As regards the kind of use and production of biomass, lettuce plant has a potential to be used as a bioreactor for producing recombinant proteins as well as edible vaccines. For this propose optimized tissue culture as well as gene transformation of Lactuca sativa are needed. Seeds of 5 cultivars namely TN-96-35, TN-96-36, TN-96-39, TN-96-41, TN-96-53, and TN-96- 54 were disinfected by being soaked in a solution of 2.5% sodium hypochlorite containing 0.1% Tween 20 for 25 minutes. After being rinsed three times with sterile distilled water, the seeds were made to germinate on filter paper in petri dishes with sterile distilled water in them. The cultures were continuously exposed to white fluorescent light at a constant temperature of 25 °C, for a minimum of 16 hours. When aseptically germinated seedlings were 48 to 72 hours old, cotyledons were excised near the cotiledonary node, and cut into 6 to 8 pieces to increase the wound needed by the explants for an initiation of callus growth.Explants were cultured on MS medium supplemented with different levels of naphthaleneacetic acid [NAA] (0.02, 0.05, 0.1 mg/l) and benzyladenine [BA] (0.1, 0.2, 0.4 mg/l). Two lettuce cultivars (TN-96-39, TN-96-41), showed the best callus production, embryogenesis, regeneration and proliferation. The best growth regulators for callus production and embryogenesis were 0.05 mg/l [NAA] and 0.2 mg/l [BA]. The most suitable growth regulator for direct regeneration as well as proliferation were 0.05 mg/l NAA, and 0.4 mg/l BA. To produce roots, shoots were transferred to MS media contaning 0.2, mg/l NAA. Plantlets of robuts roots were then planted in pots to produce seeds.