Extracellular matrix of plant callus tissue visualized by ESEM and SEM

Actinidia deliciosa endosperm-derived callus culture is stable over a long period of culture. This system was used to investigate the ultrastructure of extracellular matrix occurring in morphogenic tissue. Specimens were prepared by different biological techniques (chemical fixation, liquid nitrogen fixation, glycerol substitution, critical-point drying, lyophilization) and observed by scanning electron microscopy (SEM). Fresh and wet samples were analyzed with the use of environmental scanning electron microscopy (ESEM). Extracellular matrix was observed on the surface of cell clusters as a membranous layer or reticulated network, shrunken or wrinkled, depending on the procedure. Generally, shrunken membranous layers with a globular appearance and fibrils were noted after critical-point drying and liquid nitrogen fixation. Smoother surface layers without visible fibrils and showing porosity were typically seen by environmental scanning electron microscopy. Preservation with glycerol substitution caused wrinkled appearance of examined layer. Analysis of fresh samples yielded images closer to their natural state than did critical-point drying or fixation in liquid nitrogen, but it seems best to compare the results of different visualization methods. This is the first report of ESEM observations of plant extracellular matrix and comparison with SEM images from fixed material.