Institute of Advanced Research

Introduction, Cryo &

Organogenic
differentiation, Types of
UNIT - 5 culture
PLANT &
ANIMAL
BIOTECHN
OLOGY
BBT 609ET Ms. Pratiksha
Singh
Department of
B.SC 5 SEMESTER
Biotechnology&
Bioengineering
 Tissue culture
Tissue culture is the method of in vitro culture of Plant & animal cells,
tissue or organ on nutrient medium under aseptic condition usually in
glass container.
Tissue culture

Plant Animal
tissue tissue
culture culture
Definition/Meaning of Plant tissue culture
❖ Plant tissue culture is a collection of techniques used to maintain or
grow plant cells, tissues, or organs under sterile conditions on a
nutrient culture medium(MS) media of known composition.

❖ It makes use of parts of a plant(explant) to generate multiple copies of

the plant in a very short duration.

❖ The technique exploits the property of totipotency of plant cell which

means that any cell from any part of the plant can be used to generate
a whole new plant.
What is history?
1838 – Schwann & Schleidn put forward the theory that cell are
totipotent, and in principle regenerating into a complete plant.
1902 – Haberlandt conducted the first but unsuccessful attempt of
Tissue culture using monocotyledons.
Robbins (1922) – In vitro culture of root tips.
Laibach(1925) – Use of Embryo culture technique
Snow,Gautheret(1934-1939) - Importance of auxin as Growth regulator.
Gautheret,Nobecourt,White(1939) - Sucessful establishment of
continuously growing culture
Cont.
Skoog &Tsui(1951),Miller et al(1955) – In vitro cell differentiation,
discovery of kinetin.
Skoog &Miller (1957)- Discovery of principle of hormonal control of the
organ formation in tissue culture.
Reinert & Steward (1958-59) - First report on embryogenesis.
Murashige& Skoog (1962) –Development of MS media.
Guha & Maheswari (1964)- Production of first haploid plant by anther
culture.
Takabe et al(1971) – Regeneration of first plant from protoplast.
Fundamental Principles
1. Totipotency: It is the ability of plant cell to regenerate into a whole
Plant.

2. Plasticity : It is ability of plants to alter their metabolism, growth

and development to best suit their environment.
 What is Cryopreservation
❖ The word cryo comes from the Greek word karyos meaning
frost. It means preservation in a frozen state .
❖It is a process of cooling & storing cells tissues or organ at
very low temperature.
❖ In this technique, tissue culture can be preserved for a very
long time.
Principle –The cryopreservation principle is to bring the plant
cell & tissue culture to a 0 metabolism or non dividing state by
reducing the temperature.
Cryo preservation means the storage of germplasm at very low
temp.
Solid carbon dioxide (at-79⁰c)
Low temperature deep freezer (at -80⁰c)
In vapour phase nitrogen (at -150⁰c)
In liquid nitrogen (at-196⁰c)
Most commonly used cryopreservation is liquid nitrogen at
(-196⁰c). The cell stays in inactive & thus the cryo can be
conserved for long time.
Types of tissue culture
Every parts of plants can be grow into new Plant. Parts like
root,shoot,anther,buds etc. can be used for the growth of new plants.
On the basis of use of different parts of plant tissue culture can be
classified into following types.
• Seed culture
• Embryo Culture
• Callus Culture
• Organ Culture
• Protoplast Culture
• Cell culture
• Meristem culture
• Shoot tip culture
 Seed culture :
❖ Seed culture is a technique in which a complete plant or seedling can
be generated in vitro by taking the seed as an explant.
❖ This is a type of tissue culture that is extensively employed in the
cultivation of orchids as seed of these plant do not germinate well in in
vivo condition.
❖ Explants (seed) are extracted from an in-vitro grown plant and put in an
artificial environment where they may develop for this process.
❖ It has been reported that orchid lives in symbiotic relationship with
fungi from the moment of germination.
❖ The explant should be sterilized to prevent it from tissue damage.
Different stages of seed propagation
Embryo culture :
Embryo culture deals with the sterile isolation and invitro growth of
mature and immature embryo with an ultimate objective of obtaining a
viable plant.
Types of embryo culture
Mature embryo culture:
Mature embryo are isolated from ripe seeds and cultured invitro. Mature
embryo cultures are carried out in following condition.
• When the embryo remain dormant for long periods.
• Low survival of embryo in vivo.
• To avoid inhibition in the seed for germination.
• For converting sterile seeds to viable seedling.
• Immature embryo culture :
Involve the culture of immature embryos to rescue them from unripe or
hybrid seeds which fail to germinate.
Culture technique for embryo rescue:
 Nutritional requirements of embryo culture:
• There are two phases in embryo development and nutritional
requirement varies accordingly.
1. Heterotrophic phase
This is an early phase and the embryo is mostly dependent on the
endosperm and maternal tissue for nutrient supply.
2. Autotrophic phase:
This phase is characterized by the metabolic capability of embryo to
synthesize substances required for its growth which slowly makes
independent.
❖ In 1922 knudson was able to grow the orchid embryo into plantlets by
culturing them on agar media that contain sugar.

Callus Culture: It is the tissue culture in which large no. of plantlets

can be obtained from callus.
❖ A callus is defined as unidentified and unorganized, dividing mass of
cells. It is also known as pre-mature unorganized mass of cells.
❖ When the explants are cultured in a proper medium, the callus is
obtained. The growth of callus is followed by organ differentiation.
The culture is grown on a gel-like medium composed of agar and
specific nutrients required for the growth of the cells.
❖ It need to be sub cultured every 3 to 5 weeks in view of cell growth,
nutrient depletion and media drying
Organ Culture: In this, any organ of the plant such as shoot, leaf,
can be used as an explant.
❖ A number of methods can be used for the organ culture, such as
plasma clot method, raft method, grid method, and agar gel
method. This method is used to preserve the structure and
functions of an organism.
Cell culture :
❖ The culture of isolated individual cell.
❖ Carried out in dispersion media.
❖ Tissue and cell cultured in a liquid medium produce a suspension of
single cell clumps of few to many cells.
Liquid cultures must be constantly agitated, generally by a gyratory
shaker at 100-250rpm(revolution per minute) to facilitate aeration and
dissociation of cell clumps into smaller pieces.
Protoplast Culture: It is a cell without a cell wall because cell wall
has been removed by either mechanical or enzymatic processes.It is the
technique of plant tissue culture in which protoplasm is use to obtained
new plant.
❖ Protoplast can be obtain from various part of plant such as leaves,
stem, callus and from pollen grain too, but leaf mesophyll is the most
preferred tissue for this purpose.
❖ In protoplast culture, a number of phases can be observed:
development of cell wall, cell division, regeneration of a whole plant.
Osmoticum(shown below the arrow) is added to stabilize the protoplast and
prevent them from bursting.
Applications of protoplast culture

❖ Protoplast can also be induced to fuse cell with one another to produce
somatic or Para sexual hybrid.
❖ The induction of disease resistance in a crop by the incorporation of
selective genome into protoplast.
❖ Transplantation of foreign chloroplast into crop plant with inefficient
photosynthetic system.
❖ Serve for the transformation and cell modification studies.
Meristem culture:
Meristem culture involves the isolation and culture of apical meristem
tissue from a plant, which contains undifferentiated cells that can
differentiate into various plant tissues.
❖ The technique was first reported by scientists named Morel and Martin
in the year 1952.
❖ It retain the power of division long after embryogenesis is over.
❖ It is a type of tissue culture technique used in labs to eliminate viruses
and other parasites while growing plants in vitro.
❖ They either use the growing tips of plant shoots(0.1 mm to 0.5 mm) or
roots—sites containing meristematic cells—for the culturing process.
Reason for the escape of meristem from virus invasion are
❖ Virus readily move in plant body by vascular system which is absent
in meristem.
❖ High metabolic rate of division of meristem cell doesn't allow virus
multiplication.
❖ Virus inactivating system if any has high activity in meristem
compared to other.
❖ High endogenous auxin concentration may inhibit virus multiplication.
Steps involved in PTC
CHOICE OF EXPLANT
❖ The tissue obtained from a plant to be cultured is called an Explant.
❖ In a totipotent, explant can be collected from any part of the plant.
❖ In many plants, explants of various organs vary in their rate of
growth & regeneration.
❖ The choice of explant material also determines if the plantlets
developed via tissue culture are haploid/diploid.
Methods of Plant Tissue culture
❖ Tissue culture can be done only in two medium i.e Liquid medium &
semi solid agar medium.
❖ In case of liquid medium plant tissue should be fully immersed or
partially immersed but in the case of semi solid medium the plant
tissue are just placed on the surface of medium.
Micro propagation via axillary bud
proliferation
It is a technique used in plant tissue culture to rapidly produce new
plants from axillary buds.
The axillary buds are treated with hormones(under the high concⁿ of
Cytokinin) to break dormancy and produce shoot branches. The shoots
are then separated and rooted to produce plants.
Advantages:
• Rapid Multiplication: Produces large numbers of uniform plants in a
short time.
• Disease-Free Plants: Helps in obtaining pathogen-free plants.
• Genetic Uniformity: Ensures consistency in traits.
Organogenesis
The process of development of plant organs such as shoot, flower, and
root system from either an ex-plant or from the callus of culture is
known as organogenesis in plants.
The process of organogenesis can take place in three ways:
1. From an explant
2. From the callus culture
3. From the axillary buds
Fig :Plant regeneration via organogenesis.
 Types of Organogenesis in plants
There are two types of organogenesis in plants that are:
• Direct organogenesis
• Indirect organogenesis
1. Direct organogenesis: When buds and shoots are directly
developed from tissue and there is no need for the callus stage then
this process is known as direct organogenesis.
❖ Direct organogenesis results in the development of planting material
with no genetic variation therefore cloning.
❖ Uniformity in the planting material is ensured.
❖ It provides plants with better multiplication rates and cloning
propagation where the genetic variation is zero.
Indirect organogenesis :In this process of indirect organogenesis, a
plant’s organ is developed from the callus of an explant. The process of
indirect organogenesis is more useful in the development of a transgenic
plant.
❖ The process is profoundly used in transgenic plant production.
Fig: An illustration of the types of organogenesis.
Advantages Of Organogenesis
❖ Organogenesis is an effective technique to induce exhilaration shoots
in plants in vitro.
❖ The organs developed through Meristem has lower chances of
undergoing mutations.
❖ Direct organogenesis is an effective and efficient approach to induce
clonal propagation with high fidelity.
Disadvantages Of Organogenesis
❖ One of the major concerns of in vitro organogenesis is that it can
induce soma clonal variation in plants.
❖ Organogenesis is a time-consuming process.
❖ To work with organogenesis you need to have some hands-on
experience with the technique.
Embryogenesis
Embryogenesis refers to the process of development of plant embryos
being either asexual or sexual reproductive process.
❖ Embryogenesis may occur naturally in plant as a result of sexual
fertilization, and those embryo are called zygotic embryos and develop
into seeds, which can germinate and give rise to seedling.
❖ Plant cell can also be induced to form embryos in plant tissue culture;
these embryo are called somatic embryo.
 Types of embryogenesis:
Two type of embryogenesis
1. Zygotic embryogenesis
2. Somatic embryogenesis
Zygotic embryogenesis
The zygotic embryo is formed following double fertilization of the
ovule forming the plant embryo and endosperm which together go into
seed. This process is known as zygotic embryogenesis.
Somatic Embryogenesis
Somatic embryogenesis is a process in which single cell or small group
of cells follow a developmental pathway that leads to reproducible
regeneration of non zygotic embryo. Which are capable of producing
whole plant.
In this process somatic cells or tissues (haploid or diploids) develops
into differentiated embryos.
The somatic embryogenesis procedure is a three-step procedure, which
causes the induction of embryogenesis, development of the embryo and
its maturation.
 Two routes of somatic embryogenesis
a) Direct embryogenesis
b) Indirect embryogenesis
1. Direct embryogenesis: The embryos initiate directly from the
explant without callus formation and here some cells which are
called pre embryonic determined cells(PEDC) initiates embryonic
development only those cells need to be released.
2. Indirect embryogenesis: Here the embryos are developed through
cell proliferation i.e callus formation
Different stages of somatic and zygotic embryogenesis
 Applications of somatic embryogenesis
1. Large scale propagation compared to zygotic embryos.
2. Useful For mutagenic studies and mutant production.
3. Useful for gene manipulation technique.
4. Useful for pathogen free plant production.
 Advantages of Somatic Embryogenesis
1. High propagation rate
2. Soma clonal variation
3. Germplasm conservation
4. Artificial seed production
5. Labour savings
Disadvantages :
1. Inability to generate large no. of normal free living plantlets.
2. Plantlets are weaker
3. Incomplete embryo production
Comparison between zygotic and somatic
embryo
Zygotic embryo Somatic embryo
• Fertilized egg or zygote. • Sprophytic cells
• Contain seed coat. • Did not contain seed coat
• Produce seed. • Only from embryo
• Plantlets are healthy. • Plantlets are weaker
• Not like to mother plant. • Like to mother plant.
• Propagation is low. • Propagation is high.
Differentiation between organogenesis and
somatic embryogenesis
Organogenesis Somatic Embryogenesis
• Development and formation of organs Formation of an embryo from a single or
of an entity from embryonic cells. cluster of somatic cells. It is an artificial
process.
• More or less natural.
It occurs artificially.
• The process can be seen in animals
and plants. The process can be seen in plants.
• An entire plantlet with shoot and root A somatic embryo is formed.
is formed. The somatic embryos are not in
• Roots and shoots have a strong vascular association with their
association with them maternal callus
Advantages Of Micro propagation
1. Micro propagation helps in the propagation of a large number of
plants in a short span of time.

2. The plants produced are identical to the mother plant.

3. It leads to the production of healthier plantlets, which exhibit better

disease-resisting powers.

4. The germplasm stocks can be maintained for several years using this
technique.
5. It is a cost-effective process.

6. New varieties of species can be propagated.

7. A requirement of less space and human resources.

8. This method is independent of season and can be carried out anytime.

9. Millions of plantlets can be maintained in the cultural vials.

Disadvantages of Micro propagation
1. Micro propagation requires trained manpower, sophisticated
facilities, and expensive materials which make it a pricey technique.
2. Contamination is the major problem of micro propagation. The
cultures grown in labs are very sensitive to any microorganisms.
The source can be water, media, chemical components you use for
your cultures, air, or even your hands.
3. Some plants are difficult to maintain by micro propagation
because they start releasing growth inhibitory substances (like
phenol) in the medium. These compounds turn the medium into dark
color (it’s called brewing of the medium) and inhibit the growth of
the tissues.
4. Plants are not autotrophic

5. Acclimatization is difficult process to get high percentage

of survival of plants.

6. All plants produced via micro propagation are genetically

identical clones, leading to a lack of overall disease resilience,
as all progeny plants may be vulnerable to the same infections.

7. Some plants are very difficult to disinfect of fungal

organisms.