Red nightshade (Solanum alatum Moench.) is one of the important medicinal plants from the Solanaceae family. Tissue culture technology is a very useful application of biotechnology which can be used for plant preservation, rapid multiplication and in genetic engineering for molecular breeding or improving secondary metabolite production. In this study, plant regeneration from three different explant sources derived from young seedlings, including hypocotyl, coleoptile and leaf segments, was evaluated. In this research, efficient protocols for regeneration of red nightshade from all three explants are reported. The best shoot regeneration response (100%) was obtained at 0.5 mg/l BAP concentration for leaf explants. Regenerated plants adapted to greenhouse conditions with a high efficiency (100%) and produced normal flowers and seeds. Also, leaf explant was found to be the most efficient explants for plantlet regeneration, with a mean number of 29.93 shoots per explant. Finally, the potential of leaf explants for gene transfer and expression by means of a gene gun (at two rupture disk pressure of 1100 and 1350 psi), was evaluated using transient GUS expression, and leaf explants were found to be suitable for this purpose.