Solanum nigrum L. is a medicinal plant of solanaceae family with distinguished therapeutic properties. Traditionally, S. nigrum. had been used as an anti-tumorgenic, antioxidant, hepatoprotective, diuretic, and antipyretic agent. The most important alkaloid member in this plant is solanine. Therefore, this study was conducted to utilize tissue culture techniques for the enhancement of solanine production in the in vitro grown cultures of this promising neglected plant. For callus growth and development experimental part, the highest callus growth parameters (callus diameter (21.4 mm) and callus fresh weight (2202.4 mg) were obtained in callus grown on Murashige & Skoog MS media supplemented with 2,4-Dichlorophenoxyacetic acid (2.0 mg•L-1) plus 1.5 mg•L-1 Thidiazuron. Similar trend was also obtained in cell suspension culture experiment, as maximum growth was recorded at similar hormone combination. Moreover, High-performance liquid chromatography analysis revealed that, solanine was affected by growth regulator type and concentration. The highest solanine levels were obtained when the explants were treated with 6-benzylaminopurine at level of 2.0 mg•L-1, as solanine content reached up to (2.61, 1.53 mg.g-1) for callus and cell suspension, respectively, while, microshoot contained the highest solanine (4.52 mg.g-1 DW) at 6-benzylaminopurine level of 1.6 mg.L-1. Additionally, carbon source had positively affected solanine level, where 0.2 M sucrose resulted in production of the highest amounts (3.13, 2.03 and 1.20 mg.g-1 DW) of solanine in microshoots, callus and cell suspension, respectively. Also, exposing microshoots and callus to light intensity of (100 μmol.m-2s-1) yielded the highest solanine content (4.03 and 1.26 mg.g-1 DW, respectively),while the lowest solanine levels (1.50 and 0.48 mg.g-1 DW) were observed in plant material exposed to the lowest light intensity treatment (25 μmol.m-2s-1). Our results were promising for production of solanine especially in the microshoot (4.52 mg.g-1 DW) using 1.6 mg.L-1 of BA, as compared with previous studies which maximized production of solanine only up to 2.33 mg. g-1 DW using 0.5 mg.L-1 of cholesterol in Solanum lyratum.