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 Protoplast culture covering isolation, fusion, somatic hybrid & cybrid production, preferential chromosome elimination, role in CMS, and genetic transformation.



Protoplast Culture


1. Introduction
A protoplast is a plant cell without a cell wall, surrounded only by the plasma membrane.
Protoplast culture allows direct access to the plasma membrane and genome, making it a powerful tool for:
Somatic hybridization
Cybrid production
Genetic transformation
Cytoplasmic trait transfer (e.g., CMS)


2. Isolation of Protoplasts


2.1 Source of Protoplasts
Young leaves (mesophyll cells)
Callus tissue
Cell suspension cultures
Roots or hypocotyls
Young, actively dividing tissues are preferred due to high viability.
2.2 Methods of Protoplast Isolation
A. Mechanical Method
Cell walls removed by cutting and plasmolysis
Rarely used
Causes low yield and high damage
B. Enzymatic Method (Most Common)
Cell wall digested using enzymes:
Enzyme
Function
Cellulase
Degrades cellulose
Pectinase
Degrades middle lamella
Hemicellulase
Removes hemicellulose
2.3 Steps in Enzymatic Isolation
Surface sterilization of explant
Cutting tissue into small pieces
Incubation in enzyme solution
Gentle shaking
Filtration to remove debris
Centrifugation and washing
Collection of viable protoplasts
2.4 Osmotic Stabilizers
Prevent protoplast bursting:
Mannitol
Sorbitol
Sucrose
2.5 Viability Testing
Fluorescein diacetate (FDA) staining
Viable protoplasts fluoresce green
3. Culture of Protoplasts
3.1 Culture Media
MS medium
KM8p medium
Modified B5 medium
Supplements:
Auxin (2,4-D, NAA)
Cytokinin (BAP, kinetin)
Osmoticum
3.2 Stages of Protoplast Culture
Cell wall regeneration
First mitotic division
Micro-callus formation
Callus development
Plant regeneration (organogenesis / embryogenesis)
4. Protoplast Fusion (Somatic Hybridization)
4.1 Definition
Fusion of two genetically different protoplasts to form a hybrid cell.
4.2 Types of Fusion
A. Spontaneous Fusion
Rare
Occurs naturally during isolation
B. Induced Fusion
Most commonly used.
4.3 Methods of Protoplast Fusion
1. Chemical Fusion
Polyethylene glycol (PEG) + Ca²⁺
Most widely used
Causes membrane agglutination and fusion
2. Electro-fusion
Electric pulses align and fuse protoplasts
High efficiency
Precise control
5. Generation of Somatic Hybrids
5.1 Somatic Hybrid
Contains nuclear and cytoplasmic genomes of both parents
Produced after fusion and regeneration
5.2 Steps
Fusion of protoplasts
Selection of fused cells
Callus formation
Plant regeneration
Confirmation using molecular markers
5.3 Applications
Hybridization between sexually incompatible species
Transfer of disease resistance
Improvement of crop plants
6. Cybrids (Cytoplasmic Hybrids)
6.1 Definition
Cybrids contain:
Nuclear genome from one parent
Cytoplasm (mitochondria/chloroplasts) from both parents
6.2 Methods of Cybrid Production
Fusion of:
Normal protoplast + enucleated protoplast
Irradiation (UV or X-ray) of donor nucleus
Selective elimination of nuclear DNA
6.3 Importance
Transfer of cytoplasmic traits
Especially useful for CMS
7. Preferential Elimination of Chromosomes
7.1 Definition
Selective loss of chromosomes from one parent after fusion.
7.2 Causes
Genomic incompatibility
Asynchronous cell division
Nuclear–cytoplasmic interactions
7.3 Significance
Helps stabilize cybrids
Produces asymmetric hybrids
Useful in alien gene transfer
8. Role in Cytoplasmic Male Sterility (CMS)
8.1 CMS Definition
CMS is the inability of plants to produce functional pollen due to mitochondrial genome alterations.
8.2 Role of Protoplast Fusion
CMS cytoplasm can be transferred via cybrids
Nuclear genome from elite cultivar
Cytoplasm from CMS donor
8.3 Advantages in Hybrid Seed Production
Eliminates manual emasculation
Enhances hybrid vigor
Used in crops like:
Rice
Maize
Brassica
Sunflower
9. Role in Genetic Transformation
9.1 Why Protoplasts Are Ideal
No cell wall barrier
Direct DNA uptake
9.2 Methods of DNA Introduction
PEG-mediated DNA uptake
Electroporation
Liposome-mediated transfer
Microinjection
9.3 Expression of Transgenes
Stable integration
Transient expression studies
Reporter genes (GUS, GFP)
10. Advantages of Protoplast Culture
Direct genetic manipulation
Creation of novel hybrids
Cytoplasmic trait transfer
Useful in monocots and dicots
11. Limitations
Difficulty in plant regeneration
Genetic instability
Low efficiency in some species
High technical expertise required
12. Conclusion
Protoplast culture is a cornerstone technique in modern plant biotechnology. Its applications in somatic hybridization, cybrid formation, CMS transfer, and genetic transformation make it invaluable for crop improvement and hybrid seed technology.

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