1. Protoplast Fusion Technology Presented by MONA ALBUREIKAN 2. What is protoplast fusion? cell walls are removed.

They only have a cell membrane. can be obtained by specific lytic enzymes. It is a physical phenomenon. it is possible to transfer some useful genes. The hybrid protoplast = cytoplasm + two fused parent nuclei. important tools in strain improvement. Used for prokaryotic and eukaryotic cells. 3. Why use protoplast fusion? create strains with desired properties. industrial properties. one of the most frequently used research tools. a great potential for genetic analysis. for strain improvement. 4. Enzymes used for breaking of cell walls: Various enzymes used for this process. Cellulase and pectinase or macerozyme for plant cell wall. Bacterial cell wall are degraded by lysozyme. Fungal wall degraded by Novozyme -234 ( glucanase and chitinase). Streptomyces cell wall degraded by lysozyme and achromopeptidase 5. Methods of protoplast fusion:Protoplast fusion classified into two categories: Spontaneous fusion: - often fuse spontaneously. - fuse through their plasmodesmata. Induced fusion: - Fusion with the help of fusion inducing chemicals agent. - Normally , do not fuse with each other. 6. How the isolated protoplast can be induced to fuseby three ways;1- Mechanical fusion: Isolated protoplast are brought into intimate physical contact mechanically under microscope using micromanipulator or perfusion micropipette. 7. How the isolated protoplast can be induced to fuse2- Chemofusion: Several chemicals has been used to induce protoplast fusion. such as sodium nitrate ,polyethylene glycol, Calcium ions(Ca++ ). cause the isolated protoplast to adhere each other. It is a non specific. It is inexpensive. It can cause massive fusion product. It can be cytotoxic and non selecetive and having less fusion frequency. 8. How the isolated protoplast can be induced to fuse3- Electrofusion; Used electric stimulation. Used An electric field of low strength (10Kvm-1). Electrofusion is easy to control. It is having fusion frequency up to 100%. It is gives reproducibility. It is less cytotoxic. The equipment is sophisticated and expensive. 9. Mechanism of protoplast fusion: 10. Protoplast fusion in bacteria: In bacteria protoplast can be obtained. Fusion can be carried out with low frequency in some gram positive organisms. For gram negative bacteria it is possible to obtain protoplast but regeneration is difficult. The procedure is highly efficient and yields upto 80% transformants. 11. References; http://www.biocompare.com/images/bc/006/ArticleI mages/hybridomaguide_0304_8.jpg http://www.aidic.it/IBIC2008/webpapers/96Verma.p df http://en.wikipedia.org/wiki/Somatic_fusion http://www.ihar.edu.pl/ZIKiT/hybrids.php4 12. THANK YOU Methods of Protoplasts Fusion

Protoplasts fusion is a physical phenomenon. During fusion, two or more protoplasts come in contact and adhere with one another spontaneously or in presence of fusion inducing chemicals. An important aspect has been that incompatibility

barriers do not exist during the cell fusion process at Interspecific, Intergeneric or even inter kingdom level.

A) Spontaneous Fusion:

During the enzymatic degradation of cell walls some of the adjacent protoplasts may fuse together to form homocaryons. These plurinucleate cells sometimes contain 2-3 nuclei, a phenomenon attributed to expansion and subsequent coalescence of the plasmodesmatal connections between the cells. The protoplasts once they are freely isolated, donot fuse spontaneously with each other.

Mechanical Fusion:

The giant protoplast of Acelabularia have been fused mechanically by pushing together two protoplast. This fusion does not upon the presence of fusion inducing agents. However, in this procedure protoplasts are likely to get injury. Protoplast released from meiocytes in enzyme solutions readily fuse by gentle tapping in depression slide.

B) Induced Fusion:

Fusion of freely isolated protoplasts from different sources with the help of fusion inducing chemical agents is known as induced fusion. Normally, isolated protoplasts donot fuse with each other because the surface of the isolated protoplasts carries negative charge around the outside of plasma membrane and thus is a strong tendency for protoplasts to repel one another due to their same charges.

i) NaNO3 Treatment:

Equal densities of protoplasts from two different sources are mixed and then centrifuged at 100g for 5 minutes to get a dense pellet. This is followed by addition of 4 ml of 5.5% sodium nitrate in 10.2% sucrose solution to Resuspend the

protoplast pellet. The suspended protoplasts are kept in water bath at 35 0C for 5 minutes and again centrifuged at 200 g for 5 minutes. The pellet is once again kept in water bath at 30 0C for 30 minutes. The fusions of protoplast take place at the time of incubation. Finally, the protoplasts are plated in semisolid culture medium. The frequency of fusion is not very high in this method.

ii) High PH or Ca++ Treatment:

Kelier and Melchers ( 1973) developed a method to effectively induce fusion of tobacco protoplasts at a high temperature ( 37 0C) in media containing high concentration of Ca++ ions at a highly alkaline condition ( PH 10.5). Equal densities of protoplasts are taken in centrifuge tube and protoplasts are spun at 100 g for 5 minutes. The pellet is suspended in 0.5 ml of medium. a 4 ml of 0.05 M CaCl2, 2H2O in 0.4 M mannitol at PH 10.5 is mixed to the protoplast suspension. The centrifuge tube containing protoplast at high PH or Ca++ is placed in water bath at 30 0C for 10 minutes and is spun at 50 g for 3 to 4 minutes. This followed by keeping the tubes in water bath (37 0C) for 40- 50 minutes. About 20-30% protoplast are involved in this fusion experiment.

iii) PEG Treatement:

PEG induces protoplast aggregation and subsequent fusion. But the concentration and molecular weight of PEG are important with respect to fusion. A solution of 37.5% w/v PEG of molecular weight 1500 to 6000 aggregates mesophyll and cultured cell protoplasts during a 45 minutes incubation at room temperature. Fusion of protoplast takes place during slow elusion of PEG with liquid culture medium. Carrot protoplast can be fused by 28% PEG 1500 and fusion can be

promoted by ca++ ion at concentration of 3.5 mM. But higher concentration of Ca++ ion has been considered beneficial. In some studies, high PH/Ca++ and PEG method have been combined. The PEG method has been modified slightly to fuse higher plant protoplast as indicated below:

a) PEG is more effective when it is mixed with 10-15% dimethyl sulfoxide. b) Addition of concanvalin A to PEG increases protoplast fusion frequency. c) Sea water has been used alone or in combination with PEG to fuse protoplasts.

iv) Electro Fusion:

Recently, mild electrical stimulation is being used to fuse protoplasts. This technique is known as electrofusion of protoplasts. Two glass capillary microelectrode are placed in contact with the protoplasts. An electric field of low strength (10kv m-3) give rise to dielectrophoretic pole generation within the protoplast suspension. This lead to pearl chain arrangement of protoplasts. The number of protoplasts within a pearl chain depends upon the population density of the protoplast and the distance between the electrodes. Subsequent application of high intensity electric impulse ( 100kv m-3) for some microseconds results in the electric breakdown of membrane and subsequent fusion.

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