Initiation and Maintenance of Callus & Suspension Culture .pdf

Callus Culture
Definition
Callus culture is a plant tissue culture method where plant cells divide uncontrollably to form an undifferentiated mass known as a "callus." This technique is commonly used for genetic modification, somatic embryogenesis, and micropropagation.

Steps in Callus Culture
Selection of Explant

A small plant part (explant) is taken from leaves, stems, roots, or meristems.
Young, actively growing tissues are preferred as they respond better in culture.
Sterilization

The explant is sterilized using chemicals like ethanol, sodium hypochlorite, or mercuric chloride to remove microbial contamination.
Inoculation on Medium

The sterilized explant is placed on a nutrient-rich agar medium supplemented with essential elements, vitamins, and plant growth regulators like auxins (e.g., 2,4-D, NAA) and cytokinins (e.g., BAP, Kinetin).
These hormones help in cell division and callus formation.
Incubation

The culture is maintained under controlled temperature (25–28°C), humidity, and light conditions (16-hour light and 8-hour dark photoperiod).
Callus Formation

Within a few weeks, the explant develops an unorganized, undifferentiated mass called callus.
Subculturing and Further Applications

The callus can be subcultured on fresh media to promote growth, organogenesis (shoot and root formation), or somatic embryogenesis.
It can also be used for genetic transformation studies, plant regeneration, or secondary metabolite production.
Suspension Culture
Definition
Suspension culture is a liquid culture system where plant cells or small cell aggregates are grown in a continuously shaking medium. It allows cells to remain in a dispersed state and promotes rapid cell division, making it ideal for large-scale metabolite production.
Types of Suspension Cultures
Batch Culture: Cells grow in a fixed volume of medium and are harvested at a specific growth phase.
Continuous Culture: Fresh medium is continuously supplied, and cells are maintained in an actively growing phase for an extended period.

1. Guided By- Dr. Bharti Sahu
Presented By- Somnath
MSc 2nd Sem
Biotechnology
SPCA Nawapara
INITIATION ANDINITIATION AND
MAINTENANCE OF CALLUSMAINTENANCE OF CALLUS
AND SUSPENSION CULTUREAND SUSPENSION CULTURE

2. SynopsisSynopsis
Introduction:1.
2. Initiation of Callus Culture
A. Selection of Explant
B. Surface Sterilization
C. Nutrient Medium & Root and Shoot Formation
3. Incubation Conditions
4. Maintenance of Callus Culture
D. Solidifying Agent
5. Suspension Culture
A. Establishment of Suspension Culture
B. Maintenance of Suspension Culture
6. Conclusion:
7. References:

3. Plant tissue culture relies on the balance of plant growth regulators (PGRs), mainly cytokinins and auxins, to
control organogenesis. The appropriate concentration of these hormones determines whether shoots, roots, or
callus will form from an undifferentiated mass of cells.
Introduction:1.
Young and actively dividing plant tissues such as leaf, root, stem, hypocotyl, or cotyledon are preferred.
The choice of explant depends on the species and the objective of the culture.
A. Selection of Explant
B. Surface Sterilization
To remove microbial contaminants (fungi, bacteria), the explants undergo sequential treatment with disinfectants:
Washing: Rinse the explants thoroughly with running tap water for 10-15 minutes to remove dust and dirt.
Treatment with Detergent (Optional): Some protocols use 0.1% Tween-20 or Tween-80 for 5-10 minutes to help
remove surface contaminants.
Disinfection with Chemical Agents:
Washing with Sterile Distilled Water: After each sterilization step, explants are washed 3-5 times with sterile
distilled water to remove residues of chemicals.
Ethanol (70%) for 30-60 seconds – Helps to remove surface pathogens.
Sodium hypochlorite (NaOCl) (1-2%) for 5-10 minutes – Kills bacteria and fungi.
Mercuric chloride (HgCl₂) (0.1%) for 1-2 minutes (if needed, but highly toxic) – Provides strong sterilization but
needs thorough washing afterward.
2. Initiation of Callus Culture

4. Growth Regulator Function
Concentration
Range
Auxins (e.g., IAA, IBA,
NAA, 2,4-D) Promote root initiation
and elongation
0.5 - 2 mg/L
Cytokinins (e.g., BAP,
Kinetin, TDZ, Zeatin)
Promote shoot formation
and multiplication
0.5 - 5 mg/L
C. Nutrient Medium & Root and Shoot Formation (PGRs):
Murashige and Skoog (MS) Medium is commonly used.
The formation of roots and shoots from callus or explants depends on the balance of plant growth
regulators (PGRs), specifically auxins and cytokinins.
Promotes cell division in meristematic regions,
leading to shoot formation.
Example: BAP (2 mg/L) + NAA (0.2 mg/L)
High Cytokinin:Auxin Ratio → Shoot Induction

5. High Auxin:Cytokinin Ratio → Root Induction
Stimulates cell elongation and differentiation into roots.
Example: NAA (1 mg/L) + IBA (0.5 mg/L)
Equal Cytokinin and Auxin Ratio → Callus Formation
Maintains an undifferentiated cell mass without organ formation.
D. Solidifying Agent:
Why is the Medium Solidified?
In callus and shoot cultures, a solid medium is used instead of
a liquid medium. The main reasons are:
Support for Tissue Growth:
Solid medium provides a stable surface for tissue attachment.
Prevents callus from breaking apart, ensuring organized shoot and root development.
Better Nutrient Uptake:
Explants can absorb nutrients gradually from the medium instead of being submerged in liquid.
Preventing Excessive Oxygen Exposure:
Solid medium helps maintain controlled aeration and prevents oxidative stress.
Avoiding Shear Stress:
Liquid cultures (especially agitated ones) can damage fragile tissues.
Solid medium prevents mechanical stress on growing shoots and roots.

6. 3. Incubation Conditions
Temperature: 25-28°C
Photoperiod: 16-hour light / 8-hour dark cycle
Humidity: 50-60%
Light Intensity: 1000-3000 lux (low light to promote callus formation)
4. Maintenance of Callus Culture
Once a callus is formed, it must be maintained and subcultured periodically to sustain its growth and prevent contamination.
Key Maintenance Steps:
1. Subculturing the Callus
2. Monitoring Growth & Morphology
3. Preventing Contamination
4. Storage and Long-Term Maintenance
Callus is transferred to fresh medium every 4-6 weeks to replenish nutrients.
A small portion of callus (~1 cm²) is cut and transferred using sterile forceps.
Healthy Callus: Firm, friable (loosely held together), cream to pale yellow color.
Unhealthy Callus:
Brown/Black Callus: May indicate phenolic oxidation (treat with activated charcoal 0.2% in medium).
Hard Callus: Often due to excessive cytokinin (reduce BAP/Kinetin levels).
Watery Callus: Too much auxin; reduce 2,4-D concentration.
Use sterile tools and work under a laminar airflow hood.
Regularly check for fungal or bacterial growth.
Short-term Storage: Callus can be maintained at 4°C in low-light conditions for a few weeks.
Long-term Storage:
Cryopreservation (-196°C in liquid nitrogen) is used for germplasm conservation.
Slow growth storage at low temperatures (10-15°C) reduces metabolism and prolongs viability.

7. 5. Suspension Culture
Definition: A suspension culture consists of single cells or small aggregates grown in a liquid medium with continuous agitation.
Steps for Initiation:
A. Establishment of Suspension Culture
B. Maintenance of Suspension Culture
Callus is carefully transferred to a liquid MS medium containing 2,4-D (0.5-2 mg/L) or IAA (Indole-3-acetic acid) (0.5-1 mg/L) to
induce cell division.
1.
The culture is placed on a rotary shaker (100-150 rpm) to keep the cells in suspension and ensure aeration.2.
Filter Paper or Mesh: Large cell clumps are filtered out to maintain uniform cell suspension.3.
Agitation: Rotary shaking at 100-150 rpm prevents cell clumping and improves nutrient uptake.
Subculture:
pH Monitoring:
The optimal pH is 5.6-5.8, which is checked regularly to prevent acidification due to cell metabolism.
Performed every 7-10 days to maintain healthy cell growth.
A portion of the culture (10-20%) is transferred to a fresh medium.
Oxygenation:
Continuous shaking provides oxygen, but aeration systems (e.g., bubbling air through the medium)
can also be used for large-scale cultures.

8. 6. Conclusion:
7. References:
The formation of roots and shoots in plant tissue culture is primarily controlled by the balance between cytokinin and auxin
concentrations. A high cytokinin-to-auxin ratio promotes shoot induction, while a high auxin-to-cytokinin ratio favors root
formation. When both hormones are present in nearly equal amounts, callus formation occurs without organogenesis.
Understanding this hormonal regulation is essential for successful plant regeneration and propagation in biotechnology and
agricultural research.
Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures.
Physiologia Plantarum, 15(3), 473-497.
1.
George, E. F., Hall, M. A., & De Klerk, G. J. (2008). Plant propagation by tissue culture. Springer Science & Business
Media.
2.
Smith, R. H. (2013). Plant tissue culture: techniques and experiments. Academic Press.3.

9. T h a n k Y o u