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 as micropropagation. 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 clean 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.

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.

BACKGROUND
Tissue culture (often called micropropagation) is a special type of asexual propagation where a
very small piece of tissue (shoot apex, leaf section, or even an individual cell) is excised (cutout) and placed in sterile (aseptic) culture in a test tube, petri dish or tissue culture container
containing a special culture medium.

Overview of the Tissue Culture Process

The culture medium contains a gel (agar) with the proper mixture of nutrients, sugars, vitamins
and hormones, which causes the plant part to grow at very rapid rates to produce new
plantlets. It has been estimated that one chrysanthemum apex placed in tissue culture could
produce up to 1,000,000 new plantlets in one year. Thus, tissue culture is used for rapid
multiplication of plants. A very specialized laboratory is required for tissue culture. All the
procedures are done in a laboratory and special ventilated cabinet that is as sterile as an operating
room.

Steps in Tissue Culture

(images courtesy of Dr. Dan Lineberger, aggiehorticulture.tamu.edu/tisscult/microprop/microprop.html)
Explant: Cut-out Plant Tissue and Place in Tissue Culture Container
The first step is to obtain what is called and explant. This means to simply cut-out a
very small piece of leaf or stem tissue, or even isolate individual cells, and place them
in a tissue culture container. The tissue has to be sterilized so it will not have any
contaminating bacteria or fungus. It is then placed inside the tissue culture contain on
a gel called agar. In the agar is dissolved all the sugar, nutrients and hormones the
plant needs.

Explants can be pieces of any part of the plant (leaves, stems, flowers, etc.),
or even individual isolated cells.
Multiplication: Tissue Grows and Produces Small Plants
The tissue will begin to grow. It may make a big blob of tissue called callus, or it may
make new shoots directly from the explant tissue that was inserted in the container.

A mass of callus tissue is formed that is just starting to make new plantlets.

New plantlets (shoots with leaves) are forming.

If the conditions are right a small "forest" of plants

will develop in the tissue culture container.
Rapid Multiplication by Transfer of Cultures
Once the plantlets start developing, some can be removed and placed in new tissue
culture containers. Thus, another "forest"' of plants is produced. This results in a
rapid multiplication of the cultures and many thousand of plants can be produced in a
few months.

Some of the small plantlets can be removed and transferred to new tissue culture
containers. These will produce more shoots and fill the container.
Transplanting
When the plantlets are large enough, they can be removed from the tissue culture
container and transferred into pots with potting soil. The young plants are growth in a
greenhouse just like you would any young seedling or cutting.

When the small plant clones are removed from the culture containers, they must be
transplanted into some type of acclimation container or kept under a mist system until
the acclimate to the ambient environment.

After acclimation, the young plants can be transplanted

and grown in pots in a greenhouse to produce new plants.
TISSUE CULTURE- An Introduction
Plant tissue culture is the culture and maintenance of plant cells or organs in sterile, nutritionally and
environmentally supportive conditions (in vitro). Plant cell and tissue culture include the cultural
techniques for regeneration of functional plants from embryonic tissues, tissue fragments, calli, isolated
cells, or protoplasts. It has applications in research and commerce. In commercial settings, tissue culture
is often referred to as micro-propagation, which is in fact one of the techniques in tissue culture. Micropropagation refers to the production of whole plants from cell cultures derived from explants (the initial
piece of tissue put into culture) or meristem cells.
Haberlandt was the first scientist to produce whole plants from plant tissues and so he is popularly called
as the Father of Tissue Culture. Plant tissue culture and molecular biology form the basis for genetic
engineering.
Plant tissue culture consists of pollen culture, callus tip culture, stem tip culture, meristem tip culture and
protoplast fusion (Fig: 1).

Fig: 1. Various Tissue Culture Types

Conditions required for plant cells to grow in vitro:

Freedom from competitions particularly from microorganisms for nutrition and other factors

Availability of nutrients and removal of waste products for better growth of the tissue

A controlled environment to maintain the cultures

Tissue culture offers significant benefits over traditional propagation methods.

Much faster rates of growth can be induced in vitro than by traditional means.

Multiplication of plants which are very difficult to propagate by cuttings or other traditional
methods.

Production of large numbers of genetically identical clones in a short time

Seeds can be germinated with no risk of damping off/ predation.

Under certain conditions, plant material can be stored in vitro for considerable periods of time with
little or no maintenance

Tissue culture techniques are used for virus eradication, genetic manipulation, somatic
hybridization and other procedures that benefit propagation, crop improvement, and basic
research.

The success for plant tissue culture is based on the principle called totipotency - the ability of
undifferentiated plant tissues to differentiate into functional plants when cultured in vitro.
Plant tissue culture is used widely in plant science; it also has a number of commercial applications.
Applications include:

Micro-propagation is widely used in forestry and in floriculture. Micro-propagation can also be

used to conserve rare or endangered plant species.

A plant breeder may use tissue culture to screen cells rather than plants for advantageous
characters such as salt tolerance.

Large-scale growth of plant cells in liquid culture inside bioreactors as a source of secondary
metabolites.

To cross widely related species by protoplast fusion and regeneration of the novel hybrid called
cybrids

To cross-pollinate distantly related species and then rescue of the resulting embryo through
culture of embryos to overcome post-zygotic abortion

For production of doubled plants from haploid cultures to achieve homozygous lines more rapidly
in breeding programmes, usually by treatment with colchicine which causes doubling of the
chromosome number.

Certain techniques such as meristem tip culture may be employed that can be used to produce
clean plant material from virus infected plants.

Technique
A part or an organ of a plant is disinfected with a sterilizing agent and placed inside a test tube containing
a nutrition balanced medium under controlled environments in order to culture it into a whole plant. Many
explants, for example, stem, leaf, bud, meristem are used for this purpose. The figure shows a banana
plantlet cultured and regenerated by means of tissue culture (Fig: 2).

Fig: 2. Tissue Cultured Banana.

By means of tissue culture it is possible to produce pathogen free plantlets by mass multiplication in a
very limited amount of area from a very small sterile part of a mother plant. This method is also used to
produce/ multiply plants that are to be transported across national border and so for their faster
multiplication.

But the establishment of a tissue culturing unit needs huge financial investments, skilled
labors/technicians and required areas for its establishment are major constraints. Plant tissues grow and
multiply in the labs only when there is an uncompetitive, growing condition with uninterrupted supply of
nutrients.
Medium:
It contains all the elements that contribute the required nutrients that aid to the growth of the tissues; it is
in liquid state or semi-solid in nature. The tissues are grown on the media. It consists of 95% of water,
major and minor nutrients, plant growth hormones, vitamins, sugar rich compounds and chelating agents.
Totipotency:
It is the ability of a tissue or an organ of a plant to produce the whole plant, under the optional laboratory
conditions and this is called as Totipotency. This is the baseline over which plant tissue culture relies
upon.
Callus Culture:
When the cells divide into an undifferentiated mass it is called as callus. Any part of a plant can be used
to produce the calli. It may be a stem, leaf, meristem or any other part. It is used to produce variations
among the plantlets.
Suspension culture:
The callus produced from the explants are grown on nutrient solutions (that are semi solid) for a period of
time and they are induced to produce plants with new traits.
Embryo Culture:
The method of culturing mature and immature embryos in media is called embryo culture. By this method,
it is possible to produce plants from dormant seeds and seeds with metabolites that inhibit germination.
This method is very important in crop improvement programs.
Somatic Embryogenesis:
When the plants are grown on nutrient media, calli are formed. When these calli are subjected to growth
in cytokinin medium, somatic embryos are formed. They are circular, elongated, heart and torpedo
shaped. Among that torpedo shaped embryos produce the whole plant that is very robust (Fig: 3).

Fig: 3. Generation of Plants by Somatic Embryogenesis

a. Somatic embryos
b. Heart-shaped somatic embryos
c.

Elongated somatic embryos

d. Torpedo shaped somatic embryos

e. Whole plant
Apart from this above mentioned method, the suspensions of the callus are transferred to conical flasks
and separate somatic embryos are produced and allowed to mature. These mature embryos when
induced with cytokinin at the fourth stage produce the whole plantlets. Moreover these plants are
hardened in a green house after rooting occurs.
Somatic embryo culture is a very special method in plant tissue culture. Calcium alginate is used to
produce artificial seeds from somatic embryos. It is very useful in studying secondary metabolites from
the cell and to do research in somatic embryo culturing.
Organogenesis:
Any plant part when placed and cultured on a media, tries hard to get back its own life. In this
phenomenon, the callus are produced. These callus are subjected to cytokine treatment which produce

the shoots. This method of producing plant organs is called as organogenesis. These shoots are
transferred to root including auxin contained media and we thus get the whole plantlets.
Embryo Rescue Culture:
Some commercially important crops hindered in germination due to the constraints related to seed
anatomy and physiology. Embryo abortion is a major factor to this. These embryos of such seeds are
isolated and cultured on suitable nutrient media so as to regenerate the plants easily. Embryos obtained
after incompatible sexual mating also be rescued by this methodology.
Shoot Tip Culture:
The meristem tip of a plant is more efficient in creating a whole plant than its tissues from the stem. This
idea is made use of in the shoot tip culture.

Fig: 4. Methods of Producing Plants from Shoot Tip Culture.

1. Shoot tip in the medium
2. Shoot callus
3. Growth of stem
4. Root initiation
5. Hardening
6. Whole plant.

This method is used to produce plants free from pathogens, for meristems do not support the growth of
viral particles. Hence the plants produced by this method can be stored pathogen free for a longer period.
Meristem Culture:
Disinfected small bits of meristem tips of 0.1 to 0.5mm length are used to produce callus on suitable
media to produce whole plants. This is called as meristem culture and is used to produce pathogen free
plantlets.
Anther Culture:
When the pollen/anthers of the correct stage are collected and grown on suitable media, it is called as
anther culture. It is used to produce haploid plants.

Fig: 5. Production of Plants by means of Anther Culture.

1. Pollen on media
2. Callus formation

3. Shoot initiation
4. Root initiation
5. Whole plant.
The choice of mother plant for collecting the pollen, proper maintenance in lab, the pH balance of the
nutrient medium, incubation period for producing the plant are all considered to be major factors.
Two Stages in Anther /Pollen Culture:
The culturing of pollen consists of two stages namely direct and indirect culturing methods. In the direct
method, the pollen by themselves produce the plant directly from the medium. In the indirect method, the
pollen produces a callus from which haploid plants are produced. Diploid plants are produced from the
pollen sacs in a short period of time because the tips of these plants are always pathogen free.
Rejuvenation of Plants:
It is very feasible to produce the whole plant out of tissues collected from old plants in very short period of
time. This is called plant rejuvenation. This method was demonstrated successfully in tapioca.
Hybrid Sorting:
It is also possible to produce hybrids from incompatible species (where in the hybrids are hard to be
formed) by means of protoplasmic fusion. Hybrid may be formed by culturing the fused protoplasts in
suitable nutrient media.
Micro Propagation:
The plant tissues from shoot nodes are used in plant tissue culture to produce thousands clones of a
mother plant in a very short period of time (Fig: 6).

Fig: 6. Production of Plants through Mass Multiplication.

Synthetic Seeds:
Embryogenesis is the basis for various technologies on biotechnology.

Somatic embryos are used to produce artificial seeds. Sodium alginate and calcium chloride are used to
produce the seed coats of the somatic embryos. These are capable of producing plantlets from the soil
bed just like the natural seeds.
By this method, it is possible to multiply clonal plants and to create variations in the genetic architecture of
the tissues. This method is used to produce vegetables, especially seedless watermelon are multiplied by
this method.

Fig: 7. Production of Plants from Artificial Seeds

Protoplast Culture:
The removal of cell membrane from a cell produces a protoplast. Protoplasts are used to produce
variations in the morphology of leaves and flowers, ability/potential of the growth of the embryo, and
enhancement of disease resistance in plants.

Fig: 8. Growth of Protoplast on Nutrient Media

It is feasible to produce a whole plant from a protoplast. Two protoplasts are isolated from two parents
from any plant organ and are fused with chemicals that induce fusion between them. This induces
variations in the genes and plants are produced with the variations.

Choice of explant[edit]
The tissue obtained from a plant to be cultured is called an explant based on work with certain Model
systems[disambiguation needed] particularly tobacco it has often been claimed that a totipotent explant can be
grown from any part of the plant and may include portions
of shoots, leaves, stems, flowers, roots and single, undifferentiated cells.,[citation needed]however this has
not been true for all plants.[3] In many species explants of various organs vary in their rates of growth
and regeneration, while some do not grow at all. The choice of explant material also determines if
the plantlets developed via tissue culture are haploid or diploid. Also the risk of microbial
contamination is increased with inappropriate explants.
The first method involving the meristems and induction of multiple shoots is the preferred method for
the micropropagation industry since the risks of somaclonal variation (genetic variation induced in
tissue culture) are minimal when compared to the other two methods. Somatic embryogenesis is a
method that has the potential to be several times higher in multiplication rates and is amenable to
handling in liquid culture systems like bioreactors.
Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that
become problematic during the tissue culture process. Certain soil microflora can form tight
associations with the root systems, or even grow within the root. Soil particles bound to roots are
difficult to remove without injury to the roots that then allows microbial attack. These
associated microflora will generally overgrow the tissue culture medium before there is significant
growth of plant tissue.
Some cultured tissues are slow in their growth. For them there would be two options: (i) Optimizing
the culture medium; (ii) Culturing highly responsive tissues or varieties.[4]Necrosis could spoil
cultured tissues. Generally, plants varieties are different in tissue culture necrosis. Thus, by culturing
highly responsive varieties (or tissues) it can be managed.[4]
Aerial (above soil) explants are also rich in undesirable microflora. However, they are more easily
removed from the explant by gentle rinsing, and the remainder usually can be killed by surface
sterilization. Most of the surface microflora do not form tight associations with the plant tissue. Such
associations can usually be found by visual inspection as a mosaic, de-colorization or
localized necrosis on the surface of the explant.
An alternative for obtaining uncontaminated explants is to take explants from seedlings which are
aseptically grown from surface-sterilized seeds. The hard surface of the seed is less permeable to
penetration of harsh surface sterilizing agents, such as hypochlorite, so the acceptable conditions of
sterilization used for seeds can be much more stringent than for vegetative tissues.

Tissue cultured plants are clones. If the original mother plant used to produce the first explants is
susceptible to a pathogen or environmental condition, the entire crop would be susceptible to the
same problem. Conversely, any positive traits would remain within the line also.

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.
Advantages:
1.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 that are
beneficial to the human beings. 2.To produce plants anytime we want although the
climates are not appropriate to produce a plant. Moreover, if seed is not available, it is
possible to produce a plant with this method. 3.If there is plant with partially infected
tissue, it is possible to produce a new plant without infection. 4.Very helpful in the
genetically modified organism studies. 5.Very useful solution for the prevention of
starvation in third world countries since the process id highly efficient, by using only one
plant, it is possible to produce more than one thousand of the same plant with higher
productive if its genome changed. 6.The equipments are cheaper when compared to
the animal cell culture. Disadvantages:
1.If large scale production is being thinking, the costs of the equipments are very
expensive. 2.The procedure is very variable and it depends on the type of the species
so sometimes it needs trial-and-error type of experiments if there is not any review
about that species. 3.The procedure needs special attention and diligently done
observation. 4.There may be error in the identity of the organisms after culture.
5.Infection may continue thorough generations easily if possible precautions are not
taken 6.Decrease genetic variability.

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.[5]

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.[6]

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, [7] and in vitro
flowering studies.[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.