Mineral nutrients are one of the most basic components of plant tissue culture media. Nitrogen in the form of NH₄⁺ and NO₃^- is the dominant mineral nutrient in most plant tissue culture formulations, with effects dependent on both the proportion and the amount of NH₄⁺ and NO₃^-. The effects of nitrogen nutrition on the growth of nonembryogenic and embryogenic cell lines of sweet orange (C. sinensis (L.) Osbeck cv. 'Valencia'), tissues routinely used in citrus horticultural and plant improvement research, was explored using an experimental approach free of ion confounding that included a 2-component mixture (NH₄⁺:K⁺) and a quantitative factor [NO₃^-] crossed by the mixture, thereby providing ion-specific estimates of proportional and amount effects.

First, the linear mixture component, though only a comparison of the design space vertices, was highly significant for both tissue types and showed that NH₄⁺ was required by both tissues. Second, the NH₄⁺ * K⁺ mixture term was highly significant for both tissue types, revealing that NH₄⁺ and K⁺ exhibit strong synergistic blending and showed that growth was substantially greater at certain blends of these two ions. Third, though the interaction between the NH₄⁺:K⁺ mixture and NO₃^- amount on fresh weight accumulation for both tissue types was significant, it was substantially less than the main effect of the NH₄⁺:K⁺ mixture. Fourth, a region of the design space was identified where fresh weight growth was increased 198% and 67% over the MS medium controls for nonembryogenic and embryogenic tissues.

By designing a mineral nutrient experiment free of ion confounding, a direct estimation of ion-specific proportional and amount effects on plant tissue growth is possible. When the ions themselves are the independent factors and/or mixture components, the resulting design space can be systematically explored to identify regions where the response(s) is substantially improved over current media formulations. In addition, because the response is over a defined experimental region, a specific medium formulation is more accurately interpreted as a coordinate in the specified design geometry.