Agr232 - c8 Principles and Techniques of Tissue Culture - White

NAJ/FPA/AGR232 CHAPTER 8: PRINCIPLES AND TECHNIQUES OF TISSUE CULTURE
NO SUB-TOPIC
8.1 Principles of tissue culture for micropopagation
8.2 Techniques of in-vitro micropropagation
Terminology Definition
Aseptic Free from microorganism
Callus Undifferentiated, swollen cell mass forming under the
influence of elevated plant hormone levels
Etiolation Yellow and stretched plant; parts elongate until light is
intercepted
Explant Part of an organism used in vitro culture
IAA Indoleacctic acid; a plant hormone to increase cell
elongation
in vitro “in glass”; as in tissue culture methods
Proliferation Rapid production of a cell; rapid increase in number
Organogenesis The formation of roots, shoots or flower buds from the cells
in culture in manner similar to adventitious root or shoot
formation in cuttings
Terminology Definition
Morphogenesis Change in shape
Primordia The earliest detachable stage of an organ such as leaf,
root or root branch
Root hairs Epidermal cell extensions of young root that increase
absorptive surface area
Totipotency Plant cell have the ability to produce whole plant from
single cell.
The ability of a single cell to divide and produce all of
dedifferentiated cells in an organism.
Wound Formation of callus in wounded area
response
De novo Anew; starting from beginning
Direct Formation of organs directly on the surface of cultured
organogenesis intact explants. The process does not involve callus
formation.
Indirect Callus is first produced from explant. Organ can then be
organogenesis produced from callus tissue or from a cell suspension
produce from that callus.
Plant organ formation on callus tissues derived from
explant.
1. In a relatively short time and space a large

number of plantlets can be produced starting
from the single explant.
2. Taking an explant does not usually destroy the
mother plant, so rare and endangered plants
can be cloned safely.
3. It is easy to select desirable traits directly from
the culture setup (in vitro) thereby decreasing
the amount of space required, for field trials.
4. Once established, a plant tissue culture line
can give a continuous supply of young plants
throughout the year.
5. The time required is much shortened, no need to

wait for the whole life cycle of seed development.
For species that have long generation time, low
level of seed production, or seeds that readily do
not germinate, rapid propagation is possible.
6. In vitro growing plants usually free, from the bacterial
and fungal diseases. Virus eradication and
maintenance of plants in virus free state. This
facilitates movement of plant across international
boundaries.
7. Plant tissue banks can be frozen and then
regenerated through tissue culture. It preserves the
pollen and cell collections from which plants may
be propagated.
 Plant cells or tissue culture can be

defined as the growth of the plant cells
in vitro (glass).
 Scope of tissue culture
i. Micropropagation
ii. Crop improvement
iii. Plant pathology
iv. Production of transgenic plants
v. Conservation
vi. Secondary metabolite
 Also known as tissue culture.

 Is a practice used to propagate plants
under sterile conditions or in a controlled
environment.
 Often to produce clones of a plant.
 Tissues or cells, either as suspensions or as
solids is maintained under conditions
conducive for their growth and
multiplication.
 In vitro production of plant is generally

describe as micropropagation.
 Micropropagation can be defined as
asexual multiplication of plants in a small
area of glass vessel under controlled
physio-chemical conditions.
› Micro = small area of glass vessel.
› Propagation = to increase the numbers of
propagules.
1. Structure and utilities

2. Washing room
3. Media room
4. Glassware/plasticware
5. Transfer room
6. Growth room
7. Cold storage
8. Greenhouse
 Explants are used as source material to

establish cells and tissues in vitro.
 All operations involving the handling of
explants and their culture are carried out
in an aseptic environment under defined
conditions.
 Disinfection of explants before culture is
essential to remove surface
contamination with minimal damage to
plant cells.
Apical/terminal bud
Explant
Leaf
• Transferred tissues from
plants to nutrient medium.
• Explant may be taken
from any part of the plant
Axillary bud
like root, stem, leaf or
meristemic tissues like
cambium, anthers,
Stem stamens and etc.
• Cell, tissue or organ of a
Root
plant that is used to start in
vitro cultures.
 Culture media contain microelements,

macroelements, vitamins, other organic
components, plant growth regulator and
sucrose.
 The composition of the culture media
depends upon the plant species and the
explants.
 Available ready-made powdered
medium or stock solutions can be used
for the preparation of culture media.
Stage 0
Management of
donor plant(s)
Stage 4 Stage 1
Hardening, acclimatization Establishment of aseptic
and transfer of plants in soil cultures
Stage 2
Stage 3
Multiplication of shoots
Induction roots
and/or elongation
 To obtain suitable and responsive

explants (stem/bud/leaf/root).
 Plants are maintained in clean
environment of greenhouse or controlled
room.
 Introduction of the surface disinfected explants into

culture and followed with initiation of shoot growth.
 Type of explant:
› Shoot formation may be initiated from apical and axillary
buds
› Adventitious meristems originated from shoots, leaves,
bulb scales, flower stems or cotyledon (direct
organogenesis)
› Callus that develop at the cut surface of explants (indirect
organogenesis)
 The objectives of this stage are:
› To place selected explants into culture
› Avoiding contamination
› Providing an environment that promotes shoot production
 Shoot proliferation and multiple shoot

production
 Each explants has expended into a
cluster of small shoots
 Multiple shoots are separated and
transplanted to new culture medium
 Shoots are subculture every 2-8 weeks
 Shoot elongation and rooting

 Rooting stages prepared the regenerated
plants for transplanting from in vitro to ex
vitro conditions in controlled environment
rooms, in the glasshouse and later to their
ultimate location
 Not only on rooting of shoots but also
conditioning of the plants to increase their
potential for acclimatization and survival
during transplanting
 Transfer of regenerated plants to soil

under natural environmental conditions
 Transplantation of in vitro derived plants
to soil is often characterized by lower
survival rates
 Before transfer to of soil-rooted plants to
their final environment, they must be
acclimatized in a controlled environment
room or in the glasshouse
 It can be achieved by any one of the

following pathways:
i. Culture of apical and axillary buds
ii. Meristem and single or multiple-node
culture (shoot culture)
iii. Adventitious shoot formation
iv. Somatic embryogenesis
 Every method has its own advantage
and disadvantage
 Most frequently used micropropagation

method for commercial mass production
of plants
 Shoot apical or axillary buds contain
several developing leaf primordia
 Explants are 3-4 mm in diameter and 2
cm in length
 Development in vitro is regulated to
support the growth of shoots without
adventitious regeneration
Axillary shoot proliferation

Growth of axillary buds stimulated by cytokinin treatment; shoots
arise mostly from pre-existing meristems.
The in vitro micropropagation of plants by the axillary bud method.

 Meristems are groups of undifferentiated

cells that are established during plant
embryogenesis.
 Meristems continuously produce new
cells which undergo differentiation into
tissues and the initiation of new organs,
providing the basic structure of the plant
body.
The technique of shoot tip or meristem culture

 One of the plant regeneration pathways in

vitro.
 Adventitious meristems develop de novo
and in vitro they may arise directly on
stems, roots or leaf explants, often after
wounding or under influence of exogenous
growth regulators.
 Adventitious buds or shoots usually develop
near existing vascular tissues.
 Adventitious organs sometimes also
originate in callus that forms at the cut
surface of explants.
 Somatic embryogenesis is an artificial process in

which a plant or embryo is derived from a
single somatic cell or group of somatic cells.
 Somatic embryos are formed from plant cells that
are not normally involved in the development of
embryos, i.e. ordinary plant tissue. No endosperm
or seed coat is formed around a somatic embryo.
 This process occurs naturally in some plant species
and can be also induced in vitro in other species.
 Somatic embryogenesis may occur directly from
cells or organized tissues in explants or indirectly
through an intermediate callus stage.
Life Cycle of Plant Tissue Culture

www.jains.com/Tissue/tissueculture1.htm
1. Rapid multiplication of genetically uniform plants (clones) that

possess desirable traits.
› A single explant can be multiplied into several thousand plants
in a very short time. Once established, actively dividing cultures
are a continuous source of microcuttings which can result in
plant production under greenhouse conditions without
seasonal interruption.
2. The production of multiples of plants in the absence of seeds or

necessary pollinators to produce seeds.
3. The regeneration of whole plants from plant cells that have been
genetically modified.
 Using methods of micropropagation, the nurseryman can
rapidly introduce selected superior clones of ornamental plants
in sufficient quantities to have an impact on the landscape
plant market.
4. The production of plants in sterile containers that allows

them to be moved with greatly reduced chances of
transmitting diseases, pests and pathogens.
5. The production of plants from seeds that otherwise

have very low chances of germinating and growing..
› E.g. orchids and nepenthes.
6. To clean particular plant of viral and other infections

and to quickly multiply these plants as 'cleaned stock'
for horticulture and agriculture.
› Applications of micropropagation
1. The production of many plants that are clones of each

other.
2. Used to produce disease-free plants.
3. Produces rooted plantlets ready for growth, saving
time for the grower when seeds or cuttings are slow to
establish or grow.
4. It can have an extraordinarily high frequency rate,
producing thousands of propagules while
conventional techniques might only produce a
fraction of this a number.
5. It is the only viable method of regenerating genetically
modified cells or cells after protoplast fusion.
6. 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 can't be
stored.
7. Often produces more robust plants, leading to
accelerated growth compared to similar plants
produced by conventional methods - like seeds or
cuttings.
8. Some plants with very small seeds, including most
orchids, are most reliably grown from seed in sterile
culture.
9. A greater number of plants can be produced per
square meter and the propagules can be stored
longer and in a smaller area.
1. It is very expensive and can have a labour cost of more

than 70%.
2. 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.
3. An infected plant sample can produce infected
progeny. This is uncommon if the stock plants are
carefully screened and vetted to prevent culturing
plants infected with virus or fungus.
4. 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.

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NAJ/FPA/AGR232 CHAPTER 8: PRINCIPLES AND TECHNIQUES OF TISSUE CULTURE

NAJ/FPA/AGR232 CHAPTER 8: PRINCIPLES AND TECHNIQUES OF TISSUE CULTURE

1. In a relatively short time and space a large

5. The time required is much shortened, no need to

 Plant cells or tissue culture can be

 Also known as tissue culture.

 In vitro production of plant is generally

1. Structure and utilities

 Explants are used as source material to

 Culture media contain microelements,

 To obtain suitable and responsive

 Introduction of the surface disinfected explants into

 Shoot proliferation and multiple shoot

 Shoot elongation and rooting

 Transfer of regenerated plants to soil

 It can be achieved by any one of the

 Most frequently used micropropagation

Axillary shoot proliferation

The in vitro micropropagation of plants by the axillary bud method.

 Meristems are groups of undifferentiated

The technique of shoot tip or meristem culture

 One of the plant regeneration pathways in

 Somatic embryogenesis is an artificial process in

Life Cycle of Plant Tissue Culture

1. Rapid multiplication of genetically uniform plants (clones) that

2. The production of multiples of plants in the absence of seeds or

4. The production of plants in sterile containers that allows

5. The production of plants from seeds that otherwise

6. To clean particular plant of viral and other infections

1. The production of many plants that are clones of each

6. It is useful in multiplying plants which produce seeds in

1. It is very expensive and can have a labour cost of more

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