Callus Culture and Regeneration of Plants
Introduction
Callus culture is an in vitro plant tissue culture technique in which an unorganized mass of proliferating cells (callus) is induced from explants such as leaf, stem, root, or meristem under aseptic and controlled conditions. These callus cells retain totipotency, enabling regeneration of complete plants under suitable hormonal and nutritional conditions.
Callus culture forms the foundation of plant biotechnology, playing a crucial role in micropropagation, genetic transformation, somaclonal variation, and secondary metabolite production.
Definition
Callus is a mass of undifferentiated parenchymatous cells produced by continuous cell division of explant tissues when cultured on a nutrient medium supplemented with plant growth regulators.
Principle of Callus Culture
Based on the concept of cellular totipotency.
Dedifferentiation of mature cells occurs due to the action of auxins and cytokinins.
Redifferentiation and organ formation occur by altering the hormonal balance in the culture medium.
Explant Sources
Leaf segments
Stem nodes and internodes
Root tips
Hypocotyls
Cotyledons
Meristematic tissues
Culture Medium
The commonly used medium is Murashige and Skoog (MS) medium, containing:
Components
Macronutrients – N, P, K, Ca, Mg, S
Micronutrients – Fe, Mn, Zn, Cu, Mo
Vitamins – Thiamine, Nicotinic acid, Pyridoxine
Carbon source – Sucrose
Plant growth regulators
Auxins: 2,4-D, NAA, IAA
Cytokinins: BAP, Kinetin
Solidifying agent – Agar
Steps in Callus Culture
1. Selection of Explant
Healthy, young tissues with high meristematic activity are selected.
2. Surface Sterilization
Washing with detergent
Treatment with ethanol (70%)
Sterilization using sodium hypochlorite or mercuric chloride
Rinsing with sterile distilled water
3. Inoculation
Sterilized explants are placed aseptically on nutrient medium.
4. Incubation
Temperature: 25 ± 2°C
Photoperiod: Dark or low light
Relative humidity controlled
5. Callus Induction
High auxin concentration (especially 2,4-D) promotes callus formation.
Types of Callus
Compact callus – Hard, dense, slow growing
Friable callus – Soft, loose, fast growing
Embryogenic callus – Capable of forming somatic embryos
Non-embryogenic callus – Cannot regenerate plants
Plant Regeneration from Callus
Plant regeneration occurs through two main pathways:
1. Organogenesis
Formation of organs (shoots and roots) from callus.
Types
Direct organogenesis – Organs develop directly from explant
Indirect organogenesis – Organs develop via callus phase
Hormonal Control
High cytokinin : auxin → Shoot formation
High auxin : cytokinin → Root formation
2. Somatic Embryogenesis
Formation of embryo-like structures from somatic cells.
Stages
Globular stage
Heart-shaped stage
Torpedo stage
Cotyledonary stage
Somatic embryos germinate to form complete plantlets.
Hardening and Acclimatization
Regenerated plantlets are transferred to soil or vermiculite.
Gradual exposure to external environment.
Essential for survival under field conditions.
Factors Affecting Callus Culture
Type and age of explant
Composition of medium
Concentration of growth regulators
pH of medium (5.6–5.8)
Temperature and light
Genotype of plant
Applications of Callus Culture
Micropropagation of plants
Production of disease-free plants
Genetic transformation and transgenic plants
Somaclonal variation and crop improvement
Production of secondary metabolites
Germplasm conservation
Protoplast culture and fusion
Advantages
Rapid multiplication of plants
Year-round production
Requires small space
Useful for rare and endangered species
Limitations
Somaclonal variation
High cost and technical skill required
Risk of contamination
Not suitable for all plant species
Conclusion
Callus culture and plant regeneration represent a cornerstone of plant tissue culture technology. By manipulating growth regulators and culture conditions, whole plants can be regenerated from undifferentiated cells, demonstrating the totipotent nature of plant cells. This technique has immense significance in plant breeding, biotechnology, and conservation.
Callus is a mass of
A. Differentiated cells
B. Undifferentiated cells
C. Dead cells
D. Reproductive cells
Answer: B
Callus culture is based on the principle of
A. Differentiation
B. Totipotency
C. Mutation
D. Hybridization
Answer: B
The most suitable tissue for callus induction is
A. Xylem
B. Phloem
C. Meristematic tissue
D. Cork
Answer: C
The most widely used medium for callus culture is
A. White’s medium
B. Knop’s medium
C. Murashige and Skoog (MS) medium
D. Gamborg’s medium
Answer: C
Which hormone is essential for callus induction?
A. Cytokinin
B. Auxin
C. Gibberellin
D. Ethylene
Answer: B
2,4-D is a synthetic
A. Cytokinin
B. Auxin
C. Gibberellin
D. Inhibitor
Answer: B
High concentration of auxin promotes
A. Shoot formation
B. Root formation
C. Callus formation
D. Flower formation
Answer: C
Friable callus is
A. Hard and compact
B. Soft and loosely arranged
C. Highly differentiated
D. Dead tissue
Answer: B
Compact callus is
A. Loose and soft
B. Hard and dense
C. Embryogenic
D. Liquid
Answer: B
Embryogenic callus is capable of
A. Rooting only
B. Shoot formation only
C. Somatic embryogenesis
D. Senescence
Answer: C
Somatic embryogenesis originates from
A. Zygote
B. Somatic cells
C. Gametes
D. Pollen grains
Answer: B
The first stage of somatic embryo development is
A. Heart stage
B. Torpedo stage
C. Globular stage
D. Cotyledonary stage
Answer: C
Organogenesis is the formation of
A. Seeds
B. Callus
C. Organs
D. Protoplasts
Answer: C
Organ formation through callus is called
A. Direct organogenesis
B. Indirect organogenesis
C. Micropropagation
D. Fertilization
Answer: B
High cytokinin to auxin ratio induces
A. Roots
B. Shoots
C. Callus
D. Embryos
Answer: B
High auxin to cytokinin ratio induces
A. Shoots
B. Roots
C. Leaves
D. Flowers
Answer: B
Agar in culture media functions as
A. Nutrient
B. Growth regulator
C. Solidifying agent
D. Vitamin
Answer: C
Sucrose acts as a
A. Hormone
B. Vitamin
C. Carbon source
D. Buffer
Answer: C
The optimal pH of MS medium is
A. 3.5
B. 4.5
C. 5.6–5.8
D. 7.0
Answer: C
Ideal temperature for callus culture is
A. 15°C
B. 20°C
C. 25 ± 2°C
D. 35°C
Answer: C
Surface sterilization is done to avoid
A. Differentiation
B. Growth
C. Contamination
D. Regeneration
Answer: C
Mercuric chloride is used for
A. Nutrition
B. Sterilization
C. Hormone action
D. Solidification
Answer: B
Callus culture requires
A. Open field conditions
B. Aseptic conditions
C. Natural soil
D. Sunlight only
Answer: B
Totipotency refers to the ability of a cell to
A. Divide only
B. Form callus
C. Develop into a whole plant
D. Form roots only
Answer: C
Thiamine added in MS medium is a
A. Hormone
B. Vitamin
C. Carbohydrate
D. Enzyme
Answer: B
Non-embryogenic callus
A. Produces embryos
B. Regenerates plants
C. Cannot regenerate plants
D. Is highly organized
Answer: C
Callus culture is widely used in
A. Animal breeding
B. Plant biotechnology
C. Marine biology
D. Zoology
Answer: B
Genetic variation arising in tissue culture is called
A. Mutation
B. Hybridization
C. Somaclonal variation
D. Polyploidy
Answer: C
Which of the following is a cytokinin?
A. IAA
B. NAA
C. BAP
D. 2,4-D
Answer: C
Callus culture is useful for production of
A. Antibiotics
B. Secondary metabolites
C. Vaccines
D. Hormones
Answer: B
Hardening of plantlets is done to
A. Increase growth
B. Increase callus
C. Acclimatize plants to external conditions
D. Induce mutation
Answer: C
Callus cells are usually
A. Dead
B. Non-dividing
C. Actively dividing
D. Specialized
Answer: C
Callus induction generally requires
A. Bright light
B. Continuous light
C. Darkness or low light
D. UV light
Answer: C
Best explant for regeneration is
A. Old tissue
B. Mature tissue
C. Young meristem
D. Dead tissue
Answer: C
Plant regeneration from callus proves
A. Mutation
B. Totipotency
C. Heterosis
D. Polyploidy
Answer: B
Callus culture is a technique of
A. Ecology
B. Cytology
C. Plant tissue culture
D. Taxonomy
Answer: C
Somatic embryos differ from zygotic embryos because they
A. Undergo fertilization
B. Lack dormancy
C. Contain endosperm
D. Form seeds
Answer: B
Gibberellins mainly promote
A. Callus formation
B. Stem elongation
C. Rooting
D. Senescence
Answer: B
MS medium was developed by
A. White
B. Knop
C. Murashige and Skoog
D. Gamborg
Answer: C
Structure that develops into a complete plant in somatic embryogenesis is
A. Root primordium
B. Shoot primordium
C. Somatic embryo
D. Callus mass
Answer: C
Callus culture is useful for
A. Micropropagation
B. Genetic transformation
C. Virus-free plants
D. All of the above
Answer: D
Which factor does NOT affect callus culture?
A. Genotype
B. Medium composition
C. Growth regulators
D. Soil type
Answer: D
Protoplast culture is often initiated from
A. Seeds
B. Callus
C. Flowers
D. Fruits
Answer: B
Callus formation represents
A. Redifferentiation
B. Dedifferentiation
C. Fertilization
D. Senescence
Answer: B
Redifferentiation results in
A. Callus formation
B. Organ formation
C. Cell death
D. Variation
Answer: B
Secondary metabolites are commonly produced using
A. Seed culture
B. Root culture
C. Callus culture
D. Pollen culture
Answer: C
Balanced auxin and cytokinin concentrations favor
A. Rooting
B. Shooting
C. Callus induction
D. Flowering
Answer: C
Plant tissue culture requires
A. Sterile environment
B. Soil
C. Rainwater
D. Fertilizers
Answer: A
Callus culture is helpful in conservation of
A. Common plants
B. Rare plants
C. Endangered plants
D. Both B and C
Answer: D
The final goal of callus culture is
A. Callus formation
B. Cell multiplication
C. Whole plant regeneration
D. Mutation induction
Answer: C
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