Under the guidance of
Dept. of Pharmacognosy,
VIGNAN PHARMACY COLLEGE
(Approved by AICTE & PCI Affiliated to JNTU KAKINADA)
VADLAMUDI, GUNTUR DIST, ANDHRA PRADESH, INDIA, PIN: 522 213
PLANT TISSUE CULTURE
Preparation of medium
Sterilization of medium
Basic requirement of tissue culture laboratory
Establishment of plant tissue culture
Types of culture
PLANT TISSUE CULTURE
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
• proposed concept of totipotency
• cells cultured under right
• Callus cultured from tree
Gautheret, Nobecourt, Whire
In the 1930s.
• cells kept alive but did not
Other organic supplements :
Protein, coconut milk,yest,malt extract, orange juice, and tomato
Growth regulators :
Demineralized or distilled water
Solidifying agents :
pH adjusters :
5 - 6 it is considered to be optimum.
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.
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.
9Incubator Hot air oven
BASIC REQUIREMENT FOR A TISSUE CULTURE
For the successful achievement, the following general basic facilities are
Equipment & apparatus
Washing and storage facilities
Media preparation 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
EQUIPMENT & APPARATUS
VESSELS & GLASS WARE :
• All the glassware should be of Pyrex.
• Large test tubes,flasks,graduated pipettes etc.. are used.
• 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.
ESTABLISHMENT OF PLANT TISSUE CULTURE
In vitro culturing of plant tissue culture involves the following
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.
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 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
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.
PROLIFERATION OF CULTURE
• if callus is well developed, it should cut into small pieces &
transferred to another fresh medium containing hormones, which
•The medium used for production of more amount of callus is called
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
•It contains a uniform suspension of separate cells in a liquid medium
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.
GROWTH PROFILE OF PLANT TISSUE CULTURE
They are classified as :
Single cell 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
c) Linear phase: The growth follows a linear pattern with respect to
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
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.
Methods used to determine are..
• By counting the cell number in haemocytometer under a
•Suspension culture is preferable.
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
FRESH CELL WEIGHT
•When cells increase in number, the liquid will be turbid.
•As a result optical density altered, detected by colorimeter.
VIABLE CELL TEST
•The staining method such as fluorescein di-acetate is used for
accessing the cell viability.
•Dead cells appear as fluorescein red.
TYPES OF CULTURE
Root tip culture
Leaf or leaf primordial culture
Shoot tip culture
Complete flower culture
Anther & pollen culture
Ovule & embryo culture
Protoplast culture 31
Availability of raw materials.
Fluctuation in supplies & quantity.
Easy purification of the compound.
Modifications of chemical structure.
Disease free & desired propagule.
Immobilization of cell.
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
antitumor haploid & homozygous lines
food additives essential oil
• 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.
Who is the father of tissue culture ?
The production of secondary metabolites requires the use of ?
Synthetic seed is produced by encapsulating somatic embryo with
Hormone pair required for a callus to differentiate are ?
Auxin & cytokine
DMSO (dimethyl sulfoxide) is used as ?
The most widely used chemical for protoplast fusion, as fusogen is
Poly ethylene glycol (PEG)
Callus is ?
Unorganized actively dividing mass of cell, maintained in culture
Which of the plant cell will show totipotency ?
To obtain haploid plant, we culture ?
Growth hormone produce apical dominance ?
The vector mostly used in crop improvement ?
A media which contains chemically defined compound is called ?