Micropropagation is a technology for rapidly producing many plant clones in limited space. The process involves several stages: selecting a disease-free mother plant, establishing aseptic cultures, multiplying shoots, rooting plantlets, and hardening before transplantation. This method allows for disease-free plant production, conservation of endangered species, and efficient plant material exchange.

1. MICROPOPAGATION
By: Purbasa Maiti
MSc Microbiology

2. DEFINITION
Micropropagation is an advanced vegetative
propagation technology for producing a large number
of transplants in a limited time and space.
Clones of mother
plant

3. STAGES
Stage 0 — Mother Plant
Selection:
Stage I — Establishment of
Aseptic Culture:
Stage II — Multiplication of
shoots:
Stage III — In Vitro Rooting:
Stage IV — Transplantation
or Hardening:

4. Stage 0 — Mother Plant Selection:
 selection of suitable mother plant
is crucial
 disease free mother explant is
selected
 High potential to grow and divide
 Certain growth parameters of
mother plant can be improved by
pretreatment of mother explant
before initiation of cultures.

5. Stage I — Establishment of Aseptic Culture
• The main steps involved are:-
Surface sterilisation
Establishment of explant on appropriate culture medium.
• Plant tissues are commonly associated with bacteria and fungus.
• Therefore, adequate sterilization methods are employed to eliminate
microorganisms from explant

6. A quick dipping (5-30 sec) of
explant in 70% ethanol
Treatment with several sterilizing agents or surface disinfectants
such as:- sodium hypochlorite (2%), calcium hypochlorite and
saturated chlorine water
final treatment with sterile water to
remove traces of chemicals before
inoculating explant.
In tissue culture media, presence of sucrose as a carbon source encourages
their growth.

7. • The main objective of the stage II is the multiplication of shoot to give rise new
individual plant.
Involves three routinely used methods like:
• (a) Callus mediated multiplication
• (b) Adventitious shoots mediated multiplication
• (c) By apical or axillary shoots
Stage II — Multiplication of shoots:

8. (a) Callus Mediated Shoot Multiplication:
 Innumerable number of plants can be
grown by callus culture.
 Ratio of auxins to cytokinins could play
a decisive role in re-differentiation of
shoots from the callus.
 Callus mediated shoot multiplication
involves certain drawbacks.
1. There might be gradual decline or
total loss of regeneration potential
of callus cells.
2. callus mediated regenerated
plants generally exhibit genetic
variations which reflects on
variations of morphological
characters.

9. (b) In vitro Multiplication of Adventitious Shoots:
In this method, adventitious shoots arise directly
from the tissues of the explant like from stem,
tuber, bulb, leaf tissues other than leaf axils
remains (organogenesis).
It does not involve callus mediated regeneration.

10. (c) Axillary Shoot Proliferation:
• Axillary shoots developed from axillary
buds present in the axils of each leaf. In
leaf axils unsprouted status of axillary
buds is due to apical dominance
exhibited by growing shoot tip region at
the top. Synthesis of auxin in apical shoot
meristem is probably responsible for
apical dominance.
• However, apical dominance can be
reversed by synthesis of cytokinin in
axillary buds or entry of cytokinin into the
buds. The culture media are generally
enriched with very high concentration of
cytokinin by synthetic cytokinin like BAP
and kinetin play a prominent role in
releasing unsprouted axillary buds and its
further proliferation.

11. Stage III — In Vitro Rooting:
• Shoots or plantlets obtained during stage not
contain roots and fails to grow in soil.
Therefore, adequate steps are taken to grow
individual plantlets that can carry out
photosynthesis and survive without external
supply of carbohydrate. Therefore, in vitro
grown shoots must be transferred to a rooting
media.
• There is a clear distinction between rooting
media from shooting media. In vitro rooting
can be accomplished by adding auxins to the
culture media.
• Supplying riboflavin and other adjuvants
enhances root induction process.

12. Stage IV — Transplantation or Hardening:
• Process of transferring plantlets from aseptic culture
medium to soil.
• Immediate transfer of tissue culture plants into soil is
detrimental for survival of regenerated plants due to
desiccation, infection, and light temperature shock.
Excess of water loss was noticed from the leaves of plants
immediately after transplantation. High rate of water loss
due to lack of protective cuticle layer.
• In order to overcome these problems regenerated plants
are subjected to hardening.

13. Steps for hardening:
Removing test tube grown plantlets and washed under
distilled water to remove media sticking to the basal portion
of the shoots
Plantlets are then placed in sterilized soil or humus soil .
These are completely covered by plastic sheet or directly
placed in green house to provide 90% to 100% humidity for
upto 15 days.
Humidity is gradually reduced to 70%.
The acclimatize plants are then transferred to the field.

14. Applications:
 Small amounts of plant tissue are sufficient for the production of millions of clones in a
year using micropropagation.
 Plants in large numbers can be produced in a short period.
 Large amounts of plants can be maintained in small spaces. This helps to save
endangered species and the storage of germplasm.
 The micropropagation method produces plants free of diseases.
 Fast international exchange of plant material without the risk of disease introduction is
provided.
 Through somatic embryogenesis production of synthetic artificial seeds is becoming
popular nowadays.