SHADAN COLLEGE

OF PHARMACY
 TISSUE CULTURE

DEFINITION
 The term tissue culture may be defined as the process of in-vitro culture of explants
(pieces of living differentiated tissues) in nutrient medium under aseptic conditions.
 Plant tissue culture is fundamental to most aspects of biotechnology of plants. It is evident
now that plant biotechnology is one of the most beneficial of all the sciences. The products
of plant biotechnology are being transferred rapidly from laboratories to the fields.
 Also, the plant tissue culture has become of great interest to the molecular
biologists, plant breeders and even to the industrialists, as it helps in improving the
plants of economic importance.
 In addition to all this, the tissue culture contributes immensely for understanding the
patterns and responsible factors of growth, metabolism, morphogenesis and
differentiation of plants.

“Plant tissue culture has a great significance in plant biotechnology specially in the crop
improvement programmes.”
 TISSUE CULTURE
Three other scientists Gautheret, White and
Nobecourt also made valuable contributions
to the development of plant tissue culture
techniques.
Later on, a number of suitable culture media
were developed, for culturing plant cells,
tissues, protoplasts, embryos, anthers, root
tips, etc. The discovery and understanding of
role of plant growth hormones in the
G. Haberlandt, a German multiplication of cell also provided an extra
botanist, in 1902 aid for the development of in-vitro culture
cultured fully methods of plants.
differentiated plant cells In India, the work on tissue culture was
isolated from different initiated during 1950s at University of Delhi.
plants. This was the very This initiation is credited to Shri Panchanan
first step for the Maheshwari who was working there in the
beginning of plant cell Department of Botany. Discovery of haploid
and tissue culture. production was a land-mark in the
development of in-vitro culturing of plants.
 TISSUE CULTURE
 The first plant from a mature plant cell was regenerated by Braun in 1959

Extensive microscopic studies led to the independent and almost

simultaneous development of the cell theory by Schleiden (1838) and
Schwann (1839). Scheilden and Schwann put forward the co-called
Totipotency theory, which states that cells are autonomic, and in
principle, are capable of regenerating to give a complete plant. Their
theory was in fact the foundation of plant cell and tissue culture.
 TISSUE CULTURE
Basic Requirements of Plant Tissue Culture
The main requirements of plant tissue culture are:
(1) Laboratory Organisation
(2) Culture Media
(3) Aseptic Conditions
 Laboratory Organisation:
In a standard tissue culture lab, there must be a few basic facilities like:
i. A Media Room for preparation, sterilization and storage of culture media.
ii. Facilities for washing of lab-wares, explants, etc.
iii. Space for storage of lab-wares.
iv. Culture rooms or incubators where conditions of temperature, humidity and light etc.
can be maintained.
v. Observation and Data Collection area.
 TISSUE CULTURE

 Culture Media:
The formulation or the medium on which the explant is cultured is called culture medium. It
is composed of various nutrients required for proper culturing. Different types of plants and
organs need different compositions of culture media. A number of media have been
devised for specific tissues and organs.
Some important of them are:
MS (Murashige and Skoog) Medium
LS (Linsmaier and Skoog) Medium
B5 (Gamborg’s) Medium
White’s Medium, etc.
Important constituents of a culture medium are:
 Organic supplements:
(a) Vitamins like thiamine (B1), Pyridoxin (B6), Nicotinic Acid (B3), etc.
(b) Antibiotics like Streptomycin, Kanamycin;
(c) Amino Acids like Arginine, Asparagine.
 TISSUE CULTURE
 Inorganic Nutrients:
Micronutrients as Iron (Fe), Manganese (Mn), Zinc (Zn), Molybdenum (Mo),
Copper (Cu), Boron (B).
Macronutrients include six major elements as Nitrogen (N), Sulphur (S),
Phosphorus (P), Potassium (K), Calcium (Ca), Magnesium (Mg).
 Carbon and Energy Source:
Most preferred carbon source is Sucrose. Others include lactose, maltose,
galactose, raffinose, cellobiose, etc.
 Growth Hormones:
a. Auxins-mainly for inducing cell division.
b. Cytokinins-mainly for modifying apical dominance and shoot differentiation.
c. Abscisic Acid (ABA)-Used occasionally.
d. Gibberellins-Used occasionally.
 Gelling Agents:
These are added to media to make them semisolid or solid. Agar, Gelatin,
Alginate etc. are common solidifying or gelling agents.
 Other Organic Extracts:
Sometimes culture media are supplemented with some organic extracts also
like coconut milk, orange juice, tomato juice, potato extract, etc.
 TISSUE CULTURE
 Aseptic Conditions:
Maintenance of aseptic conditions is the most critical and difficult aspect of in-vitro culturing
experiments. Aseptic condition mean the conditions free from any type of microorganisms
(so as to prevent the loss of experiment by contamination). For this, sterilization (i.e.,
complete removal or killing of microbes) is done. The most common contaminants in culture
are fungi and bacteria.
Measures to be taken for maintaining asepsis during tissue culture are:
 Sterilization of the culture vessels using detergents, autoclaves, etc.
 Sterilization of instruments like forceps, needles etc. by flame sterilization.
 Sterilization of culture medium using filter sterilization or autoclaving methods.
 Surface sterilization of explants using surface disinfectants like Silver Nitrate (1%), H2O2
(10-12%), Bromine water (1-2%), Sodium Hypochlorite solution (0.3-0.6%), etc.
The whole procedure of plant tissue culture is to be carried out essentially under aseptic
conditions. So, the overall design of the laboratory must focus on the maintenance of aseptic
conditions. Secondly, the worker is also required to have proper knowledge of operating
various equipment’s like pH meter, balance, laminar air flow, microscope, etc.
While performing the tissue culture experiments there must present the first aid kits and fire
extinguishers in the laboratory to avoid any mishap or accident. In addition, proper attention
should be given while handling the toxic chemicals and all the chemicals should be kept in
correct labeled containers and bottles.
 TISSUE CULTURE
Basic steps for in-vitro culturing of plants:
Proper trimming of
Intact Plant Excision of explant
explant

Explant washed in
Sterilization of Surface sterilization
sterilized distilled
Glassware of Explant
water

Inoculation of
Culture medium is Explant in the Incubation under
sterilized in culture medium suitable conditions
autoclave under aseptic for proper culture
conditions

Sub culturing for obtaining

Plantlet Transfer
desired results
 TISSUE CULTURE

 Selection and Sterilization of Explant:

Suitable explant is selected and is then excised from the donor plant. Explant is then
sterilized using disinfectants.
 Preparation and Sterilization of Culture Medium:
A suitable culture medium is prepared with special attention towards the objectives of
culture and type of explant to be cultured. Prepared culture medium is transferred into
sterilized vessels and then sterilized in autoclave.
 Inoculation:
Sterilized explant is inoculated (transferred) on the culture medium under aseptic conditions.
 Incubation:
Cultures are then incubated in the culture room where appropriate conditions of light,
temperature and humidity are provided for successful culturing.
 Sub culturing:
Cultured cells are transferred to a fresh nutrient medium to obtain the plantlets.
 Transfer of Plantlets:
After the hardening process (i.e., acclimatization of plantlet to the environment), the
plantlets are transferred to green house or in pots.
 TISSUE CULTURE

Basic Aspects of Plant Tissue Culture:

 Cellular Totipotency:
 The potential of a plant cell to grow and develop into a whole new multicellular plant
is described as cellular totipotency. In other words, the property of a single cell for
differentiating into many other cell types is called as totipotency.
 This is the property which is found only in living plant cells and not in animal cells
(exception being stem cells in animals).
 The term totipotency was coined in 1901 by Morgan. During culture practice, an
explant is taken from a differentiated, mature tissue. It means, the cells in explants
are generally non-dividing and quiescent in nature.
 To show totipotency, such mature, non-dividing cells undergo changes which revert
them into a meristematic state (usually a callus state). This phenomenon of reverting
back of mature cells to dividing state is called dedifferentiation. Now, these
dedifferentiated cells have the ability to form a whole plant or plant organ. This
phenomenon is termed as re-differentiation.
 TISSUE CULTURE
 There are different methods of culturing plant material. These methods differ on the basis
of explants used and their resultant products.
 Some of the most popular and advantageous methods in plant tissue culture are discussed
below
 Cell Culture
 Suspension Culture
 Root Culture
 Shoot Culture
 Protoplast Culture

 Cell Culture
Cell culture is actually, the process of producing clones of a single cell. The clones of cell are the
cells which have been derived from the single cell through mitosis and are identical to each
other as well as to parental cell. First attempts for cell culture were made by Haberlandt in 1902.
However, he failed to culture single cell but his attempts stimulated other workers to achieve
success in this direction.
 TISSUE CULTURE
Cell Culture
It is important to note here that the cell
cultures require a suitably enriched
nutrient medium and it should be done
in dark because light may deteriorate the
cell culture. Large scale culturing of plant
cells under in-vitro conditions provides a
suitable method for production of large
varieties of commercially important
phytochemicals.

The method of cell culture is done by following three main steps:

(a) Isolation of single cell from the intact plant by using some enzymatic or mechanical methods.
(b) In-vitro culturing of the single cell utilizing micro chamber technique, or micro drop method or
Bergmann cell plating technique.
(c) Testing of cell viability done with the phase contrast microscopy or certain special dyes.
 TISSUE CULTURE
Suspension Culture
 A culture which consists of cells or cell
aggregates initiated by placing callus tissues in
an agitated liquid medium is called as a
suspension culture. The continuous agitation
of the liquid medium during a suspension
culture is done by using a suitable device
called as shaker, most common being the
platform/orbital shaker.
 Agitation with shaker is important because it
breaks the cell aggregates into single cell or
smaller groups of cells and it helps in
maintaining the uniform distribution of single
cell and groups of cells in the liquid medium.
 A good suspension is the one which has high
proportion of single cells than the groups of
cells. Changes in the nutritional composition
of medium may also serve as a useful The general technique of suspension culture
technique for breakage of larger cell clumps involves basically two types of cultures: batch
(Fig. 7). culture and continuous cultures.
 TISSUE CULTURE

 Root Culture
Pioneering attempts for root culture were made by
Robbins and Kotte during 1920s. Later on, many
workers tried for achieving successful root
cultures. In 1934, it was White who successfully
cultured the continuously growing tomato root
tips.
Subsequently, root culturing of a number of plant
species of angiosperms as well as gymnosperms
has been done successfully. Root cultures are
usually not helpful for giving rise to complete
plants but they have importance’s of their own.
They provide beneficial information regarding the
nutritional needs, physiological activities,
nodulations, infections by different pathogenic
bacteria or other microbes, etc.
 TISSUE CULTURE
Shoot Culture
Shoot cultures have great applicability in the fields
of horticulture, agriculture and forestry. The
practical application of this method was proposed
by Morel and Martin (1952) after they successfully
recovered the complete Dahalia plant from shoot-
tips cultures.
Later on, Morel realized that the technique of shoot
culturing can prove to be a potent method for rapid
propagation of plants (i.e. Micro propagation). In
this technique, the shoot apical meristem is
cultured on a suitable nutrient medium. This is also
referred to as Meristem Culture (Fig. 8).

Meristem tip culture is also beneficial for recovery of pathogen-free specially virus-free
plants through the tissue culture techniques. Various stages in this culture process are
the initiation of culture, shoot multiplication, rooting of shoots and finally the transfer of
plantlets to the pots or fields.
 TISSUE CULTURE
Protoplast Culture
A protoplast is described as a plasma membrane
bound vesicle which consists of a naked cell formed
as a result of removal of cell wall. The cell wall can
be removed by mechanical or enzymatic methods.
In-vitro culturing of protoplasts has immense
applications in the field of plant biotechnology.
It not only serves for genetic manipulations in plants
but also for biochemical and metabolic studies in
plants. For protoplast culture, firstly the protoplasts
are isolated from the plants utilizing some chemical
or enzymatic procedure.
At present, there are available a number of enzymes
which have enabled the isolation of protoplasts from
almost every plant tissue. After isolation of
protoplasts, they are purified and then tested for
their viability. Finally the purified viable protoplasts
are cultured in-vitro using suitable nutrient medium
which is usually either a liquid medium or a
semisolid agar medium.
 TISSUE CULTURE

1. To produce many copies of same plant then this may be used to produce
plants with better flowers, odour, fruits or any other properties of plants which
are beneficial to human beings.
2. To produce plants anytime we want although the climate is 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 a partially infected tissue, it is possible to produce a new plant
without infections.
4. Very helpful in the genetically modified organism studies.

1. If the large scale production is being thinking, the costs of the equipments is
very high.
2. The procedure needs special attention.
3. There may be error in the identifying of the organisms after culture.
4. Infection may continue through generations.
 TISSUE CULTURE
 The past decades of plant cell biotechnology has evolved as a new era in the field of
biotechnology, focusing on the production of a large number of secondary plant
products.
 During the second half of the last century the development of genetic engineering and
molecular biology techniques allowed the appearance of improved and new agricultural
products which have occupied an increasing demand in the productive systems of several
countries worldwide.
 Nevertheless, these would have been impossible without the development of tissue
culture techniques, which provided the tools for the introduction of genetic information
into plant cells.
 Nowadays, one of the most promising methods of producing proteins and other
medicinal substances, such as antibodies and vaccines, is the use of transgenic plants.
 Transgenic plants represent an economical alternative to fermentation-based production
systems. Plant-made vaccines or antibodies (plantibodies) are especially striking, as
plants are free of human diseases, thus reducing screening costs for viruses and bacterial
toxins.
 The number of farmers who have incorporated transgenic plants into their production
systems in 2008 was 13.3 million, in comparison to 11 million in 2007.
 TISSUE CULTURE

 Large scale production of useful compounds and secondary metabolites by using

genetically engineered plant tissue cultures.
 Technique of micro propagation for enhancing the rate of multiplication of economically
important plants.
 Eradication of systemic diseases in plants and raising disease free plants.
 Soma-clonal variations are useful sources of introduction of valuable genetic variations in
plants.
 Helps plants in imparting resistance to antibiotics, drought, salinity, diseases, etc.
 Large scale production of biomass energy.
 Plant tissue culture aids in producing the genetically transformed plants.
 Early flowering can be induced by in-vitro culturing of plants so as to attain commercial
benefits.
 Triploids as well as polyploid plants can also be produced by tissue culture techniques for
uses in plant breeding, horticulture and forestry.
 Seedless fruits and vegetables can be produced by following the endosperm culture
method which add to their commercial values.
 Increased Nitrogen fixation ability can be achieved through association of tissue culture
techniques with genetic engineering.
 TISSUE CULTURE

[1]. Akin-Idowu PE, Ibitoye DO, Ademoyegun OT(2009) Tissue culture as a plant production
technique for horticultural crops. Afr. J. Biotechnol. 8(16): 3782-3788.
[2]. Christou P, Capell T, Kohli A, Gatehouse JA, Gatehouse AMR (2006) Recent developments
and future prospects in insect pest control in transgenic crops. Trends Plant Sci. 11: 302-308.
[3]. Ferrante E, Simpson D (2001) A Review of the Progression of Transgenic Plants Used to
Produce Plantibodies For Human Usage.Bio. & Biomed. Sci. Issue 1.
[4]. https://www.intechopen.com/books/recent-advances-in-plant-in-vitro-culture/plant-
tissue-culture-current-status-and-opportunities
[5]. Thorpe T (2007) History of plant tissue culture. J. Mol. Microbial Biotechnol. 37: 169-180.
[6]. James C (2008) Global Status of Commercialized Biotech/ GM Crops. ISAAA Brief No. 39.
Ithaca, NY. 243.
[7]. Navarro-Mastache LC (2007) Large scale commercial micro propagation in Mexico. The
experience of Agromod, S.A. de C.V. Acta Horti. 748: 91-94.
[8]. Pareek LK (2005) Trends in Plant Tissue Culture and Biotechnology. Jodhpur, India.
Agrobios. 350.
[9]. Vasil IK (1994) Molecular Improvement of Cereals. Plant Mol. Biol. 25: 925-937.