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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 light 535 nm. Under fluorescence microscope dead protoplasts show bright red nucleus clearly differentiated from viable green FDA positive cells in dual viability test. This assay allows rapid quantitative assessment of protoplast isolation success and cytotoxicity of fusogen treatments. Compared to colorimetric dyes like Evans blue which stains dead cells blue but requires bright field observation, PI provides fluorescent contrast compatible with flow cytometric analysis sorting live cells for downstream culture fusion transformation. Maintaining membrane integrity crucial because only viable protoplasts can regenerate wall divide form microcallus and participate in heterokaryon formation during somatic hybridization procedures.

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 charge prevents efflux, emitting green fluorescence 520 nm upon excitation blue light 490 nm under fluorescence microscope. Dead protoplasts with compromised membranes lack esterase activity and cannot retain fluorescein thus remain non fluorescent and also allow entry of counter stain propidium iodide marking nucleus red. Assay provides rapid quantitative estimation of viability percentage and metabolic vigor as fluorescence intensity correlates with esterase activity and capacity to regenerate wall and divide in culture medium with hormones. Dual staining FDA propidium iodide enables simultaneous live dead discrimination via flow cytometry sorting heterokaryons for somatic hybridization applications.

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 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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