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 fuse
by 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 fuse
2- 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 fuse
3- 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