Standardization of electrolyte leakage data and a novel liquid nitrogen control improve measurements of cold hardiness in woody tissue

Background A variety of basic and applied research programs in plant biology require the accurate and reliable determination of plant tissue cold hardiness. Over the past 50 years, the electrolyte leakage method has emerged as a popular and practical method for quantifying the amount of damage inflicted on plant tissue by exposure to freezing temperatures. Numerous approaches for carrying out this method and analyzing the resultant data have emerged. These include multiple systems for standardizing and modeling raw electrolyte leakage data and multiple protocols for boiling or autoclaving samples in order to maximize leakage as a positive control. We compare four different routines for standardization of leakage data and assess a novel control method-immersion in liquid nitrogen in lieu of traditional autoclaving-and apply them to woody twigs collected from 12 maple (Acer) species in early spring. We compare leakage data from these samples using each of four previously published forms of data analysis and autoclaving vs. liquid nitrogen controls and validate each of these approaches against visual estimates of freezing damage and differential thermal analysis. Results Through presentation of our own data and re-analysis of previously published findings, we show that standardization of raw data against estimates of both minimum and maximum attainable freezing damage allows for reliable estimation of cold hardiness at the species level and across studies in diverse systems. Furthermore, use of our novel liquid nitrogen control produces data commensurate across studies and enhances the consistency and realism of the electrolyte leakage method, especially for very cold hardy samples. Conclusion Future leakage studies that relativize data against minimum and maximum leakage and that employ our updated liquid nitrogen control will contribute generalizable, repeatable, and realistic data to the existing body of cold hardiness research in woody plants. Data from studies conducted using a liquid nitrogen (and not an autoclaving) control can still be compared to previously published data, especially when raw data are standardized using the best-performing approach among those we assessed. Electrolyte leakage of woody twigs emerges as a useful technique for quickly assessing the probability of tissue death in response to freezing in dormant plants. Differential thermal analysis may provide different and complementary information on cold hardiness.