Suspension Cultures

Introduction:

• Suspension culture involves growing single plant cells or small cell aggregates in a liquid nutrient medium under continuous agitation.
• It is widely used for mass propagation, secondary metabolite production, and genetic studies in plant biotechnology.

 

1. Definition:

• A suspension culture is a type of in vitro culture where cells or tissues are dispersed in a liquid medium and maintained under constant stirring to keep them suspended.
• Typically derived from callus cultures, these cells grow in a homogeneous environment, allowing better nutrient uptake.

 

2. Objectives of Suspension Cultures:

1. Mass Production of Plant Cells: For large-scale production of cells.
2. Secondary Metabolite Production: Enhanced synthesis of valuable compounds like alkaloids or flavonoids.
3. Genetic Manipulation: Study of gene expression in single-cell systems.
4. Protoplast Culture: Used to regenerate plants from isolated protoplasts.

 

3. Types of Suspension Cultures:

a. Batch Culture:

·        Process: Cells are grown in a fixed volume of medium.

·        Growth Phases: Lag, exponential, stationary, and decline.

·        Advantage: Simple and easy to set up.

·        Disadvantage: Limited growth period due to nutrient depletion.

b. Continuous Culture:

·        Process: Fresh medium is continuously added while an equal volume of spent medium is removed.

·        Types:

o   Chemostat: Nutrient concentration is controlled.

o   Turbidostat: Culture density is maintained by adjusting dilution rates.

·        Advantage: Maintains cells in the exponential phase for a long time.

·        Disadvantage: Complex setup, requires constant monitoring.

 

4. Requirements for Suspension Culture:

a. Explant Source:

·        Usually derived from callus culture.

b. Culture Medium:

·        Liquid MS (Murashige and Skoog) medium is commonly used.

·        Supplements: Sucrose (carbon source), plant growth regulators (PGRs) like auxins (2,4-D) and cytokinins.

c. Agitation:

·        Done using an orbital shaker or magnetic stirrer at 100-150 rpm.

·        Purpose: Prevents cell clumping and ensures even nutrient distribution.

d. Environmental Conditions:

·        Temperature: 25-28°C.

·        Light: Generally maintained in the dark to avoid differentiation.

·        Aeration: Proper oxygen supply is crucial for cell growth.

 

5. Procedure for Establishing Suspension Culture:

1. Initiation:
• Callus Induction: Start with callus culture from an explant.
• Cell Separation: Transfer a small portion of the callus to a liquid medium.
2. Inoculation:
• Place the callus in a sterile liquid medium.
3. Agitation:
• Maintain continuous shaking at 100-150 rpm to keep cells in suspension.
4. Subculture:
• Transfer a portion of the cell suspension to a fresh medium every 7-14 days to maintain growth.

 

6. Growth Measurement:

• Packed Cell Volume (PCV): Measures the volume of settled cells after centrifugation.
• Cell Count: Using a hemocytometer or flow cytometry.
• Dry Weight: Measures biomass by drying and weighing cells.

 

7. Applications of Suspension Cultures:

1. Production of Secondary Metabolites:
• Used to produce medicinal compounds like quinine and taxol.
2. Genetic Engineering:
• Useful for introducing and expressing foreign genes.
3. Protoplast Culture:
• Protoplasts can be isolated from suspension cultures and regenerated into plants.
4. Bioreactors:
• Large-scale suspension cultures in bioreactors enable commercial production of plant products.
5. Studies on Cell Physiology:
• Understanding cell division, differentiation, and metabolism at the cellular level.

 

8. Advantages:

1. Homogeneous Growth: Uniform distribution of cells.
2. Scalability: Easy to scale up for industrial applications.
3. Nutrient Access: Better nutrient uptake than solid media.

 

9. Disadvantages:

1. Shear Stress: Cells are sensitive to mechanical agitation.
2. Contamination Risk: Higher risk due to liquid medium.
3. Clumping: Cells may aggregate, affecting growth.

 

Conclusion:

• Suspension culture is a powerful tool in plant biotechnology for mass cell production, secondary metabolite synthesis, and genetic engineering studies.
• With proper medium selection, agitation, and environmental control, it offers immense potential for research and industrial applications