Tissue Culture

The term tissue culture is commonly used in a very

wide sense to include in vitro aseptic culture of plant
cells, tissue and organs.
Is the term for the process of growing cells artifically
in the laboratory.
Involves both plant and animal cells
Tissue culture produces clones, in which all product
cells have the same genotypes (unless affected by
mutation during culture).

Haberlandt

early 1900S
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

Plant Tissue Culture
Is a practice used to propagate clones of a plant
There are various reasons this may be done:
1)To create exact copies of plants that produces
particularly good flowers or fruits.
2)To quickly produce mature plants.
3)To produce multiple of plants in the absence of seeds
or necessary pollination to produce seeds.
4)Used to regenerate the whole plants from plant cells
that have been genetically modified.


What is needed?
Tissue culture, both plant and animal has several critical requirements:
1)Appropriate tissue
-Some tissue culture better than others


2)A suitable growth medium
-Containing energy sources and inorganic salts to supply cell growth
needs. This can be liquid or semisolid.


Aseptic conditions
- Microorganisms grow much more quickly than plant and animal tissue
and can over run a culture


4)Growth regulators
-In plants, both auxins and cytokins.
-In animal, this is not as well defined and the
growth substances are provided in serum from
the cell types of interest
5)Frequent subculturing
- To ensure adequate nutrition and to avoid the
build up of waste conditions.

Aseptic Technique
Is the exclusion of invading microorganisms during
experimental procedures
Using sterile instruments and culture media
Media and apparatus are sterile by autoclaving (121C for
15 minutes)
Aseptic transfer performed in a transfer chamber such as
laminar flow hood which also preferably equipped with a
bunsen burner.
Common sterilants are ethyl alcohol an clorox with an
added surfactants.


Culturing (micropropagating) plant Tissue
the steps.
1)Selection of the plant tissue
-Plant tissue (explant) from a healthy vigorous
mother plant
-Often the apical bud, but can be other tissue



2)Sterilization
-This tissue must be sterilized to remove
microbial contamination



3)Establishment of the explant
-Establishment in a culture medium. The
medium sustain the plant cells and encourage
cell division. It can be solid or liquid.
-Each plant species has particular medium
requirements that must be established by trial
and error.



4)Multiplication
-The explant gives rise to a callus ( a mass of
loosely arranged cells) which is manipulated by
varying sugar concentrations and the auxin (low):
cytokinin (high) ratios to form multiple shoots.
-The callus may be subdivided a number of times



5)Root formation
-The shoots are transfered to a growth
medium with relatively higher auxin: cytokinin
ratios.


Benefits of plant tissue culture
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, the regenerated through tissue
culture.
Plant culture in approved media are easier to export than are soil-
grown plants, as they are pathogen free and take up little space
(most current plant export is now done in this manner)
Tissue culture allows fast selections for crop improvement-
explants are chosen from superior plants, then cloned.

Laboratory Requirements for Tissue Culture
General Organization
Localize each portion of the tissue culture procedure in a
specified place in the laboratory. An
assembly-line arrangement of work areas (such as, media
preparation, glassware washing,
sterilization, microscopy, and aseptic transfers) facilitates all
operations and enhances cleanliness.
Media (tissue culture and nutrient agar) are available from
Carolina Biological Supply Co.,
Burlington, NC. Laminar flow hoods are available from
several suppliers.
Any laboratory in which tissue culture techniques are
performed,
regardless of the specifi c purpose, must contain a
number of
basic facilities. These usually include the following:
A general washing area
A media preparation, sterilization, and storage area
An aseptic transfer area
Environmentally controlled incubators or culture
rooms
An observation/data collection area.
Washing Area
The washing area should contain large sinks, draining
boards,
and racks, and have access to deionized/distilled water.
Space
for drying ovens or racks, automated dishwashers, acid
baths,
pipet washers and driers, and storage cabinets may be
necessary
in the washing area, depending on the work being
performed
Media Preparation Area
The media preparation area should have ample storage space
for the chemicals, culture vessels and closures, and glassware
required for media preparation and dispensing. Bench space for
hot plates/stirrers, pH meters, balances, water baths, and mediadispensing
equipment should be available. Other necessary
equipment may include air and vacuum sources, Bunsen burners
with a gas source, refrigerators and freezers for storing stock
solutions and chemicals, a microwave or convection oven, and
an autoclave or domestic pressure cooker for sterilizing media,
glassware, and instruments.
In preparing culture media, analytical grade chemicals should be
used and good weighing habits practiced. To insure accuracy,
an exact, step-by-step routine should be developed for media
preparation. This routine should be contained in a complete
media preparation checklist required to be completed by all media
preparers, even for the simplest media.
The water used in preparing media should be highly purifi ed
though deionization and/or distillation. Tap water is not
recommended because it may contain undesirable salts and
dissolved gases, microorganisms (algae, fungi, bacteria), and
particulate matter (silt, oils, organic matter, etc.). Water used for
plant tissue culture should meet, at a minimum, the standards for
type II reagent grade water, i.e., be free of pyrogens, gases, and
organic matter and have an electrical conductivity less than 1.0
mho/cm.
The most common and preferred method of purifying water to
type II standards is a deionization treatment followed by one or
two glass distillations. The deionization treatment removes most
ionic impurities, and the distillation process removes large organic
molecules, microorganisms, and pyrogens. Three other methods
that will produce type II purity water are absorption fi ltration,
which uses activated carbon to remove organic contaminants
and free chlorine; membrane fi ltration, which removes particulate
matter and most bacterial contamination; and reverse osmosis,
which removes approximately 90% of the bacterial, organic, and
particulate matter as well as about 90% of the ionized impurities
Transfer Area
Under very clean and dry conditions, tissue culture techniques
can be successfully performed on an open laboratory bench.
However, it is advisable that a laminar fl ow hood or sterile transfer
room be utilized for making transfers. Within the transfer area
there should be a source of electricity, gas, compressed air, and
vacuum.
The most desirable arrangement is a small dust-free room
equipped with an overhead ultraviolet light and a positivepressure
ventilation unit. The ventilation should be equipped
with a high-effi ciency particulate air (HEPA) fi lter. A 0.3-m HEPA
fi lter of 99.97-99.99% effi ciency works well. All surfaces in the
room should be designed and constructed in such a manner that
dust and microorganisms do not accumulate and the surfaces
can be thoroughly cleaned and disinfected. A room of such
design is particularly useful if large numbers of cultures are being
manipulated or large pieces of equipment are being utilized.
Another type of transfer area is a laminar fl ow hood. Air is forced
into the unit through a dust fi lter then passed through a HEPA
fi lter. The air is then either directed downward (vertical fl ow unit)
or outward (horizontal fl ow unit) over the working surface. The
constant fl ow of microbe-free fi ltered air prevents non-fi ltered air
and particulate matter in the room from settling on the working
surface.
The simplest type of transfer area suitable for tissue culture work
is an enclosed plastic box commonly called a glove box. This
type of culture hood is sterilized by an ultraviolet light and wiped
down periodically with 70% alcohol when in use. This type of unit
is used when relatively few transfers are performed.
Culture Room
All types of tissue cultures should be incubated under conditions
of well-controlled temperature, humidity, air circulation, and light
quality and duration. These environmental factors may infl uence
the growth and differentiation process directly during culture or
indirectly by affecting their response in subsequent generations.
Protoplast cultures, low-density cell suspension cultures, and
anther cultures are particularly sensitive to environmental
condition.
Typically, the culture room for growth of plant tissue cultures
should have a temperature between 15 and 30 C, with a
temperature fl uctuation of less than 0.5 C; however, a wider
range in temperature may be required for specifi c experiments.
It is also recommended that the room have an alarm system to
indicate when the temperature has reached preset high or low
temperature limits, as well as a continuous temperature recorder
to monitor temperature fl uctuations.
The temperature should be
constant throughout the entire culture room (i.e., no hot or cold
spots). The culture room should have enough fl uorescent lighting
to reach the 10,000 lux; the lighting should be adjustable in terms
of quantity and photoperiod duration. Both light and temperature
should be programmable for a 24-hr period. The culture room
should have fairly uniform forced-air ventilation, and a humidity
range of 20-98% controllable to 3%. Many incubators, large
growth chambers, and walk-in environmental chambers meet
these specifi cations.
Water Purifi cation System; water should have
a resistivity of at least 200,000 ohms/ cm and a
conductivity 5.0 micromhos/cm
NA Purifi cation of water for media
preparation
1 Electronic Balance (0.01 g readability; 200 g
minimum capacity)
B933 295.00 Measuring out biochemicals and
media
1 pH meter (range 0-14 +/- 0.01; automatic
temperature compensation 0-600 C; one or two point
calibration)
P976 79.90 Measurement and adjustment of
media pH
1 Hot Plate/Stirrer (7 x 7 ceramic top; variable
heating range from ambient to 4000 C; variable
stirring speed from 50-150 rpm; chemically resistant)
H926 295.00 Mixing & heating media and
stock
1 Refrigerator/freezer; capable of maintaining a
refrigerator temperature of 2-60 C with a freezer
temperature of approximately 200 C
NA Storage of stock solutions,
media, hormones
1 Laminar Flow Transfer Hood; incoming air should be
HEPA fi ltered to remove 99.99% of particles larger
that 0.3m; should meet or exceed the Class 100
Clean Standard 209D; should maintain a fl ow of 90
fpm +/- 20% at static pressures of 0.6-1.2
NA Provide a sterile atmosphere to
transfer cultures
1 4 liter 70% Isopropyl alcohol (or several pint bottles
purchased from a local pharmacy)
NA Used to sterilize instruments
and work areas
1 roll Aluminum foil, heavy duty; (18 x 100 ft roll
Culture tubes, 25 x 150 mm, borosilicate glass; 500
tubes/ case
C930 146.50 Starting cultures in Stage I
1 case Culture tube racks; holds 40, 25 mm culture tubes;
withstands temperatures up to 1210 C
C908 90.50 Holding culture tubes
500 Closures, for 25 mm culture tubes, 500 each C945 139.25 Sealing culture tubes
1 case Culture vessel, baby food jar; glass culture vessel;
autoclavable; uses Magenta B Cap (C903) as
closure; 6 oz; 100/ case
autoclavable; uses Magenta B Cap (C903) as
closure; 4 oz height; 100/ case
C900 38.50 Culture vessel for
maintaining plant cultures
1 case Magenta B Caps; autoclavable closure for baby food
jars; fi ts both C904 and C900; clear polypropylene
closure; 100/ case
C903 215.50 Closure for baby food culture
vessel
2 cases Culture vessels; autoclavable culture vessel and lid
made from clear polypropylene; round vessel
measures; 250/ case
C913 173.00 Culture vessel for maintaining
plant cultures
1 gal Detergent NA Cleaning glassware
1 case Culture dishes, disposable, sterile, 100 x 15 mm D940 75.50 Sterile surface for cutting
explants (for Stage I cultures)
1 gal Chlorine bleach (sodium hypochlorite)
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 121
Domestic pressure cookers( for small amounts)

For the sterilization of glassware and metallic
equipments Hot air oven with adjustable tray is
required.
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
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 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 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 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.
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
APPLI CATI ONS
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
III. Sterilization and Use of Supplies and
Equipment:
A. Sterilizing tools, media, vessels etc.
1. Autoclaving
Autoclaving is the method most often used for
sterilizing heat-resistant items and our usual
method for sterilizing items. In order to be
sterilized, the item must be held at 121C, 15
psi, for at least 15 minutes. It is important that
items reach this temperature before timing
begins. Therefore time in the autoclave will
vary, depending on volume in individual
vessels and number of vessels in the autoclave.
Most autoclaves automatically adjust time
when temperature and psi
4
are set, and include time in the cycle for a slow
decrease in pressure. There are tape indicators
that can be affixed to vessels, but they may not
reflect the temperature of liquid within them.
There are also test kits of microorganisms
that can be run through the autoclave cycle
and then cultured.
Empty vessels, beakers, graduated cylinders,
etc., should be closed with a cap or aluminum
foil. Tools should also be wrapped in foil or
paper or put in a covered sterilization tray. It is
critical that the steam penetrate the items in
order for sterilization to be successful.
2. Autoclaving and Fiter-sterilizing Media and
Other Liquids
Two methods (autoclaving and membrane
filtration under positive pressure) are
commonly used to sterilize culture media.
Culture media, distilled water, and other heat
stable mixtures can be autoclaved in glass
containers that are sealed with cotton plugs,
aluminum foil, or plastic closures. However,
solutions that contain heat-labile components
must be filter-sterilized. For small volumes of
liquids (100 ml or less), the time required for
autoclaving is 15-20 min, but for larger
quantities (2-4 liter), 30-40 min is required to
complete the cycle. The pressure should not
exceed 20 psi, as higher pressures may lead to
the decomposition of carbohydrates and other
components of a medium. Too high
temperatures or too long cycles can also result
in changes in properties of the medium.
Organic compounds such as some growth
regulators, amino acids, and vitamins may be
degraded during autoclaving. These
compounds require filter sterilization through
a 0.22 m membrane. Several manufacturers
make nitrocellulose membranes that can be
sterilized by autoclaving. They are placed
between sections of a filter unit and sterilized
as one piece. Other filters (the kind we use)
come pre-sterilized. Larger ones can be set
over a sterile flask and a vacuum is applied to
pull the compound dissolved in liquid through
the membrane and into the sterile flask.
Smaller membranes fit on the end of a sterile
syringe and liquid is pushed through by
depressing the top of the syringe. The size of
the filter selected depends on the volume of
the solution to be sterilized and the
components of the solution.
Nutrient media that contain thermo labile
components are typically prepared in several
steps. A solution of the heat-stable
components is sterilized in the usual way by
autoclaving and then cooled to 35-50 C
under sterile conditions. Solutions of the
thermo labile components are filter-sterilized.
The sterilized solutions are then combined
under aseptic conditions to give the complete
medium.
In spite of possible degradation, however,
some compounds that are thought to be heat
labile are generally autoclaved if results are
found to be reliable and reproducible. These
compounds include ABA, IAA, IBA, kinetin,
pyridoxine, 2-ip and thiamine are usually
autoclaved.
3. Ethylene Oxide Gas
Plastic containers that cannot be heated are
sterilized commercially by ethylene oxide gas.
These items are sold already sterile and cannot
be resterilized. Examples of such items are
plastic petri dishes, plastic centrifuge tubes
etc.
4. UV Radiation
5 6
It is possible to use germicidal lamps to
sterilize items in the transfer hood when no
one is working there. We do not do this. UV
lamps should not be used when people are
present because the light is damaging to eyes
and skin. Plants left under UV lamps will die.
5. Microwave
It is also possible to sterilize items in the
microwave; we do not do this.
6. More Comments
Know which of your implements, flasks, etc.
are sterile and which are not. Sterile things will
have been autoclaved and should be wrapped
with some kind of protective covering, e.g. foil,
for transport from the autoclave to the hood.
Our usual autoclave time of 20 minutes is
intended for relatively small volumes. Large
flasks of media, water, etc. may require longer
autoclaving periods. It is preferable to put no
more than one liter of liquid in a container to
be autoclaved. Also, be sure to leave enough
room in the container so that the liquid does
not boil over.
Items that come packaged sterile, e.g. plastic
petri plates, should be examined carefully for
damage before use. If part of a package is
used, seal up the remainder and date and
label. Use up these items unless there is some
question about their sterility; they are
expensive

Two methods (autoclaving and membrane filtration under positive pressure) are commonly used to
sterilize culture media. Culture media, distilled water, and other stable mixtures can be autoclaved
in glass containers that are sealed with cotton plugs, aluminum foil, or plastic closures. However,
solutions that contain heat-labile components must be filter-sterilized.
Generally, nutrient or plant tissue culture media are autoclaved at 1.05 kg/cm
2
(15 psi) and 121C.
The time required for sterilization depends upon the volume of medium in the vessel. For small
volumes of liquids (100 ml or less), the time required for autoclaving is 15-20 min, but for larger
quantities (2-4 liter), 30-40 min is required. The pressure should not exceed 20 psi, as higher
pressures may lead to the decomposition of carbohydrates and other thermolabile components of
a medium. There is evidence that medium exposed to temperatures in excess of 121C may not
properly gel or may result in poor cell growth. The minimum times required for sterilization of
different volumes of medium are listed below.
Since many proteins, vitamins, amino acids, plant extracts, hormones, and carbohydrates are
thermolabile and may decompose during autoclaving, filter sterilization may be required. The
porosity of the filter membrane should be no larger than 0.2 microns (m). Empty glassware that is
to hold media must be sterilized in an autoclave before filter sterilization.
Nutrient media that contain thermolabile components can be prepared in several steps. That is, a
solution of the heat-stable components is sterilized in the usual way by autoclaving, then cooled to
35-50 C under sterile conditions; in a separate operation, solutions of the thermolabile
components are filter-sterilized. The sterilized solutions are then combined under aseptic
conditions to give the complete media.
indexing
It is testarious plant parts ie cuttings for theing of vfor
the presence of plant pathogen.
If the plant is pathogenic then it is subjected to various
procedures for elimanation of pathogens.
Indexing programms are being used in many
commericial labs, universities and government labs.
Normally micropropagated plants are indexed and
common crops are bannana, vannial, spices etc..
Careful evaluation of an artificial media is required
prior to initation of elimination procedures
Indexing techniques
Indexing strategies recolve around the host
crop studied as well as specific pathogens
affeciting the crop.
It need to be developed that 10exploit the
biology of the pathogen within the
host2)utilize dettection /eradiction tech that is
feconomically easible & sensitive.3)repeated
over months to reduce probability of
infection4)performed under strict sanitation
eradication
One methdod for eliminaton of systemic
fungal and bacterial plant pathogens from
geraniums is accomplised by using culture
indexing
It is the simplest,very effective method