Viability and Plating Density of Protoplasts,Respiratory Activity,Plasma Membrane,Plant Cells

	Viability and Plating Density of Protoplasts - Testing protoplast viability:Protoplast viability could be tested in the beginning of protoplast research by a variety of methods including the following: (i) presence of cytoplasmic streaming, (ii) exclusion of Evans Blue dye; (iii) change in protoplast size due to change in the level of osmoticum (osmoticum is a solution causing change in osmotic pressure); (iv) presence of photosynthetic and respiratory activity. However, protoplast viability is now most frequently tested using several dyes including fluorescein diacetate (FDA), phenosafranin and calcofluor white (CPW).
Following are some details: (i) As FDA accumulates within the plasma membrane, viable protoplasts fluoresce green/white. It should be examined within 5-15 minutes after the FDA treatment, after which FDA dissociates. (ii) Phenosafranin (0.1%) detects dead protoplasts, which turn red in its presence, the viable protoplasts remaining unstained even after 2 hours in the stain solution.
(iii) CPW (0.1% v/v) detects onset of cell wall regeneration around plasma membrane of viable protoplasts in the form of a ring of fluorescence. Minimum plating density (mpd) and culturing individual protoplasts:A minimum plating density of protoplasts is required for growth to begin, because protoplasts enrich the culture medium due to cell leakage. The mpd for tobacco is 5 x 103 to 1 x 105 protoplasts/cm3 (protoplasts/cm3 can be estimated using a haemocytometer). However, due to requirements of genetic engineering experiments, there is a need to culture individual or fewer protoplasts. Following approaches are used for this purpose:
(i) Small volume cultures (less than 1 cm3 ) may be used to reduce the number of protoplasts, although mpd is maintained. Hanging droplets (2 Ill) and other similar techniques (e.g. multidrop array) have been used to reduce the volume. (ii) Conditioned media (in which plant cells were already grown), after filtering, can be used for growing isolated protoplasts at lower densities. (iii) Feeder layers of protoplasts can be prepared by plating solid media with protoplasts followed by irradiation (which should inactivate but not kill these protoplasts). Protoplasts at lower density (5-50 protoplasts/cm3) can now be plated on this feeder layer.