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:
- Type
of Explant: Different tissues have varying resistance to sterilization
agents.
- Sterilization
Time: Overexposure may damage tissues; underexposure may not kill
contaminants.
- Agent
Concentration: Must be optimized for each explant type.
- Size
of Explant: Smaller explants are more sensitive to harsh chemicals.
6. Common Problems and Solutions:
- Contamination:
- Cause:
Inadequate sterilization or poor handling.
- Solution:
Increase sterilization time or concentration; maintain strict aseptic
conditions.
- Tissue
Browning:
- Cause:
Oxidation of phenolic compounds.
- Solution:
Use antioxidants like ascorbic acid or incorporate activated
charcoal into the medium.
- 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