1. What is a Protoplast?
A protoplast is a plant cell stripped of its cell wall, leaving a naked, membrane-bound cell. This allows for:
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Direct gene uptake
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Cell fusion
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Regeneration from a single cell
Protoplasts must be cultured in carefully controlled conditions to avoid bursting (lysis) and ensure survival.
2. Protoplast Isolation
| Step | Description |
|---|---|
| Tissue collection | Young leaves or callus used as source |
| Enzyme digestion | Cellulase and pectinase in osmotic solution |
| Filtration | Removes debris |
| Centrifugation | Separates viable protoplasts |
| Resuspension | In 0.4–0.6 M mannitol/sorbitol to maintain osmotic pressure |
Sterility is essential throughout the process.
3. Culture of Protoplasts
Once isolated, protoplasts are:
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Suspended in liquid MS medium with 2–5% mannitol
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Supplemented with NAA, 2,4-D, and BAP
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Sometimes embedded in alginate beads for immobilization
Goal: Regeneration of the cell wall → cell division → microcallus → shoots/roots → plantlets
📌 Regeneration from Protoplasts
4. Protoplast Fusion Techniques
| Technique | Mechanism |
|---|---|
| PEG-mediated fusion | Chemical method; protoplasts treated with PEG + Ca²⁺ |
| Electrofusion | Electric pulses align and fuse protoplasts |
Outcomes:
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Somatic hybrids: Between different species or genera
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Cybrids: Cytoplasmic hybrids (nucleus from one parent, organelles from another)
These hybrids are used for:
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Transferring disease resistance
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Overcoming sexual incompatibility
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Studying mitochondria or chloroplast genetics
5. Applications
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Somatic Hybridization: Combining species with no natural compatibility
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Example: Tomato + Potato (“Pomato”)
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Gene Editing: Direct delivery of CRISPR plasmids
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Metabolic Engineering: Production of unique secondary metabolites
Protoplasts serve as a plant biotechnology platform, bridging cellular and genetic techniques.