Download What is Tissue Culture

TISSUE CULTURE TECHNIQUE
LECTURE 4
Madam Noorulnajwa Diyana Yaacob
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CO2: Ability to demonstrate important recent
advance in methods and application of
biotechnology with regards to
microorganisms and plants
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Tissue culture is the growth of tissues and/or
cells separate from the organism. This is
typically facilitated via use of a liquid, semisolid, or solid growth medium, such as broth
or agar. Tissue culture commonly refers to
the culture of animal cells and tissues, while
the more specific term plant tissue culture is
being named for the plants.
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.This growth of cells and tissues is called invitro growth.
Contrasted to in-vivo growth which is growth
within the body.
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In modern usage, tissue culture generally refers
to the growth of cells from a tissue from a
multicellular organism in vitro. These cells may
be cells isolated from a donor organism, primary
cells, or an immortalised cell line. The term
tissue culture is often used interchangeably with
cell culture
Tissue culture is an important tool for the study
of the biology of cells from multicellular
organisms. It provides an in vitro model of the
tissue in a well defined environment which can be
easily manipulated and analyzed
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Cell culture refers to the removal of cells from
an animal or plant and their subsequent
growth in a favorable artificial
environment. The cells may be removed from
the tissue directly and disaggregated by
enzymatic or mechanical means before
cultivation, or they may be derived from a cell
line or cell strain that has already been
established.
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Primary culture refers to the stage of the
culture after the cells are isolated from the
tissue and proliferated under the appropriate
conditions until they occupy all of the
available substrate (i.e., reach confluence). At
this stage, the cells have to be subcultured
(i.e., passaged) by transferring them to a new
vessel with fresh growth medium to provide
more room for continued growth.
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After the first subculture, the primary culture
becomes known as a cell line or
subclone. Cell lines derived from primary
cultures have a limited life span (i.e., they are
finite), and as they are passaged, cells with
the highest growth capacity predominate,
resulting in a degree of genotypic and
phenotypic uniformity in the population.
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Tissue culture in plants is the technique of
growing plant cells, tissues and organs in an
artificial prepared nutrient medium static or
liquid under aseptic conditions.
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In vitro cultures in which isolated organs, tissues, cells,
protoplasts are used has made considerable advances in
last two decades.
Though the technique is latest one but history of this
began more than 225 years back when first callus
formation was done by Duhamel du Monceau in 1756.
Haberlandt 1898 successfully cultured somatic cells of
higher plants in simple nutrient solutions.
Although he was able to maintain the cells in nutrient
medium, the cell division was not recorded until much
later.
The first real success was made by Nobecourd, Gautheret
and White who successfully cultured cambium tissue and
maintained them for more than a year through 5 or 6 sub
segments sub cultures.
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Micro propagation: The term represents the vegetative multiplication of
plants in artificial media under aseptic conditions from tissue; organs of
plants e.g. root tip, shoot tip, embryo, stem and callus etc.
Production of disease free plants: By using tissue culture in plants
healthy disease free plants of potato, sugarcane, sweet potato, and
strawberry have been produced.
Androgenic haploids and their use in breeding: With the help of tissue
culture in plants haploid embryos or haploid plants are raised by another
culture technique.
Embryo rescue for successful hybridisation: The hybrid embryos
produced as a result of interspecific or intergeneric cross usually
collapse due to incompatibility. Such embryos are isolated from female
plants and rescued by growing them on synthetic medium.
Induction and selection of mutants: By adding chemical mutagens into
the medium for growing various traits, useful viable mutants can be
produced.
Somaclonal variation: These are variations produced in the plants
regenerated from tissue cultures involving callus formation. Vitiations
appearing during tissue culture in plants are called somaclonal variation
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Tissue culture (often called
micropropagation) is a special type of asexual
propagation where a very small piece of
tissue (shoot apex, leaf section, or even an
individual cell) is excised (cut-out) and placed
in sterile (aseptic) culture in a test tube, petri
dish or tissue culture container containing a
special culture medium.
Overview of the Tissue Culture
Process
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The culture medium contains a gel (agar) with the
proper mixture of nutrients, sugars, vitamins and
hormones, which causes the plant part to grow at
very rapid rates to produce new plantlets.
It has been estimated that one chrysanthemum
apex placed in tissue culture could produce up to
1,000,000 new plantlets in one year.
Thus, tissue culture is used for rapid
multiplication of plants. A very specialized
laboratory is required for tissue culture.
All the procedures are done in a laboratory and
special ventilated cabinet that is as sterile as an
operating room
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Explant: Cut-out Plant Tissue and Place
in Tissue Culture Container
Multiplication: Tissue Grows and Produces
Small Plants
Rapid Multiplication by Transfer of Cultures
Transplanting
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The first step is to obtain what is called and
explant.
This means to simply cut-out a very small piece
of leaf or stem tissue, or even isolate individual
cells, and place them in a tissue culture
container.
The tissue has to be sterilized so it will not have
any contaminating bacteria or fungus.
It is then placed inside the tissue culture contain
on a gel called agar.
In the agar is dissolved all the sugar, nutrients
and hormones the plant needs.
Explants can be pieces of any part of
the plant (leaves, stems, flowers, etc.),
or even individual isolated cells.
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The tissue will begin to grow. It may make a
big blob of tissue called callus, or it may
make new shoots directly from the explant
tissue that was inserted in the container.
A mass of callus tissue is formed that is just
starting to make new plantlets.
New plantlets (shoots with leaves) are
forming.
If the conditions are right a small "forest" of plants
will develop in the tissue culture container.
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Once the plantlets start
developing, some can be
removed and placed in
new tissue culture
containers.
Thus, another "forest"' of
plants is produced. This
results in a rapid
multiplication of the
cultures and many
thousand of plants can
be produced in a few
months.
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When the plantlets are
large enough, they can
be removed from the
tissue culture container
and transferred into pots
with potting soil.
The young plants are
growth in a greenhouse
just like you would any
young seedling or
cutting.
When the small plant clones are removed from the
culture containers, they must be transplanted into
some type of acclimation container or kept under a
mist system until the acclimate to the ambient
environment.
After acclimation, the young plants can be
transplanted
and grown in pots in a greenhouse to produce
new plants.
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Plant Transgenesis – transferring genes to
plants directly
◦ Development of plant vaccines, plants that produce
their own pesticides and are resistant to herbicides
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17 countries are growing more than 200
million acres of crops improved through
biotechnology
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Conventional Selective Breeding and
Hybridization
Cloning
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Protoplast fusion
Leaf fragment technique
Gene guns
Chloroplast engineering
Antisense technology
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Conventional Selective Breeding and
Hybridization
◦ Sexual cross between two lines and repeated
backcrossing between hybrid offspring and parent
 Can take years
◦ Polyploid plants (multiple chromosome sets greater
than normal)
 Increases desirable traits, especially size
 Whole chromosomes can be transferred rather than
single genes
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Cloning – growing plants from a single cell
◦ Protoplast fusion is the fusion of two protoplast
cells from different species
 Protoplast cell is a callus cell whose cell wall has been
dissolved by the enzyme cellulase
 Fusion of the two protoplast cells creates a cell that
can grow into a hybrid plant
 Examples include broccoflower
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Cloning
◦ Leaf fragment technique
 Small discs are cut from leaf
 Cultured in a medium containing genetically modified
Agrobacter (Agrobacterium tumefaciens)
 A soil bacterium that infects plants
 Bacterium contains a plasmid, the TI plasmid, that can be
genetically modified
 DNA from the TI plasmid integrates with DNA of the host
cell
 Leaf discs are treated with plant hormones to stimulate
shoot and root development
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Cloning
◦ Gene Guns
 Used to blast tiny metal beads coated with DNA into an
embryonic plant cell
 Aimed at the nucleus or the chloroplast
 Use marker genes to distinguish genetically
transformed cells
 Antibiotic resistance
 Technique is useful in plants that are resistant to
Agrobacter
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Cloning
◦ Chloroplast engineering
 DNA in chloroplast can accept several new genes at
once
 High percentage of genes will remain active
 DNA in chloroplast is completely separate from DNA
released in pollen – no chance that transformed genes
will be carried on wind to distant crops
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Cloning
◦ Antisense technology
 Process of inserting a complementary copy of a gene
into a cell
 Gene encodes an mRNA molecule called an antisense
molecule
 Antisense molecule binds to normal mRNA (sense
molecule) and inactivates it
 Example is Flavr Savr tomato
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The Flavr Savr tomato is a
genetically altered tomato
developed by Calgene.
It contains an antisense
RNA which inhibits the
expression of a gene that
normally causes fruit to
soften, therefore, the fruit
stays firm longer.
This allows producers a
greater period of time for
transportation and the
opportunity for mechanical
harvesting with little
bruising.
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Animal culture was first successfully
undertaken by Ross Harrison in 1907.
This was followed by a series of
developments that made cell culture widely
available tool for scientists including
development of antibiotics, use of trypsin to
remove cells from culture vessels,
development of standardized, chemically
defined culture media that made far easier to
grow cells.
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The term tissue culture refers to the culture
of whole organs, tissue fragments as well as
dispersed cells on a suitable nutrient
medium. It can be divided into
(1) organ culture and
(2) cell culture mainly on the basis of whether
the tissue organisation is retained or not.
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In organ cultures, whole embryonic organs or
small tissue fragments are cultured in vitro in
such a manner that they retain their tissue
architecture.
In contrast, cell cultures are obtained either
by enzymatic or mechanical dispersal of
tissues into individual cells or by
spontaneous migration of cells from
explants; they are maintained as attached
monolayers or as cell suspensions.
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Freshly isolated cell cultures are called
primary cultures; they are usually
heterogeneous and slow growing, but are
more representative of the tissue of their
origin both in cell type and properties.
Once a primary culture is subcultured, it gives
rise to cell lines, which may either die after
several subcultures (such cell lines are known
as finite cell lines) or may continue to grow
indefinitely (these are called continuous cell
lines).
1880 Roux Embryonic chicken in saline
frog embryo
1900 Harrison
• Anchorage dependent
• Nutrients
• Relative slow growth rate
• Doubling 1 day vs 20 minutes bacteria
• Contamination
characteristics for in vitro cell growth:
1. Cells require an anchor like the lymph clots (the
cover slip)
2. Cells require nutrients provided by the lymph.
3. Cells grow very slow; 20 hours doubling time
compared to 20 minutes for bacteria This means
cell cultures are vulnerable to contamination
1900 Harrison
Carrel (surgeon, 1923)
Aseptic techniques
Carrel Flask
1912-1946 Culture Chicken Embryo Fibroblast
Plasma+tissue homogenate
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Cell Cultures Cell cultures may contain the following three
types of cells:
(1) stem cells,
(2) precursor cells and
(3) differentiated cells.
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Stem cells are undifferentiated cells, which
have unlimited capacity for poliferation, and
they can differentiate under correct inducing
conditions into one of several kinds of cells;
different kinds of stem cells differ markedly
in terms of the kinds of cells they will
differentiate into.
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Precursor cells are derived from stem cells,
are committed to differentiation, but are not
yet differentiated; these cells retain the
capacity for proliferation.
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differentiated cells, usually, do not have the
capacity to divide. Some cell cultures, e.g.,
epidermal keratinocyte cultures, contain all
the three types of cells.
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Cell cultures can be grown as
(1) monolayers or as
(2) suspension cultures.
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The initiation of cell cultures may be conveniently
dealt with under the following heads:
(1) preparation and sterilization of the substrate
(culture vessels),
(2) preparation and sterilization of the medium,
(3) isolation of explant,
(4) disaggregation of the explant, and
(5) subculture and cloning.
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Anchorage – dependant
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Anchorage - independant
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Cells associated with body fluid
-blood cells
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Grown in suspension
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Will eventually need subculturing
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Most animal derived cells
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Adhere to bottom of a flask and form a
monolayer
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Eventually cover entire surface of substratum
(confluence)
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Proliferation then stops
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Need to subculture cells at this point (remove to
fresh medium)
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Proliferation can begin again
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Primary culture
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Continuous cell line
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Taken from fresh tissue
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Limited life span in culture
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Treated by proteolytic enzyme (Trypsin)
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Separate into single cells
-epithelial cells
-fibroblasts
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Derived from humans
Been transformed
-lose sensitivity to factors associated with growth control
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Produce immortalised cell lines
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Often lose their anchorage-dependence
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More easily cultured
In 1997, cloning was revolutionized
when Ian Wilmut and his colleagues at
the Roslin Institute in Edinburgh,
Scotland, successfully cloned a sheep
named Dolly. Dolly was the first cloned
mammal.
Wilmut and his colleagues transplanted
a nucleus from a mammary gland cell
of a Finn Dorsett sheep into the
enucleated egg of a Scottish blackface
ewe. The nucleus-egg combination
was stimulated with electricity to fuse
the two and to stimulate cell division.
The new cell divided and was placed in
the uterus of a blackface ewe to
develop. Dolly was born months later.