Optimization of in vitro culture conditions of grapevine phylloxera-resistant rootstock cultivar ‘Ruggeri-140’(Vitisberlandieri x Vitisrupestris) was carried out. Among the different sterilization treatments, maximum aseptic cultures were obtained for both explants apical tips and nodal segments when treated with Ca(ClO)2 at concentration of 1.5 % for 10 minutes plus 70 % ethanol for 30 s (T7). The maximum shoot proliferation was observed both in apical and nodal meristems cultured on MS medium supplemented with 1.0 mg/l BAP. MS/2 medium containing 1.0 mg/l indole-3-butric acid (IBA) gave the highest rooting percentage (100%) with the highest mean number and length of roots. The ex vitro survival of rooted micro shoots was 75.0%.