Discuss the factors affecting the yield and viability of protoplasts isolated from leaves. How are isolated protoplasts purified?

Factors Affecting Yield and Viability of Protoplasts

The yield and viability of protoplasts isolated from leaves are affected by both endogenous (plant-related) and exogenous (environmental/methodological) factors.

Endogenous Factors

• Plant Species and Genotype: Different plant species and even different genotypes within a species exhibit varying cell wall thicknesses and compositions, impacting protoplast release efficiency. For example, tobacco protoplasts are relatively easy to isolate compared to cereals.
• Leaf Age and Physiological State: Younger, actively growing leaves generally yield more protoplasts with higher viability than older, senescing leaves. Leaves subjected to stress (e.g., drought, nutrient deficiency) also show reduced protoplast viability.
• Cell Type: Mesophyll cells are the primary source of protoplasts from leaves, but their viability can vary depending on their location within the leaf and their metabolic activity.

Exogenous Factors

• Enzymatic Digestion: The choice of cell wall degrading enzymes (cellulase, pectinase, hemicellulase) and their concentrations are critical. Incomplete digestion results in intact cell walls, while over-digestion can damage the protoplasts. Optimal enzyme cocktail and incubation time need to be determined empirically for each species.
• Osmotic Pressure: The osmotic pressure of the isolation medium must be carefully controlled to prevent protoplast rupture or plasmolysis. Mannitol or sorbitol are commonly used as osmoticum.
• Temperature: Enzymatic digestion is typically performed at temperatures between 25-30°C. Higher temperatures can denature enzymes, while lower temperatures reduce their activity.
• pH: The pH of the isolation medium affects enzyme activity and protoplast stability. A pH range of 5.4-5.8 is generally optimal.
• Duration of Incubation: Prolonged incubation can lead to protoplast damage, while insufficient incubation results in incomplete cell wall removal.
• Mechanical Handling: Gentle handling is crucial to avoid mechanical damage to the fragile protoplasts.

Purification of Isolated Protoplasts

Purification is essential to remove cell debris, undigested cells, and enzymes, which can inhibit protoplast growth and division. The purification process typically involves several steps:

Step-by-Step Purification Procedure

1. Filtration: The initial step involves filtering the protoplast suspension through a series of sieves with decreasing pore sizes (e.g., 80 µm, 40 µm, 20 µm) to remove large debris and undigested cells.
2. Centrifugation: Protoplasts are gently centrifuged at low speed (e.g., 600 rpm for 5-10 minutes) to pellet them. The supernatant containing debris and enzymes is discarded.
3. Washing: The protoplast pellet is resuspended in fresh osmoticum solution and centrifuged again. This washing step is repeated several times to remove residual enzymes and debris.
4. Density Gradient Centrifugation (Optional): For higher purity, density gradient centrifugation using Percoll or Ficoll can be employed. Protoplasts migrate to a specific density band, separating them from remaining contaminants.
5. Microscopic Examination: The purified protoplast suspension is examined under a microscope to assess purity and viability. Viability can be assessed using staining with dyes like fluorescein diacetate (FDA).

Table: Comparison of Purification Methods

Method	Principle	Advantages	Disadvantages
Filtration	Size exclusion	Simple, inexpensive	Less effective for removing small debris
Centrifugation & Washing	Sedimentation based on density	Effective for removing large particles	Can damage protoplasts if speed is too high
Density Gradient Centrifugation	Separation based on density	High purity	More complex, expensive