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BCH (H) - VI Advanced Cell Biology-1

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20 views27 pages

BCH (H) - VI Advanced Cell Biology-1

Uploaded by

tara
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Tissue Culture

 Tissue culture is the term used for “the


process of growing cells artificially in the
laboratory”

 Tissue culture involves both plant and


animal cells

 Tissue culture produces clones, in which all


product cells have the same genotype
(unless affected by mutation during
culture)
History

Tissue culture had


its origins at the
beginning of the
Haberlandt 20th century with
the work of
Gottleib
Haberlandt
(plants) and
Alexis Carrel
Carrel (animals)
The First artificial medium
 The first commercial use of plant clonal
propagation on artificial media was in the
germination and growth of orchid plants, in the
1920’s
 In the 1950’s and 60’s there was a great deal of
research, but it was only after the development of
a reliable artificial medium (Murashige & Skoog,
1962) that plant tissue culture really ‘took off’
commercially
Critical requirements of Plant and
Animal Tissue culture

 Appropriate tissue (some tissues


culture better than others)
 A suitable growth medium containing
energy sources and inorganic salts to
supply cell growth needs. This can be
liquid or semisolid
 Aseptic (sterile) conditions, as
microorganisms grow much more
quickly than plant and animal tissue
and can over run a culture
Growth regulators - in plants, both
auxins & cytokinins. In animals, this is
not as well defined and the growth
substances are provided in serum from
the cell types of interest
Frequent subculturing to ensure
adequate nutrition and to avoid the
build up of waste metabolites
Plant Tissue Culture
Steps involved in Plant Tissue
Culture
 Selection of the plant
tissue (explant) from a
healthy vigorous
‘mother plant’ - this is
often the apical bud,
but can be other tissue
as well.

 This tissue must be


sterilized to remove
microbial
contaminants.
 Establishment of the
explant in a culture
medium. The medium
sustains the plant cells
and encourages cell
division. It can be solid
or liquid

 Each plant species (and


sometimes the variety
within a species) has
particular medium
requirements that must
be established by trial
and error
 Multiplication- The
explant gives rise to a
callus (a mass of
loosely arranged cells)
which is manipulated
by varying sugar
concentrations and
Dividing shoots the low auxin :
cytokinin ratios to form
multiple shoots

 The callus may be


subdivided a number
of times

Growing in warmth and good light


Root formation -
The shoots are
transferred to a
growth medium
with relatively
higher auxin:
cytokinin ratios
Young banana
plants
 The rooted shoots are
potted up (deflasked)
and ‘hardened off’ by
gradually decreasing
the humidity

 This is necessary as
many young tissue
culture plants have no
waxy cuticle to prevent
water loss
Applications:

A single explant can be multiplied into


several thousand plants in less than a
year - this allows fast commercial
propagation of new cultivars.

Taking an explant does not usually


destroy the mother plant, so rare and
endangered plants can be cloned safely.

Once established, a plant tissue culture


line can give a continuous supply of
young plants throughout the year
 In plants prone to virus diseases, virus free
explants (new meristem tissue is usually virus free)
can be cultivated to provide virus free plants.

 Plant ‘tissue banks’ can be frozen, then


regenerated through tissue culture.

 Plant cultures in approved media are easier to


export than are soil-grown plants, as they are
pathogen free and take up little space.

 Tissue culture allows fast selection for crop


improvement - explants are chosen from superior
plants, then cloned.

 Tissue culture clones are ‘true to type’ as compared


with seedlings, which show greater variability.
Animal Tissue Culture
Animal tissue/cell culture -
differences from plant tissue culture

 Animal cell lines have limited numbers of


cell cycles before they begin to degrade
 Animal cells need frequent subculturing to
remain viable
 Tissue culture media is not as fully defined
as that of plants - in addition to inorganic
salts, energy sources, amino acids,
vitamins, etc., they require the addition of
serum (bovine serum is very common, but
others are used)
 Animal tissue
cultures can pose
biohazard concerns,
and cultures require
special inactivation
with hypochlorite
followed by
incineration.

Gloves and labcoat are always worn. The pipettes


used are disposable.

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