Asimple 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 mMIAA and rooted on MS 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 commercialvalue of scopolamine than the other tropane alkaloids, tetraploidy can efficiently be used to improve scopolamine production of H. reticulatus.