Surface Sterilization and Pre-treatment of Explants

Introduction:

• Surface sterilization is a crucial step in plant tissue culture to eliminate contaminating microorganisms (bacteria and fungi) from the surface of explant tissues.
• Effective sterilization ensures a contamination-free environment for successful in vitro plant growth.
• Pre-treatment prepares explants by enhancing their survival rate and regeneration potential in culture media.

 

1. Definition of Explant:

• An explant is any part of a plant (e.g., leaf, stem, root, or embryo) used for in vitro culture.
• Explants must be sterilized before culturing to prevent microbial contamination.

 

2. Importance of Surface Sterilization:

• Prevents contamination: Eliminates surface microbes.
• Enhances tissue viability: Protects cells from infection.
• Promotes healthy growth: Ensures successful establishment of cultures.

 

3. Surface Sterilization Procedure:

a. Selection of Explants:

·        Choose young, healthy plant parts.

·        Common explants:

o   Leaves (for callus induction)

o   Stem segments

o   Root tips

o   Nodal segments

o   Seeds and embryos

b. Washing of Explants:

·        Step 1: Rinse explants under running tap water for 20-30 minutes to remove dirt and debris.

·        Step 2: Wash with a mild detergent (e.g., Tween-20) for 5-10 minutes.

·        Step 3: Rinse thoroughly with distilled water.

c. Sterilization Agents and Protocols:

d. Rinsing:

·        After chemical sterilization, rinse explants 3-5 times with sterile distilled water to remove residual chemicals.

 

4. Pre-treatment of Explants:

a. Purpose:

·        Pre-treatment prepares the explants for culture by improving their regeneration ability and reducing stress.

b. Types of Pre-treatments:

1.        Antioxidant Treatment:

o   Objective: Prevents oxidative browning of tissues (especially important for tissues rich in phenolic compounds).

o   Common Solutions:

§  Ascorbic acid (50-100 mg/L)

§  Activated charcoal (0.1-1%) in the medium.

2.        Cold Treatment:

o   Objective: Breaks dormancy in certain seeds or buds.

o   Process: Store explants at 4°C for 1-7 days before culture.

3.        Growth Regulator Pre-treatment:

o   Objective: Enhances cell division and callus formation.

o   Example: Dip explants in a solution containing auxins (e.g., IAA or 2,4-D) for a few hours.

4.        Surface Drying:

o   Dry the explant surface briefly on sterile filter paper to reduce excess moisture before inoculation.

 

5. Factors Affecting Sterilization:

1. Type of Explant: Different tissues have varying resistance to sterilization agents.
2. Sterilization Time: Overexposure may damage tissues; underexposure may not kill contaminants.
3. Agent Concentration: Must be optimized for each explant type.
4. Size of Explant: Smaller explants are more sensitive to harsh chemicals.

 

6. Common Problems and Solutions:

1. Contamination:
• Cause: Inadequate sterilization or poor handling.
• Solution: Increase sterilization time or concentration; maintain strict aseptic conditions.
2. Tissue Browning:
• Cause: Oxidation of phenolic compounds.
• Solution: Use antioxidants like ascorbic acid or incorporate activated charcoal into the medium.
3. Tissue Damage:
• Cause: Excessive exposure to sterilants.
• Solution: Reduce concentration or exposure time; rinse thoroughly.

 

7. Applications:

• Micropropagation: Mass propagation of disease-free plants.
• Genetic Engineering: Produces clean explants for transformation.
• Germplasm Conservation: Ensures contamination-free storage.

 

Conclusion:

• Surface sterilization and pre-treatment of explants are fundamental steps in plant tissue culture, ensuring a contamination-free environment for successful culture initiation.
• Careful optimization of sterilization agents, exposure times, and pre-treatment strategies enhances the survival and growth potential of explants, leading to better outcomes in plant tissue culture processes