Introduction to Micropropagation, Approaches of Micropropagation,

1. Introduction to
Micropropagation
Dr SV Suresh Kumar
Professor of Pharmacognosy
CES College of Pharmacy, Kurnool
Andhra Pradesh

2. Micropropagation
 Plants can be propagated by sexual (through generation of
seeds) or asexual (through multiplication of vegetative parts)
means.
 Clonal propagation refers to the process of asexual
reproduction by multiplication of genetically identical copies
of individual plants, where the term clone is used to
represent a plant population derived from a single individual
by asexual reproduction.
 In vitro clonal propagation through tissue culture is referred
to as micro propagation.
 Micropropagation is the practice of rapidly multiplying
stock plant material to produce a large number of progeny
plants, using modern plant tissue culture methods.

3.  Micropropagation is used to multiply noble plants such as
those that have been genetically modified or bred through
conventional plant breeding methods. It is also used to
provide a sufficient number of plantlets for planting from a
stock plant which does not produce seeds, or does not
respond well to vegetative reproduction.
 Technique of Micropropagation:
 Micro propagation is a complicated process and mainly
involves 5 stages (stage 0 and IV) for more comprehensive
representation of micro- propagation.
 Stage 0:This is the initial step in micropropagation, and
involves the selection and growth of stock plants for
about 3 months under controlled conditions.
 Stage I: In this stage, the initiation and establishment of
culture in a suitable medium is achieved. Selection of
appropriate explants is important.The most commonly
used explants are organs, shoot tips and axillary buds.

4.  Stage II: In this stage, the major activity of micro
propagation occurs in a defined culture medium. Stage II
mainly involves multiplication of shoots or rapid embryo
formation from the explant.
 Stage III:This stage involves the transfer of shoots to a
medium for rapid development into shoots. Sometimes,
the shoots are directly planted in soil to develop roots. In
vitro rooting of shoots is preferred while simultaneously
handling a large number of species.
 Stage IV:This stage involves the establishment of
plantlets in soil.This is done by transferring the plantlets
of stage III from the laboratory to the environment of
greenhouse. For some plant species, stage III is skipped,
and un-rooted stage II shoots are planted in pots or in
suitable compost mixture.

5.  StageV: In this stage, the major activity of micro
propagation occurs in a defined culture medium. Stage II
mainly involves multiplication of shoots or rapid embryo
formation from the explant.

6. Stages involved in micropropagation

7. Approaches involved in Micropropagation
1. Multiplication by axillary buds/apical shoots.
2. Multiplication by adventitious shoots.
Besides the above two approaches, the plant regeneration
processes namely organogenesis and somatic embryo-
genesis may also be treated as micro propagation.
3. Organogenesis:The formation of individual organs such
as shoots, roots, directly from an explant or from the
callus and cell culture induced from the explant.
4. Somatic embryogenesis:The regeneration of embryos
from somatic cells, tissues or organs.

8.  1 Multiplication by axillary buds/apical shoots.
 Actively dividing meristems are present at the axillary
buds and apical shoots (shoot tips).
 The axillary buds located in the axils of leaves are capable
of developing into shoots.
 By means of induced in vitro multiplication in micro
propagation, it is possible to develop plants from
meristem and shoot tip cultures and from bud cultures.

9.  Shoot tip cultures-
 Apical meristem is a dome of tissue located at the
extreme tip of a shoot.The apical meristem along with the
young leaf primordia constitutes the shoot apex.

10.  Meristem or shoot tip is isolated from a stem by aV-
shaped cut.The size (frequently 0.2 to 0.5 mm) of the tip
is critical for culture. In general, the larger the explant
(shoot tip), the better are the chances for culture survival.
For good results of micro propagation, explants should be
taken from the actively growing shoot tips, and the ideal
timing is at the end of the plants dormancy period.
 The most widely used media for meristem culture are
MS medium andWhite’s medium.

12.  In stage I, the culture of meristem is established. Addition
of growth regulators namely cytokinins (kinetin, BA) and
auxins (NAA or IBA) will support the growth and
development.
 In stage II, shoot development along with axillary shoot
proliferation occurs. High levels of cytokinins are required
for this purpose.
 Stage III is associated with rooting of shoots and further
growth of plantlet.
Consequently, stage II medium and stage III medium should
be different in composition.The optimal temperature for
culture is in the range of 20-28°C (for majority 24-26°C).
Lower light intensity is more appropriate for good micro
propagation.

13.  Bud Cultures-
 The plant buds possess quiescent or active meristems
depending on the physiological state of the plant.Two types
of bud cultures are used— single node culture and axillary
bud culture.
 Single node culture:This is a natural method for vegetative
propagation of plants both in vivo and in vitro conditions. A
bud along with a piece of stem is isolated and cultured to
develop into a plantlet. Closed buds are used to reduce the
chances of infections. In single node culture, no cytokinin is
added.

15. A). Shoot initiation from nodal part. (B). Multiplication & elongation of
shoots. (C). Rooting of in vitro formed shoots (D & E). In vitro hardening of
rooted shoots. (F). Ex vitro hardening. (G). 1 year old plant in pot.

16.  Axillary bud culture: In this method, a shoot tip along with
axillary bud is isolated.The cultures are carried out with high
cytokinin concentration. As a result of this, apical dominance
stops and axillary buds develop. A schematic representation
of axillary bud culture for a rosette plant and an elongate
plant is given as follows.

17.  For a good axillary bud culture, the cytokinin/ auxin ratio is
around 10: 1.This is however, variable and depends on the
nature of the plant species and the developmental stage
of the explant used. In general, juvenile explants require
less cytokinin compared to adult explants. Sometimes,
the presence of apical meristem may interfere with
axillary shoot development. In such a case, it has to be
removed.

18.  2 Multiplication by adventitious shoots .
 The stem and leaf structures that are naturally formed
on plant tissues located in sites other than the normal
leaf axil regions are regarded as adventitious shoots.
 There are many adventitious shoots which include
stems, bulbs, tubers and rhizomes.The adventitious
shoots are useful for in vivo and in vitro clonal
propagation.
 The meristematic regions of adventitious shoots can be
induced in a suitable medium to regenerate to plants.

19.  Adventitious shoots and roots developed from an immature leaf explant on
induction medium containing IAA (a) NAA (b).Adventitious shoots developed
from an immature leaf explant on induction medium containing BA (c)

20.  3 Organogenesis
 Organogenesis is the process of morphogenesis
involving the formation of plant organs i.e. shoots,
roots, flowers, buds from explant or cultured plant
tissues. It is of two types i.e direct organogenesis and
indirect organogenesis.
 Direct organogenesis-Tissues from leaves, stems, roots
and inflorescences can be directly cultured to produce
plant organs. In direct organogenesis, the tissue
undergoes morphogenesis without going through a callus
or suspension cell culture stage.
 Indirect organogenesis-When the organogenesis occurs
through callus or suspension cell culture formation, it is
regarded as indirect organogenesis. Callus growth can be
established from many explants (leaves, roots,
cotyledons, stems, flower petals etc.) for subsequent
organogenesis.

21. Micro Propagation of plant by organogenesis-a) Direct organogenesis
b) Indirect organogenesis through callus c) Indirect organogenesis
through suspension culture

23.  In general, the bigger the explant the better the chances for
obtaining viable callus/cell suspension cultures. It is
advantageous to select meristematic tissues (shoot tip, leaf,
and petiole) for efficient indirect organogenesis.This is
because their growth rate and survival rate are much better.
 For indirect organogenesis, the cultures may be grown in
liquid medium or solid medium. Many culture media (MS, B5
White’s etc.) can be used in organogenesis.The
concentration of growth regulators in the medium is critical
for organo- genesis.

24.  4. Embryogenesis.
 The process of regeneration of embryos from somatic
cells, tissues or organs is regarded as somatic (or asexual)
embryogenesis.
 Somatic embryos are structurally similar to zygotic
(sexually formed) embryos, and they can be excised from
the parent tissues and induced to germinate in tissue
culture media.
 Two routes of somatic embryogenesis are known — direct
and indirect.
 Direct Somatic Embryogenesis-When the somatic
embryos develop directly on the excised plant (explant)
without undergoing callus formation, it is referred to as
direct somatic embryogenesis .This is possible due to the
presence of pre-embryonic determined cells (PEDQ found
in certain tissues of plants.

25.  Indirect Somatic Embryogenesis- In indirect
embryogenesis, the cells from explant (excised plant
tissues) are made to proliferate and form callus, from
which cell suspension cultures can be raised. Certain cells
referred to as induced embryo genic determined cells
(IEDC) from the cell suspension can form somatic
embryos.
 Embryogenesis is made possible by the presence of
growth regulators (in appropriate concentration) and
under suitable environmental conditions.

27.  Somatic embryogenesis (direct or indirect) can be
carried on a wide range of media (e.g. MS,White’s).The
addition of the amino acid L-glutamine promotes
embryogenesis.The presence of auxin such as 2, 4-
dichlorophenoxy acetic acid is essential for embryo
initiation. On a low auxin or no auxin medium, the
embryo genic clumps develop into mature embryos.
 Indirect somatic embryogenesis is commercially very
attractive since a large number of embryos can be
generated in a small volume of culture medium.The
somatic embryos so formed are synchronous and with
good regeneration capability.

28. Applications of Micro propagation
 Micro propagation has become a suitable alternative to
conventional methods of vegetative propagation of
plants.There are several advantages of micro
propagation.
 High Rate of Plant Propagation- Through micro
propagation, a large number of plants can be grown from
a piece of plant tissue within a short period. Another
advantage is that micro propagation can be carried out
throughout the year, irrespective of the seasonal
variations.
 Production of Disease-free Plants- It is possible to
produce disease-free plants through micro propagation.
Meristem tip cultures are generally employed to develop
pathogen-free plants .

29.  Cost-effective Process- Micro propagation requires
minimum growing space.Thus, millions of plant species
can be maintained inside culture vials in a small room in
a nursery.
 Production of seeds in some crops- Micro
propagation, through axillary bud prolife- ration
method, is suitable for seed production in some plants.
This is required in certain plants where the limitation for
seed production is high degree of genetic conservation
e.g. cauliflower, onion.
 It is the only viable method of regenerating genetically
modified cells after protoplast fusion.
 Micropropagation often produces more robust plants,
leading to accelerated growth compared to similar plants
produced by conventional methods - like seeds or
cuttings.

30. Disadvantages of Micropropagation
 Contamination of Cultures- During the course of micro
propagation, several slow-growing microorganisms (e.g.
Eswinia sp, Bacillus sp) contaminate and grow in cultures.
The microbial infection can be controlled by addition of
antibiotics or fungicides.
 Brewing of Medium- Micro propagation of certain plants
(e.g. woody perennials) is often associated with
accumulation of growth inhibitory substances in the
medium. Chemically, these substances are phenolic
compounds, which can turn the medium into dark colour.
Phenolic compounds are toxic and can inhibit the growth
of tissues. Brewing of the medium can be prevented by
the addition of ascorbic acid or citric acid or polyvinyl
pyrrolidone to the medium.

31. Disadvantages of Micropropagation
 GeneticVariability -When micro propagation is carried
out through shoot tip cultures, genetic variability is very
low. However, use of adventitious shoots is often
associated with pronounced genetic variability.
 Vitrification-During the course of repeated in vitro shoot
multiplication, the cultures exhibit water soaked or almost
translucent leaves. Such shoots cannot grow and even
may die.This phenomenon is referred to as vitrification.
This can be be prevented by increasing the agar
concentration (from 0.6 to 1%) in the medium. However,
increased agar concentration reduces the growth rate of
tissues.
 For some micro propagation techniques, expensive
equipment, sophisticated facilities and trained manpower
are needed.This limits its use.