This document discusses micropropagation, which is the rapid multiplication of plant materials using tissue culture methods. It involves taking explants like shoot tips or buds and culturing them on growth media to produce many new plantlets. The process involves initiation, multiplication, rooting, and acclimatization stages. Approaches include multiplication from axillary buds/shoots or adventitious shoots. Applications are high rate propagation of disease-free plants, seed production in some crops, and cost effectiveness. Automation using bioreactors and robots can increase production scale but reduces flexibility.

1. Allie N U,
MSc Biotechnology,

2. Introduction
 Micropropagation is a practice of rapidly
multiplying stock plant material to produce a large
number of progeny plants using modern PTC
methods.
 Used to multiply novel plants, that are genetically
modified or bred through conventional plant
breeding methods.
 Also used to provide no. of plantlets from a stock
plant that does not produce seeds, or does not
respond well to vegetative propagation.

3. Clonal propagation
 Process of asexual reproduction by multiplication
of genetically identical copies of individual plants.
 Clone refers to plant population derived from a
single plant by asexual reproduction.
 Asexual reproduction through multiplication of
vegetative parts is the method for invivo
propagation of certain plants (as no viable seed is
produced).
 Successfully applied for propagation of apple,
potato, tuberous and several ornamental plants.

4. Invitro clonal propagation
 Invivo clonal propagation of plants is expensive
and frequently unsuccessfull
 Invitro clonal propagation through tissue culture –
micropropagation.
 Use of tissue culture technique for
micropropagation- Morel(1960) for orchids.
 Used for rapid multiplication of plants.

5. Technique
 It’s a complicated process.
 Involves 3 stages (I,II and III). Sometimes two more
stages are added (0 and IV).
Stage 0:-
 Initial step in micropropagation.
 Involves the selection and growth of stock plants for
about 3 months under controlled conditions.
Stage I:-
 Initiation and establishment of culture in suitable
medium.
 Commonly used explants are organs, shoot tips and
axillary buds.
 Also explant is surface sterilized and washed before
use.

6. Stage II:-
 Activity of micropropagation occurs in defined culture
medium.
 Involves multiplication of shoots or rapid embryo
formation from the explant.
Stage III:-
 Involves the transfer of shoots to a medium for rapid
development.
 Sometimes, shoots are directly planted in soil to
develop roots.
 Invitro shooting is preferred while handling large
number of species.

7. Stage IV:-
 Involves establishment of plantlets in soil.
 Done by transferring plantlets of stage III from lab
to environment of green house.
 In some plants stage III is skipped and stage II
shoots are planted in pots or in suitable compost
mixture.
 These stages are useful for comparison between
two or more plant systems, besides better
understanding.

10. Approaches
 Two approaches:
1. Multiplication by axillary bud/apical shoots.
2. Multiplication by adventitious shoots.
 Besides these two approaches, plant
regeneration processes namely ‘organogenesis’
and ‘somatic embryogenesis’ may also be used.

11. Multiplication by axillary bud and
apical shoots
 Actively dividing meristems are present at axillary
and apical shoots.
 Axillary buds located in axile of leaves are
capable of developing into shoots.
 In the invitro state only limited no.of axillary
meristem can form shoots.
 Possible to develop plants from
1. meristem and shoot tip cultures ,
2. bud cultures

12. Meristem and shoot tip
cultures
 Apical meristem- dome of tissue located at extreme
tip of a shoot.
 Apical meristem along with young leaf primordia
constitute the shoot apex.
 For disease free plants, meristem tips are cultured.
Procedure :-
 Meristem or shoot tip is isolated from a stem by a V
shaped cut.
 Size (0.2 to 0.5mm) of tip is critical for culture.
 For good results, explants should be taken from
actively growing shoot tips, ideal timing is at end of
plant dormancy period.
 Widely used media are MS media and White’s

13. Stages of growth
Stage I:-
 Culture of meristem is established.
 Addition of growth regulators, cytokinins(kinetin, BAP) and
auxins(NAA, IBA) support growth and development.
Stage II:-
 Shoot development along with axillary shoot proliferation
occurs.
 High level of cytokinins are reqiured.
Stage III:-
 Associated with rooting of shoots and further growth.
 Facilitated by low cytokinin and high auxin concentration.
Thus both medium will be different in composition.
 Optimum temperature- 20- 28ºC and lower light intensity.

14. Bud cultures
 Plant bud possess active meristems depending
on physiological state of plant.
 Two types of bud cultures-
1. Single node culture:- natural method for
vegetative propagation of plants.
 Bud found in axil of leaf is comparable to stem
tips, for ablility in micropropagation.
 A bud along with piece of stem is isolated and
cultured to develop into plantlet.
 Closed buds used to reduce infection.
 No cytokinin is added here.

15. 2. Axillary bud culture:- shoot tip along with
axillary bud is isolated.
 Cultures carried out with high cytokinin
concentration. Thus apical dominance stops
and axillary bud develop
 Good axillary bud culture – cytokinin/ auxin ratio
is 10:1.

16. Multiplication by adventitious
shoots
 Adventitious shoots include stems, bulbs, tubers
and rhizomes.
 Useful for invivo and invitro clonal propagation.

19. Applications
1) High rate of plant propagation:-
 Large no.of plants can be grown from single piece of
plant tissue within short period.
 Can be carried out throughout the year irrespective of
seasonal variations.
 Small sized propagules obtained in micropropagation
can be easily stored for many years.
2) Production of disease free plants:-
 Meristem tip cultures are generally employed to
develop pathogen free plants
 Successfully used for production of virus free plants of
sweet potato, cassava and yam

20. 3) Production of seeds in some crops:-
 Through axillary bud proliferation seed production
can be done in some plants.
 Required in certain plants where limitation for
seed production is high degree of genetic
conservation.
4) Cost effective process:-
 Requires minimum growing space.
 Production cost is relatively low.

21. Automated micropropagation
 It is now possible to automate micropropagation
at various stages.
 Bioreactors have been set up for large scale
multiplication of shoots and bulbs.
 Some workers employ robots thus to reduce
production cost.
 Commmercial exploitation of tissue culture
technology is limited because of high cost of
production.
 This can be reduced by devising systems.
Bioreactors can be used for somatic
embryogenesis.

22. Robotics
 Robotics can be used , for example, in media
preparation, cutting microshoots and placing
them back on fresh medium.
 First commercial automated tissue culture robot
was introduced by Australian company for bio
robotics in 1997. target species- eucalypts,
acacia teak and pine.
 Miiwa 1991 – fully automated robotic
multiplication system.

23. Types
1) Total automation:-
With use of image analysis system and a robot or
other device
Little or no human interventions, except to control
the computer and repair parts
Eg: Toshiba ptc robot
2) Semi automation:-
Involvement of human operators contributing to any
stage of process.
Most commonly researched type of automation

24. Advantages
1) Management – management of machine will be
much easier.
2) Contamination – reduction of contamination can
be expected by using automation.
3) Operation time – two or three shifts become
possible.this increase the utilization of capacity
and reduce the depreciation costs per unit
4) Uniformity – machines act uniformly.

25. Disadvantages
 Flexibility – plant growth and shape are affected
by uncontrollable environmental factors. Once
production is started on non-uniform stock, it
would be difficult to coorect it in machines.
 Correction ability- application of automation
naturally decrease opportunity to detect
contamination.