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#plant cell wall

4 public questions tagged with this topic.

Plant tissue culture 3

Cellulase and pectinase are used for:

Plant cell walls composed of cellulose microfibrils embedded in hemicellulose and pectin matrix prevent direct membrane manipulation. Protoplast technology demands complete wall removal without lysing protoplasm. Cellulase complex from Trichoderma reesei hydrolyzes β-1,4 glycosidic bonds of cellulose, while pectinase or macerozyme from Rhizopus degrades polygalacturonate of middle lamella, loosening intercellular adhesion. Digestion carried out in isoosmotic solution of mannitol or sorbitol at pH 5.4 to 5.8, supplemented with calcium chloride for membrane stabilization, releases spherical protoplasts within 4 to 12 hours. Yield improves with purified enzymes, gentle shaking, and young leaf tissue. Viability assessed by fluorescein diacetate retention confirms intact plasmalemma. This enzymatic approach underpins somatic hybridization via PEG-induced fusion, electroporation-mediated transformation, and studies of cell wall regeneration and transient gene expression in plant molecular biology. Enzyme mixture optimization includes 2 percent cellulase RS and 0.5 percent macerozyme R-10 with 0.6 M mannitol and calcium chloride. Vacuum infiltration improves enzyme penetration. After digestion, protoplasts purified by sucrose gradient centrifugation and washed to remove enzyme residues. High viability essential for subsequent PEG-mediated transfection or somatic hybridization via electrical fusion techniques.

Ref: Cocking 1960 protoplast isolation; Bhojwani & Dantu Ch 14 enzymatic; NCBI PMC3248752 cellulase macerozyme protocol; Gamborg & Phillips Methods.

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Plant tissue culture 2

Protoplasts are plant cells that lack:

Protoplasts are plant cells that completely lack cell wall, defined as protoplasm enclosed only by plasma membrane after removal of cellulose hemicellulose and pectin components. Isolation employs enzyme mixture cellulase hydrolyzing beta 1,4 glucan backbone of cellulose microfibrils and pectinase macerozyme dissolving middle lamella pectin crosslinking cells, often adding hemicellulase driselase; incubation performed in osmoticum mannitol sorbitol 0.4-0.6 molar to prevent lysis due to loss of wall counteracting turgor. After digestion spherical protoplasts released from leaf mesophyll or cell suspension appear intact with visible chloroplasts. They retain nucleus vacuole mitochondria capable of resynthesizing new cell wall within 24-72 hours mediated by cellulose synthase complexes delivering glucan chains, re-entering cell cycle forming microcallus. Absence of wall makes protoplasts ideal recipients for direct gene transfer via electroporation PEG mediated DNA uptake and for somatic hybridization because membranes of different species can be forced to coalesce unrestricted by wall barrier. Viability assessed by fluorescein diacetate FDA green fluorescence. Totipotency retained ensures whole plant regeneration from single protoplast under appropriate division promoting medium.

Ref: Cocking 1960 protoplast isolation; NCBI Bookshelf protoplast manual.

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BASICS of PLANT+PYQS

Wall pressure is exerted by:

Pick B: Cell wall on protoplast. Thinking through BASICS of PLANT step by step rules out the lookalikes and leaves Cell wall on protoplast. Skip these: A) Protoplast on wall; C) Vacuole on wall; D) Plasma membrane. For hormones and genes, match the molecule or gene to its real role, not a neighboring pathway.

Ref: Best CSIR NET Plant Physiology books: Master Unit 6 with Taiz & Zeiger and Salisbury & Ross. Crack Part C experimental questions with top textbooks.

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