Protoplast – unexplored aspects

Tech de’carta

By-Tarang Joshi(B.tech 3rd year)

Rashi Jain(B.tech 3rd year)

SIR PADAMPAT SINGHANIA UNIVERSITY

UDAIPUR

Protoplast Fusion Technology

Protoplasts are the cells of which cell walls are removed and cytoplasmic membrane is the outermost layer in such cells.

Protoplast can be obtained by specific lytic enzymes to remove cell wall.

Protoplast fusion is a physical phenomenon, during fusion two or more protoplasts come in contact and adhere with one another either spontaneously or in presence of fusion inducing agents.

Diagrammatic representation

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Protoplast fusion is an important tool in strain improvement for bringing genetic recombinations and developing hybrid strains in filamentous fungi.

Cont…

This technique will be one of the most frequently used research tools for tissue culturists,molecular biologists and biotechnologists

Enzymes used for breaking of cell walls:

For protoplast fusion it is important that the cell wall of plant and microorganisms is degraded .

Various enzymes used for this process.Cellulase and pectinase or macerozyme acting on plant cell wall.

Bacterial cell wall are degraded by the action of lysozyme.

Methods of protoplast fusion

Protoplast fusion can be broadly classified into two categories:

Spontaneous fusionInduced fusion

The isolated protoplast can be induced to fuse by three ways:

Mechanical fusionChemofusionElectrofusion

Mechanism of protoplast fusion

When the protoplasts are brought into close proximity this is followed by an induction phase thereby changes induced in electrostatic potential of the membrane results in fusion .After the fusion,the membranes stabilizes and the surface potential returns to their former state.

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When the protoplasts are closely adhered ,the external fusogens cause disturbance in the intramembranous proteins and glycoproteins.This increases membrane fluidity and creates a region where lipid molecule intermix,allowing coalescence of adjacent membranes.The negative charge carried by protoplast is mainly due to intramembranous phosphate groups .The addition of Ca ++ ions causes reduction in the zeta potential of plasma membrane and under this situation protoplasts are fused.

Biotechnological applications:

Protoplasts contained all the intracellular organelles of cells and form a vital link in transferof micromolecules between cyto organelles,currently most of the laboratories engaging in fungal genetics are using gene manipulation procedures based on protoplasts

The process involves protoplast mutagenesis, transformation and protoplast fusion.

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Protoplast fusion was perfomed between Saccharomyces diastatiacus and a brewing polyploidy

Saccharomyces uvarum strain.Some clones derived from fusion products can produce low carbohydrate beer of acceptable flavor.

Electrically induced protoplast fusion employed for hybrid construction in ergosterol producing yeast strains.Some fusion products proved to be hybrid with respect to ergosterol content and to remain stable over several generations.

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Effects of protoplast fusion on δ- endotoxins production in Bacillus thuringiensis species (H14) found that Bacillus thuringiensis fusants have 1.48 time more δ-endotoxins than wild type.

Fusants of Penicillium chrysogenum and Cephalosporium acremonium produced a novel lactam antibiotic.

Protoplast fusion was caried between different strains of Tolypocladium inflatum .One of the recombinants

produced cyclosporine 2.8 times more than parental strain.

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Diploid strains of Torulaspora delbrueckii were tested for industrial application.

Fermentation activity and tolerance to freeze-thawing in dough were succesfully inherited by the diploid strains .

SANK 50268 cells required 4.5 h for saline pretreatment, while YL3 cells required only 1 h.

With respect to dehydration, YL3 cells were processed 2.5 times faster than SANK 50268 cells.

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YL3 did not require a frequent change of the starch filter on the dehydrator drum as did SANK 50268.

Thus, the dehydration process was greatly improved with YL3 with regard to labor ,time and economy, such as the amount of electricity needed.

Agriculture field

To produce germplasm previously unavailable to the plant breeder.

To transfer traits such as disease and herbicide resistance.

To produce hybrids and cybrids.

Strain Improvement

Phaffia rhodozyma Astaxanthin - used in the aquaculture industry which is

limited because of the low carotenoid content from natural isolates.

Parental strains produce approx. 1600 μg carotenoid/g yeast.

After protoplast fusion hybrid of P.rhodozyma was stable and consistently produce more than 2000 μg carotenoid/g yeast.

Targeting of xylanase to increase protein accumulation in plant cells

Fungal xylanase of Trichoderma reesei was chosen as the model protein.

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When xylanase was targeted to chloroplasts and peroxisomes simultaneously the amount of xylanase accumulated was 160% of that in chloroplasts and 240% of that in peroxisomes.

The growth stage of the transgenic plants also affected the

total amount of xylanase; the highest level of accumulation

occurred at the time of flowering.

Intergeneric Hybrids by Protoplast Fusion

Saccharomyces cerevisiae

Zygosaccharomyces fermentati

Characteristics of hybrid

Resistance to certain drugs.

Ability to grow with either cellobiose or lactic acid as the main carbon source.

Stable under nonselective conditions.

Can tolerate high temperature so as to produce maximum amount of ethanol efficiently.

Conclusion

They serve as excellent starting material for:

Cell cloning

Development of mutant lines.

Protoplasts used as tool in physiological and biochemical studies.

Strain development of industrially important microorganisms.

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Isolated plant protoplasts have several biotechnological applications, including their fusion to generate novel somatic hybrid and cybrid plants and the production of transgenic plants expressing specific characteristics.

Diploids obtained through protoplast regeneration express their genetic characteristics perfectly.

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