Hairy roots were induced from shoot buds and seedling hypocotyls of Holostemma by infection with agropine type Agrobacterium rhizogenes strains. Type of explant, Agrobacterium rhizogenes strain used for infection, co-culture time and photoperiod influenced the transformation frequencies. Hairy roots were induced from seedling hypocotyls and shoot bud explants upon infection with agropine type Agrobacterium rhizogenes strains. The hairy roots were thin, whitish in colour and showed negatively geotropic growth. The transformed nature of hairy roots was confirmed by opine analysis.