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#protoplast fusion

12 public questions tagged with this topic.

Plant tissue culture 2

Fusion of cytoplasm of two cells with nucleus from one parent is called:

Fusion product containing cytoplasm of two cells but nucleus from only one parent is termed cybrid cytoplasmic hybrid, distinction from true somatic hybrid which contains both nuclei yielding allotetraploid. Cybrids arise when protoplasts of two sources fuse forming heterokaryon with two nuclei and mixed organelles followed by selective loss or inactivation of one nucleus. Inactivation can be engineered by pre treating one partner with gamma irradiation X ray or metabolic inhibitor iodoacetate that damages nucleus while preserving cytoplasm viability. During subsequent mitosis heterokaryon resolves uninucleate cybrid retaining nuclear genome of one parent determining morphological traits while cytoplasmic organelles mitochondria chloroplasts may be biparental, recombined, or sorted singly. This outcome is deliberately exploited to transfer extra nuclear encoded traits. Unlike somatic hybrid with combined chromosome sets, cybrid is diploid nuclear background with novel cytoplasm enabling creation of novel cytoplasmic genomic interactions. Molecular analysis using mitochondrial RFLP chloroplast SSR confirms organelle identity. Cybrid formation is major tool for cytoplasmic genetic engineering overcoming strict maternal inheritance barriers in sexual crossing impossible for organelle traits.

Ref: Sidorov et al., 1981 cybrid concept; Gleba 1979 Cytoplasmic inheritance.

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Plant tissue culture 2

Electrofusion induces fusion by:

Electrofusion induces protoplast fusion by applying controlled electrical pulses that create reversible membrane breakdown at points of contact allowing lipid bilayers to merge and cytoplasms to coalesce, offering higher fusion frequency viability and selectivity than chemical PEG method. Protocol consists of two electrical phases: first low strength high frequency alternating current 50 to 200 kilohertz field causes dielectrophoresis polarizing protoplasts forming induced dipoles aligning them in pearl chains bringing membranes into intimate contact at poles. Second short high intensity direct current pulses 0.5 to 2 kilovolts per centimeter lasting microseconds generate transient pores dielectric breakdown in contacting membranes that upon resealing fuse into single membrane encircling two protoplasts. Post fusion alternating current field maintained briefly stabilizes heterokaryons. Advantages include ability to monitor individual pair fusion under microscope enabling one to one fusion selection, reduced chemical toxicity preserving division capacity, and high heterokaryon yield up to 60 percent. Parameters voltage duration number of pulses optimized per species to avoid Joule heating irreversible electroporation and cell death. Electrofusion combined with fluorescence activated sorting and microfluidic devices now facilitates precise cybrid and somatic hybrid production for citrus potato Brassica rootstock improvement and organelle transfer programs.

Ref: Zimmermann & Vienken 1982 electrofusion; Tempelaar et al., Plant Sci electroporation.

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Plant tissue culture 2

Common fusogen used for induced protoplast fusion is:

Common chemical fusogen employed to induce protoplast fusion is polyethylene glycol PEG polymer with molecular weight typically 1500 to 6000 daltons applied at concentration 15 to 40 percent weight per volume combined with calcium ions 50 millimolar and alkaline pH 9 to 10. PEG induces fusion through multiple mechanisms: it is highly hydrophilic binding water molecules dehydrating intermembrane space reducing hydration repulsion between negatively charged phospholipid bilayers, promotes close apposition of membranes via calcium bridging between phosphate head groups and creates molecular disorder increasing membrane fluidity and probability of lipid mixing and coalescence upon gradual dilution washing out PEG. Efficiency of heterokaryon formation ranges 10 to 30 percent depending on species and concentration exposure time 15 to 30 minutes. Although PEG causes some cytotoxicity protein denaturation requiring thorough washing after treatment, it remains cheapest most accessible method for somatic hybridization compared to electrofusion equipment requiring electroporators. Other chemical fusogens include high calcium alkaline pH alone and inactivated Sendai virus with fusogenic hemagglutinin proteins. PEG fusion nonspecifically generates multinucleate products but selection via fluorescence markers or complementation enables recovery of desired hybrids for crop improvement.

Ref: Kao & Michayluk 1974 PEG fusion; NCBI protoplast fusion review.

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Plant tissue culture 2

Somatic hybridization involves fusion of:

Somatic hybridization involves fusion of protoplasts isolated from somatic vegetative tissues of different species cultivars or genera, bypassing sexual incompatibility barriers that prevent conventional crossing due to pre zygotic pollen stigma incongruence or post zygotic endosperm abortion. Protoplasts are obtained by enzymatic digestion of cell walls using cellulase Onozuka R-10 and macerozyme releasing naked cells bounded solely by plasma membrane maintaining totipotency to regenerate wall and divide. Mixed protoplast populations labeled with different fluorescent markers or selectable complementation traits are induced to fuse using polyethylene glycol PEG with calcium at high pH causing membrane dehydration and coalescence or by electrofusion using alternating current for alignment and direct current pulses for reversible membrane breakdown forming heterokaryons containing mixed cytoplasms and nuclei. Fusion product nuclear fusion may produce symmetric somatic hybrid allotetraploid containing both parental chromosome sets or after chromosome elimination asymmetric hybrids carrying partial genomes. This technique enabled transfer of polygenic traits disease resistance abiotic tolerance cytoplasmic male sterility from wild relatives to crops, exemplified by pomato potato plus tomato fusion and citrus somatic hybrids for rootstock improvement.

Ref: Gleba & Sytnik 1984 somatic hybridization; Evans et al., Handbook Plant Cell Culture.

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