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Transformed hairy root cultures of Valeriana officinalis were established by infection with Agrobacterium rhizogenes strain ATCC 15834. To determine the effect of different media on the growth of V. officinalis hairy roots, MS, B5 media (1.0X and 0.5X strength), N6 medium and a modified MS medium without phytohormones were used. In addition, different NH4+ to NO3- ratios in MS medium were studied. The effects of these treatments were evaluated after 21 days of culture in relation to hairy root growth. B5 and ½ B5 media were the best basal media for hairy root growth. MS medium supplemented with a 20:20 ratio (mM) of NH4+ to NO3- displayed highest growth rates and biomass yield in hairy root cultures. The present study demonstrated that the composition of culture medium and the ratio of different nitrogen sources have significant impact on the growth of V. officinalis hairy roots.