Introduction to tissue culture, culture types, micropropagation benefits and disadvantages, cryopreservation utilization of plant tissue culture,

1. PLANT TISSUE
CULTURE
METHODS & APPLICATIONS
By
OJUEDERIE Omena Bernard
Department of Biotechnology
College of Food Sciences
Bells University of Technology
Ota.

2. Introduction
Tissue culture
“… A method of biological research
in which fragments of tissues from
an animal or plant are grown in vitro
in artificial medium under aseptic
conditions and continue to survive and
function.”

3. Introduction
What is plant tissue
culture?
“… the aseptic culture of plant
protoplasts, cells, tissues or
organs under aseptic conditions
which lead to cell multiplication or
regeneration of organs or
whole plants.”

4. Basic concepts of plant
tissue culture(PTC)Two concepts, are central to understanding
plant cell, tissue, organ culture and
regeneration.
Plasticity.
-ability to initiate cell division from almost any
tissue of the plant.
-ability to regenerate lost organs or undergo
developmental pathways in response to
particular stimuli.
Totipotency.
-each cell has the capacity to regenerate the
entire plant.

5. Basic concepts of plant
tissue culture(PTC)
Cells lines differentiate to form
specialized tissues and organs.
Unlike animal cells, living plant cells
re-differentiate.
Therefore, tissue can be regenerated
from explants such as cotyledons,
hypocotyls, leaf, ovary, protoplast,
petiole, root, anthers, etc.

6. Short history of Plant
Tissue Culture
A German plant physiologist Gottlieb
Haberlandt (1902) cultured isolated
single palisade cells from leaves in
Knop's salt solution enriched with
sucrose.
Haberlandt is regarded as the father of
plant tissue culture.

7. Short history of Plant
Tissue Culture
Toward Commercial Micropropagation 1950s
Morel & Martin 1952 used Meristem-tip culture for
disease elimination. They recovered for the first time,
virus- free Dahlia plants

8. Short history of Plant
Tissue Culture
Morel 1960 Disease
eradication
Wimber 1963 & in vitro
production of
orchids

9. Short history of Plant
Tissue Culture
Commercialization of Micropropagation
Murashige and Skoog(1962) Developed a universally used
high salt medium containing mineral salts, vitamins, an
energy source and growth hormone (MS medium)
Murashige(1974) Broad commercial application

10. What is required?
Tissue culture has several critical
requirements:
Appropriate tissue.
A suitable growth medium which
can be liquid or semisolid.
Aseptic (sterile) conditions, as
microorganisms grow much more
quickly than plant and animal
tissue and can over run a culture.

12. Tissue culture
environment
General Laboratory and media
preparation room
Transfer area
Culturing facilities/Growth
rooms
Washing and storage rooms
Greenhouses and containment
room

13. General laboratory & media

14. Transfer area (GRC IITA IBADAN)

15. Design of a Laminar flow
hood

16. A section of a culture room
Timer switch to
control
photoperiod
Fluorescent tube
placed above shelf
for lighting
A level of shelf
containing in vitro
cultures

17. Hardening process( Acclimatization)

18. Micropropagation
“… the art and science of multiplying
plants in vitro
It implies
- regeneration
- multiplication
- uniformity ??

19. Starting material for
Micropropagation
(Explant)
Propagation from
plant cells, tissues or
organs under aseptic
conditions in synthetic
medium in vitro.
Many different explants
can be used for
micropropagation, but
axillary buds and
meristems are most
commonly used
Tip bud
Leaf
Axillary
bud
Internode
Root

20. Stages of
Micropropagation
Stage I - Selection & preparation of the
mother plant – sterilization of the plant tissue
takes place 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

21. Stages of micropropagation
STAGE IV: Transfer to Natural
Environment
STAGE III:
Pretransplant
(rooting)
STAGE II: Shoot
Production
STAGE I - Sterilization

22. Growth medium
The growth medium used depends on the
plant species being grown. The medium
contains the following components:
all of the minerals and vitamins needed for
plant growth and differentiation;
a carbon/energy source such as sugar as the
explant cannot usually photosynthesize;
various growth regulators to encourage cell
enlargement, division and differentiation;
agar which is used to solidify the medium.

23. Plant growth regulators
Two major hormones affect Plant
Differentiation:– Auxins: 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

24. Hormonal balance
Auxin Cytokinin
High Low
Root formation on cuttings
Embryogenesis
Adventitious root formation in callus
Callus initiation
Adventitious shoot formation
Axillary shoot growth
Low High

25. Methods of
micropropagation
Clonal propagation involves the
multiplication of genetically identical
lines by asexual reproduction.
Micropropagation involves the use of
bud culture, meristem and shoot-tip
culture techniques to introduce plants in
vitro by induction to form adventitious
buds, shoots and entire plants.

26. Methods of
micropropagation
cont’d
Bud culture is the culture of plant buds
which contains active meristems.
Meristem culture is the culture of apical
meristems which are capable of active
cell division and differentiation into
specialized and permanent tissue such
as shoots and roots

27. Methods of
micropropagation
cont’d
Single node culture is done on a
hormone free medium.
Axillary bud culture is done using
excised shoot tips cultured on medium
amended with high cytokinin
concentration 6 Benzyl amino
purine(BAP)/Benzyl adenine(BA)

28. Bud culture by single
nodes
Surface sterilization
in NaOCL
Rinsing with
SDW
Transfer into sterile
container for 2nd
sterilization
Steps in single node culture
1 2 3
4
5

29. Methods of
micropropagation
cont’d
Micro cuttings of yam during and after
subculture
3 4

30. In vitro plantlet
regeneration via
meristem & shoot tip
culture

31. Meristem culture
A meristem is an undifferentiated but
determined tissue, the cells of which
are capable of active cell division and
differentiation into specialized and
permanent tissues such as shoots and
roots.
It has the capacity of producing a
complete plant in vivo and in vitro
(Totipotent)

32. Shoot apical meristem

33. Meristem excison using stereomicroscopes

34. Dumet et al., 2008

35. Shoot tip culture
Clonal in vitro propagation by repeated
enhanced formation of axillary shoots
from shoot-tips cultured on media
supplemented with plant growth
regulators, usually cytokinins.
The size of the shoot tip is between 0.5
to 5mm and consists of primordia
leaves.

37. Stages in acclimatization of Cassava
1 2 3
4 5

38. Acclimatization of Yam using constructed humidity
chamber
Acclimatization process using polyethylene

40. Problems in
Micropropagation
There is problem of genetic variability due to
Somaclonal variation in some cultures.
Contamination is a major problem which could
cause high losses within a short period.
It requires expensive equipments and well
trained manpower.
Vitrification may occur which reduces the rate
of growth multiplication of the plant and
eventually causes death.

41. Benefits of
micropropagation
Rapid multiplication of superior clones
can be carried out through out the year,
irrespective of seasonal variations.
Multiplication of disease free plants e.g.
virus free plants of sweet potato
(Ipomea batatas), cassava (Manihot
esculenta) e.t.c.
It is a cost effective process as it
requires minimum growing space.

42. Growth Media– Minerals, Growth factors,
Carbon source, Hormones
Environmental Factors– Light, Temperature,
Photoperiod.
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.
Factors affecting Tissue
Culture

43. Applications of plant
tissue culture
Agriculture
Anther & pollen culture-Production of
haploid plants.

44. Applications of plant
tissue culture
Production of virus-free plants for safe
germplasm transfer.
Screening of in vitro lines for stress and
disease resistance.
Somaclonal Variations has been used
in plant breeding programmes where
the genetic variations with desired or
improved characters are introduced into
the plants.

45. Somaclonal variation
Somaclonal variation is a general
phenomenon of all plant regeneration
systems that involve a callus phase.
There are two general types of
Somaclonal Variation:
Heritable, genetic changes (alter the
DNA).
Stable, but non-heritable changes
(alter gene expression).

46. Germplasm conservation
An extension of micropropagation techniques
through two methods:
Slow growth techniques e.g.: ↓ Temp., ↓
Light, media supplements ( growth
retardants).Medium-term storage (1 to 4
years)
Cryopreservation.Ultra low temperatures in
liquid nitrogen at -196o
C.
Stops cell division & metabolic processes
Very long-term (indefinite?)

47. Clonal germplasm flow from medium-
term storage to long term storage
D.Dumet, 2009. IITA

48. Cryopreservation
Micropropagation Unit, Royal Botanic Gardens, Kew UK.
Cryopreservation is a valuable technology for the cost-
effective,sustained long-term conservation of plant genetic material.

49. Cryopreservation
Requirements
Preculturing– Usually a rapid growth rate to create
cells with small vacuoles and low water content.
Cryoprotection– Glycerol, DMSO, PEG, etc…, to
protect against ice damage and alter the form of ice
crystals.
Freezing– The most critical phase; one of two
methods is used:
Slow freezing which allows for cytoplasmic
dehydration.
Quick freezing which results in fast intercellular
freezing with little dehydration.

50. Cryopreservation
Requirements
Storage – Usually in liquid nitrogen (-196o
C)
to avoid changes in ice crystals that occur
above -100o
C
Thawing– Usually rapid thawing to avoid
damage from ice crystal growth.
Recovery (don’t forget you have to get a
plant)
– Thawed cells must be washed of
cryoprotectants and nursed back to normal
growth.
– Avoid callus production to maintain genetic
stability.

51. Applications of plant
tissue culture
Horticulture and Forestry:
Micropropagation method is used for rapid
multiplication of ornamental plants as well as
important trees yielding high fuel, pulp, fruits or oil
at a large scale.

52. Applications of plant
tissue culture
Today's high yielding oil palms on plantations
in Malaysia were generated in the 1960s by
tissue culture methods.
These palms produce 30% more oil than
normally cultured palms.
Improvement of economically important forest
trees is being done through genetic
transformation and rapid Micropropagation
.e.g in vitro regeneration and genetic
transformation of conifers.

53. Applications of plant
tissue culture
Industries:
Plant cell culture is used for biotransformation
(modification of functional groups of organic
compounds by living cells).
Food and agricultural biotechnologists are involved
in using tools of molecular biology to enhance the
quality and quantity of foods and economic crops.
For example, Golden Rice was genetically
enhanced with added beta carotene, which is a
precursor to Vitamin A in the human body.
Plant cells can be cultured in fermenters for the
industrial production of secondary
Metabolites using cell culture.

54. PLANT SPECIES AND
SECONDARY METABOLITES
OBTAINED FROM THEM USING
TISSUE CULTURE TECHNIQUES
Product Plant source Uses
Artemisin Artemisia spp Antimalarial
Capsaicin Capsicum
annum
Cures
Rheumatic pain
Codeine Papaver spp. Analgesic
Camptothecin Campatotheca
accuminata
Anticancer
Quinine Cinchona
officinalis
Antimalarial

55. At Bells University
Plant tissue culture
practicals
Capsicum annum (Red
Pepper) regenerated via seed
culture
Apple regenerated via
embryo culture
Apple subcultured into test
tubes using

56. At Bells University
Yam
micropropagation
Cassava
micropropagation

57. Possible areas for research
Somatic embryogenesis of white
Guinea yam(Dioscorea rotundata).
In vitro regeneration of Bambara
groundnut (Vigna subterranea
L.Verdc) via mersitem culture.
In vitro micropropagation of medicinal
plants.

58. Conclusion
The ability to regenerate entire plants from
cells or tissues has been invaluable to plant
biotechnology due to the totipotent nature of
plant cells.
From the sole objective of demonstrating the
totipotency of differentiated plant cells, the
technique now finds application in both basic
and applied researches in number of fields of
enquiry.
Plant tissue culture has generated intense
interest among molecular biologists, plant
breeders and commercial horticulturists.
This technique should therefore be
encouraged and supported by African
countries as it could aid in improving food
security in Africa.

60. References
D.Dumet,A.Adeyemi,O.B.Ojuederie (2008). Yam
invitro genebanking. Genebank manual.
http://www.iita.org/genebank/manual
D.Dumet,A.Adeyemi,O.B.Ojuederie (2008).Cassava
in vitro processing and the genebanking.Genebank
manual. http://www.iita.org/genebank/manual
HORT689/AGRO689 Biotechniques in Plant Breeding
H.S Chawla .2002 Introduction to Plant Biotechnology
2nd edition. Oxford & IBH Publishing C./ Pvt. Ltd New
Delhi India
Murashige T. and Skoog F. (1962). A revised medium
for rapid growth and bioassay withTobacco tissue
culture. Physiologia plantarum 15: 473-497.