Protoplast fusion technology involves removing the cell walls from cells, leaving only the cell membrane. This allows the fusion of the cell membranes and cytoplasm of two different cell types, transferring genes. There are three main methods of inducing protoplast fusion - mechanical fusion using microtools, chemical fusion using substances like polyethylene glycol, and electrofusion using electric fields. Enzymes like cellulase and pectinase are used to break down plant cell walls, while lysozyme degrades bacterial cell walls. Protoplast fusion has applications in strain improvement for industrial purposes by creating cells with desired properties.

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