Key message Manipulation of PGR'S significantly enhanced in vitro tuberization from noncorm bud explants. Elicitor treatment with AlCl3 enhanced the production of colchicine. HPLC analysis revealed significantly higher colchicine content in in vitro raised plants compared to field grown tubers. The study will help in mass propagation, conservation, and commercialization of Gloriosa superba L. for large scale production of colchicine. Gloriosa superba L. tubers are a rich source of commercially important colchicine and due to overexploitation, the species has become vulnerable. In the present investigation, in vitro tuber productions were carried out for its propagation and conservation. The in vitro and field-grown tubers were assessed for their colchicine content and antimicrobial activities. Maximum callusing was obtained when the medium was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylaminopurine (kinetin). Among different auxins tested for in vitro tuberous root production, IAA (1.5 mg/L) induced 78.2% tuberous root per callus. In vitro micro-tuber raised in media supplemented with 1.5 mg/L thidiazuron (TDZ) recorded the highest response (72.9%) with 28.4 tubers per explant. Sucrose (6%) with TDZ (1.5 mg/L) produced significantly more micro-tubers per callus. Elicitor treatment with AlCl3 at 125 mu M and 150 mu M resulted in a significant increase in the micro-tuber and tuberous root production respectively. N6-(2-Isopentenyl) adenine (2ip) (1.0 mg/L) induced the highest frequency of in vitro micro-tuber sprouting and tuber formation compared to 6-benzylaminopurine (BAP) and 1-naphthalene acetic acid (NAA). The elicitor-treatments with AlCl3 significantly increased the colchicine content of in vitro tuberous root and tubers than that of the field grown tubers. The anti-microbial activity of in vitro raised tubers, tuberous roots and AlCl3 treated samples were significantly higher compared to the field grown samples. An optimized tissue culture system for mass propagation of G. superba with conservation aspects and the production of high-value colchicine is presented here, which can be used in various medicinal systems.