What is Plant tissue culture?
Plant tissue culture is a collection of techniques used to maintain or
grow plant cells, tissues or organs under sterile conditions on a nutrient
culture medium of known composition.
Basics of Plant tissue culture
Ø Totipotency
Ø Morphogenic potential
Totipotency:
·
It is the ability of single cell to divide and re-divide and produce all
of the differentiated cells in an organism.
·
Totipotency is the genetic
potential of a plant cell to produce the entire plant.
·
Totipotency is the cell
characteristic in which the potential for forming all the cell types in the
adult organism is retained.
·
The cells are obtained
from stem, root or other plant parts and are allowed to grow in culture medium
containing mineral nutrients, vitamins and hormones to encourage cell division
and growth.
·
The cells in culture will produce an
unorganized proliferative mass of cells which is known as callus tissue.
Morphogenic potential:
It includes cell divisions and
cellular differentiations:
Cell divisions:
·
Cell division is
the process of splitting a parent cell into two daughter
cells.
Cellular differentiations:
·
Cellular
differentiation is the process of forming a variety of cell types that have
specific functions.
Cellular differentiations may be
·
Differentiation
The cells derived from root apical meristem (RAM) and shoot
apical meristem (SAM) and cambium differentiate, mature to perform specific
functions. This act leading to maturation is termed differentiation. They
undergo a few or major structural changes both in their cell walls and
protoplasm.
·
Dedifferentiation
In
plants, the living differentiated cells can regain the capacity to divide
mitotically under certain conditions. The sum of events, that bestow this
capacity to divide once again, are termed dedifferentiation. A dedifferentiated
tissue can act as meristem (e.g., interfascicular vascular cambium, wound
meristem, cork cambium).
·
Redifferentiation
The products of dedifferentiated cells/tissue which lose the
ability to divide are called redifferentiate cells/tissues and the event is
redifferentiation.
Applications of Plant Tissue Culture
·
Micropropagation
It is a plant tissue culture
technique used for production
of plantlets, in
which the culture of aseptic small sections of tissues and organs in vessels
with defined culture medium and under controlled environmental conditions.
Micropropagation is the technique of multiple
production of plants in
vitro.
·
Genetic Engineering
It is the direct genetic
modification or genetic manipulation of an organism’s gene by biotechnology.
·
Polyploidy
Polyploids
act as a source of genetic variation in plant tissue culture.
Multiple genomes alter gene frequency; induce
a permanent hybridity, genetic buffering and evolutionary flexibility (esp.
Allopolyploids)
Autopolyploid
typically have larger cell sizes, resulting in larger, lusher plants than the
diploid version .
Chromosome
doubling occurs naturally in all plants at low frequency as a result of mitotic
failure.
Can be induced by chemicals (colchicines from
Colchicum autumnale (saffron), vincristine and vinblastine from vinea rosea)
applied to meristematic tissue.
Young zygotes respond best; vegetative tissue
usually results in mixoploid chimeras.
·
Secondary metabolite production
These are not directly
involved in normal growth, development and reproduction but having ecological
function.
Five secondary metabolites are:
I.
Terpenoids and steroids
II.
Fatty acids derived substances and polyketides
III.
Alkaloids
IV.
No ribosomal polypeptides and
V.
Enzyme cofactor
TECHNIQUES OF PLANT TISSUE
CULTURE
Micro propagation
Micro
propagation starts with the selection of plant tissues (ex-plant) from a
healthy, vigorous mother plant.
Any part of the plant (leaf, apical meristem,
bud and root) can be used as ex-plant.
Stages of Micropropagation:
The whole process can be summarized into the
following stages:
I.
Stage
0: Preparation of donor plant
§ Any
plant tissue can be introduced in vitro.
§ To
enhance the probability of success, the mother plant should be ex vitro
cultivated under optimal conditions to minimize contamination in the in vitro
culture.
II.
Stage
1: Initiation stage
§ Ex-plant
is surface sterilized and transferred into nutrient medium.
§ Combined
application of bactericides and fungicides products is suggested.
§ Selection
of products depends on the type of ex-plant to be removing contaminants with
minimal damage to plant cells.
§ Disinfectants
like sodium hypochlorite, calcium hypochlorite, ethanol and mercuric chloride
(HgCl2) are used.
§ Cultures
are incubated in growth chamber either under light or dark conditions according
to the method of propagation.
III.
Stage
2: 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 plant is attained.
IV.
Stage
3: Rooting stage
§ Rooting
stage may occur simultaneously in the same culture media used for
multiplication of the ex-plant.
§ 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.
V.
Stage
4: Acclimatization stage
§ The
in vitro plants are weaned and hardened.
§ Hardening
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 hardened under greenhouse.
BASIC REQUIREMENTS
OF PLANT TISSUE CULTURE
In
plant tissue culture techniques, there are some basic requirements,
a)
Aseptic condition
b)
Control of temperature
c)
Proper culture media
d)
Sub-culturing
Aseptic conditions
§ The
tissue culture laboratory should have aseptic conditions.
§ It
means it should be well sterilizes against pathogen.
§ A
pathogen free environment will help in maintaining good health of the callus,
cell or protoplast cultures resulting in recovery of healthy plants from such
cultures.
§ The
explants and glassware should be properly sterilized before their entry into
the tissue culture laboratory.
Control of
Temperature
§ Air
conditioning of the tissue culture laboratory is essential.
§ Generally,
temperature between 18-25OC must be maintained.
§ However,
this temperature varies from species to species.
§ High
temperature adversely affects the growth of the callus.
§ Thermohygrometer is used to measure the temperature of the laboratory.
Proper Culture
Medium
§ Culture
media have been developed by various workers for different crop species.
§ The
medium has to be modified as per as the requirement of a species.
§ The
culture media developed by Murashige and Skoog (1962) is used with some
modification in various crop species.
Sub-Culturing
Transfer
of tissues or callus from old culture media to fresh culture media is called
sub-culturing.
It
is essential to maintain good health of the callus or tissues, because after
some period, some nutrients are depleted in the culture media and change of
media become essential.
Apparatus
·
Graduated cylinder
·
Beaker
·
Spatula
·
Forceps
·
Electronic balance
·
PH meter
·
Aluminum foil
·
Jug
·
Magnetic foil
·
Micropipette
·
Magnetic heat stirrer
·
Autoclave
·
Microwave
·
Laminar air flow
·
Magnetic stirrer
·
Tip box
·
Jars
·
Indicator tape
·
Polypropylene bags
·
Rubber bands
Material
·
Sugar/sucrose
·
MS (Murashige and Skoog) media
·
Agar
·
Distilled water
Hormones
·
Cytokinin
·
Auxin
For PH maintenance
·
HCL
·
NaOH
Important steps for
plant tissue culture
Plant
tissue culture techniques generally consist of five important steps:
i.
Media preparation and sterilization
ii.
Ex-plants selection and collection
iii.
Explants inoculation
iv.
Culture incubation
v.
Rooting and Acclimatization
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