Genetic transformation systems require protocols that allow regenerating transgenic plants from transformed tissues. This study aimed to establish a protocol for indirect organogenesis in leaf explants of a Eucalyptus grandis × E. urophylla AEC 224 clone. During callogenesis stage, several concentrations of NAA and then NAA or 2,4-D combined with TDZ were tested in JADS culture medium for 30 days, followed by subculture of the explants in the regeneration medium, containing 5.0μM BA and 0.5μM NAA for another 30 days. In these media,the explant oxidation rate was high(95 %). Thus, in order to reduce oxidation, different culture media were compared: WPM, MS, JADS and modified QL, followed by explant transfer onto regeneration medium. The highest percentage of regeneration and the lowest oxidation rate were achieved on WPM medium. Then, NAA and 2,4-Dwere tested in combination with TDZ and also TDZ and BA combined with NAA in WPM medium. The most efficient culture media in terms of shoot regeneration were WPM supplemented with 0.25μM TDZ and 0.1μM NAA during 30 days for callus induction and then with 5.0μM BA and 0.5μM NAA for another 30 days. This protocol yielded a regeneration rate of 43 %, with a low oxidation of tissues. A rooting experiment was conducted using half strength MS medium and comparing three concentrations of IBA(2.46, 4.90 and 7.35μM). The highest rooting percentage(35 %) was obtained on medium containing2.46μM IBA. Once the shoots were rooted, acclimatization in a greenhouse was not challenging and plant survival reached 100 %. Genetic transformation systems require protocols that allow to regenerate transgenic plants from transformed tissues. This study aimed to establish a protocol for indirect organogenesis in leaf explants of a Eucalyptus grandis × E. urophylla AEC 224 clone. During caliogenesis stage, several concentrations of NAA and then NAA or 2,4-D combined with TDZ were tested in JADS culture medium for 30 days, followed by subculture of the explants in the regeneration medium, containing 5.0 μM BA and 0.5 μM NAA for another 30 days. In these media, the explant oxidation rate was high (95%). Thus, in order to reduce oxidation, different culture media were compared: WPM, MS, JADS and modified QL, followed by explant transfer onto regeneration medium. The highest percentage of regeneration and the lowest oxidation rate were Then, NAA and 2,4-Dwe tested in combination with TDZ and also TDZ and BA combined with NAA in WPM medium. The most efficient culture media in terms of shoo This process yielded a regeneration rate of 43%, with a low oxidation of the tissues . A rooting experiment was conducted using half strength MS medium and comparing three concentrations of IBA (2.46, 4.90 and 7.35 μM). The highest rooting percentage (35%) was obtained on medium containing 2.46 μM IBA. Once the shoots were rooted, acclimatization in a greenhouse was not challenging and plant survival reached 100%.