In vitro methods provide a variety of tools to supplement traditional methods for collecting, propagating and preserving endangered plant species. In this study, an efficient protocol was developed for in vitro propagation of Colutea gifana, a rare and endangered plant species with limited reproductive capacity that grows in a very narrow area of Iran. Single nodes explants were used for a series of experiments to select appropriate disinfection method and growth regulators for establishment, proliferation and rooting stages. Explants showed the highest establishment percent after 15 min treatment with 2% sodium hypochlorite (NaOCl) cultured in MS medium plus 2.2 μM 6-benzylaminopurine (BAP) and 1 μM indole-3-butyric acid (IBA). BA was more effective cytokinine in comparison to Thidiazuron (TDZ) and Kinitin (Kn) in proliferation stage and the best result was obtained with 8.8 μM BA. In vitro rooting of proliferated shoots were induced in half-strenght MS medium with both tested auxins i.e. IBA and α-naphthaleneacetic acid (NAA). Eighty percent of the plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development. These plantlets can be used to replenish declining populations in the wild to conserve C. gifana from extinction and also for further studies about this species

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