Sound Practices to Effectively Perform Electrolyte Leakage Assays

The effectiveness of electrolyte leakage assays was tested in order to develop simple and sound procedures and to evaluate cold tolerance in fruit tree research studies. Divergences in specific methodological details that may impact the accuracy of these types of assays have been found in the literature. In this methodological study, leaves from grapefruit trees grown in the field were used to evaluate: 1) the adequate ratio between the number of leaf disks and the volume of water in a sample tube; and 2) the addition of ice chips. These trials showed that using less than one leaf disk per ml of water per tube resulted in too much variability in electrolyte leakage, which decreased the effectiveness of this method to estimate the lethal freezing temperature. The addition of ice chips into test tubes to nucleate the water-soaked tissues did not have any effect on electrolyte leakage compared to the non-addition of ice chips when this addition was done after the immersion of the test tubes in the refrigerated bath; however, electrolyte leakage was different when the ice chips were added before the immersion of the tubes in the refrigerated bath since the ice melted before leaf disks were subjected to freezing temperatures.