A simple and efficient procedure was established for in vitro propagation of lattice henbane (Hyoscyamus reticulatus L.) using shoot tip explants cultured on Murashige and Skoog (MS) medium supplemented with N6-benzylaminopurine (BAP) and indolyl-3-acetic acid (IAA). In vitro-induced autotetraploid plants of H. reticulatus L. exhibiting high yield of scopolamine were successfully induced by different concentrations of colchicine solutions (0.00, 0.05, 0.1, 0.2 and 0.5 %) which were applied for 24, 48 and 72 h. Treated shoot tips were regenerated on MS medium supplemented with 8.8 mM BAP and 2.2 mM IAA and rooted on ½MS medium containing 2.2 mM indolyl-3-butyric acid, then were acclimatized and transferred to soil. According to the results, 0.1 % (w/v) of colchicine for 48 h can be effective for induction of polyploidy in H. reticulatus L. The induced tetraploid plants represented a different structural, physiological and biochemical characteristics. Autotetraploid plants of H. reticulatus also showed a higher conversion of hyoscyamine to scopolamine as scopolamine content was increased from 0.23 in diploids to 8.66 % in the induced tetraploid plants. Regarding the higher commercial value of scopolamine than the other tropane alkaloids, tetraploidy can efficiently be used to improve scopolamine production of H. reticulatus.