This Research has been performed in the laboratory of the National Commission of Biotechnology (NCBT) in Damascus / Syria during the period between 2020-2022. The aim of this work was to develop a protocol for micropropagation of Vitex agnus-castus as in order to produce hardened rooted plants ready for transfer to the permanent land. The optimal concentration of sodium hypochlorite (NaOCl) and the appropriate time for surface disinfection in primary cultivation were studied. The effect of some growth regulators was studied during the propagation and rooting stages of this plant. Explants cuttings 1–1.5 cm at the propagation stage were cultured on nutrient medium of MS, WPW, SH and B5 with different concentrations of plant growth regulators, where seven different concentrations( 0,0.5, 1, 1.5, 2, 2.5, and 3 mg.l-1) of benzyl adenine (BA) were used. The concentration of cytokinin BA 1 mg.l-1 with respect to the average number of growths, was significantly higher than the rest of the concentrations with a value of (2.02 growth), The control was significantly superior to the average growth length over the rest of the concentrations with a value of (4.87 cm(. As for the average number of leaves, the control was significantly superior to the rest of the concentrations with a value of (10.49 leaves. plant-1(. The shoots resulting from vegetative propagation were grown on nutrient media MS, WPW, SH and B5, which were supplemented with two types of auxins: Naphthalene acetic acid NAA and Indole butiric acid IBA at the following concentrations (0,0.25, 0.5, 1, 1.5, 2 mg.l-1).To obtain the highest rooting rate and good quality roots.To rooting percentage, the concentration of 0.5 mg.L-1 of the MS medium was significantly with a ratio (95.92%) for the IBA and (80.94%) for the NAA at the same concentration. For to the number of roots the concentration of 0.5 mg L-1 of the WPM medium was significantly with a value of (9.86 root.plant-1) for IBA and (7.86 root.plant-1) for NAA at the same concentration. For root elongation, the concentration of 0.5 mg.l-1 of the medium SH was significantly with a value of (8.87 cm) for auxin IBA and (8.14 cm) for the NAA at the same concentration. The success rate of the acclimatization process was 94% for Vitex agnus-castus.
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