As many problems with plant cells are due to their rigid cell wall, an obvious solution is to remove it. Cells devoid of their walls are called protoplasts. They are characterized by circular shape and therefore do not disturb the laminar flow. Protoplasts can be prepared from plant tissues and cells by hydrolyzing the components of cell wall under isotonic conditions. It should be noted, however, that the preparation of protoplasts is even more difficult and laborious than the preparation of cells. Moreover, only a few types of tissues (typically leaf mesophyll) are suitable, and the isolated protoplasts are fragile and require a careful handling.
Despite being more suitable for FCM than intact cells, protoplasts share many of their disadvantages. These include the large size, low permeability of the plasma membrane for some compounds, autofluorescence, presence of secondary metabolites, and non-specific binding of fluorescent probes. If viable protoplasts are not needed, some of these difficulties may be overcome by appropriate fixation. As it happens, some disadvantages may be turned into advantages. For example, autofluorescence of chlorophyll may be useful for identifying protoplasts from autotrophic tissues containing chloroplasts (Buiteveld et al. 1998) or to measure the content of a secondary metabolite (Brown et al. 1984). To conclude, the use of protoplasts provides a solution if FCM analysis and/or sorting of whole plant cells is required. Different options should be pursued in other cases.
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