Botany
assignment
TOPIC- PLANT TISSUE
CULTURE
NAME- AKANKSHA PRAJAPATI
CLASS- BSc. LIFE SCIENCES
SEMESTER- VI SECTION-A
ROLL NO.-3663/ 17081583028
SUBMITTED TO- DR. ANURADHA
PLANT TISSUE CULTURE
EXPLANATION-
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. It is widely used to produce clones of a plant in
a method known as MICROPROPAGATION. Different
techniques in plant tissue culture may offer certain
advantages over traditional methods of propagation,
including:
• The production of exact copies of plants that produce
particularly good flowers, fruits, or have other desirable
traits.
• To quickly produce mature plants.
• The production of multiples of plants in the absence of
seeds or necessary pollinators to produce seeds.
• The regeneration of whole plants from plant cells that
have been genetically modified.
• The production of plants in sterile containers that allows
them to be moved with greatly reduced chances of
transmitting diseases, pests, and pathogens.
• The production of plants from seeds that otherwise have
very low chances of germinating and growing,
i.e. orchids and nepenthes.
• To clean particular plants of viral and other infections
and to quickly multiply these plants as 'cleaned stock' for
horticulture and agriculture.
Plant tissue culture relies on the fact that many plant cells
have the ability to regenerate a whole plant (totipotency).
Single cells, plant cells without cell walls (protoplasts),
pieces of leaves, stems or roots can often be used to generate
a new plant on culture media given the required nutrients
and plant hormones.
HISTORY
HABERLANDT
❖ Early 1900’s
❖ Proposed concept of totipotency
❖ cells cultured under right conditions
❖ callus cultured from tree cambium
GAUTHERET, NOBECOURT, WHIRE
❖ In the 1930’s
❖ Cells kept alive but did not develop.
NUTRIENT’S REQUIREMENT-
➢ Antibiotics- Stereptomycin, kanamycin activated
charcoal.
➢ Other organic supplement- protein, coconut milk,
yeast, malt extract, organic juice, and tomato juice.
➢ Growth regulators- auxins, cytokinins
➢ Water- demineralized or distilled water
➢ Solidifying agents- agar, gelatin
➢ pH adjusters- 5-6 it is considered to be optimum
PREPARATION OR CULTURE MEDIA
✓ STOCK SOLUTION 1- MgSo4, KH2Po3, KNO3,
NH4No3, CaCl2
✓ STOCK SOLUTION 2- H3Bo3, MnSo4, ZnSo4, CuSo4,
CoCl2
✓ STOCK SOLUTION 3- FeSo4, sodium EDTA
✓ STOCK SOLUTION 4- ionositol, thiamine,
pyridamine, nicotine acid, glycine
To prepare 1 liter of medium-
Take 50 ml of stock solution 1+ 5ml of stock solution
2&4 in a beaker. The stock solution 3 prepared
separately in a other 450ml flask by adding double
distilled water and heat with constant stirring. Mix two
solutions and adjust pH to 5.5.
STERILISATION OF MEDIA
[Link] prepared media should be sterilized by ISI mark
Autoclave (for large amount) at 1210 Domestic
pressure cookers (for small amounts)
2. For the sterilization of glassware and metallic
equipments Hot air oven with adjustable tray is
required.
BASIC REQUIREMENT FOR A
TISSUE CULTURE LABORATORY
For the successful achievement the following general
basic facilities are required-
Equipment and apparatus
Washing and storage facilities
Media preparation room
Sterilization room
Aseptic chamber for culture
Culture rooms or incubators fully equipped with
the temperature, light and humidity control
devices
Observation or recording area well equipped with
computer for data processing
EQUIPMENT AND APPARATUS
❖ VESSELS ANS GLASSWARE
-All the glassware should be to pyrex.
-Large test tubes, flasks, graduated pipettes etc. are used.
❖ EQUIPMENT
-Scissors, scaples, foreceps are used for explants
preparation.
-A spirit burner for flame sterilization
-Hot air oven
-A pH meter
-A BOD incubator
-Laminar air flow chamber
-A balance to weigh nutrients
-Data collection and recording room
ESTABLISHMENT OF PLANT TISSUE
CULTURE-
In vitro culturing of plant tissue culture involves the
following steps.
Collecting and sterilization of glassware tools/vessels.
Preparation of explant
Surface sterilization of explant
Production of callus from explant
Proliferation of culture
Sub culturing of callus
Suspension culture
EXPLANT PREPARATION-
EXPLANT- It is defined as a portion of plant body, which
has been taken from the plant to establish a culture.
Explant may be taken from any part of the plant like
root, stem, leaf, or meristematic tissue like cambium,
floral parts like anthers, stamens etc.
Age of the explant
Homozygous plants are preferred.
SURFACE STERILIZATION OF
EXPLANT-
For surface sterilization chromic acid , HgCl (0.11%),
calcium hypochlorite, sodium hypochlorite (1-2%),
alcohol(70%) are used. Process depends on the type of
explant.
SEED- absolute ethyl alcohol→ calcium
hypochlorite → bromine water → sterile water
FRUIT- ethyl alcohol→ sodium hypochlorite →
sterile water
STEM- running water→ sodium hypochlorite→
sterile water
LEAF- surface clean→ HgCl→ sterile water →
dried
PRODUCTION OF CALLUS FROM
EXPLANT
Sterilized explant is transferred aseptically onto
defined medium.
Transfer to BOD incubator
Temperature (25-32o) and light is necessary for callus
production.
Callus produced with in 3-8 days.
PROLIFERATION OF CULTURE
If callus is well developed , it should cut into small
pieces and transferred to another fresh medium
containing hormones, which supports growth.
The medium used for production of more amount of
callus is called proliferation medium.
SUBCULTURING OF CALLUS
After sufficient growth of callus it should be
periodically transferred to fresh medium to maintain
viability of cells.
This subculture will be done at the interval of 4-6
weeks.
After a maximum growth transfer into a potting soil
under required conditions.
SUSPENSION CULTURE
It contains a uniform suspension of separate cells in a liquid
medium.
Callus
Liquid medium
Agitated continuously
Finally cells separated
Sub- culture the cells
This can be achieved by the rotatory shaker attached within
the incubator at a rate of 50-150 rpm.
GROWTH PROFILE OF PLANT TISSUE
CULTURE
They are classified as –
*Single cell culture
*Callus culture
SINGLE CELL CULTURE-
The single cell culture exhibits various stages of growth.
o LAG PHASE- tissues starts to grow
o EXPONENTIAL PHASE- this phase is characterized by
rapid cell multiplication.
o LINEAR PHASE- the growth follows a linear pattern
with respect to time.
o PROGRESSIVE DECLARATION PHASE- as the aging of
culture increases the cell division decreases.
o STATIONARY PHASE- no growth of cells occur
Rate of production of cells=rate of their death
o SENESCENT PHASE- cell death occurs to lack of
nutrients.
IN CALLUS CULTURE-
o LAG PHASE- in this phase cell trying to adjust the new
environment condition.
o EXPONENTIAL PHASE- by utilizing nutrients rapid
multiplication occurs
o DECLINE PHASE- due to starvation some cells leads to
decline in the callus culture
o STATIONARY PHASE- no growth is evident, requires
sub culturing.
MICROPROPAGATION-
Micropropagation is the practice of rapidly multiplying
stock plant material to produce many progeny plants, using
modern plant tissue culture methods.
Micropropagation is used to multiply plants such as those
that have been genetically modified or bred through
conventional plant breeding methods. It is also used to
provide a sufficient number of plantlets for planting from a
stock plant which does not produce seeds, or does not
respond well to vegetative reproduction.
Cornell university botanist Frederick Campion
Steward discovered and pioneered micropropagation
and plant tissue culture in the late 1950s and early 1960s.
Micropropagation have 5 steps, discussed as bellow:-
STAGE 0- Selection and maintenance of stock plants for
culture initiation.
STAGE 1- Initiation and establishment of aseptic
culture(main steps explant isolation, surface sterilization,
washing, and establishment on appropriate culture
medium).
STAGE 2- Multiplication of shoots or rapid somatic embryo
formation using a defined culture medium.
STAGE 3- Germination of somatic embryos and rooting of
regenerated shoots in vitro.
STAGE 4- Transfer of plantlets to sterilized soil for
hardening under green house environment(in a few cases
this stage may also include in vivo rooting of regenerating
shoots by skipping stage 3).
TYPES OF CULTURE-
♠ Callus culture
♠ Suspension culture
♠ Root tip culture
♠ Leaf or leaf primordial culture
♠ Shoot tip culture
♠ Complete flower culture
♠ Anther and pollen culture
♠ Ovule and embryo culture
♠ Protoplast culture
ADVANTAGES
☻ Availability of raw material.
☻ Fluctuation in supplies and quantity.
☻ Patent rights.
☻ Political reasons.
☻ Easy purification of the compound.
☻ Modification of chemical structure.
☻ Disease free and desired propagule.
☻ Crop improvement.
☻ Biosynthetic pathway.
☻ Immobilization of cell.
APPLICATION-
THANK YOU
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