Micropropagation is the process of rapidly multiplying plant materials in an aseptic laboratory environment. It involves culturing small pieces of plant tissue on nutrient media containing hormones and sugars. The ratio of auxin and cytokinin hormones determines whether shoots or roots develop. Micropropagation has several advantages over traditional propagation methods, including producing many identical clones of plants that are disease-free and genetically uniform. The process involves initiation, multiplication, rooting, and transfer to soil stages. Common micropropagation techniques are meristem culture, callus culture, and embryogenesis.

1. MICROPROPAGATION

2. DEFINITION
Production of whole plant from a
section of plant such as stem tip,
node, meristem, embryo or even a
seed.
“… the art and science of
multiplying plants in vitro.”

3. BASIS FOR MICROPROPAGATION
 Two Hormones Affect Plant Differentiation:
 Auxin: Stimulates Root Development
 Cytokinin: Stimulates Shoot Development
 Generally, the ratio of these two hormones can
determine plant development:
  Auxin &↓Cytokinin = Root Development
  Cytokinin & ↓Auxin = Shoot Development
 Auxin = Cytokinin = Callus Development

4. CONTROL OF IN VITRO CULTURE
Cytokinin
Auxin
Leaf strip
Adventitious
Shoot
Root
Callus

5. THREE FUNDAMENTAL ABILITIES OF
PLANTS
 Totipotency
The potential or inherent capacity of a plant cell to
develop into an entire plant if suitably stimulated.
It implies that all the information necessary for
growth and reproduction of the organism is contained
in the cell
 Dedifferentiation
Capacity of mature cells to return to meristematic
condition and development of a new growing point,
follow by redifferentiation which is the ability to
reorganise into new organ
 Competency
The endogenous potential of a given cells or tissue to
develop in a particular way

6. HISTORY OF PLANT TISSUE
CULTURE
1838-39 Cellular theory (Cell is
autonomous and totipotent)
Schleiden-
Schwann
1902 First attempt of plant tissue
culture
Harberlandt
1939 Continuously growing callus
culture
White
1946 Whole plant developed from
shoot tip
Ball
1950 Organs regenerated on callus Ball
1954 Plant from single cell Muir
1960 Protoplast isolation Cocking

7. 1962 MS media Murashige -
Skoog
1964 Clonal propagation of orchids Morel
1964 Haploids from pollen Guha
1970 Fusion of protoplasts Power
1971 Plants from protoplasts Takebe
1981 Somaclonal variation Larkin
FREDERICK C. STEWARD

8. CHARACTERISTICS OF PLANT
CELLS
 Large (10-100mM long)
 Tend to occur in
aggregates
 Shear-sensitive
 Slow growing
 Easily contaminated
 Low oxygen demand (kla
of 5-20)
 Will not tolerate
anaerobic conditions
 Can grow to high cell
densities (>300g/l fresh
weight).
 Can form very viscous
solutions

9. MICROPROPAGATION
 Embryogenesis
 Direct embryogenesis
 Indirect embryogenesis
 Organogenesis
 Organogenesis via callus formation
 Direct adventitious organ formation
 Microcutting
 Meristem and shoot tip culture
 Bud culture

10. SOMATIC EMBRYOGENESIS
 The production of
embryos from somatic or
“non-germ” cells.
 Usually involves a callus
intermediate stage which
can result in variation
among seedlings

11. Peanut somatic embryogenesis

12. ORGANOGENESIS
 The production of roots,
shoots or leaves.
 These organs may arise
out of pre-existing
meristems or out of
differentiated cells.
 Like embryogenesis, may
involve a callus
intermediate, but this
may often occurs without
callus.

14. MICROCUTTING PROPAGATION
 This is a specialized form of organogenesis
 It involves the production of shoots from
pre-existing meristems only.
 Requires breaking apical dominance
 Microcuttings can be one of three types:
 Nodal
 Shoot cultures
 Clump division

15. FACTORS AFFECTING PLANT TISSUE
CULTURE
 Growth Media
 Minerals, Growth factors, Carbon source, Hormones
 Environmental Factors
 Light, Temperature, Photoperiod, Sterility, Media
 Explant Source
 Usually, the younger, less differentiated the
explant, the better for tissue culture
 Genetics
 Different species show differences in amenability
to tissue culture
 In many cases, different genotypes within a species
will have variable responses to tissue culture;

16. STEPS 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
 Stage II - Multiplication
 explant transferred to shoot media; shoots can be constantly
divided
 Stage III - Rooting
 explant transferred to root media
 Stage IV - Transfer to soil
 explant returned to soil; hardened off

18. •The plant tissues are removed
from an intact plant in a sterile
condition.
• Clean stock materials that are
free of viruses and fungi are
important in the production of
the healthiest plants.
•Explants used can be stem tips,
anthers, petals, pollen and
others plant tissues.
•The explant material is then
surface sterilized, usually in
multiple courses of bleach and
alcohol washes, and finally rinsed
in sterilized water.
Stage 0 – Selection & preparation
of the mother plant

19. •A small portion of plant tissue, sometimes
only a single cell, is placed on a growth
medium, typically containing sucrose as an
energy source and one or more plant growth
regulators (plant hormones).
• Usually the medium is thickened with agar
to create a gel which supports the explant
during growth.
Stage I - Initiation of culture

21. Multiplication is the taking of tissue samples produced
during the first stage and increasing their number.
Establishment stage is followed by multiplication.
 Through repeated cycles of this process, a single
explant sample may be increased from one to hundreds
and thousands of plants.
 Depending on the type of tissue grown, multiplication
can involve different methods and media. If the plant
material grown is callus tissue, it can be placed in a
blender and cut into smaller pieces and recultured on the
same type of culture medium to grow more callus tissue.
If the tissue is grown as small plants called plantlets,
hormones are often added that cause the plantlets to
produce many small offshoots.
Stage II - Multiplication

24. After the formation of multiple shoots, these shoots
are transferred to rooting medium with a high
auxincytokinin ratio. After the development of roots,
plantlets can be used for hardening.
Stage III - Rooting
Stage IV - Transfer to soil
"Hardening" refers to the preparation of the plants for
a natural growth environment. Hardening typically
involves slowly weaning the plantlets from a high-
humidity, low light, warm environment to what would be
considered a normal growth environment for the species
in question.
In the final stage of plant micropropagation, the
plantlets are removed from the plant media and
transferred to soil or (more commonly) potting compost
for continued growth by conventional methods.

26. METHODS OF
MICROPROPAGATION
1.Meristem culture
2.Callus culture
3.Suspension culture
4.Embryo culture
5.Protoplast culture

27. 1. Meristem culture
In Meristem culture the Meristem and a few
subtending leaf primordial are placed into a suitable
growing media.
 An elongated rooted plantlet is produced after some
weeks, and is transferred to the soil when it has
attained a considerable height.
 A disease free plant can be produced by this method.
 Experimental results also suggest that this technique
can be successfully utilized for rapid multiplication of
various herbaceous plants.

30. 2. Callus culture
A callus is mass of undifferentiated parenchymatous
cells. Equimolar amounts of auxin and cytokinin stimulate
cell division. Leads to a mass proliferation of an
unorganised mass of cells called a callus.
 The callus growth and its organogenesis or
embryogenesis can be referred into three different
stages.
•Stage I: Rapid production of callus after placing the
explants in culture medium
•Stage II: The callus is transferred to other medium
containing growth regulators for the induction of
adventitious organs.
•Stage III: The new plantlet is then exposed gradually to
the environmental condition.

31. 3. Suspension culture
A cell suspension culture refers to cells and or groups of
cells dispersed and growing in an aerated liquid culture
medium. Callus is placed in a liquid medium and shaken
vigorously and balanced dose of hormones.
When callus pieces are agitated
in a liquid medium, they tend to
break up.
Suspensions are much easier to
bulk up than callus since there
is no manual transfer or solid
support.

32. INTRODUCTION INTO SUSPENSION
+
Plate out
Sieve out lumps
1 2
Pick off
growing
high
producers
Initial high
density
Subculture
and sieving

33. EMBRYO CULTURE
 Embryo culture developed from the need to rescue
embryos (embryo rescue) from wide crosses where
fertilization occurred, but embryo development did
not occur
 These techniques have been further developed for
the production of plants from embryos developed by
non-sexual methods (haploid production discussed
later)

35. Protoplast Culture
In protoplast culture, the plant cell can be isolated
with the help of wall degrading enzymes and growth in
a suitable culture medium in a controlled condition for
regeneration of plantlets.
 Under suitable conditions the protoplast develops a
cell wall followed by an increase in cell division and
differentiation and grows into a new plant.
 The protoplast are fist cultured in liquid medium at
25 to 28 Ć with a light intensity of 100 to 500 lux or
in dark and after undergoing substantial cell division,
they are transferred into solid medium congenial for
morphogenesis.

37. Protoplasts Isolation and Culture

38. Advantages
Micropropagation has a number of advantages over
traditional plant propagation techniques:
•The main advantage of micropropagation is the production
of many plants that are clones of each other.
•Micropropagation can be used to produce disease-free
plants.
•It can have an extraordinarily high fecundity rate,
producing thousands of propagules while conventional
techniques might only produce a fraction of this number.
•It is the only viable method of regenerating genetically
modified cells or cells after protoplast fusion.

39. •Some plants with very small seeds, including most orchids,
are most reliably grown from seed in sterile culture.
•A greater number of plants can be produced per square
meter and the propagules can be stored longer and in a
smaller area.
•It is useful in multiplying plants which produce seeds in
uneconomical amounts, or when plants are sterile and do
not produce viable seeds or when seed cannot be stored
•Micropropagation often produces more robust plants,
leading to accelerated growth compared to similar plants
produced by conventional methods - like seeds or cuttings.

40. DISADVANTAGES
Micropropagation is not always the perfect means of
multiplying plants. Conditions that limits its use include:
•It is very expensive, and can have a labour cost of more
than 70%.
•A monoculture is produced after micropropagation,
leading to a lack of overall disease resilience, as all progeny
plants may be vulnerable to the same infections.
•An infected plant sample can produce infected progeny.
This is uncommon as the stock plants are carefully
screened and vetted to prevent culturing plants infected
with virus or fungus.

41. •Not all plants can be successfully tissue cultured, often
because the proper medium for growth is not known or the
plants produce secondary metabolic chemicals that stunt
or kill the explant.
•Sometimes plants or cultivars do not come true to type
after being tissue cultured. This is often dependent on
the type of explant material utilized during the initiation
phase or the result of the age of the cell or propagule
line.
•Some plants are very difficult to disinfect of fungal
organisms.

42. Anther/Microspore Culture

43. FEATURES OF MICROPROPAGATION
 Clonal reproduction
 Way of maintaining heterozygozity
 Multiplication Stage can be recycled many times
to produce an unlimited number of clones
 Routinely used commercially for many ornamental
species, some vegetatively propagated crops
 Easy to manipulate production cycles
 Not limited by field seasons/environmental influences
 Disease-free plants can be produced
 Has been used to eliminate viruses from donor plants

44. Rapid micropropagation and callus
induction of Terminalia bellerica Roxb. -
An endangered plant
Jitendra Mehta*, Monika Sain, Banwari Lal Mathuriya, Ritu Naruka,
Ambika Kavia and Dev Ratan
Plant Tissue Culture Laboratory & Department of Biotechnology,
Vital Biotech Research Institute, University of Kota, Kota (Rajasthan), India.
______________________________________________________________________
____________________ ABSTRACT An in vitro micropropagation system has been
developed for Terminalia bellerica Roxb., an important Indian medicinal plant. Nodal
segments obtained from 15-d-old aseptically grown seedlings were used as explants.
MS medium containing 2.0 mg/l BAP was found most suitable for culture initiation.
Although shoot multiplication was achieved on MS medium containing BAP and Kn,
the maximum number of shoots was obtained with 3.5 mg/l BAP+ 0.5 mg/l Kn. Best
rooting response was observed on medium containing quarter strength MS salts,
0.8% agar and 1.0 mg/l IBA. Plantlets were hardened initially in culture room
conditions and then transferred to misthouse. Maximum callus induction response
was observed on MS medium supplemented with 0.25 mg/l 2,4-D+ 0.3 mg/l Kn within
4 weeks from leaf petiole.

45. 1.^B N SATHYANARAYAN. (2007). Plant Tissue
Culture: Practices and New Experimental
Protocols. I. K. International. pp. 106–.
ISBN 978-81-89866-11-2.
2. ^S. S BHOJWANI.; M K RAZDAN. (1996). Plant
tissue culture: theory and practice (Revised.).
Elsevier. ISBN 0-444-81623-2
REFERENCE