Introduction:
- Callus culture is a form of plant
tissue culture where plant cells or tissues are induced to form a mass
of undifferentiated cells called a callus.
- It plays a crucial
role in plant propagation, genetic engineering, and secondary
metabolite production.
1. Definition:
- A callus is an
unorganized, growing mass of parenchyma cells that develops from
plant explants under specific conditions.
- These cells can be
induced to differentiate into various tissues or organs (roots, shoots) in
the presence of appropriate plant growth regulators (PGRs).
2. Objectives of Callus Culture:
- Micropropagation: Mass production of
plants from a small tissue sample.
- Genetic
Transformation:
Insertion of foreign genes into plant cells.
- Somatic Hybridization: Fusion of protoplasts
from different species.
- Production of
Secondary Metabolites:
Callus cultures can produce valuable compounds like alkaloids and flavonoids.
- Disease Resistance
Studies:
Developing disease-resistant plant lines.
3. Requirements for Callus Culture:
a.
Explant Selection:
·
Any
part of a plant, such as leaf, stem, root, or cotyledon.
·
Young
tissues
are preferred due to their higher regeneration potential.
b.
Culture Media:
·
Basic
Medium:
Murashige and Skoog (MS) medium are commonly used.
·
Key
Components:
o Macronutrients: Nitrogen, phosphorus,
potassium.
o Micronutrients: Iron, magnesium, zinc.
o Carbon Source: Sucrose (2-3%).
o Agar: Solidifying agent (0.8%).
c.
Plant Growth Regulators (PGRs):
·
Auxins
(e.g., 2,4-D, IAA, NAA):
Promote callus induction.
·
Cytokinins
(e.g., BAP, kinetin):
Control cell division and differentiation.
·
Auxin-to-Cytokinin
Ratio:
High auxin and low cytokinin concentrations promote callus formation.
d.
Sterilization:
·
Explant
sterilization:
Surface sterilization using ethanol and sodium hypochlorite.
·
Media
sterilization:
Autoclaving at 121°C, 15 psi.
4. Steps in Callus Culture:
- Selection and
Preparation of Explant:
- Choose a suitable
plant part and sterilize it.
- Inoculation:
- Place the explant on
the sterile culture medium inside a laminar air flow (LAF)
cabinet.
- Incubation:
- Maintain cultures at 25°C,
under dark conditions or low light.
- Callus Induction:
- Observe the formation
of callus within 2-4 weeks.
- Subculture:
- Transfer the callus
to a fresh medium for further proliferation or differentiation.
- Differentiation
(Optional):
- Transfer the callus
to a medium with different PGR ratios to induce organogenesis (root/shoot
formation).
5. Factors Affecting Callus Formation:
- Type of Explant: Different tissues
have varying potential to form callus.
- Nutrient Composition: Proper balance of
macronutrients, micronutrients, and vitamins is crucial.
- Plant Growth
Regulators (PGRs):
The ratio of auxins to cytokinins determines callus induction and
differentiation.
- Environmental
Conditions:
- Temperature: Optimal range is 25-28°C.
- Light: Dark or low-light
conditions are generally preferred for initial callus formation.
6. Applications of Callus Culture:
- Plant Regeneration: Generates whole
plants from single cells.
- Genetic Engineering: Introduces and
expresses foreign genes.
- Secondary Metabolite
Production:
Mass production of valuable compounds (e.g., anti-cancer drugs from
Catharanthus roseus).
- Somaclonal Variation: Generates genetic
variability for crop improvement.
- Cryopreservation: Stores callus tissues
for long-term preservation.
7. Limitations:
- Genetic Stability: Callus cultures may
undergo mutations (somaclonal variations).
- Contamination Risk: Maintaining aseptic
conditions is challenging.
- Low Differentiation
Potential:
Some plant species have low regeneration ability from callus.
Conclusion:
- Callus culture is a fundamental
technique in plant tissue culture with extensive applications in plant
biotechnology and crop improvement.
- By understanding and
controlling factors such as explant selection, growth regulators,
and environmental conditions, successful callus formation and
differentiation can be achieved for various purposes