In order to provide a rapid and useful method for propagation of Withania coagulans medical plant under in-vitro condition was conducted based on completely randomized design experience in tissue culture laboratory of Agricultural Research Center of Khorasan Razavi Province. At first, sterilized seeds are placed in MS media (collected from natural habitat in Sistan and Baluchistan Province). Then for shoot regeneration internode subculture placed in MS media, including Benzylaminopurine (BAP) in 5 levels (0, 0. 5, 1, 1. 5, 2 mg/L). In step 2, for root regeneration placed in MS media in including indole-3-butyric acid (IBA) in 4 levels 0. 5, 1, 1. 5, 2 mg/L and Naphthaleneacetic acid (NAA) in 0. 5, 1, 1. 5, 2 mg/L), also, control treatment (non-application of hormone) was considered. Finally, in order to adapt to seedling, they were kept in an adaptive room for 30 days. The highest proliferation index, number of branches, and number of leaf belong to 0. 5 mg/L BAP treatment and the best concentration of growth regulators with 90% marcotting was in the treatment of IBA at 0. 5 mg/L concentration. Finally, by transfer of regenerated explants to the soil, 80 percent of them survived. In general, for the production of strong and succulent seedlings, the use of BAP and IBA at 0. 5 mg/L concentration is recommended for branching and marcotting, respectively.