Subculture and Repeated Transfer of Explants and Cultures

Introduction:

• Subculture (also called passaging) is the process of transferring plant explants or tissue cultures from one medium to another to maintain growth, prevent contamination, and promote differentiation.
• It is essential for sustaining cell viability, multiplication, and organogenesis in in vitro plant tissue culture.

 

1. Definition of Subculture:

• Subculture involves transferring growing tissues, callus, or cells to fresh nutrient media periodically to maintain their growth and metabolic activity.
• It is commonly performed to:
• Prevent nutrient depletion.
• Reduce toxic metabolite buildup.
• Avoid overcrowding of cells.

 

2. Types of Subcultures:

a. Explant Subculture:

·        Transferring original explants to fresh media to continue growth.

b. Callus Subculture:

·        Transferring callus tissues to a fresh medium for continued proliferation or to induce differentiation.

c. Cell Suspension Subculture:

·        Transferring a portion of a cell suspension culture to a fresh liquid medium to maintain cell growth.

 

3. Importance of Subculture:

1. Maintains Growth and Viability: Provides fresh nutrients for continuous growth.
2. Prevents Senescence: Reduces the risk of cell aging and death.
3. Supports Organogenesis: Promotes the formation of roots, shoots, or embryos.
4. Facilitates Multiplication: Enhances the mass production of plantlets.
5. Reduces Contamination Risks: Prevents microbial overgrowth by regularly refreshing the medium.

 

4. Procedure for Subculture:

1. Preparation:
• Sterilize tools, fresh medium, and culture vessels.
• Work under a laminar airflow cabinet to maintain aseptic conditions.
2. Selection of Culture Material:
• Choose healthy, actively growing tissues or callus.
• Avoid necrotic or contaminated regions.
3. Cutting and Transferring:
• Use a sterile scalpel to cut the desired portion of the explant or callus.
• Transfer to fresh media using sterile forceps.
4. Inoculation:
• Place the tissue on the surface of the fresh medium (solid or liquid).
• For suspension cultures, pipette a portion of the cell suspension into a new flask.
5. Incubation:
• Place the subcultured tissues in the growth chamber under optimal conditions (temperature, light, humidity).

 

5. Frequency of Subculture:

• Depends on the culture type:
• Callus culture: Every 2-4 weeks.
• Cell suspension culture: Every 7-14 days.
• Organ culture: When the medium is depleted or when organ development requires fresh nutrients.

 

6. Challenges and Solutions:

a. Contamination:

·        Cause: Poor aseptic techniques.

·        Solution: Strictly follow sterile procedures and monitor cultures regularly.

b. Genetic Variability (Somaclonal Variation):

·        Cause: Repeated subculturing over long periods.

·        Solution: Limit the number of subcultures and use fresh explants periodically.

c. Tissue Browning:

·        Cause: Phenolic compound oxidation.

·        Solution: Use antioxidants (e.g., ascorbic acid) and transfer tissues to fresh media regularly.

 

7. Applications of Subculture:

1. Micropropagation: Produces large numbers of plants from a small tissue sample.
2. Germplasm Conservation: Maintains genetic resources by long-term subculture or cryopreservation.
3. Genetic Transformation: Provides healthy tissues for gene transfer experiments.
4. Secondary Metabolite Production: Enhances metabolite yield in callus or suspension cultures.

 

8. Repeated Transfers:

• Purpose: Maintain cultures for extended periods without losing their regenerative potential.
• Effect: Prevents senescence and somaclonal variation but requires careful monitoring to avoid loss of genetic fidelity.

 

Conclusion:

• Subculture and repeated transfer are essential processes in plant tissue culture for maintaining healthy, growing cultures.
• Proper techniques ensure sustained growth, prevent contamination, and enhance regeneration potential, making them vital for successful micropropagation and biotechnological applications