using Murashige and Skoog MS basal medium with supplementing 1.0 mg/L BAP and 0.5 mg/L of Kinetin. The best shoot
multiplication was 98% in 18.2 days of inoculation using MS media with 1.0 mg/L BAP only. For root induction, MS media which was supplemented with 1.0 mg/L NAA and 2.0 mg/L IBA gave the best results (100% in 6.8 days). The best supporting agent was found to be cotton swab while the best media for hardening and acclimatization of well-developed plants was a combination of sand, soil, and peat in an equal ratio (1:1:1), leading to 80% survival rate of plants. The results obtained in this study may better help grow Gladiolus plants on an industrial scale with a lower cost of production.