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PLANT TISSUE CULTURE

By

Dr.M.Sangeetha

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CONTENTS:

 Definition
 History
 Nutrient’s requirement
 Preparation of medium
 Sterilization of medium
 Basic requirement of tissue culture laboratory
 Establishment of plant tissue culture
 Growth profile
 Growth determination
 Types of culture
 Advantages
 Applications
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PLANT TISSUE CULTURE

Definition
Tissue culture is in vitro cultivation of plant cell or tissue under
aseptic and controlled environmental conditions, in liquid or on
semisolid well defined nutrient medium for the production of
primary and secondary metabolites or to regenerate plant.

Tissue culture relies on three fundamental abilities of plant there


are:
Totipotency
Dedifferentiation
competency

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HISTORY
Haberlandt

early 1900’S
• proposed concept of totipotency
• cells cultured under right
conditions
• Callus cultured from tree
cambium

Gautheret, Nobecourt, Whire

In the 1930s.
• cells kept alive but did not
develop
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NUTRIENT’S REQUIREMENT

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INORGANIC & ORGANIC SUPLLEMENTS
COUMPOUNDS Mg/Ml
NH4NO3 1,650.00
KNO3 1,900.00
CaCl2 (anhyd) 332.20
MgSO4 (anhyd) 180.70
KH2PO4 170.00
Na2EDTA 37.25
FeSO4.7H2O 27.80
H3BO3 6.20
MnSO4.H2O 16.90
ZnSO4.H2O 5.37
KI 0.83
Na2Mo4.2H2O 0.25
Sucrose 30,000.00
i-Inositol 100.00
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Thiamine.HCl 0.40
Antibiotics :
Stertomycin,kanamycin
Activated charcoal

Other organic supplements :


Protein, coconut milk,yest,malt extract, orange 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.
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 PREPARATION OF CULTURE MEDIA:

Stock solution 1 : MgSo4,KH2Po4,KNO3,NH4No3,CaCl2

Stock solution 2 : H3Bo3,MnSo4,ZnSo4,CuSo4,Cocl2

Stock solution 3 : FeSo4,sodium EDTA

Stock solution 4 : ionositol,thiamine,pyridamine,nicotinic 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.

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 STERILISATION OF MEDIA

•The prepared media should be sterilized by ISI mark Autoclave( for


large amounts) at 121º Domestic pressure cookers( for small amounts)

•For the sterilization of glassware and metallic equipments Hot air


oven with adjustable tray is required.

Incubator Hot air oven 9


 BASIC REQUIREMENT FOR A TISSUE CULTURE
LABORATORY

For the successful achievement, the following general basic facilities are
required:

 Equipment & apparatus


 Washing and storage facilities
 Media preparation room
 Sterilization room
 Aseptic chamber for culture
 Culture rooms or incubators fully equipped with temperature, light
and humidity control devices
 Observation or recording area well equipped with computer for data
processing

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TISSUE CULTURE LABORATORY

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EQUIPMENT & APPARATUS

 VESSELS & GLASS WARE :

• All the glassware should be of Pyrex.


• Large test tubes,flasks,graduated pipettes etc.. are used.

 EQUIPMENT :

• Scissors,scapels,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.
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Knife

Laminar air flow chamber

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DATA COLLECTION & RECORDING ROOM

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 ESTABLISHMENT OF PLANT TISSUE CULTURE
In vitro culturing of plant tissue culture involves the following
steps.
 Collecting & 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

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 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.

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flower

leaf

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 SURFACE STERILISATION OF EXPLANT
For surface sterilization chromic acid, Hgcl(0.11%),calcium
hypochlorite, sodium hypochlorite(1-2%),alcohal(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 Hgcl2 sterile water dried

explant
 PRODUCTION OF CALLUS FROM EXPLANT

• Sterilized explant is transferred aseptically onto defined medium.


• Transfer to BOD incubator.
• Temperature (25 ± 2 ͦ) and light is necessary for callus production.
• Callus produced with in 3-8 days.

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 PROLIFERATION OF CULTURE

• if callus is well developed, it should cut into small pieces &


transferred to another fresh medium containing hormones, which
supports growth.
•The medium used for production of more amount of callus is called
proliferation medium.

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15 DAYS  CALLUS GROWTH

30 DAYS 50 DAYS 80 DAYS 21


 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 pottling soil under required


condition.
 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 rotary shaker attached within the incubator at


a rate of 50-150 rpm.

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 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.

a) Lag phase: Tissue starts to grow.

b) Exponential phase: This phase is characterized by rapid cell


multiplication.

c) Linear phase: The growth follows a linear pattern with respect to


time 24
d) Progressive declaration phase:
Aging of culture increase cell division decreases
e) Stationary phase:
No growth of cells occur
Rate of production of cells = rate of their death
f) Senescent phase:
Cell death occurs to lack of nutrients

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 IN CALLUS CULTURE

a) Lag phase:
In this phase cell trying to adjust the new environment condition.
b) Exponential phase :
By utilizing nutrients rapid multiplication occurs.
c) Decline phase :
Due to starvation some cells leads to decline in the callus culture.
d) Stationary phase :
No growth is evident, requires sub culturing.

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 GROWTH DETERMINATION
Methods used to determine are..

 CELL NUMBER
• By counting the cell number in haemocytometer under a
microscope.
•Suspension culture is preferable.

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 PACKED CELL VOLUME

•Cell suspension is transfer to graduated centrifuge.


•Centrifuged at 2000 rpm for 5mints.
•Cell will form pellets called biomass volume, expressed by ml-1

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 FRESH CELL WEIGHT

•When cells increase in number, the liquid will be turbid.


•As a result optical density altered, detected by colorimeter.

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 VIABLE CELL TEST

•The staining method such as fluorescein di-acetate is used for


accessing the cell viability.
•Dead cells appear as fluorescein red.

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 TYPES OF CULTURE
Callus culture

Suspension culture

Root tip culture

Leaf or leaf primordial culture

Shoot tip culture

Complete flower culture

Anther & pollen culture

Ovule & embryo culture

Protoplast culture 31
Callus culture Suspension Pollen culture
culture

Ovule culture Root tip culture Shoot tip culture

Protoplast culture Leaf primordial culture Flower culture

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 ADVANTAGES :

Availability of raw materials.

Fluctuation in supplies & quantity.

Patent rights.

Political reasons.

Easy purification of the compound.


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Modifications of chemical structure.

Disease free & desired propagule.

Crop improvement.

Bio-synthetic pathway.

Immobilization of cell.

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 APPLICATIONS
alkaloids virus-free plants forest trees

saponins apical meristem culture fruit crops

secondary metabolite PTC in industry micro propagation

steroids anther or pollen culture vegetatively


propagated plants
antitumor haploid & homozygous lines

plantation crop
Bio pesticides

food additives essential oil


yielding plants

chemicals 35
REFERENCES :

• Trease and evans pharmacognosy.


W.C evans-page num :72,73,74.

•A textbook of industrial pharmacognosy by A.N Kalia.


Page num-105 to 114.

• A textbook of pharmacognosy by M.K Gupta & P.K


Sharma. Page num-171 to 185.

• Pharmacognosy and photochemistry-part 2 by Vinod


D Rangari. Page num-51 to 56.

• Internet source. 36
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Who is the father of tissue culture ?
 Haberlandt

The production of secondary metabolites requires the use of ?


Cell suspension

Synthetic seed is produced by encapsulating somatic embryo with


Sodium alginate

Hormone pair required for a callus to differentiate are ?


Auxin & cytokine

DMSO (dimethyl sulfoxide) is used as ?


Cryoprotectant

The most widely used chemical for protoplast fusion, as fusogen is


Poly ethylene glycol (PEG)
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Callus is ?
Unorganized actively dividing mass of cell, maintained in culture

Which of the plant cell will show totipotency ?


Meristem

To obtain haploid plant, we culture ?


Entire anther

Growth hormone produce apical dominance ?


Auxin

The vector mostly used in crop improvement ?


Agro bacterium

A media which contains chemically defined compound is called ?


Synthetic medium
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