Plant Tissue Culture Types and Techniques

2. Tissue Culture
 The process of growing cells artificially in the
laboratory
 Involves both plant and animal cells
 Produces clones, in which all product cells have
the same genotype
(unless affected by mutation during culture)

3. Requirements
Tissue culture has several critical requirements:
 Appropriate tissue
 A suitable growth medium
 Aseptic (sterile) conditions
 Growth regulators
 Frequent subculturing

4. What is plant tissue culture?
Plant tissue culture is a technique of growing plant
cells, tissues, organs, seeds or other plant parts in a
sterile environment on a nutrient medium

5. How?
Adult plant cells are totipotent, meaning they have the ability to give rise to a fully
differentiated plant. Because of this, it is possible to collect cells from a mature plant
and use those cells to produce clones of that plant.

6. Basic Tissue Culture Procedures

7. Micropropagation
 The art and science of plant multiplication in vitro
 Usually derived from meristems (or vegetative buds)
without a callus stage
 Tends to reduce or eliminate somaclonal variation,
resulting in true clones
 Can be derived from other explant or callus (but
these are often problematic)

8. 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

9. An unspecialized and unorganized,
growing and dividing mass of cells,
produced when explants are cultured on
the appropriate solid medium (with both
an auxin and a cytokinin and correct
conditions)
During callus formation there is some
degree of dedifferentiation both in
morphology and metabolism, resulting
in the lose the ability to photosynthesis
Callus Culture

10. Callus cultures may be compact or friable
Compact callus shows densely aggregated cells
Friable callus shows loosely associated cells and the
callus becomes soft and breaks apart easily
Habituation: it is the lose of the requirement for auxin
and/or cytokinin by the culture during long-term culture
Callus

11. Cell-suspension cultures
 When friable callus is placed into the appropriate liquid medium and
agitated, single cells and/or small clumps of cells are released into the
medium and continue to grow and divide, producing a cell-suspension
culture
 The inoculum used to initiate cell suspension culture should neither be
too small to affect cells numbers nor too large too allow the build up of
toxic products or stressed cells to lethal levels
 Cell suspension culture techniques are very important for plant
biotransformation and plant genetic engineering

12. Embryo Culture
 Embryos are allowed to grow for some time in an
artificial medium
 Embryo culture developed from the need to rescue
embryos from wide crosses where fertilization occurred,
but embryo development did not occur
 Artificial embryo culture media basically contain
glucose, pyruvate, and energy-providing components,
but the addition of amino acids, nucleotides, vitamins,
and cholesterol improve the performance of embryonic
growth and development

14. Somatic Embryogenesis
 When embryos regenerate from somatic cells or tissues
(which are haploid,diploid etc.) it is termed as Somatic
Embryogenesis.
 Somatic embryogenesis is a process by which the somatic
cells or tissues develops into differentiated embryos.

15. Somatic Embryogenesis
Types:
1. Direct somatic embryogenesis:
 The embryo is formed directly from a cell or small group of cells
such styles or pollen without the production of an intervening
callus.
 Direct somatic embryogenesis is generally rare
2. Indirect somatic embryogenesis
 Callus is first produced from the explant and then embryos are
produced from the callus tissue or from a cell suspension cultures.

17. Organogenesis
Definition: The production of organs, either directly from an
explant or from a callus culture.
 Organogenesis depend on adventitious organs arising either
from a callus culture or directly from an explant or on the
formation of axillary bud to regenerate whole plants from
some types of tissue culture.

19. Shoot and Root Culture
 Shoot culture is promoted by
cytokinins and auxins like NAA
(naphthalene acetic acid)
 The shoot and root cultures are
generally controlled by auxin-
cytokinins balance
 Usually an excess of auxin promotes
root culture, whereas that of
cytokinin promotes shoot culture

20. Meristem Culture
 Cultivation of axillary or apical meristems is called
"Meristem culture".
 Meristem culture involves development of already
existing shoot meristem.
 Technique is used for quick vegetative propagation
of a large number of plant species in a short
period.
 Meristem culture produces virus free callus and
eventually plantlet.

22. Anther culture
 Anther culture is a technique by which the developing
anthers at a precise and critical stage are excised
aseptically from unopened flower bud
 Cultured on a nutrient medium where the
microspores within the cultured anther develop into
callus tissue or embryoids
 Give rise to haploid plantlets either though
organogenesis or embryogenesis.

24. Pollen Culture
 Pollen or microspore culture is an in vitro technique
by which the pollen grains preferably at the
uninucleated stage ,are squeezed out aseptically from
the intact anther
 then cultured on nutrient medium
 develop into haploid embryoids or callus tissue that
give rise to haploid plantlets by embryogenesis or
organogenesis

25. Protoplast
The living material of a plant or
bacterial cell, including the
protoplasm and plasma membrane
after the cell wall has been removed.

26. Ovule culture
 Utilized for raising hybrids which
normally fail to develop due to
abortion of the embryo in early stages
 Ovules can be easily excised from the
ovary and cultured on the basal
medium
 Loss of hybrid embryo due to
premature abcission of fruits may be
prevented by ovary culture
 Addition of fruit/vegetable juice
increase the initial growth

27. Ovary culture
 Sucessfully employed to interspecific
hybrids between sexually
incompatible species
 Ovaries are excised from the flower
and cultured at the zygote or two
celled proembryo stage for obtaining
for obtaining normal development
on culture medium
 Coconut milk added as supplement
to medium promote formation of
fruits
 In anethum, addition of kinetin in
the medium caused polyembryonony
which gave rise to multiple shoots

28. Advantages
 Mass production of various plant cultivars
 6 million plants per year from one explant.
 Much higher production rate than other asexual
propagation methods.
 Especially beneficial for:
 Plants in high demand or valuable plants.
 Plants that are slow or difficult to propagate.
 Endangered species.

29. Advantages
 Production of pathogen-free plants
 Maintaining disease-free plants by micropropagation.
 Germplasm preservation
 Germplasm: the DNA of a species
 In the past: seeds
 limited shelf-life
 don’t preserve uniform characteristic (variability)

30. Advantages
 Continuous year round production
 Unaffected by climate
 Propagated in controlled lab conditions
 The ability to change specific conditions to meet the
needs of a particular plant species.
 Mainly, nutrient, light and temperature requirements.

31. Advantages
 The original plant is not
destroyed in the process - a
factor of considerable
importance to the owner of a
rare or unusual plant.

32. Disadvantages
 Specialized equipment required
 Laminar flow cabinets
 Autoclave
 Water purification systems
 Glassware etc…
 High labor cost is the most limiting factor
 Skilled labor required

33. Disadvantages
 Contamination risks
 Maintenance of aseptic (sterile) environment difficult.
 Rapid spread of contaminants = widespread loss.
 Risk of mutation arising
 Artificial environment induces mutations.
 Responses to tissue culture conditions varies
 Trial and error to determine optimum media or conditions。

34. Factors affecting tissue culture
 The areas in which tissue culture techniques can be used
are very wide.
 The choice of technique is dependent on what one wants
to achieve. It may be mass production, breeding of
new varieties, or producing virus-free plants.
 To be able to successfully propagate plants in vitro,
understanding how and why these factors affect plant
growth in an in vitro environment is crucial.

35. Factors affecting tissue culture
 The in vitro growth and development of a plant is
determined by a number of factors:
 The genetic make-up of the plant
 Source of explants
 Nutrients
 Environmental factors: light, temperature, pH, O2 and
CO2 concentrations.

36. Factors affecting tissue culture
 The genetic make-up of the plant.
 The genetic make-up is a decisive factor at every stage in
the plant.
 It determines, for example, if a plant is a monocotyledon
or dicotyledon, or which temperature is optimal for
growth.
 The type of in vitro environment that must be created in
the lab to ensure that growth and development of the
explant takes place, is totally dependent on the genotype
of the plant.

37. Factors affecting tissue culture
 Source of explant
 Young explant vs. old explant
 Usually the younger, less differentiated explant, the
better for tissue culture
 Type of explant – leaf, stem, root, meristem, etc.

38. Factors Affecting Tissue Culture
 Growth medium (Artificial)
 Nutrients
 Plant hormone
 Vitamins
 Environmental factors (Controlled)
 Light intensity
 Photoperiod
 Temperature
 Sterility