Iranian seedless barberry is a very recalcitrant species in in vitro culture which does not show appropriate growth on standard culture media. Response surface methodology was employed to evaluate the effects of changing macronutrients concentrations on establishment and proliferation phases. KNO3 and NH4NO3 macronutrients at 0.3 to 1.5× MS medium levels and CaCl2, MgSO4 and KH2PO4 macronutrients in a range of 0.5 to 1.5× MS medium concentrations were tested in a response surface design with 30 treatments. Many significant interactions were found among the macronutrients. High concentrations of KNO3, NH4NO3 and CaCl2 improved the growth rate in the establishment phase. The growth rate in media containing high KNO3 and low CaCl2 was high. Reduced concentrations of CaCl2 and KNO3 decreased hyperhydricity. The greatest hyperhydricity was induced when both NH4NO3 and CaCl2 were used at 1.5× MS level. The number of hooked leaves decreased as KH2PO4 increased and MgSO4 reduced. In the proliferation phase, there were many significant interactions among the macronutrients. Increased concentration of NH4NO3 and reduced concentration of KH2PO4 improved the growth rate. Proliferation rate increased in media containing high concentration of KNO3 and low to moderate concentrations of NH4NO3. The greatest production of new tissues and organs was seen in media with high KNO3 and moderate to high CaCl2. High concentration of NH4NO3 and low concentration of KH2PO4 also increased production of new tissues and organs. No shoot apical meristem was seen when CaCl2 level was high and KNO3 level was low. Formation of shoot apical meristem required high KH2PO4 concentration and low CaCl2 concentration. Finally, low concentration of KNO3 and low to moderate concentrations of NH4NO3 increased phenol exudation.

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