Download SOMATIC CELL FUSION

SOMATIC CELL FUSION
(PROTOPLAST FUSION)
Group Members
•
•
•
•
•
•
Muhammad Ahmad
Aghna Naveed
Suniza Dar
Sarmad Basit
Kiran Ali
Maria Gillani
What is protoplast?
• A protoplast is a plant, bacterial or fungal cell
that had its cell wall completely or partially
removed using either mechanical or enzymatic
means.
What is PROTOPLAST Fusion ?
• Protoplast fusion is a type of fusion in
which two protoplasts (I.e a cell without
cell wall) are fused or combined together(under
in vitro conditions) in such a way that the new
hybrid cell is formed as a result of the fusion
,which consist of characteristics of both
the cells due to genetic modification.
Types of protoplast fusion:
1)Spontaneous fusion: Spontaneous fusion
also known as mechanical fusion is the type of
fusion in which the two protoplasts are fused
together by bringing them closer and disrupting
their cell membranes.
2)Induced fusion: induced fusion is the type of
fusion in which either chemical agents (I.e
chemofusion) or electric current (I.e
electrofusion) is used to fuse the two protoplasts.
HISTORY
• In 1847 Theodore Schwann observed that in
certain cells the walls and cavities of the cells
coalesce(fuse) together. It was this observation
that provided the first hint that cells fuse.
• The word protoplast was first introduced in 1880
by Hastein.
• First isolation of the cell wall from from plant cell
to create protoplast was achieved by Klercker in
in 1892 by mechanical method.
• The second isolation of cell wall from the plant
cell was done by cocking in 1960 through
enzymatic method.
• The first plant regeneration was done by
Nagata and Takebe in 1971.
• The first induced fusion ( I.e chemo fusion)
was introduced by Kao and Michayluk in
1974.
SOMATIC HYBRIDIZATION
TECHNIQUE
1
2
3
4
• Isolation of protoplast
•Fusion of the protoplasts of desired
species/varieties
• Identification and Selection of somatic hybrid cells
• Regeneration of hybrid plants
Protoplast isolation
Outer most covering in plant cell is cell wall which is made
up of cellulose, hemicellulose, pectin, etc which is rigid and
is not easily breakable.
So we have two approaches to get a cell which only contains
protoplast.
1) Mechanical:In this process a force is applied to the cell
and the cell wall breaks. For example in almonds and
peanuts.
2) Enzymatic: In this case, certain enzymes are used which
dissolve the cell wall. The enzymes used are cellulose,
pectinase, etc. And example is egg.
• Enzymatic approach is more used than the
mechanical approach because in
mechanical approach the force/pressure
can sometimes damage the cell and the
pressure does not remain constant for all
the cell walls.
• Isolation of protoplasts is readily achieved by treating
cells/tissues with a suitable mixture of cell wall
degrading enzymes.
• Usually, a mixture of pectinase or macerozyme (0.11.9%) and cellulose (1-2%) is appropriate for most plant
materials. Hemicellulase may be necessary for some
tissues.
• The pH of enzyme solution is adjusted between
4.7 and 8.0 and the temperature is kept at 2530°C.The osmotic concentration of enzyme mixture and
of subsequent media is elevated to stabilize the
protoplasts and to prevent them from bursting.
• Protoplasts have been isolated from virtually all plant
parts, but leaf mesophyll is the most preferred
tissue, at least in case of dicots, for this purpose
Fusion of the protoplasts of
desired species/varieties
• Polyethylene glycol (PEG) induced protoplast fusion is the
most commonly used as it induces reproducible high
frequency fusion accompanied with low toxicity to most cell
types. The protoplast mixture is treated with 28-50% PEG
(MW 1,500-6,000) for 15-30 min, followed by gradual washing
of the protoplasts to remove PEG; protoplast fusion occurs
during the washing.
• Electrofusion:
A more selective and less drastic approach is the
electrofusion technique, which utilizes low voltage
nonuniform alternating electric current pulses to bring
the protoplasts in close contact. Fusion of protoplasts is
brought about by a short pulse of high voltage.
Selection of Hybrid Cells
The protoplast suspension recovered after a treatment
with a fusion inducing agent (fusogen) consists of the
following cell types:
(i) unfused protoplasts of the two species/strains,
(ii)products of fusion between two or more protoplasts
of the same species (homokaryons),
(iii)‘hybrid’ protoplasts produced by fusion between
one (or more) protoplast(s) of each of the two
species (heterokaryons)
• In somatic hybridization experiments, only the heterokaryotic
or ‘hybrid’ protoplasts, particularly those resulting from
fusion between one protoplast of each of the two fusion
partners, are of interest.
• However, they form only a small proportion of the population
(usually 0.5-10%). Therefore, an effective strategy has to be
employed for their identification and isolation. This step is
called the selection of hybrid cells, is the most critical, and is
still an active area of investigation.
• Some visual markers, e.g., pigmentation, of the parental
protoplasts may be used for the identification of hybrid cells
under a microscope; these are then mechanically isolated and
cultured.
• Several workers have attempted to devise systems,
which specifically select for hybrid cells. In simple
words, these systems exploit some properties (usually,
deficiencies) of the parental species, which are not
expressed in the hybrid cells due to complementation
between their genetic systems.
• Phytotoxins, antibiotics, specific amino acids can also
be used to identify hybrids.
• A more general and widely applicable strategy, but
demanding more work than the previous approach, is
to culture the entire protoplast population without
applying any selection for the hybrid cells. All the types
of protoplasts form calli; the hybrid calli are later
identified on the basis of callus morphology,
chromosome constitution, protein and enzyme
banding patterns, etc
Regeneration of Hybrid
Plants
• Once hybrid calli are obtained, plants are induced to
regenerate from them since this is a prerequisite for their
exploitation in crop improvement. Further, the hybrid plants
must be at least partially fertile, in addition to having some
useful property, to be of any use in breeding schemes. The
culture techniques have been refined to a state where plant
regeneration has been obtained in a number of somatic
hybrids
CHARACTERISTICS OF HYBRIDS
AND CYBRIDS
Somatic hybridization produces symmetric or asymmetric hybrids, or
cybrids.
1) Symmetric Hybrids:
• These contains the somatic chromosomes of both the parental
species.These are very significant as they show all the properties
exhibited by parent species.
• Many symmetric hybrids have been produced between sexually
incompatible species. Some of these possess and express useful
genes and are fertile; these have been ordered into breeding
programs. Symmetric hybrids provide the following opportunities.
i.
Hybrids between Nonflowering and Male Sterile
Strains:
• Somatic hybrids can be produced between a nonflowering and a
male sterile strain of a crop species. Such hybrids have been
produced in potato: the fusion parents were a male sterile and a
nonflowering dihaploid clone, while some of the somatic hybrids
were male fertile.
ii. Superior Somatic Hybrids:
• Some somatic hybrids may possess such desirable features that
may make them commercially useful. For example, Datura hybrids D
.Their alkaloid content is 20-25% higher than those in the parent
species hence they are industrially useful for producing
scopolamine.
• It can also generate some novel materials that may be very useful in
genetic, physiological, biochemical and other studies.
• Asymmetric Hybrids:
• These are those hybrids which preserve the
genetic material of one parent organism. The
chromosome content of other parent species is
lost.
• For example, protoplasts of a nitrate reductase
mutant of Nicotiana plumbaginifolia were fused
with irradiated protoplasts of Atropa belladona-,
the somatic hybrids were selected on nitrate
medium. The hybrids contained 4 or 6 N.
plumbaginifolia genomes plus 6-26 A. belladona
chromosomes (many of these were deleted
chromosomes).
• Somatic Cybridization:
•
The process of protoplast fusion resulting in the
development of cybrid is called as Cybridization.
• CYBRID:
Plants or cells which containing nucleus of
one species but cytoplasm from both the parental
species. For production of a cybrid cell uses two
parental cells but should be one complete cell
(cytoplasm with nucleus) and other should be
without nucleus or in active nucleus.
Aims of Cybridization:
 To obtain biological information on intra- and
intergeneric transfer of organelles .
 To obtain cytoplasm gene of one species to another
species which have nucleus and cytoplasm
 This is also a way for those species which do not
perform sexual reproduction, so this process provides
opportunity to make a desire species with combination
of both species.
Protoplast fusion in Animals
• Protoplast fusion or Hybrid cells can be produced by fusing different
types of somatic cells from two different tissues or species in a cell
culture media. Chromosome or gene mapping via somatic cell
hybridization technique is based on fusion of human somatic cell
and mouse somatic cell. Usually human somatic cells like fibrocyte
or leucocyte are fused with continuous cell lines of mouse.
When somatic cells of human and mouse origin or cells of any two
species of mammals or two cells of same species are mixed,
spontaneously cell fusion occurs at very low rate. Therefore by
adding ultraviolet inactivated Sendai (parainfluenza) virus or
chemical compound known as polyethylene glycol (PEG)
increases the somatic cell fusion by 100 to 1000 times.
Procedure:
1. Required type of human and mouse somatic cells are
selected and then mixed together along with inactivated Sendai virus or
PEG (polyethylene glycol); hence they enhance the cell fusion process.
2. After some time, mixture of all three types of cell such as human
cell, mouse cell and hybrid cells are plated on a selective media.
Commonly used selective media is known as HAT medium. This
selective media only supports the multiplication or cell proliferation of
hybrid cells.
3. Some clones are extracted from the selective media and are tested
for both cytogenetic and appropriate biochemical analysis for the
detection of investigating biological molecule, such as enzyme or
protein or any traits.
4. Now the result is correlated with the presence or
absence of a trait with the presence or absence of
human chromosome or gene in the somatic hybrid
cells.
5. Depending upon the correlation between the
presence and absence of a human chromosome or
gene with that of a trait in somatic hybrid cells, can
be used in locating the gene present in the
concerned chromosome or genetic material.
APPLICATIONS IN ANIMAL CELLS
1. Hybrid cells are used to study the gene expression.
2. Hybrid cells are used to study the basics of cell division
3. Hybrid cells can be used to study the transformation of normal
cells into malignant cells.
4. Hybrid cells are used to obtain viral replication
5. Hybrid cells are used for chromosome or gene mapping
6. Hybrid cells are also used in the production of monoclonal
antibodies
ADVANTAGES
1. Symmetric hybrids can be produced between species, which cannot be
hybridized sexually. These hybrids can be readily used in breeding
programmes for transfer of useful genes to crops or may be useful as new
crop species.
2. Hybrids can be produced even between such strains, which are completely
sterile, e.g., monoploids
3. Cytoplasm transfers can be affected in one year, while backcrossing may
take 5-6 years. Even where backcrossing is not applicable, cytoplasm transfers
can be made using this approach.
4. Mitochondria of one species can be combined with chloroplasts of another
species. This may be very important in some cases, and is not achievable by
sexual means even between easily crossable species.
5. Recombinant organellar genomes, especially of mitochondria, are
generated in somatic hybrids and hybrids. Some of these recombinant
genomes may possess useful features.
LIMITATIONS
1. Techniques for protoplast isolation, culture and fusion are not
available for many important crop species like many cereals and
pulses.
2. In many cases, chromosome elimination occurs from somatic
hybrids leading to asymmetric hybrids. Such hybrids may be
useful, but there is no control on chromosome elimination.
3. Many somatic hybrids show genetic instability, which may be
an inherent feature of some species combinations.
4. Many somatic hybrids either do not regenerate
or give rise to sterile regenerants. Such hybrids
are useless for crop improvement. All interfamily
somatic hybrids are genetically unstable and/or
morphologically abnormal, while intergeneric
and intertribal hybrids are genetically stable but
produce abnormal and/or sterile plants or only
teratomata.
5. No confirmation of expression of particular
trait in somatic hybrids.
ANY QUESTIONS ?
Quiz
• Q1. Who observed that in certain cells the
walls and cavities of the cells coalesce(fuse)
together.
a. Theodore schwann
b. Paul gilbert
c. Marie Curie
d. Michael Roberts
Q2. Name two steps of protoplast fusion.
Q3. Why is enzymatic approach more preffered
than the mechanical approach?
Q4. Give two advantages of somatic
hybridization
Q5. What are the limitations of somatic fusion?
Any two