Transcript Tissue culture - UniMAP Portal

TECHNIQUES IN
BIOTECHNOLOGY: TISSUE
CULTURE
Department of Chemical Engineering Technology,
UniMAP
PTT 104: Introduction to Biotechnology
[email protected]
COURSE OUTCOME

CO2: Ability to demonstrate important recent
advance in methods and application of
biotechnology with regards to microorganisms
and plants
WHAT IS TISSUE CULTURE
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Tissue culture is the growth of tissues and/or
cells separate from the organism. This is
typically facilitated via a liquid, semi-solid, 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.
.This growth of cells and tissues is called in-vitro
growth.
 Contrasted to in-vivo growth which is growth
within the body.

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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WHAT IS CELL CULTURE
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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.
PRIMARY CULTURE
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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.
CELL LINE
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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.
TISSUE CULTURE IN PLANT
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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.
HISTORY
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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 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.
APPLICATION OF TISSUE CULTURE IN
PLANT
Micropropagation: 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. It is used for rapid
multiplication of stock plant material to produce
large no of progeny plants. (plants that have been
genetically modified/ gone thru selective
breeding)
 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.
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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. The source of
variation due to chromosomal rearrangements.
variations appearing during tissue culture in plants
are called somaclonal variation. Benefit of somaclonal
variation: improvements of genetics. Plants that
resist to pesticides/herbicides.
TISSUE CULTURE METHOD: CLONING
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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
STEP IN TISSUE CULTURE
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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EXPLANT
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 are dissolved sugar, nutrients and
hormones the plant needs.
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Explants can be pieces of any part of the
plant (leaves, stems, flowers, etc.),
or even individual isolated cells.
MULTIPLICATION
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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.
RAPID MULTIPLICATION
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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.
TRANSPLANTING
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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.
THE NEXT REVOLUTION
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Plant Transgenesis – transferring genes to
plants directly
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Development of plant vaccines, plants that produce
their own pesticides and are resistant to herbicides
17 countries are growing more than 200 million
acres of crops improved through biotechnology
 Plants have a natural pathogen, Agrobacterium
tumefeciens, that causes tumours. A. tumefeciens
inserts its own DNA into the plant, making it a
natural vector.
 Genes can be put into A.tumefeciens surrounded
by the recognition splice sequences, and the
bacteria will introduce them into the host.
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METHODS USED IN PLANT TRANSGENESIS
Conventional Selective Breeding and
Hybridization
 Cloning
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Protoplast fusion
Leaf fragment technique
Gene guns
Chloroplast engineering
Antisense technology
METHODS USED IN PLANT TRANSGENESIS
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Conventional Selective Breeding and
Hybridization
Sexual cross between two lines and repeated backcrossing
between hybrid offspring and parent
 For example, a mildew-resistant pea may be crossed with a
high-yielding but susceptible pea, the goal of the cross
being to introduce mildew resistance without losing the
high-yield characteristics.
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Can take years or unsuccessful
Polyploid plants (multiple chromosome sets greater than
normal, usually more than 2n)
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Increases desirable traits, especially size. For example many
seedless fruit varieties are seedless as a result of polyploidy..
Whole chromosomes can be transferred rather than single
genes esults in production of enlarged fruits.
METHODS USED IN PLANT TRANSGENESIS
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Cloning – growing plants from a single cell
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Protoplast fusion is the fusion of two protoplast
cells from different species/different varieties from
same 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
form a hybrid plant
 Hybrids have been produced either between the different
varieties of the same species (e.g. between non-flowering
potato plants and flowering potato plants) or between two
different species (e.g. between wheat triticum and rye
secale to produce Triticale).
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* callus- when plant injured, callus may grow over the side of
the wound. Callus can redifferentiate into shoots and roots.
PROTOPLAST FUSION METHOD
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The somatic/protoplast fusion process occurs in four
steps:
The removal of the cell wall of one cell of each type of
plant using cellulase enzyme to produce a somatic cell
called a protoplast
The cells are then fused using electric shock
(electrofusion) or chemical treatment to join the cells
and fuse together the nuclei. The resulting fused
nucleus is called heterokaryon.
The somatic hybrid cell then has its cell wall induced
to form using hormones
The cells are then grown into calluses which then are
further grown to plantlets and finally to a full plant,
known as a somatic hybrid.
METHODS USED IN PLANT TRANSGENESIS
METHODS USED IN PLANT TRANSGENESIS
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Cloning
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Leaf fragment technique
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Small discs are cut from leaf
Discs are cultured in the to start a new plant
Early in the regeneration process, the bacterium
Agrobacterium tumefaciens carrying a Ti plasmid is
introduced into the culture
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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
The plasmid DNA combines with the plant chromosome
Discs are treated with hormones to encourage shoot and
root development and then the new plant is planted in the
soil
AGROBACTERIUM TUMEFACIENS AS A VECTOR FOR
TRANSFERRING FOREIGN GENES INTO PLANT
CHROMOSOME
This bacterium naturally infects plant cells
causing cancerous growths - crown gall disease
 Infection (vir) genes carried on Ti plasmid
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INFECTION PROCESS
Vir genes copy T-DNA
 Open channel in bacterial cell
membrane for T-DNA to pass
through
 T-DNA enters plant through
wound, integrates itself into
plant chromosome
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http://www.bio.davidson.edu/people/kabernd/seminar/2002/method/dsmeth/ds.ht
m
METHODS USED IN PLANT TRANSGENESIS
METHODS USED IN PLANT TRANSGENESIS
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Cloning
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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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GENE GUNS
stopped
Gene
gun
METHODS USED IN PLANT TRANSGENESIS
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Cloning
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Chloroplast engineering
DNA in chloroplast can accept several new genes at once
 High percentage of genes will remain active
 Advantage: 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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METHODS USED IN PLANT TRANSGENESIS
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Cloning
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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
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Example is Flavr Savr tomato
Enzyme polygalacturonase breaks down structural
polysaccharide pectin in wall of a plant.
This is part of the natural decay process in a plant
Monsanto identified the gene than encodes the enzyme and
made another gene that blocked the production of the enzyme.
METHODS USED IN PLANT TRANSGENESIS
FIRST COMMERCIAL TRANGENIC PLANT
PRODUCT: THE 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.
TISSUE CULTURE IN ANIMAL
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.
In organ cultures, whole embryonic organs or
small tissue fragments are cultured in vitro in
such a manner that they retain their tissue
architecture.
 intent to study their continued function or
development.
 In contrast, cell cultures is when cells are
removed from the organ fragments prior to, or
during cultivation, thus disrupting their normal
relationships with neighboring cells
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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).
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HISTORY OF ANIMAL
TISSUE CULURE
HISTORY
1880 Roux maintained Embryonic chicken in saline
frog embryo
1900 Harrison
• Frog neuroblast in lymph medium
•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 culture techniques were advanced significantly in
the 1940s and 1950s to support research in virology.
Growing viruses in cell cultures allowed preparation
of purified viruses for the manufacture of vaccines.
The injectable polio vaccine developed by Jonas Salk
was one of the first products mass-produced using cell
culture techniques.
This vaccine was made possible by the cell culture
research of John Franklin Enders, Thomas Huckle
Weller, and Frederick Chapman Robbins, who were
awarded a Nobel Prize for their discovery of a method
of growing the virus in monkey kidney cell cultures.
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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.
STEM CELL
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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.
PRECURSOR CELL
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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.
DIFFERENTIATE CELL
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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.
INITIATION OF CELL 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.
CELLS ARE EITHER….
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Anchorage – dependant
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Anchorage - independant
ANCHORAGE – INDEPENDANT CELLS
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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
ANCHORAGE – DEPENDANT CELLS
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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
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.
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