Protoplast isolation and fusion

Hanstein first introduced the term protoplast in 1880 that refers to the naked plant cell
all components of the plant cell excluding the cell wall.
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Protoplast isolation and fusion

Mechanical Plasmolysis of the plant cells Dissect tissue De-plasmolysis expansion & release the protoplasts from cut ends of cells

1960 enzymatic isolation of protoplasts was demonstrated

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Protoplast isolation

Cocking used a concentrated solution of cellulase to degrade the cell walls of tomato root cells.
1968 Takabe Enzymatic isolation of protoplasts from tobacco 1971 regenerated plants from the isolated tobacco protoplasts

Steps in protoplast isolation; enzymatic method: 1. Selection of starting materials (and surface sterilization); Leaves, petioles, shoot apices, roots, fruits, coleoptiles, hypocotyls, stem, embryos, microspores, & callus, cell suspension cultures
The physiological condition of the source plant is important; since the condition will influence the yield and viability of the isolated protoplasts.
plants should be grown under controlled conditions fully expanded leaves of young plants or new shoots is used (mesophyll cells) leaves are sterilized, lower epidermis is peeled off and cut into small pieces callus and suspension culture cells collect at the early 4 exponential phase of growth

Protoplast isolation
2. Enzyme treatment: 3 enzyme types are required

-Pectinase mainly degrades the middle lamina -Cellulase & hemicellulase degrade the cellulosic and hemicellulosic components of plant cells
30 min- 20 hr treatment Incubation temperature, 25 to 30oC pH of the enzyme solution, 4.7 to 6.0 An osmoticum is always included as a stabilizer since protoplasts released into the standard cell culture medium will burst (mannitol or sorbitol)

 3. Filtration: pass the solution through a stainless steel or nylon mesh (50-100 mm) Centrifugation: mix filtrate with sucrose to a final concentration of 20% and centrifuge Washing: remove protoplast band, mix with sucrose & centrifuge, repeat three times Suspend protoplasts in a volume of cell medium to an appropriate cell density

Isolation of protoplasts from Arabidopsis (14-dy-old seedlings yielded 5 106-107 protoplasts

4. Protoplast viability testing: This involves staining of the purified protoplasts.

Fluorescein diacetate

(FDA)

accumulates in the plasmalemma of viable protoplasts can be detected by fluorescence microscopy.

Evans blue
the stain

intact protoplasts are capable of excluding

Cells and protoplasts stained with Evans blue. (a) Non-stained cells (viable). (b) Viable protoplast surrounded by blue cellular aggregates (c) Cell with blue cytoplasm. (d) Control of Non-viable dead cells

5. Culture of protoplasts: Regeneration of the cell walls in liquid culture Transfer to agar media (agarose) Incubate at 20 to 28oC in low light or darkness

Within 3 weeks cell colonies are visible

organogenesis or embryogenesis shoots plantlets 8

Protoplast isolation and fusion

Uses? Studies on as a single cell system section of mutant cell lines, cloning of cell populations

Study cell wall biosynthesis

Somatic hybridization Genetic engineering

Protoplast isolation and fusion

Somatic hybridization
the fusion of two protoplasts
same species different species between different genera

cross between sexually incompatible species transfer of nuclear or cytoplasmic characters

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Fusion products
HETEROKARYON hybrid or cybrid
- a cell with more than one nucleus formed by the fusion of two cells

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Fusion products
Hybrid a mononucleate cell that results from the fusion of 2 different cells, leading to the formation of a synkaryon (a hybrid cell that results form the fusion the nuclei it carries) Cybrid a cytoplasmic hybrid produced by the fusion of a whole cell with a cytoplast. Cytoplasts are enucleated cells.

cybrid

heterokaryon

hybrid

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Protoplast fusion
Isolated protoplasts are negatively charged electrostatic repulsion between adjacent protoplast

Efficient fusion is obtained with PEG, high [Ca2+] and a relatively high pH of 5.5-6.5

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Protoplast fusion: 3 phases

3 scientists were instrumental in the development of the PEG method in 1974 Kao, Michayluk and Wallin
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2 3 Phase 1: Agglutination or adhesion two or more protoplasts are brought together in close proximity PEG, high calcium ions, high pH

Phase 2: Plasma membrane fusion at localized sites Fusion (high Temp) Formation of cytoplasmic bridges between the protoplasts

Phase 3: Formation of heterokaryon rounding off of the fused protoplasts spherical heterokaryon

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Protoplast fusion
Phase 1: Agglutination or adhesion Phase 2: Plasma membrane fusion Phase 3: Formation of heterokaryon

selection of hybrid between 1-10% of the protoplasts actually undergo fusion (A + B A, B, AB, AA, BB)
Cells of Petunia sp. leaves (with chloroplasts). Left cell was merged with a protoplast from Impatiens neuguinea petals 15 (with coloured vacuole).

Identification and selection of hybrid cells

1.Observation of visual characteristics
visual identification of the hybrid cells mechanically isolate the hybrid cells based on growth of the hybrid cells based on morphology of cells

2.Hybrid cell display of genetic complementation for recessive mutations

chlorophyll deficient mutants

3.Physiological complementation for in vitro growth requirements

- nitrate reductase mutants
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Verification and characterization of somatic hybrids

1.Morphology; the morphological characteristics are intermediate between both parents leaf shape, leaf area, trichomes, petiole size root morphology flower shape, color, size & structure pollen viability

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Verification and characterization of somatic hybrids

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Verification and characterization of somatic hybrids

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Verification and characterization of somatic hybrids

2.Isoenzyme analysis; Electrophoretic variability of isoenzymes
Isoenzymes = different molecular forms of an enzyme that catalyzes a reaction Aminotransferase, amylase, lactate dehydrogenase, super oxide dismutase (SOD)

Somatic hybrids may display isoenzyme bands of certain enzymes specific to one parent or to both parents simultaneously.

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Isoenzyme analysis
1 2 3 4 5

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Verification and characterization of somatic hybrids

3.Chromosomal constitution;
counting the chromosomes in presumed hybrid cells can be an easy and reliable method to verify hybrid cells. Should be the sum of chromosome number of two parent protoplasts used for fusion
C. sinensis and P. trifoliata 2n=18 chromosome number in one of the hybrid plants was 36, sum of C. sinensis (2n=18) and P. trifoliata
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Verification and characterization of somatic hybrids

4.Molecular techniques;
specific molecular markers of chloroplast and mitochondrial DNA have been used to characterize the nature of hybrids RFLP, AFLP, RADP, microsatellites

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Verification and characterization of somatic hybrids

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Verification and characterization of somatic hybrids

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Genetic traits transferred by somatic hybridization

Nicotiana tabacum + N. nesophila resistance to Tobacco mosaic virus Solanum tuberosum + S. chacoense resistance to Potato virus X S. tuberosum + S. commersonii frost tolerance Citrus sinensis + Poncirus trifoliate resistance against Phytophthora S. tuberosum + S. bulbocastanum resistance against root knot nematode Citrullus lanatus + Cucumis melo club rot resistance
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Cybrids
Or cytoplasmic hybrids Cells or plants containing the nucleus of one species but the cytoplasm from both parental species Produced in protoplast fusion experiments when:
Enucleate protoplast or inactivated nucleus Elimination of the nucleus from a heterokaryon Gradual elimination of the chromosomes of one species from a hybrid during mitotis

Male sterility and herbicide resistance

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Limitations
Requirement of an efficient plant regeneration protocol An effective method of selection of the fused product The regenerated plants after somatic hybridization are unbalanced & are not viable; genetic stability during protoplast culture is poor

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