For Pharmacy students

1. Lecture-4
By
Dr. Ahmed Metwaly
PROTOPLAST
FUSION

2. Objectives:
Techniques of plant tissue culture
Protoplast fusion

3. Introduction
■ Protoplast is a naked cell (without cell wall) surrounded by a plasma
membrane. It can regenerate cell wall, grow and divide.
■ Spheroplast cells have their cell wall only partially removed.
■ Is fragile but can be cultured and grow into a whole plant.
■ Cells can originate from any type of tissue (Mesophyll tissue - most
suitable source ).
■ Can be applied in somatic hybridization.
■ Can be applied in biotechnology and microbiology.
■ Somatic hybridization is the development of hybrid plants through the
fusion of somatic protoplasts of two different plant species/ varieties.
■ Somatic Hybridization was firstly introduced by Carlson in Nicotiana
glauca.
■ In 1960, E.C Cocking contributed to the enzymatic isolation and culture
of protoplast.

4. 1. Isolation of protoplast
2. Fusion of the protoplasts of desired species/varieties
Or with a desired DNA
3. Identification and Selection of somatic hybrid cells
4. Culture of the hybrid cells
5. Regeneration of hybrid plants
Steps of protoplast fusion

5. Protoplast isolation
■ Refers to the separation of protoplast from plant tissue
■ Important to isolate viable and uninjured protoplast as gently and
as quickly as possible
Involves two methods:
■ Mechanical
■ Enzymatic

6. Mechanical method
■ Tissue is immersed in 1.0 M sucrose until protoplasm shrunk away
from their enclosing cell wall (Plasmolysis).
■ Plasmolysed tissue is cut with a sharp knife at such thickness that
only cell walls are cut.
■ Undamaged protoplast in strips are released by osmotic swelling
when placed in a low concentration of sucrose solution

7. ■ Used for vacuolated cells like onion bulb scale, radish and beet
root tissues
■ Low yield of protoplast
■ Tedious process
■ Low protoplast viability

9. Enzymatic method
■ Refers to the use of enzymes to dissolve the cell wall for releasing
protoplasts.
■ The plant cell wall is mainly composed of cellulose, hemicellulose and
pectin which are respectively degraded by the enzymes cellulase,
hemicellulase and pectinase. In plant cells we mainly uses these enzymes
(cellulase, hemicellulase and pectinase).
■ Advantages:
■ Used for variety of tissues and organs such as fruits, roots, petioles,
leaves…
■ Osmotic shrinkage is minimum
■ Cells remain intact and not injured
■ High yield of protoplast
■ Easy to perform
■ More protoplast viability .

10. Leaf sterilization, removal of
epidermis
Plasmolysed cellsPlasmolysed cells
Pectinase +cellulase Pectinase
Protoplasm released Release of isolated cells
cellulase
Protoplasm
released
Isolated Protoplasm

11. Procedure
■ Incubation of leaf segments overnight in enzyme solution at
pH 4.5-6.0 & temperature 25-30 0C .
■ Mixture is filtered and centrifuged
■ Protoplast forms pellet
■ Then washed with sorbitol and re-centrifuged
■ Clean protoplasts float
■ They are pipetted out

12. Purification of protoplast
● Protoplasts are purified by removing:
○ Undigested material (debris)
○ Bursts protoplasts
○ Enzymes
● Debris are removed by filtering the preparation through a nylon
mesh
● Enzymes are removed by centrifugation whereby the protoplasts
settle to the bottom of the tube and the supernatant removed with
the help of a pipette
● Intact protoplasts are separated from broken protoplasts through
centrifugation and removed by a pipette as they are collected at
the top of tube

13. Purification of protoplast

14. Protoplast Culture
● Isolated protoplast can be cultured in an appropriate
medium to reform cell wall and generate callus
● Optimal culture conditions:
1. Optimal density to the culture.
2. Optimal auxin to cytokinin ratio, glucose and sucrose.
3. Maintain osmoprotectant in the medium
4. Temperature: 20-28°C, pH: 5.5-5.9.

15. Culture of protoplasts
● Protoplasts cultured in suitable nutrient media first generate a new
cell wall
● The formation of a complete cell with a wall is followed by an
increase in size, number of cell organelles, and induction of cell
division
● The first cell division may occur within 2 to 7 days of culture
resulting in small clumps of cell, also known as micro colony, within 1
to 3 weeks From such clumps, there are two routes to generate a
complete plant (depending on the species)
1. Plants are regenerated through organogenesis from callus masses
(Micropropagation)
2. The micro calli can be made to develop into somatic embryos
(somatic embryogenesis), which are then converted into whole plant
through germination

16. Importance of Protoplast Culture
(without fusion)
1. Gene Transfer
2. Biological examinations
● Study of Osmotic behavior
● Study of Plasma lemma
● Study of Cell wall formation
● Organelle isolation
● Study of Morphogenesis
● Virus uptake and replication
● Study of photosynthesis

18. Factors affecting protoplast culture
1. Plant species and varieties
Small genetic difference leads to varying protoplast responses to
culture conditions
2. Plant age and organ
Age of donor plant and its developmental stage (smaller better)
3. Pre-culture conditions
Climatic factors affect the yield of protoplast and response when
cultured
4. Pre-treatment to the tissue before isolating protoplasts
Cold treatment, plasmolysis and hormone increases the chance of
recovery of viable protoplasts and their plating efficiency
5. Density
It influences plating efficiency and surviving of protoplasts. At higher
density, protoplasts compete with one another while at lower density
losses of metabolites from protoplasts is more.

19. Protoplast Fusion (Somatic
hybridization)
Protoplast fusion techniques:
1. Electrofusion
2. Polyethene glycol -
induced fusion (PEG)
3. High Ca2+ , high pH
4. NaNO3 treatment
5. Mechanical fusion

20. FUSION PRODUCTS - THE HYBRIDS AND CYBRIDS
. The nuclei of two protoplasts may or may not fuse together even
after fusion of cytoplasms.
•The binucleate cells are known as heterocyte .
•When nuclei of two different sources are fused the cells are known
as hybrid.
•Only cytoplasms fuse and genetic information from one of the two
nuclei is lost is known as cybrid i.e. cytoplasmic hybrid.
•Some of the protoplasts of the same type may undergo fusion to
produce homocytes each with 2-40 nuclei.

22. Electrical fusion
If Protoplasts are placed into a small culture vessel
containing electrodes and a potential difference is applied,
then the protoplasts will line up between the electrodes.
If now an extremely short, electric shock is applied,
protoplasts can be induced to fuse.

23. PEG (Polyethylene glycol) Fusion
● It has a high molecular weight
about 1500-6000.
● Usually a PEG solution of about
28-50% is used for protoplast
fusion.
● This polymer binds to the lipid
membrane of cells and thus
induces fusion
● Fusion takes place for 45 min in
incubation .

24. Mechanical fusion
In this the isolated protoplast are
brought into intimate physical
contact mechanically. Under
microscope and using
micromanipulator or perfusion
micropipette.

26. Hybrid identification- Based on difference between
the parental cells and hybrid cell with respect to
•Pigmentation
•Cytoplasmic markers
Fluorochromes like FITC (fluoroscein isothiocyanate) and RITC
(Rhodamine isothiocyanate) are used for labelling of hybrid cells
•Presence of chloroplast
•Nuclear staining
•Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-
orcein stain
•Regeneration
Plants are induced to regenerate from hybrid calli.
These hybrid plants must be at least partially fertile, in addition to
having some useful property, to be of any use in breeding schemes.

27. Application of Protoplast
Protoplasts can be used:
● In the production of Cybrid
● For Somatic Hybridization to overcome sexually incompatible
species
● Ingesting “Foreign” material into cytoplasm
● For DNA transformation
● Used to study wall synthesis and decomposition
● Studied as Single Cell System

28. Production of Cybrid
Cybrid contain nuclear and cytoplasmic genome of one parent and
only the cytoplasmic genome of the second.

29. Ingesting “Foreign” material into cytoplasm
Protoplast being wall-less show high pinocytic activity and can ingest
biological active foreign bodies such as DNA, plasmids, bacteria ,
viruses etc.
• results into modified cells.
Advantageous to plant breeder in getting more efficient crop varieties
in near future.

30. Somatic hybridization
Fusion of protoplast that facilitates
the mixing of 2 whole genomes and
could be exploited in crosses at:
intergeneric, interkingdom and
interspecific levels
Somatic hybridization is used to
produce hybrids from sexually
incompatible species.
This method could also be used to
study selection procedures.

31. Advantages of Protoplast fusion
1. It facilitates the mixing of two genomes and can be used
in crosses at interspecific, intergeneric or even intraspecific
levels
2. To create new strains with desired properties and for
strain improvement
3. Mixing two genomes opens the door to gene transfer and
a study of gene expression, stability of several traits and cell
genetic changes

32. Disadvantages of Protoplast Fusion
During the mechanical method of isolation of protoplasts:
1. It yields a very small amount of protoplasts after a rather tedious
procedure
2. It is not suitable for isolating protoplasts from meristematic and
less vacuolated cells
● During and subsequent to digestion of the cell wall, the protoplast
becomes very sensitive to osmotic stress. Thus, cell wall and
protoplast storage must be done in an isotonic solution to prevent
rupture of the plasma membrane.