Transcript Lecture 2 - Lectures For UG-5

PRINCIPLES OF CROP PRODUCTION
ABT-320
(3 CREDIT HOURS))
LECTURE 02
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, L-asparagin,
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
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The major steps involved in the method of in vitro culture of
an explant are;
Surface Sterilization of the Explant
Preparation of Nutrient Medium
Inoculation
Callus Growth
Subculturing
Organogenesis
Direct Regeneration
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