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 of known composition. Plant tissue culture is widely used to produce clones of a plant in a method known asmicropropagation. Different
techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:

The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits.

To quickly produce mature plants.

The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.

The regeneration of whole plants from plant cells that have been genetically modified.

The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests,
and pathogens.

The production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and Nepenthes.

To clear particular plants of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture.

Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (totipotency). Single cells, plant cells without cell
walls (protoplasts), pieces of leaves, stems or roots can often be used to generate a new plant on culture media given the required nutrients and plant
hormones.

Applications[edit]
Plant tissue culture is used widely in the plant sciences, forestry, and in horticulture. Applications include:

The commercial production of plants used as potting, landscape, and florist subjects, which uses meristem and shoot culture to produce
large numbers of identical individuals.

To conserve rare or endangered plant species.[6]

A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicide resistance/tolerance.

Large-scale growth of plant cells in liquid culture in bioreactors for production of valuable compounds, like plant-derived secondary
metabolites and recombinant proteins used as biopharmaceuticals.[7]

To cross distantly related species by protoplast fusion and regeneration of the novel hybrid.

To rapidly study the molecular basis for physiological, biochemical, and reproductive mechanisms in plants, for example in vitro selection for
stress tolerant plants.[8]

To cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die (Embryo
Rescue).

For chromosome doubling and induction of polyploidy,[9] for example doubled haploids, tetraploids, and other forms of polyploids. This is
usually achieved by application ofantimitotic agents such as colchicine or oryzalin.

As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants.

Certain techniques such as meristem tip culture can be used to produce clean plant material from virused stock, such as potatoes and
many species of soft fruit.

Production of identical sterile hybrid species can be obtained.

TISSUE CULTURe
Tissue culture is the growth of tissues or cells separate from the organism. This is typically
facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. Tissue
culture commonly refers to the culture of animal cells and tissues, with the more specific term plant
tissue culture being used for plants. The term "tissue culture" was coined by American
pathologist Montrose Thomas Burrows, M.D.
[1]

In modern usage, tissue culture generally refers to the growth of cells from a tissue from a multicellular organism in vitro. These cells may be cells
isolated from a donor organism, "primary cells", or an immortalised cell line. The cells are bathed in a culture medium, which contains essential
nutrients and energy sources necessary for the cells' survival. [8] The term tissue culture is often used interchangeably with cell culture

The literal meaning of tissue culture refers to the culturing of tissue pieces, i.e. explant culture.
Tissue culture is an important tool for the study of the biology of cells from multicellular organisms. It provides an in vitro model of the tissue in a well
defined environment which can be easily manipulated and analysed.
Plant tissue culture in particular is concerned with the growing of entire plants from small pieces of plant tissue, cultured in medium [9]

What is Tissue Culture?

How does AgriForest go about turning one plant with the desired characteristics into tens of thousands of identical plants in as fast as one
year? Through its patented tissue culture protocols that it has developed over its twenty years in business.
Tissue culture is a process that involves exposing plant tissue to a specific regimen of nutrients, hormones, and light under sterile, in vitro
conditions to produce many new plants, each a clone of the original mother plant, over a very short period of time. AgriForest's tissue culture
plants are characterised by disease free growth, a more fibrous, healthier root system, a bushier branching habit, and a higher survival rate.
There are three main steps to the tissue culture process. Following these three stages, the plants are then moved from the laboratory to the
greenhouses for acclimatisation and further development.

STAGE I is the initiation phase. It concerns the establishment of plant tissue in vitro by sterilizing the material
and initiating it into culture.

STAGE II is the multiplication phase. At this stage, the in vitro plant material is re-divided and placed in a
medium with plant growth regulators that induce the proliferation of multiple shoots. This process is repeated
many times until the number of plants desired is reached.

STAGE III is the root formation phase. It involves the introduction of hormones to induce rooting and the
formation of complete plantlets.

When Can Tissue Culture Help With Your Growing Needs?

Plant tissue culture technology has proven itself to be an effective and viable option for growers to seriously consider in a variety of different situations.
When large-scale propagation of new or superior plant varieties is required for early introduction to market
Following a decision to release a new variety into the market, the key to success for growers is rapid scale up and production on a commercial level. Tissue culture
is often the fastest and most economical means to achieve this goal. We suggest that growers continually compare the cost of conventional propagation methods
to micropropagation, especially when conducted by an established tissue culture company with a proven track record of mass propagation such as AgriForest.
Give us a call or email us for detailed pricing information. We are certain that you will be pleased with our competitive rates!
When mass multiplication is needed for varieties which are difficult to regenerate by conventional methods of propagation
It is often the case that new or highly valued plant varieties are also the most difficult to propagate using traditional means. Here again, tissue culture technology
can be very helpful to growers.
The speed of plant multiplication and the quality and uniformity achieved by AgriForest's micropropagation process can be considerably superior to conventional
methods.
When disease-free plant propagation is important
One of the inherent requirements of the tissue culture process is that it be conducted in sterile, aseptic conditions. This results in plants that are generally free of
bacterial and fungal diseases. This aspect of tissue culture is especially helpful for growers that are propagating plant varieties that have major systemic disease
problems.

AgriForest's Tissue Culture Plants

The tissue culture process takes place in sterile conditions, and uses hormones that have been shown to have a carryover effect once plants are moved to
external conditions. Furthermore, tissue culture leads to the regeneration of whole plants, with their own full root systems and vigorous top growth like young

seedlings. As a result, AgriForest's tissue culture plants have many visible benefits. Our plants:
are guaranteed to be disease free

have a more fibrous, healthier root system free of any root rot problems

exhibit a denser, bushier branching habit

are characterised by more vigorous growth after transplanting

have a higher survival rate

are ready for re-sale in a shorter time

These benefits of AgriForest's tissue culture plants result in significant cost savings for growers due to a reduction in growing space and time,
and a decrease in the labour required to yield a marketable product!.

Tissue culture, a method of biological research in which fragments oftissue from an animal or plant are transferred
to an artificial environment in which they can continue to survive and function. The cultured tissue may consist of a
single cell, a population of cells, or a whole or part of an organ. Cells in culture may multiply; change size, form, or
function; exhibit specialized activity (muscle cells, for example, may contract); or interact with other cells.

HISTORICAL DEVELOPMENTS
An early attempt at tissue culture was made in 1885 by German zoologistWilhelm Roux, who cultivated tissue from a
chick embryo in a warm salt solution. The first real success came in 1907, however, when American zoologist Ross
G. Harrison demonstrated the growth of frog nerve cellprocesses in a medium of clotted lymph. French
surgeon Alexis Carrel and his assistant Montrose Burrows subsequently improved upon Harrisons technique,
reporting their initial advances in a series of papers published in 191011. Carrel and Burrows coined the term tissue
culture and defined the concept. Thereafter, a number of experimenters succeeded in cultivating animal cells, using
as culture media a variety of biological fluids, such as lymph, blood serum, plasma, and tissue extracts. In the 1980s
and 90s, methods were developed that enabled researchers to successfully grow mammalian embryonic stem
cells under artificial conditions. Those breakthroughs ultimately enabled the establishment and maintenance of

human embryonic stem cell lines, which advanced researchers understanding of human biology and greatly
facilitated progress intherapeutics and regenerative medicine.

CULTURE ENVIRONMENTS
SIMILAR TOPICS

in vitro fertilization (IVF)

Cells may be grown in a culture medium of biological origin such as blood serum or tissue extract, in a chemically defined
synthetic medium, or in a mixture of the two. A medium must contain proper proportions of the necessary nutrients for the cells
to be studied and must be appropriately acid or alkaline. Cultures are usually grown either as single layers of cells on a glass or
plastic surface or as a suspension in a liquid or semisolid medium.
To initiate a culture, a tiny sample of the tissue is dispersed on or in the medium, and the flask, tube, or plate containing the
culture is then incubated, usually at a temperature close to that of the tissues normal environment. Sterile conditions are
maintained to prevent contamination with microorganisms. Cultures are sometimes started from single cells, resulting in the
production of uniform biological populations called clones. Single cells typically give rise to colonies within 10 to 14 days of
being placed under culture conditions.

PRIMARY CULTURES AND ESTABLISHED CELL LINES

There are two main types of cultures: primary (mortal) cultures and cultures of established (immortal) cell lines. Primary cultures
consist of normal cells, tissues, or organs that are excised directly from tissue collected by biopsyfrom a living organism. Primary
cultures are advantageous in that they essentially model the natural function of the cell, tissue, or organ under study. However, the
longer the samples are maintained in culture, the more mutations they accumulate, which can lead to changes in chromosome
structure and cell function. In addition, primary cultures generally are mortal. Cells undergo an aging process whereby they
multiply for only 50 to 100 generations, after which the rate decreases markedly. The point at which cells in primary cultures stop
growing, or undergo replicative senescence, marks the so-called Hayflick limit (named for its discoverer, American
microbiologist Leonard Hayflick).

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By contrast, established cell lines can be perpetuated indefinitely. Such cell lines generally are derived from tumour biopsies from
patients, or they may be generated from primary cells that have undergone mutations that enabled them to overcome the Hayflick
limit and continue replicating. Similar to cells in primary cultures, cells in established lines accumulate mutations over time that
can change their character. Thus, in order for researchers from different laboratories to be able to compare results from
experiments using the same cell lines, they must confirm the identity of the cells that they are working with. Cell identity is
verified through a process known as authentication, in which the DNA profile of the cultured cells is compared against the known
or standard profile for that cell line.

PROCESSING OF CULTURED CELLS AND TISSUES

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Live cultures may be examined directly with a microscope, or they may be observed by means of photographs and motion
pictures taken through the microscope. Cells, tissues, and organs may also be killed, fixed (preserved), and stained for further
examination. Following fixation, samples can also be embedded (e.g., in a resin) and cut into thin sections to disclose additional
details under a light or electron microscope.
Cells in tissue culture are subjected to a broad range of experimental treatment. For example, viruses,
drugs, hormones, vitamins, disease-causingmicroorganisms, or suspected cancer-producing chemicals may be added to the
culture. Scientists then observe the cells, looking for global changes in cell behaviour or function or for changes in specific
molecules, such as alterations in the expression of a particular protein or gene.

BIOLOGICAL INSIGHTS
Tissue culture has enabled numerous discoveries in the biological sciences. It has revealed, for example, basic information about
cells regarding their composition and form; their biochemical, genetic, and reproductive activity; their nutrition, metabolism,
specialized functions, and processes of aging and healing; the effects on cells of physical, chemical, and biological agents
(drugs and viruses, for example); and the differences between normal cells and abnormal cells, such as cancer cells. Work with
tissue cultures has helped to identify infections, enzyme deficiencies, and chromosomal abnormalities, to classify tumours, and to
formulate and test drugs and vaccines.
Since the discovery that certain viruses also grow in tissue culture, the technique has been used to produce vaccines against
poliomyelitis, influenza, measles, mumps, and other infectious diseases. Cell cultures have also produced viral inhibitors,
including interferon. Hormones are also produced from cultures of cells or organs. Cultured white blood cells from two
individuals can be used to determine compatibility between potential donors and recipients of tissue transplants. By removing and
culturing cells from a pregnant woman, it is possible to tell whether her fetus has certain chromosomal defects, such as those
associated with Down syndrome and other trisomies.
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The identification and diagnosis of chromosome abnormalities and inherited disorders has been greatly enhanced by the
development of somatic cell genetics. Tissue culture techniques have been used to culture many kinds of hybrid cells that contain
chromosomes from different species in the same cell, allowing the functions of individual chromosomes to be separately defined.
Tissue culture studies have clarified the genetic causes of certain hereditary diseases, and methods have been developed for
detecting environmental substances that may cause gene damage. The nature of certain cancers has been elucidated by the
discovery of specific genes and chromosomal aberrations that are associated with the disease. The methods of somatic cell

genetics have also been applied to plant cells; for example, such methods have been used in efforts to develop new strains of
cereal crops with improved nutritional properties.

chat FEEDBACK

Theory

The theoretical basis for plant tissue culture was proposed by Gottlieb Haberlandt, German Academy of science in1902 on his experiments on the culture
of

single

cell.

The

first

true

cultures

were

obtained

by

Gautheret

from

cambial

tissue

of

Acer

pseudoplatanus.

The term plant tissue culture (Micro propagation) is generally used for the aseptic culture of cells, tissues, organs and their components under defined
chemical and physical conditions in vitro. The basic concept of the plant body can be dissected into smaller part termed as explants and any explants
can be developed into a whole plant. It is a central innovative areas of applied plant science, including agriculture and plant biotechnology. This
technique is effective because almost all the plants cell are totipotent; In each cell possesses the genetic information and cellular machinery necessary to
generate the whole organism. Since, this technique can be used to produce a higher number of plants that are genetically similar to a parent plant as well
as

to

another.

Two concepts, plasticity and totipotency, are the central processes to understand the regeneration and plant cell culture. Plants, due to its longer

life

span and sessile nature, have developed a greater ability to overcome the extreme conditions. Most of the processes inculed in plant development and
the growth, adapt to environmental conditions. When the plant cells and tissues are cultured in vitro, most of them

are generally exhibit a very high

degree of plasticity, which allows one type of organ or tissue to be initiated from another type. Like this way, the whole plant can be subsequently
regenerated.

These

maintenance

of

genetic

potential

is

called

totipotency.

The plant tissue culture medium is an artificial nutrient supplement of organic and inorganic nutrients used for cultivation of plant tissue media. The
appropriate composition of the medium largely determines the success of the culture. The culture media used for the in vitro cultivation of the plant cells
are

composed

1)

Essential

2)
3)

An

elements
organic

of
(normal

three
ions)

supplied

supplements

providing

basic

as

a
vitamins

complex

components.
mixture

and

of

salts.

amino

acids.

A source of fixed carbon which is usually supplied as sucrose.

When cultured in an appropriate medium having auxin and cytokinin, explants will give rise to an unorganized, growing and dividing mass of cells called
callus. Callus cultures are initiated from a small part of an organ or tissue segment called the explants on a growth supporting solidified nutrient medium
under sterile conditions. Any part of the plant organ or tissues may be used as the explants. At the time of callus formation, there is some degree of
dedifferentiation happens both in morphology and metabolism. One of the major consequences of this dedifferentiation is that most plant cultures lose
their ability to perform photosynthesis. The necessitates of the addition of other components such as carbon and vitamins source to the culture media,
in

addition

to

the

Morphology

unusual

mineral

of

nutrients.

callus:

Callus varies considerably in appearance and texture, ranging from hard nodular cell masses to friable soft ones. They maybe white or creamish, orange,
green either in whole or part as a result of chloroplast development. The shape of individual cells within the callus mass ranges from the near spherical or
markedly

elongated.

A typical unorganized plant callus initiated from a new explants or piece of previously initiated calli has three stages of development.

1.
2.

The induction of cell division.

A period of active cell division during which differentiated cells lose specialized features they may have acquired and become
differentiated. Cell division usually occurs in the outer layer of the explants.

3.

Period when cell division slows down on ceases and when within the callus, there is increasing cellular differentiat

de

Callus culturing is performed in the dark while light can be encourage the differentiation of the callus. At the time of long term culture, the culture may
loss the requirement for cytokinin and auxins. Manipulation of the auxins to cytokinin ratio in the medium can leads to the development of shoots, roots
or somatic embryos from which the plant can be subsequently produced.

Callus

culture
1

Callus
2.

3.

It

is

is

is
the

It
useful

starting

helps
for

the

useful
material

in

synthesis

of

for

the

the
starting

for
suspension

production

compounds

that

are

many
culture

of
subsequently

which

purposes.

cells

secondary
modified

to

are

separated.

plant
yield

the

products.
desired

product.

4. It is the starting materials for vegetative propagation of plants.

Based on the availability of the various invitro techniques, the dramatic increase in their application to various problems in basic biology, agriculture,
horticulture,

and

forestry.

The

applications

can

divide

conveniently

into

five

broad

areas;

1.Cellbehavior.
2.Plantmodification.
3.Germplasm

storage

and

pathogen

free

4.Clonal
5.Product

plants.
propagation.
formation.

6. Improved varities.

How is Tissue Culture Done?

Tissue culture is an important component of transforming plants with new genes.

During this procedure, plant cells can be removed from various parts of a plant and
placed on media in petri plates. The media does not contain the growth hormones
normally present in a plant that tell the cells which tissue to develop into. As a result, the
cells do not differentiate and instead form a mass of cells called a callus that are not
differentiated into at the tissue level.

Tissue culture is an important component of transforming plants with

new genes.
During this procedure, plant cells can be removed from various parts of a
plant and placed on media in petri plates. The media does not contain the
growth hormones normally present in a plant that tell the cells which tissue to
develop into. As a result, the cells do not differentiate and instead form a
mass of cells called a callus that are not differentiated into at the tissue level.

Cells are taken from plants and

Immature embryos are removed
Callus are masses of
grown into undifferentiated masses from seeds and placed on media.
undifferentiated cells.
called callus.
Callus cells will then begin to grow
from them.

Since plant cells are totipotent, growth hormones can be added to the media
triggering the callus cells to develop roots, shoots and eventually entire plants.
Plants regenerated from tissue culturewill be clones genetically identical to the
cell they originated from. The only animal cells that have this totipotent
characteristic are fertilized eggs.

Single cells can be regenerated into Growth hormones can be added to Regenerated plants are then
entire plants.
the media and the cells will begin to transferred into test tubes. Once
divide and differentiate into plants. they have reached a certain size,
they will be transplanted into soil.

Advantages and Disadvantages of Tissue Culture

Plant tissue is also known as micro propagation and it is the rapid multiplying of plant material to produce a large number of progeny plants, using the techniques of plant tissue
culture methods. It is a practice used for plant propagation under sterile conditions to produce plant clones.

Advantages

and

Disadvantages

of

Tissue

Culture

are

as

follows:

Advantages of Tissue Culture:

These techniques have certain advantages over traditional methods of propagation.

They produce exact copies of plants required that have desirable traits.

They produce mature plants quickly.

Multiple plants are produced in the absence of seeds or necessary pollinators to produce seeds.

Whole plants are produced regenerated from plant cells that are genetically modified.

Many plants that are clones of each other can be produced.

Diseases resistant plants are produced by micro propagation.

High rate of fecundity is obtained.

This is the only method that is viable method of regenerating genetically modified cells even after protoplast fusion.

This method is useful which produce seeds insufficient amounts, or when plants are sterile and they do not produce viable seeds or when the seeds
cannot be stored.

Some plants like orchids have very small seeds and the seeds are more reliably grown from seed in sterile culture.

A larger number of plants can be produced and propagule can be stored for longer in a smaller area.

Disadvantages of Tissue Culture:

Micro propagation is not a method of multiplying plants.

It is labor intensive and expensive process.

A monoculture that is produced after micro propagation which leads to the lack of disease resistance, all the progeny plants may be vulnerable to the
same infections.

An infected sample plant can produce an infected progeny. All plants cannot be successfully tissue cultured. It is usually because the medium for
growth is not known.

Some plants are very difficult to be disinfected from fungal organisms.

TISSUE CULTURE- A BOON OR A BANE

issue culture is a process that involves exposing plant tissue to a specific regimen of nutrients, hormones under sterile or in artificial conditions outside the mother plant to
produce many new plants. Each new plant is a clone of the original mother plant. This process is done over a very short period of time. Tissue culture is the growth of tissues or
cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid or solid growth medium such as broth or agar. Tissue culture plants are characterized
by disease free growth, a more fibrous, healthier root system, a bushier branching habit and a higher survival rate.

Plant tissue culture is a widely used procedure in plant biology in which organism is planted from the explants of the living plants in a nutrient medium under aseptic
conditions. There are both advantages and disadvantages of plant tissue culture.

Types of Cultures:

(a) ORGAN CULTURE: These are cultures of isolated plant organs including cultures derived from root tips, stem tips, leaf primordial or immature part of flowers and immature
fruits.

(b) EMBRYO CULTURE: These are culture of isolated immature or mature embryos.

(c) CALLUS OR TISSUE CULTURE: These are culture of tissue arising from disorganized proliferation of cells from segment of plant organs. Tissue or callus culture is generally
grown on solid medium as amass of cells.

(d) SUSPENSION CULTURE: These are often called cell cultures, as they represent a lower level of organization than tissue or callus culture. Suspension culture is in vitro
cultures of isolated cells and very small cell groups remaining dispersed as they grow in excited liquid media.

Positives of tissue culture

To produce many copies of the same plants then which may be used to produce plants with better flowers, odors, fruits or any other properties of the plants which
are beneficial to the human beings.

To produce plants anytime we want even if the climate is not appropriate to produce the plant. Moreover, if seed is not available, it is possible to produce a plant
with this method.

If there is plant with partially infected tissue, it is possible to produce a new plant without infection.

Very useful solution for the prevention of starvation in third world countries since the process is highly efficient i.e. by using only one plant it is possible to produce
more than one thousand copies of the same plant with higher productivity if its genome is changed.

The equipments are cheaper when compared to the animal cell culture.

It helps to eliminate plant diseases through careful stock selection and sterile techniques

The time required is quite less hence there is no need to wait for the whole life cycle of seed development.

Negatives of tissue culture:

In case of large scale production , the cost of the equipments is very high.

The procedure is quite variable and also it depends on the type of the species so sometimes it needs trial-and-error type of experiments if there is no
available review about that species.

The procedure needs special attention and diligently done observation.

There may be error in the identity of the organisms after culture.

Infection may continue through generations easily if possible precautions are not taken

Decrease genetic variability.

Plant tissue cultures can be initiated from almost any part of a plant. The physiological state of the plant does have an influence on its response to attempts to initiate tissue
culture. The parent plant must be healthy and free from obvious signs of disease or decay. Younger tissue contains a higher proportion of actively dividing cells and is more
responsive to a callus initiation programme. The plants themselves must be actively growing, and not about to enter a period of dormancy.
Tissue culture and plant regeneration are an integral part of most plant transformation strategies, and can often prove to be the most challenging aspect of a plant
transformation protocol. Key to success in integrating plant tissue culture into plant transformation strategies is the realization that a quick (to avoid too many harmful effects on
the new cultured one) and efficient regeneration system must be developed.