PRINCIPLES OF CROP

PRODUCTION
ABT-320
(3 CREDIT HOURS))

LECTURE 3
LECTURE-WISE COURSE BREAKUP
TISSUE CULTURE
MAJOR COMPONENTS OF TISSUE CULTURE MEDIA
STERILIZATION METHODS
THE TECHNIQUE OF TISSUE CULTURE

TISSUE CULTURE
Tissue culture is the technique of enhancing
vegetative plant formation from in vitro cultured
explants. Direct regeneration of plantlets from
cultured plant parts (explants) or regeneration
through callus are employed for the purpose. For both
the purposes, plant parts are grown in culture media
under aseptic conditions. Hormonal proportions are
altered so as to obtain callus formation or direct
regeneration. The callus or plantlets formed are
transferred to hardening media for hardening and
acclimatization followed by field transfer.

MAJOR COMPONENTS OF
TISSUE CULTURE MEDIA
AGAR- Agar is a galactan, a complex carbohydrate with
galactose molecules . Agar is used to solidify the medium.
ORGANIC COMPOUNDS- These are used as the source of
carbon and energy. Sucrose is used usually in all standard
media.
INORGANIC COMPOUNDS- These include micronutrients and
macronutrients.
N, P, K, Ca, Mg, S are important
macronutrients whereas B, Mo, Cu, Zn, Mg, Fe etc are
micronutrients.
GROWTH HORMONES Hormones such as cytokinins, auxins
and gibberellins are used to regulate growth in tissue culture.
Cytokinins promote cell division and regulate growth. The
most widely used auxins are adenine, kinetin, zeatin and
benzyl adenine. Auxins stimulate shoot elongation.

MAJOR COMPONENTS OF
TISSUE CULTURE MEDIA
Auxins include indole acetic acid (IAA), naphthalene
acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid
(2,4-D). Gibberellins are of less importance. However,
gibberellic acid (GA3) is used in apical meristem
culture.
VITAMINS Vitamins regulate the metabolic activities
of cells. They are required in minor quantities. Vitamin
B1 (thiamine) is used for all types of tissue culture.
Nicotinic acid, riboflavin, pyridoxin, ascorbic acid,
biotin and cyanocobalamin are also used in different
cases.
AMINO ACIDS Amino acids such as L-aspartic acid, Lasperagin, L-glutamic acid, L-glutamine etc are used in
tissue culture as sources of nitrogen.

STERILIZATION METHODS
USE OF CHEMICALS: Chemicals such as Chromic acid,
Mercuric chloride, Sodium hypochlorite, Calcium hypochlorite
and alcohol are used for the sterilization of glassware, work
tables and source materials of explants.
USE OF OVEN: A dry heat oven is used to sterilize glassware,
metallic instruments etc by hot air (200-300C) for 1 hour.
USE OF AUTOCLAVES: Autoclaving is done to sterilize nutrient
media, distilled water etc with the help of steam (121C for 30
minutes)
ULTRA FILTRATION: Vitamins, hormones etc are unstable at
high temperatures. They are sterilized using millipore
membrane filter etc.
USE OF UV LIGHT: UV light is used in the incubation chamber
to make it germ-free.

THE TECHNIQUE OF
TISSUE CULTURE
The major steps involved in the method of
in vitro culture of an explant are;
1.Surface Sterilization of the Explant
2.Preparation of Nutrient Medium
3.Inoculation
4.Callus Growth
5.Subculturing
6.Organogenesis
7.
Direct Regeneration
8.Acclimatization and transfer to the field

SURFACE STERILIZATION
OF THE EXPLANT
The plant part which is used for in vitro culture is
known as explant. Explants are taken from the
most appropriate part of the parent plant.
Development of a tissue is the result of cell
division, cell elongation and cell differentiation.
Therefore, explants from young and healthy plants
are usually used. The presence of parenchyma is
the first consideration, because parenchyma
quickly responds to culture conditions. Explants
are usually 2-5mm in size. They are sterilized with
1-2% solution of sodium or calcium hypochlorite or
0.1% solution of mercuric chloride. All the
operations in tissue culture are done under aseptic
conditions.

PREPARATION OF
NUTRIENT MEDIUM
An appropriate nutrient medium is prepared
in advance. The medium may be solidified
by using agar (6gm/l) or may be used as a
liquid, depending on the requirement. It is
poured to culture vials under aseptic
conditions.

INOCULATION
Inoculation is the transfer of explants to
culture vials. It is done in the inoculation
chamber under aseptic conditions.

CALLUS GROWTH
The explant, a 2-5 mm sterile segment excised from the
appropriate part of the plant, is transferred to the
nutrient medium and incubated at 25-28C in an alternate
light and dark regime (usually 12 hrs each). Nutrient
medium, supplemented with auxins, induces cell division.
As a result, the upper surface of the explant gets covered
by an amorphous mass of loosely arranged thin-walled
cells. This mass of tissue is called callus. It is
characterized by abnormal growth and has the potential
to produce roots, shoots and embryoids.
Callus formation is controlled by the source of the
explant, composition of the medium and environmental
factors. Explants obtained from meristematic regions
develop more rapidly than those of other tissues.

CALLUS GROWTH
Usually, callus growth is completed in three
stages, namely induction, cell division and
cell differentiation. During callus induction,
the metabolic rate of the cells increases and
cells get packed with metabolic products.
This is followed by the rapid division of such
cells.
In
the
later
stage,
cellular
differentiation starts.

SUBCULTURING
When the callus has grown for some days
(say 28 days), it is essential to subculture it
on a fresh medium. Otherwise, nutrient
depletion, accumulation of toxic metabolites
and paucity of water are sure to occur,
leading to the death of the callus.

ORGANOGENESIS
Organogenesis starts in the callus in response to
the stimulation given by the chemicals in the
medium. Organogenesis takes place in two
stages, namely caulogenesis or shoot initiation
and rhizogenesis or root initiation. Both types of
organogenesis are controlled by the hormones
present in the medium. Skoog and Miller (1957)
demonstrated that a high auxin:cytokinin ratio
may induce shoot formation. In 1966, Torrey
proposed the hypothesis of organogenesis.
According to this hypothesis, organogenesis
starts with the development of a group of
meristematic cells called meristemoids, which
initiate the formation of a primordium. Depending
on the factors within the system, this primordium
develops into either shoot, root or embryoid.

ORGANOGENESIS
Generally, in dicots, buds develop when
cytokinin:auxin ratio is 1:100. On the other
hand, callus production will be favored by
an
auxin:cytokinin
ratio
1:100.
The
formation of an embryoid from the callus is
called somatic embryogenesis. If the
hormonal conditions are correctly balanced,
an entire plantlet can be induced to grow on
the culture medium. This process is called
regeneration.

DIRECT REGENERATION
In many plants, subculturing of callus
results in undesired variations of clones
(somaclonal variations). To avoid this, direct
regeneration of the explants into plantlets
can be tried. This has been achieved in
many plant species by altering the
hormonal combination of the culture media.

OTHER METHODS OF IN
VITRO CULTURING
Plant parts like anther, pollen grains,
embryo and endosperm are also grown in
vitro for different purposes. Pollen culture
and anther culture are practiced to produce
haploid plants. in vitro culture of embryos is
very useful in some plant species in which
the embryo gets aborted under in vivo
(natural) conditions. In many orchids,
embryos are rescued and grown in artificial
media to ensure proper development of
seeds.

ACCLIMATIZATION &
TRANSFER TO THE FIELD
In the last stage, the rooted plantlets are
subjected to acclimatization, so that they can
easily adjust to the field conditions. The
plantlets are taken out from the medium,
washed thoroughly in running water to remove
the agar and are then put in a low mineral salt
medium (LMSM) for 24-48 hours. These plantlets
are
then
transferred
to
pots
containing
autoclave sterilized mixture of clay, sand and
leaf molds in 1:1:1 proportion. The pot is usually
covered with transparent polythene to maintain
humidity. It is kept undisturbed for 15-30 days.
At this stage, the plant becomes fully
acclimatized. Finally, these fully acclimatized
plantlets can be transferred to the field.

THE END