Measuring Freezing Tolerance of Leaves and Rosettes: Electrolyte Leakage and Chlorophyll Fluorescence Assays

Quantitative assessment of freezing tolerance is essential to unravel plant adaptations to cold temperatures. Not only the survival of whole plants, but also impairment of detached leaves or small rosettes after a freeze-thaw cycle can be used to accurately quantify plant freezing tolerance in terms of LT₅₀ values. Here we describe two methods to determine the freezing tolerance of detached leaves or rosettes using a full or selected set of freezing temperatures and an additional method using chlorophyll fluorescence as a different physiological parameter. Firstly, we illustrate how to assess the integrity of (predominantly) the plasma membrane during freezing using an electrolyte leakage assay. Secondly, we provide a chlorophyll fluorescence imaging protocol to determine the freezing tolerance of the photosynthetic apparatus.