Hyoscyamus muticus is cultivated in Egypt, India, Pakistan and USA for the production of the medicinally important tropane alkaloids (TA) hyoscyamine and scopolamine. The TA pathway provides an excellent model to test the feasibility of transgenic strategies to modify secondary metabolic flux. However, H. muticus transformation and regeneration has been proven difficult. We report a successful experience obtaining several independent fertile plant lines, genetically engineered to redirect the TA metabolic flux into alternative directions. cDNA from H. niger encoding tropinone reductase enzymes (TR1 or TR2), which catalyse the reduction of tropinone into scopolamine and hyoscyamine or calystegines, respectively, were incorporated in a silencing vector, followed by Agrobacterium tumefaciens transformation. Most explants produced large amounts of calli. Shoot formation occurred in the majority of the calli, being ready for rooting after an average of 9 months. On average, 45% of the shoots formed roots after 4 months. However, most rooted shoots died during ex vitro acclimatization, regardless of substrate type and cultivation conditions. This problem was overcome by the development of a specialized protocol (cultivation on soil like substrate under sterile conditions) that allowed the recovery of a reasonable percentage (44%) of fertile plants per rooted shoot. In this study were obtained 11 tr2 lines (25 plants), three tr1 lines (3 plants), four empty vector lines (13 plants) and three untransformed-regenerated lines (3 plants); a significant result compared to previous attempts to stable transform H. muticus.