protoplast unxplored aspects
-
Upload
guest800e180 -
Category
Technology
-
view
2.137 -
download
2
description
Transcript of protoplast unxplored aspects
![Page 1: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/1.jpg)
Protoplast – unexplored aspects
Tech de’carta
By-Tarang Joshi(B.tech 3rd year)
Rashi Jain(B.tech 3rd year)
SIR PADAMPAT SINGHANIA UNIVERSITY
UDAIPUR
![Page 2: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/2.jpg)
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.
![Page 3: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/3.jpg)
Diagrammatic representation
![Page 4: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/4.jpg)
Cont…
Protoplast fusion is an important tool in strain improvement for bringing genetic recombinations and developing hybrid strains in filamentous fungi.
![Page 5: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/5.jpg)
Cont…
This technique will be one of the most frequently used research tools for tissue culturists,molecular biologists and biotechnologists
![Page 6: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/6.jpg)
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.
![Page 7: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/7.jpg)
![Page 8: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/8.jpg)
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
![Page 9: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/9.jpg)
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.
![Page 10: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/10.jpg)
Cont…
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.
![Page 11: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/11.jpg)
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.
![Page 12: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/12.jpg)
Cont…
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.
![Page 13: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/13.jpg)
Cont…
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.
![Page 14: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/14.jpg)
Cont…
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.
![Page 15: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/15.jpg)
Cont…
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.
![Page 16: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/16.jpg)
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.
![Page 17: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/17.jpg)
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.
![Page 18: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/18.jpg)
Targeting of xylanase to increase protein accumulation in plant cells
Fungal xylanase of Trichoderma reesei was chosen as the model protein.
![Page 19: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/19.jpg)
Cont…
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.
![Page 20: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/20.jpg)
Intergeneric Hybrids by Protoplast Fusion
Saccharomyces cerevisiae
Zygosaccharomyces fermentati
![Page 21: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/21.jpg)
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.
![Page 22: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/22.jpg)
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.
![Page 23: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/23.jpg)
Cont…
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.
![Page 24: protoplast unxplored aspects](https://reader035.fdocuments.us/reader035/viewer/2022081508/556574bed8b42a95028b4a50/html5/thumbnails/24.jpg)
THANK YOU