Allium stamineum, an endangered medicinal plant in Iran, requires conservation efforts through in vitro culture techniques. This study established an effective protocol for callus induction and bulblet regeneration in A. stamineum using various explant types (radicle, basal plate, and cotyledon) and growth regulators. The results indicated that the best callus formation from cotyledon explants occurred in media containing 1 mg/L 2,4-D, 1 mg/L 2,4-D with 0.5 mg/L BA, 2 mg/L 2,4-D with 0.5 mg/L kinetin, and 1 or 2 mg/L 2,4-D with 1 mg/L kinetin. In terms of regeneration, cotyledon explants showed the highest regeneration rate compared to radicle and basal plate explants, with 3.33 regenerations per explant in MS medium supplemented with 1 mg/L kinetin and 1 mg/L NAA. Additionally, the highest bulblet regeneration rate (11 per explant) was obtained from callus on PGR-free medium. The maximum number of roots (7.90) and root length (10.9 mm) were observed on MS medium containing 3 mg/L IBA. Rooted bulblets were successfully acclimatized in pots filled with a cockpit mixture (3:1 v/v) with a 100% survival rate. This study not only provides a successful in vitro propagation technique for A. stamineum but also facilitates its breeding program. Manuscript profile