Protoplast fusion and Somatic Hybridization

2. PLANT TISSUE CULTURE
PROTOPLAST FUSION AND SOMATIC HYBRIDIZATION
Presented By: Ayesha Farooq

3. CONTENTS
Introduction Of Somatic Hybridization
Process Of Somatic Hybridization
Isolation Of Protoplast
Protoplast Fusion
• Spontaneous Fusion
• Induced Fusion
Selection Of Somatic Hybrids
• Biochemical Method
• Cytometric Method
• Visual Method
Applications Of Somatic Hybridization

4. WHAT IS PROTOPLAST?
 A plant, bacterial or fungal cells in which the cell wall is
removed through enzymatic degradation called
protoplast.
 The protoplast is “totipotent” in nature.

5. PROTOPLAST FUSION
Somatic fusion also known as protoplast fusion is a type of genetic modification in
plants by which two distinct species of plants (protoplast) are fused together to form
new hybrid plant with the characteristics of both a somatic hybrid.
IMPORTANCE
Sexual hybridization performed only within the specie or closely related species. In
this method ant improvement is not possible.
To overcome this problem protoplast fusion technique is used to produce viable
hybrids.

6. DIFFERENCE BETWEEN SOMATIC AND SEXUAL HYBRIDIZATION
SOMATIC HYBRIDIZATION
It involves fusion of somatic cell
(protoplast).
It requiers tissue culture technique.
Cytoplasm is contributed by both
parents.
Segregation does not occur.
Crosses between unrelated species can
be easily made.
SEXUAL HYBRIDIZATION
It involves fusion of male and female
gametes.
It does not require tissue culture
technique.
Cytoplasm is strictly contributed by
female parent.
Segregation occurs.
Crosses between unrelated species are
incompatible.

7. PROCESS OF SOMATIC HYBRIDIZATION
In somatic hybridization 4 steps are involved which are as follows:
• Removal of the cell wall using cellulase enzyme to produce a somatic cell called a
protoplast.
• The cells are then fused using:
 Electric shock (electrofusion)
 Chemical treatment
• The formation of the cell wall is then induced using hormones.
• Identification and selection of somatic hybrid.
• Somatic cells are then grown into calluses which then are further grown to plantlets
and finally to a full plant, known as a somatic hybrid.

9. ISOLATION OF PROTOPLAST
The word “PROTOPLAST” was coined by “Hanstein” in 1880.
Isolation of protoplast can be done by three methods:
• Mechanical method (Non enzymatic)
• Enzymatic method
 Sequential enzymatic (Two step)
 Mixed enzymatic (Simultaneous)

10. Mechanical method
Mechanical method of protoplast isolation was first done by “Klercker” in 1982.

11. Enzymatic method
This method was first used by “Takebe” and other in 1968 in two steps.

12. Protoplast Fusion
Isolated protoplasts are devoid of cell walls, there in vitro fusion becomes relatively
easy. There are no barriers of incompatibility.
Protoplast fusion that involves mixing of protoplasts of two different genomes can
be achieved by
• Spontaneous Fusion
• Mechanical Fusion
• Induced fusion

13. Spontaneous Fusion
• Protoplast fuse spontaneously during isolation process mainly due to
physical contact.
• Fuse through their plasmodesmata.
Mechanical Fusion
• The protoplasts can be pushed together mechanically to fuse.
• Mechanical fusion may damage protoplasts by causing injuries.

14. Induced fusion
Freshly isolated protoplasts can be fused by induction. Induced fusion are of two types:
• Chemo-fusion
Induced by chemicals also known as fusogens.
Such as; PEG, NaNo3, Ca 2+ ions, Polyvinyl alcohol.
It can be cytotoxic and non-selective and having less fusion frequency.
• Electro-fusion
Fusion induced by electronic stimulation.
Used an electric field of low strength (10kv m-1).
Electro-fusion technique is simple, quick and efficient and hence preferred by many
workers.
Less cytotoxic and having fusion frequency up to 100%.

15. Identification and Selection of hybrid cell
Different methods are used to identify and selection of hybrid cells.

16. Biochemical method
For selection of hybrid cells are
based
on the use of biochemical
compounds in
the medium (selection medium).
1. Selection of hybrid on the
basis of Drug Sensitivity

17. 2. Selection of hybrid on the basis of Auxotrophic mutants:

18. VISUAL METHOD
Visual selection of hybrid cells,
although tedious is very efficient.
1. Visual selection coupled with
differential media growth:
There exist certain natural
differences in the sensitivity of
protoplasts to the nutrients of a
given medium.

19. CYTOMETRIC METHODS:
• In this method flow cytometry and fluorescent activated cell sorting technique is
used.
• Cytometry: Method of the measurement of the characteristics of cells e.g.
 Size
 Cell Content
 Morphology
 DNA Content
 Existence of proteins in cytoplasm

20. MECHANICAL METHOD
• Visually identified in microscope
• Which is then isolated by pipette namely “Drummond pipette”.

21. APPLICATION OF SOMATIC HYBRIDIZATION
• Disease Resistance
• Environmental Tolerance
• Quality Characters
• Help to study the cytoplasmic genes
• unique nuclear-cytoplasmic genetic combination
• Somatic hybridization can be done in plants that are still in juvenile phase.