Vitality of tree fine roots: reevaluation of the tetrazolium test

Reduction of triphenyltetrazolium chloride (TTC) by tissue to the red-colored insoluble triphenylformazan (TF) is directly linked to the activity of the mitochondrial respiratory chain. Thus, only living tissues should reduce TTC to TF. However, TF production can be detected when the TTC test is applied to control tissues boiled in water. This artifact is mainly the result of the hot ethanol extraction step, which is used to disintegrate the cells and solubilize the TF. We observed that cell wall materials such as cellulose and pectin interact with TTC at temperatures above 60 degrees C, reducing the TTC to TF. By replacing the hot ethanol extraction step with an extraction procedure that involved grinding the boiled tissues and extracting the TF with ethanol at room temperature, the formation of TF was almost entirely eliminated. Application of the modified TTC assay to fine roots of Norway spruce from forest topsoil indicated that the extent of TTC reduction was related to root morphological class as: white fine roots > brown fine roots > black fine roots > boiled fine roots, corresponding to formation of 10.8, 6.1, 0.2 and 0.1 mM TFg(-1)DW, respectively. No significant differences in TTC reduction were recorded between fresh and frozen tissues (frozen in liquid N2) for any fine root class. Application of the modified TTC assay to seedling roots stressed either by drying or by exposure to the toxic metals Cd or Al resulted in significant decreases in TF formation that were related to both the duration of stress and the concentrations of toxic substances, indicating that the modified TTC assay can be used to assess the physiological condition of roots.