Types, Techniques and Process
What is Tissue Culture?
In biological research, tissue culture refers to
a method in which fragments of a tissue (plant or animal tissue) are introduced
into a new, artificial environment, where they continue to function or grow.
While fragments of a tissue are often used, it is important to note that entire
organs are also used for tissue culture purposes. Here, such growth media as
broth and agar are used to facilitate the process.
* While the term tissue culture may be used for
both plant and animal tissues, plant tissue culture is the more specific term
used for the culture of plant tissues in tissue culture.
Types of Tissue Culture
Seed culture is the type of tissue culture that
is primarily used for plants such as orchids. For this method, explants (tissue
from the plant) are obtained from an in-vitro derived plant and introduced in
to an artificial environment, where they get to proliferate. In the event that
a plant material is used directly for this process, then it has to be sterilized
to prevent tissue damage and ensure optimum regeneration.
Embryo culture is the type of tissue culture
that involves the isolation of an embryo from a given organism for in vitro
*Note, the term embryo culture is used to refer
to sexually produced zygotic embryo culture.
Embryo culture may involve the use of a mature
of immature embryo. Whereas mature embryos for culture are essentially obtained
from ripe seeds, immature embryo (embryo rescue) involves the use of immature
embryos from unripe/hybrid seeds that failed to germinate. In doing so, the
embryo is ultimately able to produce a viable plant.
For embryo culture, the ovule, seed or fruit
from which the embryo is to be obtained is sterilized, and therefore the embryo
does not have to be sterilized again. Salt sucrose may be used to provide the
embryo with nutrients. The culture is enriched with organic or inorganic
compounds, inorganic salts as well as growth regulators.
*Callus - This is the term used to refer to
unspecialized, unorganized and a dividing mass of cells. A callus is produced
when explants (cells) are cultured in an appropriate medium - A good example of
this is the tumor tissue that grows out of the wounds of differentiated
In practice, callus culture involves the growth
of a callus (composed of differentiated and non- differentiated cells), which
is the followed by a procedure that induces organ differentiation.
For this type of tissue culture, the culture is
often sustained on a gel medium, which is composed of agar and a mixture of
given macro and micronutrients depending on the type of cells. Different types
of basal salt mixtures such as murashige and skoog medium are also used in
addition to vitamins to enhance growth.
Organ culture is a type of tissue culture that
involves isolating an organ for in vitro growth. Here, any organ plant can be
used as an explant for the culture process (Shoot, root, leaf, and flower).
With organ culture, or as is with their various
tissue components, the method is used for preserve their structure or
functions, which allows the organ to still resemble and retain the
characteristics they would have in vivo. Here, new growth (differentiated
structures) continues given that the organ retains its physiological features.
As such, an organ helps provide information on patterns of growth,
differentiation as well as development.
There are number of methods that can be used for
organ culture. These include;
- Plasma clot method - Here, the method involves
the use of a clot that is composed of plasma and chick embryo extract (or any
other extract) in a watch glass. This method is particularly used for the
purposes of studying morphogenesis in embryonic organ rudiments and more
recently for studying the actions of various hormones, vitamins and carcinogens
of adult mammalian tissues.
- Raft method - For this method, the
explant is placed on a raft of lens paper/rayon acetate and floated on a serum
in a watch glass.
- Agar gel method - The medium used for this
method is composed of a salt solution, serum as well as the embryo extract or a
mixture of various amino acids and vitamin with 1 percent agar. The explant has
to be subcultured every 5 to 7 days. The method is largely used for the study
of developmental aspects of normal organs and tumors.
- Grid method - Grid method, as the name
suggests involves the use of perforated stainless steel sheet, on which the
tissue of interest is placed before being placed in a culture chamber
containing fluid medium.
*Protoplast -cells without cell walls. A
protoplast is the term used to refer to cell (fungi, bacteria, plant cells etc)
in which the cell wall has been removed, which is why they are also referred to
as naked cells.
Protoplasts may be cultured in the following
- Hanging-drop cultures
- Micro culture chambers
- Soft agars matrix
Once a protoplast has regenerated a cell wall,
then it goes through the process of cell division to form a callus, which may
then be subcultured for continued growth.
Protoplast culture is an important method that
provides numerous cells (single cells) that can be used for various studies.
- Protoplast culture
regenerated into a whole plant
- Development of hybrids
- Cell cloning
- Genetic transformations
- Membrane studies
In protoplast culture, a number of phases can be
observed. These include;
- Development of a cell wall
- Cell division
- Continuous growth or
regeneration to a whole plant
For plants, some of the special requirements
- Less amounts of iron and
zinc and no ammonium
- Higher concentration of
- High auxin/kinetic ratio
for cell division and high kinetin/auxin ration for regeneration
- Glucose and vitamins
Some of the other types of tissue culture
- Single cell culture
- Suspension culture
- Anther culture
- Pollen culture
Major Steps of Tissue Culture (Plants)
Initiation Phase (Stage 1)
The initiation phase is the first phase of
tissue culture. Here, the tissue of interest is obtained and introduced and sterilized
in order to prevent any microorganism from negatively affecting the process. It
is during this stage that the tissue is initiated in to culture.
Multiplication Phase (Stage 2)
The multiplication phase is the second step of
tissue culture where the in vitro plant material is re- divided and then introduced
in to the medium. Here, the medium is composed of appropriate components for
growth including regulators and nutrients. These are responsible for the
proliferation of the tissue and the production of multiple shoots.
*This step is often repeated several times in
order to obtain the desired number of plants
Root formation (Stage 3)
It is at this phase that roots are formed. Here,
hormones are required in order to induce rooting, and consequently complete
Plant Tissue Culture
Tissue culture is applied in plant research for such
purposes as the growing of new plants, which in some cases undergo genetic
alterations. Here, the plant of interest is taken through the tissue culture
process and grown in a controlled environment.
The Process of Plant Tissue Culture
This process involves the use of small pieces of
a given plant tissue (plant of interest). Once the tissue is obtained, it is
then cultured in the appropriate medium under sterile conditions so as to
prevent various types of microorganisms from affecting the process.
The following is a general procedure for plant
- The appropriate mixture
(such as the MS mixture) is mixed with distilled water and stirred while adding
the appropriate amount of sugar and sugar mixture. Here, sodium hydroxide or
hydrochloric acid is used to adjust the pH - Contents used here will depend on
the plant to be cultured and the number of tissues to be cultured.
- Agar is added to the
mixture, heat and stirred to dissolve
- After cooling, the warm
medium is poured into polycarbonate tubes (to a depth of about 4 cm)
- With lids sitting on the
tubes, the tubes are placed in a pressure cooker and sterilized for 20 minutes
- Cut the plant part in to
small pieces (e.g. cauliflower can be cut to florets of about 1cm across). On
the other hand, such parts as the African violet leaves can be used as a whole.
- Using detergent and water,
wash the plant part for about 20 minutes
- Transfer the plant part in
to sterilizing Clorox solution, shake for a minute and leave to sock for 20
- Using a lid, gently discard
the Clorox and retain the plant part in the container and then cap the
Transferring the plant material to a tissue
* 70 percent alcohol should be used for the
sterilization of the equipment used and containers
- Open the container and pour
sterile water to cover half the container
- Cover with a sterile lid
again and shake the container for 2 to 3 minutes in order to wash the tissue
and remove the bleach
- Pour the water and repeat
this three times
- Using sterilized gloves,
remove the plant part from the container and on to a sterile Petri dish
- Using a sterile blade cut
the plant material to smaller pieces of about 2 to 3 mm across avoiding the parts
that have been damaged by bleach
- Using sterile forceps,
place a section of the plant in to the medium
Cauliflower - partly submerged in medium with
flower bud facing up
Rose with shoots at level with medium surface
African violet leaf laid directly in surface of
*depending on the plant used, it is important to
check and find out how it should be placed in the medium
Replace the lid/cap and close tightly
This procedure will result in the development of
a callus, which then produces shoots after a few weeks. Once the shoots
develop, then the plant section may be placed in the right environment (well
lit, warmth etc) for further growth.
* Plant materials should be sterilized so as to
remove any bacteria or spores that may be present.
For plants, the medium culture acts as a greenhouse
that provides the explant with the idea environment for optimum growth. This
includes being free of microorganisms, nutrients as well as the right balance
of chemicals and hormones. Such media as BAP, TDZ are used while such hormones
as IBA and IAA are used to induce growth. Some of the major reasons tissue
culture is used for plants include;
- To produce large quantities
of a given plant
- To accelerate the
production of new varieties of a plant
- To maintain a virus free
stock of the plant of interest
Technique for Plant In Vitro Culture
Micropropagation - This technique is used
for the purposes of developing high- quality clonal plants (a clone is a group
of identical cells). This has the potential to provide rapid and large scale
propagation of new genotypes.
Somatic cell genetics - Used for haploid production
and somatic hybridization
Transgenic plants - Used for expression of
mammalian genes or plant genes for various species it has proved beneficial for
the engineering of species that are resistant against viruses and insects.
In reality, there are numerous methods used for
tissue culture given that there are different types of tissues that require
specific conditions for the culture process yield desired results. Both plant
and animal tissue can be used for tissue culture purposes for a wide range of
purposes. For instance, animal tissue culture may serve such purposes as
preservation of an organ/tissue, studying the tutors or given tissues or for
On the other hand, plant tissue culture may be used for
cloning purposes, genetic modification of a given plant or simply to accelerate
or increase yield of the plant of interest.
Tissue culture is therefore of
great significance in biological studies due to its wide range of applications.
The processes involved in tissue culture may be complex, requiring a lot of
care to avoid such effects as contamination. Because of the complexities that
may be involved in some of the steps, this may not be an experiment for
Here, learn more about Cell Culture, Cell Division, Cell Differentiation and Cell Staining as well as Gram Stain. And check out information on Cell Theory.
Return From Tissue Culture to MicroscopeMaster Home
Siya S (2016) Top 6 Types of Tissue Culture | Biotechnology.
Carrel, Alexis and Montrose T. Burrows “Cultivation of Tissues in Vitro and its Technique"; Journal of Experimental Medicine.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed
Lorraine Mineo (1990) Plant Tissue Culture Techniques. Chapter 9.