Garlic (Allium satium L.) is categorized as an important medicinal plant worldwide which was known for its very high therapeutic value. Several biotic and abiotic factors may affect its sustainable yield and quality. Viral diseases of garlic Asexual mode of propagation may cause of spread of viral diseases in garlic which may be eliminated through in vitro techniques. Therefore, research was carried out to determine effect of different sterilization methods, carbon source and explant on in vitro production of disease-free garlic. The highest numbers of clean cultures were obtained using treatment combination of 70% ethanol 1 min-1 and 55% Sodium Hypochlorite (NaOCl) 20 minutes-1. All other treatments either fail to produce clear cultures or reduced the efficiency of germination. Sugar and maltose were used as carbon source and no significant variation was observed among them during germination of cloves Two commercial varieties NARC G1 and White garlic were used as source of explant. Calli mass were assessed after interval of every 10-15 days and subsequently sub-cultured. Data was obtained after 60 days. Both accessions produced maximum callus (59.73% and 58.39% respectively) when used shoots were used explant. The calli were yellowish white in hue and had a dense and nodular morphology. Different concentrations of 2, 4-D showed significant variations in the average frequency of induced calli, germinated shoots explant of NARC G1 variety of garlic produced the highest calli at plant growth regulator concentration of 3.0 mg L-1 of 2,4-D. The result of present studies may provide a source of disease-free material for genetic manipulation of garlic genotypes.