Cryo-scanning electron microscopy observations of vessel content during transpiration in walnut petioles. Facts or artefacts?

The reliability of the cryo-SEM technique for stabilizing and quantifying embolisms in vessels of transpiring plants has recently been criticized, on the grounds that the embolisms observed are artefacts of the freezing. One of the tests used was a comparison of the embolisms found in rachises of transpiring walnut leaves (Juglans regia L.) frozen intact on the tree, with both cryo-SEM images of vessel contents and the measured hydraulic conductivity of similar samples whose xylem pressure had been returned to atmospheric pressure by cutting the leaves off under water. Embolisms found in intact frozen rachises were not present in cut rachises, and the high rachis hydraulic conductivities indicated a similar absence of embolisms. We show that the authors' conclusions are wrong because their test produces a different artefact. When the petiole is cut under dye solution it is obvious that the immersing solution is drawn into the embolized vessels and fills them. Thus, the cryo-SEM images and the high hydraulic conductivities of specimens prepared by this technique do not indicate the contents of the xylem in the intact plant. In fact, this artefact may perhaps be used (with the dye) to measure the embolisms in the intact plant. The embolisms seen in the cryo-SEM are most unlikely to be artefacts. The published work that also shows embolisms and their refilling in transpiring plants by techniques involving no freezing is reviewed.