Micropropagation of Vitex negundo L., a woody aromatic medicinal shrub, through high frequency axillary shoot proliferation

A protocol is described for rapid and large-scale propagation of the woody aromatic and medicinal shrub Vitex negundo by in vitro culture of nodal seg ments from mature plants. Of the three different cytokinins - N-6-benzylade nine (BA), kinetin, and thidiazuron - evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal concentration of 2.0 mg/l was most effective in inducing bud break. Although callus-free multiple-shoot forma tion was a function of cytokinin activity alone, faster bud break coupled w ith an enhanced frequency of shoot development (92%) and internode elongati on were dependent on the synergistic influence of gibberellic acid (GA(3)) when used at an optimal concentration (0.4 mg/l) along with BA (2.0 mg/l). The frequency of shoot proliferation was markedly influenced by the explant ing season. By repeated subculturing of nodal segments harvested from the i n vitro-formed axenic shoots on MS containing 1.0 mg/l BA and 0.4 mg/l GA(3 ), prolific shoot cultures free from proximal callusing and showing a high- frequency multiplication rate were established. The percentage shoot multip lication (98-100%) as well as the number of shoots per node (six to eight) were highest during the first three culture passages, after which there was a gradual decline in shoot development. Rooting was best induced (94%) in shoots excised from proliferated shoot cultures on half-strength MS medium augmented with an optimal combination of indole-3-acetic acid and indole-3- butyric acid each at 1.0 mg/l. Vermi-compost was the most suitable planting substrate for hardening inside a plant growth chamber and its use ensured high-frequency survival (93%) of regenerated plants prior to outdoor transf er. Micropropagated plants established in garden soil were uniform and iden tical to the donor plant with respect to growth characteristics as well as vegetative and floral morphology.