The dye gentian violet was added to culture medium in order to distinguish in vitro and ex vitro-formed roots during acclimatization of micropropagated plantlets. Shoots of the grapevine rootstock Kober 5BB were rooted on media containing the dye (0.3 and 0.15 mg·l−1) for 3 weeks. The dye coloured the roots. Root length was reduced by the presence of the dye, but root number and shoot growth were not affected. Most in vitro-formed roots continued to grow during acclimatization, and 3 weeks after the transfer to soil the root system was 60% composed of in vitro-formed roots. Our results suggest that in grapevine Kober 5BB, the in vitro-formed roots contribute to plantlet growth at least during acclimatization.