Developing hazelnut tissue culture medium free of ion confounding

Abstract

The general approach for tissue culture medium optimization is to use salts as factors in experimental design and analysis. However, using salts as factors leads to ion confounding, making it difficult to detect the effects of individual ions on particular growth responses. This study focused on testing ions as factors for the medium optimization process. NH₄ ⁺, Ca²⁺, Mg²⁺, SO₄ ²⁻ and PO₄ ³⁻ ions were used as factors in a D-optimal design. K⁺ and NO₃ ⁻ ions were used to bring the pH to neutral, and were also factors in the statistical analysis. The Chi-squared automatic interaction detection (CHAID) data mining algorithm was used to analyze shoot growth responses of ‘Barcelona’, ‘Jefferson’ and ‘Wepster’ hazelnuts. The CHAID analysis decision trees revealed significant variables and their interactions, and provided exact cut-off amounts for optimizing each of the ions. K⁺, NO₃ ⁻, and NH₄ ⁺ had significant effects on shoot quality. NH₄ ⁺ was of primary significance for shoot length followed by Mg²⁺, NO₃ ⁻ and Ca²⁺. Multiplication was mainly affected by Ca²⁺ and genotype. For the least callus formation, NH₄ ⁺ >33.3 mM was required, but this higher concentration range did not provide good shoot quality or elongation. The critical cut-off values for good shoot quality, elongation, multiplication and medium callus formation for hazelnut are suggested to be: NO₃ ⁻ ≤88 mM, NH₄ ⁺ ≤20 mM, Ca²⁺ ≤5 mM, Mg²⁺ >5 mM and K⁺ ≤46 mM.