MICROPROPAGARION

A tissue culture technique for plant propagation in vitro in which tissue is

taken from a plant and grown in a laboratory to produce large number of
plantlets that are genetically identical to the parent.

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 MICROPROPAGARION

This consists of utilizing the technique and plant cell, tissue and organ
culture.

• A small piece of excised tissue from –plant part is grown in nutrient

media under controlled aseptic microbe free condition in glass
containers (test tube , flask, jars)

• The tissue soon grows and provides three fates:

1. Large number of plants directly arising from the tissue

2. First forms an unorganized mass of cells called callus, then
regeneration of plants from callus via shoot and root.
3. Formation of callus and then differentiation into somatic embryos,
shoot and root together

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(1) Explant –to-multiple plants

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Mostly axillary buds remain dormant: Why?

Apical
dominance of
Auxin

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Pruning

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 Micropropagation by Adventitious Shoot Proliferation

Adventitious shoot proliferation in plant cell and tissue culture, in response to

hormonal manipulation of the culture medium, require de novo differentiation of
meristematic region, randomly, all over the tissue other than the pre-existing
meristem. It is a multistep process and a series of intracellular events,
collectively called induction that occurs before the appearance of
morphologically recognizable organs. Micropropagation via adventitious shoot
regeneration may occur directly or indirectly via an intervening callus phase

Root differentiation indirectly via callus 7

Direct shoot proliferation from leaf-disc culture
 Induction of organogenic differentiation

A schematic representation of in vitro adventitious shoot proliferation from leaf-

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disc culture
 Induction is IRREVERSIBLE?

The cells induced to form a specific organ and would continue to develop into
that organ even if the inductive growth regulators are removed. Hence,
induction favours the irreversible commitment of cells to follow a particular
developmental pathway. For example, Brassica juncea, undergoes the
induction of organogenic differentiation where a cytokinin, BAP induces shoot-
bud differentiation at the cut end of the cotyledon petiole. In the absence of
BAP (basal medium) only roots are formed at the same site. The cotyledons
transferred to basal medium after 11 days of incubation on BAP leads to the
development of only shoots and no roots. Similarly, the cotyledons lose the
potential to form shoots on BAP medium if they are pre-cultured on BAP free
medium for more than 7 days

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 Micropropagation by Axillary Shoot Proliferation

Axillary buds are usually present in the axil of each leaf and every bud has the

potential to develop into a shoot. In nature these buds remain dormant for

various periods. The species with strong apical dominance show the growth of

axillary buds into shoot only if the terminal bud is removed or injured. The

application of cytokinin to the axillary buds can overcome the apical dominance

effect.

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How to break dormancy of axillary bud: in vitro?

Apply cytokinins
(4.5 - 25 uM)

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 Explant –to-multiple plants

Explant: Axillary bud Multiple plants

Advantage: Incipient shoot has

already differentiated in
vivo………….It only needs division
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 Explant –to-callus-multiple plants

Competence
Extra

Differentiation

Division 13
 Explant –to-callus-multiple plants

Disadvantages:
• Chances of high somaclonal variation
• Plant may not be identical to a single mother plants
• Differentiation may vary from to cell-to-cell of same callus.

Advantages:
• Easy to grow
• Easy to store
• Comparatively large number of explants

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Explant –to-somatic embryo-multiple plants

Shoot & Root together

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 Meristem Culture For Virus Elimination

A group of identical cells which are in a continuous state of cell division. Some
of the cells from the meristematic tissue stops dividing and exhibit certain
changes to become permanent tissues of the plant

I. Apical meristem
II. Intercalary meristem
III. Lateral meristem

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 Establishment of pathogen free plants

The apical meristem of a shoot is the portion lying distal to the youngest leaf
primordium, it measures up to about 100µm in diameter and 250µm in length.
The apical meristem together with one to three young leaf primordia, measuring
100-500µm, constitutes the shoot-apex which is a source of getting virus free
plants

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 Why Apical meristem is free from viruses?

(a) Viruses readily move in a plant body through the vascular system which
is absent in the meristem.

(b) The alternative method of cell-to-cell movement of the virus through

plasmodesmata is rather too slow to keep pace with the actively growing tip.

(c) High metabolic activity in the actively dividing meristem cells does not
allow virus replication.

(d) The ‘virus inactivating systems' in the plant body, if any, has higher
activity in the meristem than in any other region. Thus, the meristem is
protected from infection.

(e) A high endogenous auxin level in shoot apices may inhibit virus
multiplication.

(f) Absence of vacuolated cells in the meristem.

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Stages of Micropropagation

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 Stages of Micropropagation

Stage 0 – Selection & preparation of the mother plant sterilization of the

plant tissue takes place

Stage I - Initiation of culture, explant placed into growth media (High

cytokinin (BA = 4.5 – 25 uM). Some time minor auxin should be added to
suppress the inhibitory effect of high cytokinin on normal shoot growth).

Stage II – Multiplication, explant transferred to shoot media; shoots can be

constantly divided (high cytokinin)

Stage III – Rooting, explant transferred to root media (NAA or IAA)

Stage IV - Transfer to soil, explant returned to soil; hardened off

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 Advantages of Micropropagation
1. Clonal mass propagation - extremely large numbers of plants can be
produced. Culture is initialized from small parts of plants – so no need of
much space
2. Production of disease and virus free plantlets. This leads to simplification of
international exchange of plants
3. Micropropagation enables growers to increase the production of plants that
normally propagate very slowly such as Narcissus and other bulbous crops.
4. Vegetative propagation of sterile hybrids can be used as parent plants for
seed production. Eg. Cabbage
5. One of the rapid methods for cloning of disease free trees.
6. In vitro cultures can be stored for long time through cryopreservation.
7. Breeding cycle can be shortened.
8. No seasonal barrier

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 Disadvantages of Micropropagation

1. Plants are not autotrophic

2. Poor Acclimatization to the field is a common problem (hyperhydricity)

3. Risk of genetic changes if 'de novo' regeneration is used

4. Mass propagation cannot be done with all crops to date. In cereals much
less success is achieved

5. Regeneration is often not possible, especially with adult woody plant

material.

6. More problems in inducing rooting

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