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#protoplasts

3 public questions tagged with this topic.

Plant tissue culture 2

Dead protoplasts are stained red by:

Dead protoplasts are stained red by propidium iodide PI nucleic acid intercalating fluorescent dye that distinguishes membrane compromised cells. PI is cationic molecule unable to cross intact lipid bilayer of viable protoplasts due to charge and size exclusion, so viable population excludes dye remaining unstained. In dead cells membrane integrity lost through enzymatic damage or physical stress, PI diffuses entering cytoplasm nucleus where it intercalates between double stranded DNA and RNA base pairs with strong affinity, enhancing red fluorescence emission at 617 nm when excited green ligh

Ref: Jones 1987 PI dead staining; Molecular Probes Handbook viability dyes.

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Plant tissue culture 2

Viable protoplasts fluoresce green when stained with:

Viable protoplasts fluoresce bright green when stained with fluorescein diacetate FDA, vital fluorescent dye used routinely for viability assessment before fusion or transformation to ensure batch quality above 80 percent. FDA itself is non fluorescent lipophilic molecule that diffuses across intact plasma membrane permeable only to living cells with intact barrier. Inside cytoplasm active non specific esterases present exclusively in living cells hydrolyze ester bonds releasing fluorescein highly fluorescent polar molecule that accumulates inside membrane intact vesicle because its negative c

Ref: Widholm 1972 FDA viability; Larkin 1976 fluorescein diacetate Plant Physiol.

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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

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

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