micropropagation, a method in plant tissue culture.

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.