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Suspension culture and development - methodology, kinetics of growth and production formation, elicitation methods, hairy root culture. Detailed notes


Suspension culture and development - methodology, kinetics of growth and production formation, elicitation methods, hairy root culture. Detailed notes




1. Introduction

Suspension culture is a type of plant tissue culture in which single cells or small cell aggregates are grown in liquid nutrient medium under continuous agitation. It is mainly used for:
Large-scale biomass production
Secondary metabolite production
Cell physiology and biochemical studies
Genetic manipulation and selection.


2. Methodology of Suspension Culture


2.1 Source of Explant

Usually initiated from friable callus
Callus derived from:
Leaf
Stem
Root
Hypocotyl
Friable callus is preferred as it disintegrates easily into single cells.

2.2 Preparation of Cell Suspension


Friable callus is transferred into liquid MS medium
Medium contains:
Carbon source (usually sucrose)
Auxins (2,4-D commonly used)
Culture maintained in:
Conical flasks
Orbital shaker (100–150 rpm)


2.3 Culture Conditions


Parameter
Requirement
Temperature
25 ± 2°C
pH
5.6 – 5.8
Light
Dark or diffused light
Aeration
Provided by shaking
Agitation
Prevents cell clumping


2.4 Subculturing


Periodic transfer to fresh medium (7–14 days)
Maintains exponential growth
Prevents nutrient depletion


2.5 Types of Suspension Cultures

Batch culture
Continuous culture
Immobilized cell culture

3. Kinetics of Growth and Product Formation


3.1 Growth Curve of Suspension Culture
Suspension culture follows a sigmoid (S-shaped) growth curve:


1. Lag Phase
Cells adapt to new environment
Little or no division
Enzyme synthesis occurs


2. Exponential (Log) Phase
Rapid cell division
High metabolic activity
Maximum biomass accumulation


3. Linear Phase
Growth rate slows
Nutrients start declining


4. Stationary Phase
Cell division stops
Biomass remains constant
Secondary metabolite production is maximum

5. Decline Phase


Cell death occurs
Toxic metabolites accumulate


3.2 Measurement of Growth
Fresh weight
Dry weight
Packed cell volume (PCV)
Cell number (haemocytometer)


3.3 Product Formation Kinetics
Secondary metabolites are classified based on growth association:


1. Growth-associated products
Produced during exponential phase
Example: Proteins, enzymes


2. Non-growth-associated products
Produced during stationary phase
Example: Alkaloids, phenolics


3. Mixed growth-associated products
Produced throughout growth cycle


4. Elicitation Methods
4.1 Definition
Elicitation is the process of stimulating secondary metabolite production in plant cell cultures using elicitors.


4.2 Types of Elicitors
A. Biotic Elicitors
Derived from biological sources
Examples:
Fungal cell wall extracts
Yeast extract
Chitosan
Polysaccharides


B. Abiotic Elicitors


Non-biological
Examples:
Heavy metals (Ag⁺, Cd²⁺)
UV radiation
Temperature stress
Salicylic acid
Jasmonic acid


4.3 Mechanism of Elicitation


Elicitor binds to cell membrane receptor
Activation of signal transduction pathways
Increase in intracellular Ca²⁺
Activation of defense genes
Enhanced secondary metabolite synthesis


4.4 Advantages of Elicitation
Increased metabolite yield
Reduced culture time
Cost-effective production
5. Hairy Root Culture
5.1 Definition
Hairy root culture is a genetically transformed root culture induced by infection with Agrobacterium rhizogenes.


5.2 Mechanism of Hairy Root Induction


A. rhizogenes transfers Ri plasmid T-DNA
rol (root locus) genes integrate into plant genome
Causes:
Profuse root branching
Fast growth
Hormone-independent growth


5.3 Methodology


Plant explant infected with A. rhizogenes
Co-cultivation for 24–48 hours
Transfer to antibiotic medium to remove bacteria
Selection of transformed roots
Maintenance in hormone-free liquid medium


5.4 Characteristics of Hairy Root Cultures
Genetic stability
Rapid growth rate
High biosynthetic capacity
No need for growth regulators


5.5 Applications
Production of secondary metabolites:
Alkaloids (e.g., atropine)
Terpenoids
Phenolics
Study of root metabolism
Biotransformation reactions


6. Advantages of Suspension and Hairy Root Cultures
Uniform growth
Easy scale-up in bioreactors
Controlled conditions
Consistent metabolite yield


7. Limitations
Genetic instability in long-term cultures
Shear sensitivity of cells
High operational cost


8. Conclusion
Suspension culture and hairy root culture are powerful tools in plant biotechnology for large-scale production of biomass and valuable secondary metabolites. The use of growth kinetics studies and elicitation strategies significantly enhances productivity.



50 MCQs: Suspension Culture & Hairy Root Culture


1. Suspension culture is best initiated from
A. Hard callus
B. Compact callus
C. Friable callus
D. Meristem
Answer: C
2. Suspension cultures are grown in
A. Solid medium
B. Semi-solid medium
C. Liquid medium
D. Agar medium
Answer: C
3. Continuous shaking in suspension culture helps in
A. Cell differentiation
B. Aeration and uniform cell distribution
C. Callus formation
D. Organogenesis
Answer: B
4. Ideal agitation speed for suspension culture is
A. 10–20 rpm
B. 50–60 rpm
C. 100–150 rpm
D. 300 rpm
Answer: C
5. Commonly used auxin for suspension culture
A. IAA
B. NAA
C. 2,4-D
D. Kinetin
Answer: C
6. pH of suspension culture medium is usually
A. 4.0
B. 4.5
C. 5.6–5.8
D. 7.0
Answer: C
7. The growth curve of suspension culture is
A. Linear
B. Parabolic
C. Sigmoid
D. Exponential only
Answer: C
8. During lag phase
A. Rapid cell division occurs
B. Secondary metabolites form
C. Cells adapt to new environment
D. Cell death occurs
Answer: C
9. Maximum biomass accumulation occurs in
A. Lag phase
B. Exponential phase
C. Stationary phase
D. Decline phase
Answer: B
10. Secondary metabolites are mainly produced during
A. Lag phase
B. Log phase
C. Stationary phase
D. Decline phase
Answer: C
11. Packed cell volume (PCV) measures
A. DNA content
B. Protein level
C. Cell biomass
D. Metabolite concentration
Answer: C
12. Which is a growth-associated product?
A. Alkaloids
B. Phenolics
C. Terpenoids
D. Proteins
Answer: D
13. Non-growth associated products are produced in
A. Lag phase
B. Exponential phase
C. Stationary phase
D. Decline phase
Answer: C
14. Elicitation increases production of
A. Primary metabolites
B. Secondary metabolites
C. DNA
D. RNA
Answer: B
15. An elicitor is
A. Growth hormone
B. Nutrient
C. Stress-inducing agent
D. Antibiotic
Answer: C
16. Yeast extract is an example of
A. Abiotic elicitor
B. Chemical elicitor
C. Biotic elicitor
D. Physical elicitor
Answer: C
17. Salicylic acid acts as
A. Nutrient
B. Abiotic elicitor
C. Antibiotic
D. Growth inhibitor
Answer: B
18. Heavy metals used as elicitors include
A. Na⁺
B. K⁺
C. Ag⁺
D. Ca²⁺
Answer: C
19. Signal transduction during elicitation involves
A. DNA methylation
B. Protein degradation
C. Calcium influx
D. Cell lysis
Answer: C
20. Hairy root culture is induced by
A. Agrobacterium tumefaciens
B. Agrobacterium rhizogenes
C. Rhizobium
D. Bacillus
Answer: B
21. Hairy roots arise due to transfer of
A. Ti plasmid
B. Ri plasmid
C. pBR322
D. BAC
Answer: B
22. rol genes are responsible for
A. Shoot induction
B. Callus formation
C. Root proliferation
D. Flowering
Answer: C
23. Hairy root cultures grow
A. With high auxin
B. With cytokinin
C. Without growth regulators
D. Only on solid medium
Answer: C
24. A major advantage of hairy root culture is
A. Slow growth
B. Genetic instability
C. High metabolite production
D. Need for hormones
Answer: C
25. Hairy roots show
A. Poor branching
B. Profuse lateral branching
C. No growth
D. Callus formation
Answer: B
26. Hairy root cultures are mainly used for production of
A. Vitamins
B. Carbohydrates
C. Secondary metabolites
D. Lipids
Answer: C
27. Antibiotics are used in hairy root culture to
A. Induce roots
B. Eliminate Agrobacterium
C. Increase growth
D. Prevent differentiation
Answer: B
28. One example of alkaloid produced in hairy root culture is
A. Morphine
B. Atropine
C. Nicotine
D. All of the above
Answer: D
29. Immobilized cell culture involves
A. Free cells
B. Entrapment in a matrix
C. Solid callus
D. Root culture
Answer: B
30. Batch culture is characterized by
A. Continuous medium supply
B. Closed system
C. Constant nutrient addition
D. No growth phases
Answer: B
31. Continuous culture maintains cells in
A. Lag phase
B. Log phase
C. Stationary phase
D. Decline phase
Answer: B
32. Shear sensitivity is a problem in
A. Solid culture
B. Suspension culture
C. Seed culture
D. Root nodules
Answer: B
33. Bioreactors are used for
A. Small-scale culture
B. Large-scale suspension culture
C. Seed germination
D. Organ culture
Answer: B
34. Hairy root cultures are genetically
A. Unstable
B. Stable
C. Variable
D. Temporary
Answer: B
35. Chitosan is a
A. Growth regulator
B. Antibiotic
C. Biotic elicitor
D. Nutrient
Answer: C
36. UV radiation is classified as
A. Biotic elicitor
B. Abiotic elicitor
C. Chemical elicitor
D. Hormonal elicitor
Answer: B
37. Primary metabolites are mainly produced during
A. Stationary phase
B. Decline phase
C. Exponential phase
D. Lag phase
Answer: C
38. A major limitation of suspension culture is
A. Uniform growth
B. Easy scale-up
C. Genetic instability
D. Controlled conditions
Answer: C
39. Haemocytometer is used to measure
A. Metabolites
B. Protein
C. Cell number
D. DNA
Answer: C
40. Friable callus is preferred because it
A. Is hard
B. Grows slowly
C. Easily disperses into single cells
D. Forms shoots
Answer: C
41. Stationary phase is characterized by
A. Rapid cell division
B. Nutrient limitation
C. Cell elongation
D. DNA synthesis
Answer: B
42. Hairy roots differ from normal roots because they
A. Grow slowly
B. Need hormones
C. Grow rapidly and autonomously
D. Are genetically unstable
Answer: C
43. Jasmonic acid is used as
A. Nutrient
B. Growth hormone
C. Elicitor
D. Antibiotic
Answer: C
44. Agitation in suspension culture prevents
A. Cell division
B. Cell aggregation
C. DNA replication
D. Metabolite synthesis
Answer: B
45. The decline phase is due to
A. Nutrient abundance
B. Toxic metabolite accumulation
C. Increased oxygen
D. Hormone addition
Answer: B
46. Secondary metabolite production is part of
A. Growth metabolism
B. Defense response
C. Cell division
D. DNA replication
Answer: B
47. The most suitable medium for suspension culture is
A. White’s medium
B. Gamborg’s B5
C. Murashige and Skoog (MS)
D. N6 medium
Answer: C
48. Hairy root cultures are maintained in
A. Solid agar
B. Semi-solid medium
C. Liquid hormone-free medium
D. Soil
Answer: C
49. Biotransformation refers to
A. Cell division
B. Gene transfer
C. Conversion of substrates into products
D. Callus formation
Answer: C
50. Main advantage of suspension culture over callus culture
A. Slower growth
B. Uniform exposure to nutrients
C. Less oxygen
D. Genetic variation
Answer: B






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