Testing of the viability of isolated protoplasts

Staining Methods for Assessing Protoplast Viability

Staining methods are essential for determining the viability and functional status of protoplasts. The most commonly used methods include:

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1. Fluorescein Diacetate (FDA) Staining Method

• Principle:
  FDA is a dye that passes through the plasma membrane of viable cells and accumulates inside. Once inside, it is hydrolyzed by cellular esterases, producing a fluorescent product.
• Procedure:
• Dissolve FDA in acetone to make a 0.01% solution.
• Add FDA to the protoplast suspension.
• Observe under a fluorescence microscope within 5–15 minutes.
• Result:
• Viable Protoplasts: Fluoresce yellow-green.
• Non-Viable Protoplasts: Do not fluoresce.
• Limitation:
• Fluorescence diminishes after 15 minutes due to dissociation from membranes.

 

2. Phenosafranine Staining

• Principle:
  Phenosafranine selectively stains dead protoplasts by penetrating compromised membranes, while viable cells remain unstained.
• Procedure:
• Use a 0.01% solution of phenosafranine.
• Mix with the protoplast suspension.
• Result:
• Dead Protoplasts: Appear red.
• Viable Protoplasts: Remain unstained.

 

3. Calcofluor White (CFW) Staining

• Principle:
  CFW binds specifically to beta-linked glucosides in the newly forming cell wall of protoplasts, indicating the onset of cell wall synthesis.
• Procedure:
• Mix 0.1 mL of protoplast suspension with 5.0 μL of a 0.1% w/v solution of CFW.
• Observe under a fluorescence microscope.
• Result:
• Newly synthesized cell walls appear as a fluorescent ring around the plasma membrane.

 

Comparison of Staining Methods

Method	Viability Indicated	Result Observed	Specificity
FDA	Viable protoplasts	Yellow-green fluorescence	Viable cells only.
Phenosafranine	Dead protoplasts	Red color	Dead cells only.
CFW	Cell wall formation onset	Fluorescent ring around cells	New cell wall formation.

 

Conclusion

Staining techniques like FDA, phenosafranine, and CFW provide quick and reliable ways to assess protoplast viability and functional status. These methods are integral in optimizing protoplast isolation protocols and monitoring their regeneration into complete cells or tissues.