Testing Protoplast Viability, Cytoplasmic Streaming, Exclusion of Evans, Blue Dye, Protoplast Size, Osmoticum solution Causing, Fluorescein Diacetate

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Home >> Plant Biotechnology and Genomics >> Protoplast Culture Regeneration and Somatic Hybridization >> Testing Protoplast Viability

Viability and Plating Density of Protoplasts

Protoplasts viability could be tested 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 (FCW).

Following are some details;

(i) As FDA accumulates within the plasma membrane, viable protoplasts fluoresce gree/white. It should be examined within 5-15 minutes after the FDA treatment, 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) CFW (0.1% v/v) detects onset of cell wall regeneration around plasma membrane of viable protoplasts in the form of a ring of fluorescence.

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