Minimum Plating Density MPD and Culturing Individual Protoplasts, Cell Leakage, Haemocytometer, Fewer Protoplasts, Small volume Cultures

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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 5×103 to 1 ×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 of 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 ml) 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 these feeder layers.

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