49. Choose the CORRECT sequence of steps involved in cytoplast production.
(A) Digestion of cell wall → protoplast viability → cybrid formation → osmotic stabilizer
(B) Osmotic stabilizer → digestion of cell wall → protoplast viability → cybrid formation
(C) Protoplast viability → osmotic stabilizer → digestion of cell wall → cybrid formation
(D) Osmotic stabilizer → digestion of cell wall → cybrid formation → protoplast viability
Cytoplast production involves isolating cytoplasm without the nucleus from plant protoplasts, a key technique in somatic hybridization for cybrid creation in plant biotechnology. The correct sequence prioritizes cell wall removal in a stabilized environment to ensure protoplast integrity before further applications like cybrid formation.
Correct Answer
Option (B): Osmotic stabilizer → digestion of cell wall → protoplast viability → cybrid formation.
This follows the logical workflow: osmotic support prevents lysis during wall digestion, enabling viable protoplasts (or cytoplasts via enucleation) for fusion into cybrids.
Option Analysis
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(A) Digestion of cell wall → protoplast viability → cybrid formation → osmotic stabilizer: Incorrect, as digestion without prior osmotic stabilizer causes protoplast bursting due to hypotonic shock; stabilizer must precede enzymatic treatment.
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(B) Osmotic stabilizer → digestion of cell wall → protoplast viability → cybrid formation: Correct, matching standard protocol—stabilizer (e.g., sorbitol, mannitol) maintains turgor, enzymes (cellulase, pectinase) remove wall, viability is assessed, then cytoplasts fuse for cybrids.
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(C) Protoplast viability → osmotic stabilizer → digestion of cell wall → cybrid formation: Incorrect, viability testing cannot precede isolation steps; stabilizer is initial.
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(D) Osmotic stabilizer → digestion of cell wall → cybrid formation → protoplast viability: Incorrect, cybrid formation requires prior viability confirmation; viability follows isolation, not vice versa.
Introduction to Cytoplast Production Steps Sequence
Cytoplast production steps sequence is a cornerstone of plant biotechnology, enabling nucleus-free cytoplasmic isolation for cybrid formation. This process supports genetic engineering by fusing cytoplasts with intact protoplasts, bypassing sexual barriers in crop improvement.
Detailed Cytoplast Production Process
The cytoplast production steps sequence begins in a hypertonic medium with osmotic stabilizers like 0.5-1M mannitol to balance internal pressure. Cellulase and macerozyme then digest the cell wall, yielding fragile protoplasts. Viability (via FDA staining) confirms >80% intact cells before micromanipulation enucleates nuclei, producing cytoplasts for PEG/electrofusion into cybrids.
Key reagents include:
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Osmoticum: Mannitol or sorbitol.
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Enzymes: 1-2% cellulase, 0.1% pectinase.
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Viability test: Fluorescein diacetate under UV.
Applications in Cybrid Formation
Cytoplasts drive cybrid production, transferring cytoplasmic male sterility (CMS) for hybrid seeds. Examples include Brassica cybrids combining chloroplasts and mitochondria from donor cytoplasts.
Common Exam Mistakes
Students often reverse stabilizer and digestion, ignoring lysis risks. Focus on osmotic stabilizer first in cytoplast production steps sequence for accuracy.
