Q.53 The two enzymes commonly used for isolation of protoplasts from plants are
(A) Cellulase and Lipase (B) Cellulase and Amylase
(C) Pectinase and Cellulase (D) Pectinase and Lipase
Plant Protoplast Isolation: Correct Enzymes Revealed
Protoplasts are plant cells without cell walls, vital for somatic hybridization, gene transfer, and regeneration in biotechnology. Question 53 asks for the two enzymes routinely used to digest cell walls mechanically or enzymatically. This is a staple in plant molecular biology and tissue culture curricula.
Correct Answer: (C) Pectinase and Cellulase
Pectinase and cellulase are the standard enzymes for protoplast isolation.
Pectinase (e.g., macerozyme) degrades pectin in the middle lamella, separating cells. Cellulase (e.g., Onozuka R-10) then hydrolyzes cellulose and hemicellulose in the cell wall proper. Protocols use sequential (pectinase first, then cellulase) or simultaneous methods, yielding viable protoplasts for culture.
Why Other Options Fail: Detailed Analysis
Each wrong choice pairs an irrelevant enzyme, testing cell wall composition knowledge.
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(A) Cellulase and Lipase: Lipase breaks down lipids, not plant cell walls (which lack major lipids). Useless for protoplast release; targets membranes instead.
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(B) Cellulase and Amylase: Amylase digests starch (polysaccharides in cytoplasm/plastids), irrelevant to cellulose/pectin walls. No role in wall degradation.
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(D) Pectinase and Lipase: Pectinase works, but lipase again misses the mark—fats aren’t structural in walls. Incomplete digestion leaves wall remnants.
Enzyme Action Comparison Table
| Option | Enzyme 1 | Enzyme 2 | Targets in Cell Wall? | Viable for Protoplasts? |
|---|---|---|---|---|
| (A) | Cellulase | Lipase | Cellulose; Lipids (No) | No |
| (B) | Cellulase | Amylase | Cellulose; Starch (No) | No |
| (C) | Pectinase | Cellulase | Pectin; Cellulose | Yes |
| (D) | Pectinase | Lipase | Pectin; Lipids (No) | No |
Practical Insight
Yields reach 10^6–10^7 protoplasts/g tissue with 1–2% enzyme mixes at 25–30°C for 2–4 hours, pH 5.5–5.8. Common in solanaceous plants like tobacco. Master this for lab protocols or exams—cell wall = pectin (middle lamella) + cellulose (wall).
