Freezing tolerance in alpine plants as assessed by the FDA-staining method

We established a method employing fluorescein diacetate (FDA)-staining with a laser scan microscope for evaluation of the freezing tolerance of leaf mesophyll cells, and applied it to assessments of freezing tolerance in leaves of nine alpine plants, Arcterica nana, Cassiope lycopodioides, Diapensia lapponica, Empetrum nigrum, Loiseleuria procumbens, Phyllodoce nipponica, Rhododendron aureum, Schizocodon soldanelloides, and Vaccinium vitis-idaea, which naturally occur on Mt. Iwo (36°00′N, 138°22′W, 2760 m a.s.l.) in 2001. The results obtained with the FDA-staining method were strongly correlated with those of the electrolyte-leakage test, suggesting that this method is highly reliable. Leaf mesophyll cells in all these plants collected in early September survived after freezing at -5°C, but did not survive below -30°C. The survival ratio at subzero temperatures, ranging from -5 to -16°C, varied among the species. On the other hand, all the mesophyll cells of the plants collected in the middle of November survived freezing at -30°C. However, the survival ratio decreased after freezing at -80°C in Phyllodoce nipponica and Empetrum nigrum. Using the FDA-staining method, we were able to confirm the increase of the freezing tolerance of alpine plants from September to November. Advantages of the new method are discussed