Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. Phytohormone treatment is required for the induction of adventitious shoots in most species. Whether adventitious shoots can be induced is determined by the balance between auxin and cytokinin (CK) levels. Much effort goes into determining optimum concentrations and combinations of phytohormones in each tissue used as explants and in each plant species. In ipecac, however, adventitious shoots can be induced on internodal segments in culture medium without phytohormone treatment. This allows the inherent plasticity of ipecac for cell differentiation to be evaluated. To induce adventitious shoots in ipecac, we cultured internodal segments at 24 °C under 15 µmol m^-2 s^-1 of light in a 14-h light/10-h dark cycle on phytohormone-free B5 medium solidified with 0.2% gellan gum for 5 weeks. To investigate phytohormone dynamics during adventitious shoot formation, we measured endogenous indole-3-acetic acid and CKs in the segments by liquid chromatography-tandem mass spectrometry LC-MS/MS. This method allows analysis of endogenous indole-3-acetic acid and CKs levels in a simple manner. It can be applied to investigate the dynamics of endogenous auxin and CK during organogenesis in other plant species.