PLANT TISSUE CULTURE

Plant Tissue Culture

(OREVIEW)
 LEARNING OUTCOMES

1. Define Tissue culture

2. Requirements of tissue culture

3. Know explant

4. Qualities of explant

5. Qulaities of good explant

6. Browning of explants

7. Steps in tissue culture

8. Factors affecting tissue culture

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 Tissue culture is the culture and maintenance
of plant cells or organs in sterile, nutritionally and
environmentally supportive conditions (in vitro).

Tissue culture produces clones, in which all

product cells have the same genotype (unless
affected by mutation during culture).

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 What conditions do plant cells need
to multiply in vitro?
Tissue culture has several critical requirements:

• Appropriate tissue (some tissues culture better than others)

• A suitable growth medium containing energy sources and

inorganic salts to supply cell growth needs. This can be
liquid or semisolid

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• Aseptic (sterile) conditions, as microorganisms grow much
more quickly than plant and animal tissue and can
overrun a culture.

• Growth regulators - in plants, both auxins & cytokinins.

• Frequent subculturing to ensure adequate nutrition and to

avoid the build-up of waste metabolites
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 EXPLANT

✓ Explants: Cell, tissue or organ of a plant that is used

to start in vitro cultures.

✓ Many different explants can be used for tissue

culture, but axillary buds and meristems are most
commonly used.
 Appropriate tissue (Explant)

✓ The explants must be sterilized to remove

microbial contaminants.

✓ Chemical sterialization without injuring the

plant cells beyond recovery

.
• Explants with high mitotic activity are good for
callus initiation.

• Immature tissues are more plastic than mature

ones.

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• The size and shape of the explants is also
important.

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 Choice of explant
Desirable properties ✓ Shoot tips
of an explant ✓ Axillary buds
✓ Easily sterilisable ✓ Seeds
✓ Juvenile
✓ Hypocotyl (from
✓ Responsive to culture germinated seed)
✓ Leaves
APPLICATION OF TISSUE CULTURE
✓ AVAILABILITY OF RAW MATERIAL- some
plants are difficult to cultivate and are also not
available in abundance.

✓ FLUCTUATION IN SUPPLIES AND QUALITY-

The production of crude drugs is subject to
variation in quality due to changes in climate, crop
diseases and seasons.

✓ PATENT RIGHTS- By this method ,it is possible

to obtain a constant supply and new methods can
be developed for isolation and improvement of
yield , which can be patented.
✓ EASY PURIFICATION OF THE COMPOUND- The
natural products from plant tissue culture may be
easily purified because of the absence of significant
amounts of pigments and other unwanted
impurities.

✓ CROP IMPROVEMENT- Plant tissue culture is

advantageous over the conventional cultivation
techniques in crop improvement by somatic
hybridization or by production of hybrids.
✓ Tissue culture techniques are used for virus
eradication, genetic manipulation, somatic
hybridization and other procedures that benefit
propagation, plant improvement and basic research.

✓ In a relatively short time and space a large number

of plantlets can be produced starting from the single
explant

✓ Taking an explant does not usually destroy the

mother plant, so rare and endangered plants can be
cloned safely
THREE MAIN METHODS GENERALLY USED IN
TISSUE CULTURE

MICRO PROPAGATION THROUGH THE

ENHANCED MULTIPLICATION OF
AXILLARY BUD.

ORGANOGENESIS.

SOMATIC EMBRYOGENESIS
 AXILLARY BUD

The axillary bud (or lateral bud) is an embryonic or

organogenic shoot located in the axil of a leaf. Each
bud has the potential to form shoots, stems,
branches or flowers.

Using optimum concentration of cytokinin or

combination of cytokinin and Auxin the
dormancy of the axillary buds can be broken.
Once the dormancy is broken, they develop
into shoots
 Organogenesis

The ability of non-meristematic plant tissues to

form various organs roots, shoots or leaves.

These organs may arise out of pre-existing

meristems or out of differentiated cells.
 Plant Organogenesis
Indirect:
Explant → Callus → Meristemoid →
Primordium

This pathway includes a callus stage.

Callus: Undifferentiated tissue that
develops on or around an injured or
cut plant surface or in tissue culture.

Direct:
It bypasses a callus stage. The cells
in the explant act as direct
precursors of a new primordium
An organ or a part in its most
rudimentary form or stage of
development
Central Dogma of organogenesis
High cytokinin:auxin ratio promotes shoots and
inhibits roots
High auxin:cytokinin ratio promotes roots and/or
callus formation while inhibiting shoot formation
Auxin/cytokinin 10:1-100:1 induces roots.
1:10-1:100 induces shoots
Intermediate ratios around 1:1 favor callus growth.
 SOMATIC EMBRYOGENESIS

The process of initiation and development of

embryos or embryo-like structures from somatic
cells (Somatic embryogenesis).
 Plant growth regulators
(Body building Plants)
Auxins:
- induces cell division, cell elongation, swelling of tissues,
formation of callus, formation of adventitious roots.
- inhibits adventitious and axillary shoot formation
- 2,4-D, NAA, IAA, IBA, pCPA…
Cytokinins: - shoot induction, cell division
- BAP, Kinetin, zeatin, 2iP…
Gibberellins: plant regeneration, elongation of internodes
- GA3…
Abscisic acid: induction of embryogenesis
- ABA
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 STEPS IN PLANT TISSUE
CULTURE
STAGE 1: Initiation phase

✓ Selection of the plant tissue (explant) from a

healthy vigorous ‘mother plant

✓ Sterilized in order to prevent any microorganism from

negatively affecting the process.
STAGE 2: Multiplication stage

The plant material is introduced in to the medium. Responsible for

the proliferation of the tissue and the production of multiple
shoots.

Each plant species has particular medium requirements

that must be established by trial and erro
STAGE 3: Root formation

It is at this phase that roots are formed. Here,

hormones are required in order to induce rooting,
and consequently complete plantlets.
 FACTORS AFFECTING PLANT
TISSUE CULTURE
1. Source of explant

2. Type of explant

3. Orientation of explant

4. Light

5. Temperature
 Fundamental abilities of plants

Totipotency
the potential or inherent capacity of a plant cell to develop into an
entire plant if suitable stimulated.
It implies that all the information necessary for growth and
reproduction of the organism is contained in the cell
Dedifferentiation
The capacity of mature cells to return to meristematic condition and
development of a new growing point, followed by
redifferentiation which is the ability to reorganize into new
organs
Competency
the endogenous potential of a given cell or tissue to develop in a
particular way
 RFERENCE

I. Aardsma, Gerald E., Radiocarbon and the Genesis Flood , El Cajon, CA, Institute for
Creation Research, 1991.

II. Ackerman, Paul D., In God’s Image After All: How Psychology Supports Biblical
Creationism, Grand Rapids, Baker, 1990.

III. Allen, Keith, & Briggs, Derek, Evolution and the Fossil Record, Washington,
Smithsonian, 1989.

IV. Andrews, E H; Werner, Gitt; Ouweneel, W J and European Creationist Congress,

Concepts in Creationism, Hertfordshire, Evangelical, 1986