3rd lecture about micropropagation and protoplast fusion techniques

1. Lecture 3
By
Dr. Ahmed Metwaly
TISSUE CULTURE

2. Objectives:
Techniques of plant tissue culture
■ Micropropagation
■ Protoplast fusion

3. Micropropagation
■ Micropropagation starts with the selection of plant
tissues (explant) from a healthy, vigorous mother plant.
Any part of the plant (leaf, apical meristem, bud and
root) can be used as explant.

4. Stage 0: Preparation of donor plant
■ This stage involves the preparation of mother plants to
provide quality explants for better establishment of
aseptic cultures in stage 1.
■ To reduce the contamination problem in the subsequent
stages, mother plant should be grown in a glasshouse,
this will also reduce the need for a harsh sterilization
treatment.
■ Stage 0 also includes exposing the stock plants to suitable
light, temperature and growth regulator treatments to
improve the quality of explants.
■ Stage 0 give a complete idea about the most suitable
conditions for plant propagation.

5. Stage I: Initiation stage
Explant: choosing:
■ Meristematic tissues, located either on terminal or axillary buds, are
induced to proliferate in response to hormonal treatments.
■ If the objective is to produce virus-free plants from an infected
individual it becomes necessary to start with sub-millimeter shoot tips
and away from the infection.
■ If the objective is an enhanced auxiliary branching, only the explants
which carry a pre-formed vegetative bud will be suitable.

6. ■ Explant Sterilization
■ In this stage an explant is surface sterilized and transferred into
nutrient medium.
■ Generally, the combined application of bactericide and fungicide
products is suggested. The selection of products depends on the
type of explant to be introduced.
■ The surface sterilization of explant in chemical solutions is an
important step to remove contaminants with minimal damage to
plant cells .
■ The most commonly used disinfectants are sodiumhypochlorite,
calcium hypochlorite , ethanol and mercuric chloride (HgCl2).
■ The cultures are incubated in growth chamber either under light or
dark conditions according to the method of propagation.

7. Stage II: Multiplication stage
■ The aim of this phase is to increase the number of
propagules. The number of propagules is multiplied by
repeated subcultures until the desired (or planned)
number of plants is attained.

8. Stage III shoot production
■ Selection of cytokinin type and concentration production
determined by:
■ •Shoot multiplication rate
■ •Length of shoot produced
■ •Frequency of genetic variability

9. Stage IV: Rooting stage
■ The rooting stage may occur simultaneously in the
same culture media used for multiplication of the
explants by increasing auxins ratio. However, in some
cases it is necessary to change media, including
nutritional modification and growth regulator
composition to induce rooting and the development of
strong root growth

10. Stage V: Transfer to natural
environment (Transplantation)
■ Growing quality plants from in vitro to ex vitro
conditions.
■ IT is done gradually from high to low humidity and
from low light intensity to high light intensity. The
plants are then transferred to an appropriate
substrate (sand, peat, compost etc.) and gradually
done under greenhouse.

11. ■ Advantages
Micropropagation has a number of advantages over traditional
plant propagation techniques:
■ The main advantage of micropropagation is the production
of many plants that are clones of each other.
■ Micropropagation can be used to produce disease-free
plants.
■ It can have an extraordinarily high fecundity rate, producing
thousands of propagules while conventional techniques
might only produce a fraction of this number.
■ It is the only viable method of regenerating genetically
modified cells or cells after protoplast fusion.
■ It is useful in multiplying plants which produce seeds in
uneconomical amounts, or when plants are sterile and do
not produce viable seeds or when seed cannot be stored.
■ Micropropagation has an accelerated growth compared to
similar plants produced by conventional methods - like
seeds or cuttings.

12. ■ Disadvantages
■ Micropropagation is not always the perfect means of
multiplying plants. Conditions that limits its use
include:
■ It is very expensive.
■ A monoculture is produced after micropropagation,
leading to a lack of overall disease resilience.
■ An infected plant sample can produce infected
progeny. This is uncommon as the stock plants are
carefully screened.
■ Not all plants can be successfully tissue cultured.
■ Some plants are very difficult to disinfect of fungal
organisms.

13. Introduction for protoplast fusion
■ 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 hybridisation.
■ Can be applied in biotechnology and microbiology.
■ Somatic hybridisation 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.

14. 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

15. 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

16. Mechanical method
■ Tissue is immersed in 1.0 M sucrose (high concentration)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

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

18. 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 .

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

20. 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

21. 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

22. Purification of protoplast

23. 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

24. 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.

26. 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.

27. 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 .

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

29. 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.

30. 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.

31. 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

32. 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

34. 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

35. 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

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

37. 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.

38. 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.

39. 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

40. 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.